Scientific Reports of the
Hokkaido Salmon Hatchery
No. 50: 1-99, November 1996

The Pathobiology of Ectoparasitic Protozoans
on Hatchery-Reared Pacific Salmon*
Shigehiko Urawa
Research Division,Hokkaido Salmon Hatchery, Fisheries Agency of Japan
2-2 Nakanoshima, Toyohira-ku, Supporo 062, Japan

Abstract. - The present study was conducted to determine the occurrence and geographical distribution of external protozoan parasites on hatchery-reared Pacific salmon (chum, pink, maw and sockeye salmon) in northern Japan, and to clarify their potential impact on fish populations, and host-parasite interactions such as the defense mechanism of host fish. A flagellate Ichthyobodo necator and two
ciliates Trichodina truttae and Chilodonella piscicola arc widespread in salmon hatcheries. The infection experiments indicate that these thrce protozoan parasites are cpidemic pathogens capable of causing large economic losses both in hatchery and wild conditions during the course of artificial salmon
ranching. Especially, I. nerator infection has high potential to cause mass mortalities among anadromous salmonids during their initial marine life. The virulence of the ectoparasitic protozoans is affected by the mode of attaching and feeding, parasite intensity, host condition, and environments. The
ideal goal should be to control parasite infections in hatcheries on the basis of these biological information.

Contents],Background ...............................................

2-- - 5
3.1 Morphology, taxonomy and seawater adaptability - - - - 1.5
3.2 Host-,yxasite interactions ............................
23
3.3 Effects on seawater survival of anadromous fish
- - - - - 33

2. Host range and geographical distribution of ectoparasites
3 . Ichthyohodn necator-* * * * *

* *

*

*

* *

*

*

*

3.4 Effects of rearing conditions. .........................
40
3.5 Controls.
46
4. Trichodina truttae
51
4.1 Morphology and taxonomy.
4.2 Pathogenicity and host-parasite interactions
*
*
* *
57
5 . Chilodonella piscicola
5.1 Morphology and pathogenicity. * * * . * * * * * * * * * * * 66
76
6, Concluding remarks
Summary
78
Acknowledgments
80
References
80
Appendixes
88.........................................

..........................

-- - - - - -- - - .......................................

..................................................
..........................................
.................................................
................................................

Abstractin Japanese.........................................

*A thesis submitted for the degree of Doctor of Philosophy.

Published papers are reproduced with kind permission fromthe publishers (refer tn Acknowledgments).

Contribution A No. 354 from the Hokkaido Salmon Hatchery

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2 If . I-fAWf$$SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

1. Background
Pacific salmon
The Pacific salmon (genus Oncorhynchus) are
one of the most valuable biological and economic
resources in the North Pacific region. They are composed of seven species: pink (0. gorbuscha), chum
( 0 . k e t a ) , sockeye ( 0 . n e r k a ) , chinook (0.
tshawytscha), coho (0.kisutch), maw (0.masou),and amago (= biwamasu, 0. rhodums) salmon (Grootand Margolis, 1991). The first five species reproduce
both in the Asia and North America, and the last two
occur only in Far East Asia. Pacific salmon havebeen transplanted beyond their natural distribution in
the Northern Hemisphere and also in the Southern
Hemisphere.
Life history of Pacific salmon varies withspecies. However, they spawn in gravel beds in fresh
water, and young salmon generally migrate to sea
after an early freshwater life, being widely distributedin the North Pacific Ocean and Bering Sea. After several years of the marine life, they migrate to theirnatal streams or lakes, where spawning takes place
between late summer and early winter. After which,most of spawners die.
Pacific salmon are harvested commercially inthe coastal waters and high seas of the North Pacific
Ocean and Bering Sea. They are also caught by sport
fishermen in coastal waters. Due to overfishing andhabitat deterioration, salmon stocks were depleted
early in this century. Thus, salmon enhancement programs have been rapidly developed for the last two
decades in the North Pacific rim nations (USA,
Canada, Japan and Russia) to supplement natural production (Mahnken et al., 1986; Urawa andKaeriyama, 1986). Production of farmed Pacificsalmon is also increasing in Japan, Canada and USA,and also in the Southern Hemisphere such as Chileand New Zealand.

hatchery releases: more than two billion chum fry are
annually released from about 300 hatcheries in northern Japan. Pink salmon are enhanced along the
Okhotsk Sea and Pacific coasts of Hokkaido withannual returns of 2-4 millions. Both species are
released from hatcheries after several months of feeding, immediately go downstream to marine estuaries,
and migrate to the high seas (Salo, 1991; Heard,1991). The period of their marine life varies from two
to five years in chum salmon, but only one year inpink salmon.
In addition to these species, anadromous masu
and sockeye salmon enhancement has been recently
developing in Hokkaido. Both fish species exhibit agreater variety of life history patterns than pink andchum salmon (Burgner, 1991; Kato, 1991). The life
history of masu salmon is different between anadromous and nonanadromous types. Anadromous maw
migrate seaward after one or two years of freshwater
life in rivers, and return to home stream after the oneyear marine life (Kato, 1991). Thus, young masu
salmon are released in rivers during the juvenile stage(age 0.0, age designation follows to the European
system defined by Koo, 1962) within one year ofrearing or the smolt stage (age 1.0) after more than
one-year rearing (Mayama, 1992). Although sockeye
salmon are primarily anadromous, there are distinct
populations called kokanee which reproduce in freshwater lakes without any marine life. Anadromous
sockeye smolts (age 1.0) are released only i n
Hokkaido (Urawa, 1991; Kaeriyama et aI., 1992), andkokanees are enhanced in several lakes in Honshu
and Hokkaido (Kaeriyama, 1991).
While salmon enhancement has been wellextended, the major number of released salmonids
still die in wild environment before their harvest or
spawning. It is an important task of fisheries biologists to determine factors contn buting to natural mortality in salmonid populations.

Salmon enhancement in Japan
Five species (pink, chum, sockeye, masu, andamago salmon) are native in Japan and artificially
propagated, and coho salmon have been introduced
from North America mainly for marine net farming.
Chum salmon is most abundant among them,and its recent harvest has reached to approximately
sixty million adults in the Japanese coastal waters(Kaeriyama, 1989; Kaeriyama and Urawa, 1992).
This increase in harvest is attributable to intensive

Parasites and disease problems
The rapid development of salmon enhancement
increases the risk of disease outbreaks in hatcheries
and probably also in natural waters. The scientificaspect of salmon culture has been greatly advanced
during the last three decades. This has resulted inconsiderable progress in the development and application of various scientific principles in preventing orcontrolling many disease conditions (Wood, 1979).
Most of these researches seem to have focused on-2

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

viral or bacterial diseases such as infectious
hematopoietic necrosis (IHN) caused by a rhabdovirus or furunculosis by Aeromonas salmonicida.
In addition, the impact of infectious agents on wild
salmonid populations is poorly known.
Although more than 175 species of parasites
have been recorded from Pacific salmon within theirnative range (Margolis, 1982), relatively little attention was paid on these parasites from the aspect of
disease agents. The principal parasitic diseases ofhatchery-reared salmonids appear to be caused byprotozoans (Molnir, 1987). Ectoparasitic protozoans
often bring about severe problems in intensive culture
because they can rapidly multiply and be directlytransmitted from fish to fish in such conditions.
Detection of external protozoans may be relatively
easy, but it does not directly lead to a decision making for control or treatment due to their complex relationship with host fish and lack of taxonomic andbiological information for determining theirpathogenic potentials. Thus, the decisions are stilllargely dependent on the experience and techniquesof the personnel involved.

gills of salmonids, resulting in host mortalities(Robertson et al., 1981). Robertson (1985) consideredthat Ichthyobodo infection is probably the major
cause of acute mortality of cultured salmonid fry in
Scottish fish farms rather than viral infection. Despite
the economical importance of this parasite, there has
been relatively little research carried out on aspectsof its biology. Our knowledge is poor about host-parasite interactions such as the parasite's potential
impact on fish populations and the defense mechanism of host fish. The environmental factors affecting
the outbreaks of these parasite diseases have also notbeen understood yet. In addition, there are some confusion on the taxonomy of I. necator in relation to its
seawater survivability, since similar bodonid flagellates occur on marine flatfish (see Urawa et al, 1991)
and also on octopus (Forsythe et al., 1991).
The trichodinid T. tvutfae (Ciliophora:
Peritrichida) has been found on salmonids reared athatcheries along the coasts of the North Pacific
Ocean and adjacent seas (Davis, 1947, 1953;
Bogdanova, 1963, 1967, 1977; Arthur and Margolis,
1984). Mortalities associated with heavy infections
were recorded in hatchery-reared juveniles(Bogdanova and Shtein, 1963; Takeda et al., 1969;
Takeda, 1971; Hoskins et al., 1976), while
MacMillan (1991) considered that trichodinids areprobably least harmful among external protozoans
that commonly appear on cultured fishes. No infection experiment has been conducted to confirm thepathogenicity of trichodinids and the host-parasite
interactions.
The holotrichous ciliate C. piscicola (= C. cyprini) (Ciliophora: Cyrtophorida) is distributed throughout the world and commonly found on the gills andskin of various freshwater fishes (Hoffman, 1978).
The parasite occasionally causes severe proliferationof the gill epithelium, but some doubts have been caston its obligate pathogenicity. Several workers suspected that the parasite causes disease conditions
only after the host has been significantly stressed(Lom and Nigrelli, 1970; Egusa, 1983; MacMillan,
1991). Thus, its pathogenic potentials need to beevaluated by transmission experiments.

Ectoparasitic protozoans
There have been few studies to examine the parasite fauna of hatchery-reared salmonids in Japan,
although several protozoans were reported fromfarmed salmonids (Sano, 1966, 1970a, 1970b; Hara,
1972) and Awakura (1989) reviewed the parasite
fauna of wild masu salmon. The present intensive
survey indicates that the ectoparasite fauna of hatchery-reared salmonids is predominated by three protozoans: a flagellate Ichthyobodo necator (Henneguy,1883), and two ciliates Trichodina truttae Mueller,
1937 and Chilodonellapiscicola (Zacharias, 1894).
The bodonid flagellate I. necator (= Costia
necatrix) (Sarcomastigophora: Kinetoplastida) is
widely distributed in the Northern Hemisphere andinfects a wide variety of freshwater fishes and marine
salmonids. In Japan, this parasite was first recorded
from cyprinids by Suzuki (1938, 1942) and later
found also on farmed salmonids (see Nagasawa et al.,1987). While several authors reviewed a biology ofthis parasite (Bauer, 1959; Becker, 1977; Robertson,1985), in general, its importance seems to be underestimated because it is so often missed by scientists
and fish farmers due to its small size and absence ofapparent symptoms in infected fish. Heavy infections
cause severe epidermal destruction in the skin and

The objectives of the present study
The first purpose is to determine the host range
and distribution of external protozoan parasites onhatchery-reared Pacific salmon in Japan. Second, thepathogenic effects of these parasites is evaluated pre--3-

3 I f . 2 bAWfk SCI REP HOKKAIDO SALMON HATCHERY, NO 50,1996

some useful information that could make it possible
to avoid major losses of salmonids in both wild andhatchery conditions.

cisely by infection experiments. Third, biological factors important for control of these protozoans areinvestigated. This framework study will provide-4

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

2. Host Range and Geographical
Distribution of Ectoparasites
Abstract. - The occurrence of Ichthyobodo necator, Trichodina truttae and Chilodonella piscicola
was investigated on juvenile Pacific salmon (chum, pink, masu and sockeye salmon) reared at 204
hatcheries in northern Japan. These ectoparasitic protozoans were widespread in the investigated area
at water temperatures between 2"and 15°C. Ichthyohodo necator was most common among the threeparasites, being recorded from all four salmonid species. Chilodonelka piscicola was found on thc former three fish species, and T. truttae was encountered on only chum salmon. The percentage of positive hatcheries was 37.3% for I. necator, 15.2% for T. trultae, and 8.8% for C. piscicola. The occurrence of Ichthyohodo infections was not related to type of water supply (spring, well, infiltrate, or riverwaters), which implies that there are other mechanisms to spread the parasite among host populations
besides dircct transmission. In contrast, the majority of Trichodina and Chilodonella infections
occurred at hatcheries supplied with river water, suggesting that wild fish may serve as the rescrvoirsof infection. Statistical comparisons indicated the existence of a possible interference cornpctition
betwcen 1. necator and T. truttae on the host body surface.

lations.

Introduction

Materials and Methods

Ectoparasitic protozoans often bring about
severe problems in intensive fish culture because they
can rapidly multiply and be directly transmitted insuch conditions. T h e y i n c l u d e the f l a g e l l a t eIchthynhodo necator (Henneguy, 1883), and the ciliates T r i c h o d i n a t r u t t a e Mueller, 1 9 3 7 a n d
Chilodonella piscicola (Zacharias, 1 894). These
ectoparasitic protozoans are important pathogens ofhatchery-reared salmonids, responsible for mass mortalities and large economic losses. Their epizootiology has, however, received relatively little attention.
The present study describes the host range and geographical distribution of the ectoparasites infecting
hatchery-reared salmonids in northern Japan, and discusses their dispersal mechanisms among host popu-

F i s h w e r e sampled f r o m 2 0 4 f r e s h w a t e r
hatcheries in six prefectures of northern Japan during
the period of March 1982 - May 1991 (Appendix
tables 1 & 2). A total of 9,449 fish were collected,
comprising 8,660 chum (Oncorhynchus keta), 391
pink (0.gorbuscha), 65 sockeye (0.nerka, including
kokanees) and 333 masu (0. rnasou) salmon (Table
2.1). The majority of these samples were underyearling juveniles within 3 months from the first feeding.
The fish were immediately fixed in 10% formalin at
the hatcheries and preserved for later parasitologicalexaminations in the laboratory (Hokkaido Salmon
Hatchery). The type of water supply and water temperatures in rearing ponds were also recorded. The

Table 2.1. The occurrence of cctoparasitic protozoans on 4 species of salmonids reared at hatcheries in Hokkaido and northern

Honshu.

Host species

No of positive hatchencs*'

No. ofhatcheries
studiedIchthyobodo
necatorTrichodina
truttae

Chilodonella
prscicola

Toid noof fishexamincd

Hokkaido

Chum salmon
Pink salmon
Masu salmon
Sockeye salmon*'41 (34.7)
3 (17.6)
9 (75.0)19 (16.1)

17
12

3

7s

118

6 (5.1)l(S.9)

5,209

0 (0)

0 (0)

5 (41 -7)

333l(33.3)

0 (0)

0 (0)

65

30 (40.0)

12 (16.0)

7 (9.3)

3,451

39 1

HonshuChum salmon"'Number of hatcheries where each parasite was recorded (proportionof positive hatcheries in W )"'Sockeye salmon includes kokanees.

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2 i f . 3 TAM%. SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

classification of water supply was in accordance withthe concept defined by Shimizu (1984). Ichthyobodo
necator is most abundant on the fins (Urawa, 1992a),and C. piscicolu is usually defined to the gills (Urawa
and Yamao, 1992). After measurements of fish size
(folk length and wet weight), the dorsal and anal finsand gills were removed from fish and examined forparasites with a light microscope at magnifications of
X 200-400. In addition, sediment in the bottom of
the sample bottles was examined for T. truttae and C.
piscicola because these ciliates are easily detachedfrom their hosts in formalin solution. Parasitologicalterms follow the definitions given by Margolis et al.

(1 982).

ResuItslchthyohodo necator
Host range. This parasite was recorded from all
four species, chum, pink, masu and sockeye salmon,
but there was a slight difference in its occurrence
among the host species (Table 2.1). The prevalenceof infection averaged 43.0% in masu, 27.0% in sockeye, 24.3% in chum, and only 1.7% in pink salmon(Appendix tables 1 & 2).
Distribution. The parasite was quite widely distributed in northern Japan (Figs. 2.1 & 2). It was

14 %Fig. 2.1. The geographical distribution of Ichthyobudo necator at salmon hatcheries in Hokkatdo. Solid circles indicate the locations ofpositive hatcheries where the parasite was recorded, and open circles indicate the locations of negative hatcheries. Numerals referto hatcheries listed in Appendix table I. Large arrows indicate the boundaries between regions. Percentages express the proportjonof positive hatcheries in each coastal region.

6 -URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

found at 46 hatcheries (36%) studied in Hokkaido,
and at 30 hatcheries (40%) in northern Honshu(Table 2.2). The proportion of Zchthyobodo-positive
hatcheries was relatively high (48-64%) along the
Pacific coasts of Hokkaido (regions 5 & 6) and theJapan Sea coasts of Honshu (regions 7 & 9). In particular, the infection was most prevalent along thecoasts of inlets such as Volcano Bay (Uchiura wan)in the Pacific west region of Hokkaido and Mutsu
Bay in Aomori Prefecture, where the proportion of
positive hatcheries was 66.7% and 75.0%, respective46'
N

ly. Annual surveys confirmed that infections occurred
every year at several hatcheries (e.g., Chitose, Yoichi
and Hiroo hatcheries; Appendix tables 1 & 2).
Environment. The parasite infections broke out
in hatcheries with all the types of water supply (Table
2.3). The frequency of infections (number of parasitepositive groups/total number of sample groups) wasapproximately the same (18.2-28.6%) among individUdl type of water supply (spring, well, infiltrate, or
river waters), but increased to 44.4-71.4% when riverwater was mixed with other types of water supply.

140' 142* 144- 146'E

Tsugaru
Strait A

44'

42'
40"

38'

27%

i

40krn

- O

Fig. 2.2. The geographical distribution of lchthyobodo necatur at salmon hatcheries in northern Honshu. Solid and open circles indicatc the locations of positivc and negative hatcheries, respectively. Numerals refer to hatcheries listed in Appendix table 2.Large
arrows and dotted lines indicate the boundaries between regions and prefectures, respectively. Percentages express the proportionof positive hakhenes in each coastal region.

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2 - F A m l SCI. REP HOKKAIDO SALMON HATCHERY, NO. 50,1996

Table 2.2. The occurrence of ectoparasitic protosoans on juvenile salmonids at hatcheries in different coastal regions of Hokkaido andnorthcrn Honshu.

__~

No. of positive hatcheries*

No. ofRegionhatcheries

Ichthyobodn

Trichodina

Chilodonella

necatortnittaepiscicola

21
27
29
I
21
24
129

7 (33.3)
8 (29.6)
8 (27.6)
1 (14.3)
12 (57.1)
10 (41.7)
46 (35.7)

7 (33.3)
2 (7.4)7(24.1)3 (14.3)

2 (8 3 )
19 (14 7)

0 (0)
0 (0)
2 (8.3)
1 I (8.5)

11
37
27
75

7 (63.6)
10 (27.0)
13 (48.1)
30 (40.0)

2 (18.2)
5 (13.5)5 (18.5)
12 (16.0)

2 (18.2)2 (5.4)3 (11.1)
7 (9.3)204

76 (37.3)

31 (15.2)

18 (8.8)

studied

Hokkaido
1 Nemnro Strait
2. Sea of Okhotsk
3. Sea of Japan
4. Tsugaru Strait
5. Pacific Ocean (west)
6. Pacific Ocean (east)
Subtotal

Honshu7. Tsugam Strait
8. Pacific Ocean
9. Sea of Japan
Subtotal
Totall(3.7)

5 (17.2)0 (0)1 (4.8)*Number of hatcheries where each parasite was recorded (proportion of positive hatcheries in %)Table 2.3. The frequency (%) of ecloparasitic protozoans on hatchery-reared juvenile salmonids in northern Japan with dirkrent types
of water supply.
Frequency* (%jType ofwater supply
Spring water (S)Well water (W)s + w
Infiltrate water (I)
W+I

S+I
River water (R)

S+R
W+R

S+W+R
I+R
S + Lake water

Water tempcrature ("CjIchthyobodo

Trichodina

Chilodonella

necatortruttae18.2 (lo/%*)24.4 (20/82)
25 0 (2/8)
28 6 (6/21)
57.1 (4/7)
100.0 (1/1)
25.0 (11/44)
45.6 (26/57)
53.4 (39/73)
7 1.4 (5/7)
44 4 (4/9)
100.0 (1/1)

3.6 (2/55)
1.2 (1/82)
25.0 (2/8)
9.5 (2/21)
14.3 (1/7)
0 (0/1)
20.5 (9/44)
21.1 (12/57)
15.1 (11/73)
0 (0/7)
11.1 (1/9)
0 (0/1)

Discicola
3.6 (2/55)
6.1 (5/82)
0 (OIR)0 (0/21)14.3 (1/7)
0 (0/1)2.3 (1/44)
12.3 (7/57)
16.4 (12/73)
14.3 (1/7)
0 (0/9)0 (011)

Mean
8.0

8.1
7.8
8.5
9.6
5.5
7.3
5.8
6.9
6.4
9.8
1.8

*Number of groups in which each parasite was recorded/total numbcr of sample groups examined

x-

Range
5.0-10.4
2.0-13.6
7.0-9.0
6.0-10.4
8.4-11.7

3.0- 15.0
3.5-10.5
2.6-13.0
6.0-7.2
6.0- 12.4

URAWA - PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

2okq

Infections were found at a wide range of water temperatures (1.8"-13.6"C) (Fig. 2.3). The mean watertemperature recorded when infections were observed
was 6.1"C in Hokkaido and 8.7"C in Honshu.

30r

HOKKAIDO

lchthyobodo

HONSHUrlchthyobodo

100Trichodina truttae
Host range. This ciliate was recorded only fromchum salmon (Table 2.1).
Distribution. The parasite was found at 14.7% of
the hatcheries in Hokkaido and 16.0% of those innorthern Honshu (Table 2.2, Figs. 2.4 & 5). The proportion of Trichodina-positive hatcheries was almost
uniform (13.5-18.5%) throughout the regions ofnorthern Honshu, but varied considerably fromregion to region in Hokkaido, where it was high in
hatcheries along the shores of Nemuro Strait (region
1, 33.3%) and the Sea of Japan (region 3, 24.1%) but
low (0-8.3%) in those along other coastal regions(Table 2.2).
Environment. The distribution of T. truttae varied significantly with the type of water supply (Table
2.3). More than 80% of infections were observed inhatcheries where river water, solely or with otherwaters, was used. The water temperatures in which
the parasite was found were between 3.0"C and 15.0"
C, averaging 7.0"C in Hokkaido and 9.8"C in
Honshu (Fig. 2.3).

?!

Trichodina

Trichodina

Chilodonella

Childonella

10

Y

0
30

n

[

0

4

8

12

1 6 0

4

8

12

16

Water temperature ("C)

Fig. 2.3. The frequency histograms showing the occurrenceof Ichthyobodo necutor, Trichodina truttae, andChilodonella piscicola on hatchely-reared salmonids in
Hokkaido and northern Honshu at differcnt water temperatures. Total number of sample groups is 90 in
Hokkaido and 74 in Honshu. Solids and opens indicate
the percentage of infected and uninfected sample
groups, respectively. Arrowhcads represcnt the meanwater temperature at which the parasite was observed.

Chilodonella piscicola
Host range. This ciliate parasite was recorded
from chum, pink and masu salmon (Table 2.1). The

Table 2.4. The observed and expected coexistence among Ichthyobodo necutor, Trichodinu truttae, and Chilodonella piscicolu on
juvenile chum salmon reared at hatcheries using river water. The table includes three cases of concurrent infections with three
species.
No. of infections*'

Total
no. ofParan tes

Ichthyobodo
necutorTrichodina
truttae
Chilodonella
piscicola

Observed
Expected
Observed
Expected
Observed
Expected

Ichthyobodo
58 (72.5"')
48 (60.0)
12 (33.3)
21 (58.3)
10 (52.6)
11 (57.9)

Trichodina
12 (15.0)
21 (26.3)
17 (47.2)
10 (27.8)
7 (36.9)
5 (26.3)

ChilodonellulO(12.5)
11 (13.7)
7 (19.5)
5 (13.9)
2 (10.5)
3 (15.8)

infections
XO(100.0)
SO(100.0)36 (100.0)
36 (100.0)
19 (100.0)
19 (100.0)"'Expected coexistencc is calculated by the hypothesis that each parasite's probability of infection is essentially random, being notaffected by the presence of other parasite species. The number of expected cases or coexistence of parasite A with B (EC,,) is given by
the formula: EC4, = (ONBIN) X ONA,where ONA= total number of observed infections of parasiie A, ONB= total number of observed
infections of parasite B, and N = total number of observed infections of parasites (ONA+ ON, + ...).
*'Proportion (%) of infections.

9S if . 3 $A%% SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

proportion of Chilodonella-positive hatcheries in
Hokkaido was highest (41.7%) for maSu salmon, as
compared to only 5.1% and 5.9% for Chum and Pink
salmon, respectively. Most infections of maSu salmon
occurred during their first feeding period.
Distribution. This parasite was the least cornmon of the three species of ectoparasites, being foundin only 8.8% of the hatcheries surveyed in Hokkaido
and Honshu (Table 2.2, Figs. 2.6 & 7). There were noapparent regional differences in the proportion of
Chilodonella-positive hatcheries except for the
Hokkaido coasts of Tsugaru Strait (region 41 andPacific Ocean (region 5), where the ciliate was notrecorded.

Environment. The occurrence of outbreaks washighest (14.6%) under rearing conditions where riverwater was mixed with spring andor well water, as
compared with the other types of water supply (Table
2.3). The parasite was recorded at water temperatures
ranging from 3.0" C to 12.3" C, with an average of
6.5" C and 10.5"C in Hokkaido and Honshu, respectively (Fig. 2.3).
Interspecific competition
Table 2.4 compares the observed and expected
frequency of coexistence among the parasite species
on chum salmon reared at hatcheries with river water.
Distinct differences between predicted and observed

- O

40 km

0%
Fig. 2.4. The geographical distribution of Trichodina truttae at salmon hatcheries in Hokkaido. Solid and open circles indicate thelocations of positive and negative hatchenes, respectively. Numerals refer to hatcheries listed in Appendix table 1. Large m o w s
indicate the boundaries between regions. Percentages express the proportion of positive hatcheries in each coastal region.

10 -URAWA- PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

frequencies exist for the co-occurrence of I. necatorand T. truttae but not in other cases (C. piscicola vs I.
necator or T. truttae). The number of observed infections of I. necatnr concurrent with T truttae was 43%
lower than expected.
Other ectoparasites
A monogenean Gyrodactylus masu Ogawa,1986 was occasionally found on the fins of juvenile
chum, sockeye and maw salmon at Chitose Hatchery.
In addition, unidentified sessile peritrichs (Peri46'

140- 142- 144' 146'E

trichida) attached to the fins of chum salmon fry
reared at Asahi Hatchery on Rishiri Island, northern
Hokkaido, but the prevalence of infections was low(8.7%).

Discussion
The ectoparasite fauna of hatchery-reared
salmon juveniles examined here was predominantly
composed of three protozoan species, I. necator, T.
truttae, and C. piscicola. This simple fauna may be

Tsugaru
Strait n

N

44'
42'
40'

38'

14%

I

40 km

-O

Fig. 2.5. The geographical distribution of Trichodrnu frutfueat salmon hatcheries in northern Honshu. Solid and open circles indicate
the locations of positive and negative hatcherics, respectively. Numerals refer to hatcheries listed in Appendix table 2. Large
arrows and dotted lines indicate the boundaries between regions and prefectures, respectively. Percentages express the proportionof positive hatcheries in each coastal region.

11 -

2 If

d -d-J$f% SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

closely related to the short rearing period of the fish:
the majority of juvenile chum and pink salmon are
released after 2-3 months of feeding. It also seems to
be limited by low rearing water temperatures below
15°C. For example, the large ciliate Ichthyophthirius
rnultz$liis occasionally causes considerable problems
at trout and salmon farms in North America (Wood,1979) and Europe (Bauer, 1959; Bogdanova, 1976;
Valtonen and Keraen, 1981), but in northern Japan
this ciliate was not observed perhaps due to the cold
rearing water, whose temperature is considerably
below the parasite's optimum (24" to 26"C, accord-46"

ing to Bauer, 1959).
The three protozoan species were widely recorded in northern Japan at water temperatures ranging
from 2" to 15°C. Although there were distinct differences in rearing temperatures between Hokkaido andHonshu, the frequency of infections with each parasite species was similar in both areas. Bauer (1959)
mentioned that C. piscicola divides most actively at
5-10°C. This is almost consistent with the temperature range for Chilodonella infections observed in thepresent survey. On the other hand, Becker (1977)
noted that Zchthyobodo survives at temperatures from140' 142' 144- 146'E

N

4 4"
42'

40
38'

u /o
Fig. 2.6. The geographical distribution of Chilodonella piscicoh at salmon hatcheries in Hokkaido. Solid and open circles indicate thelocations of positive and negative hatcheries, respectively. Numerals refer to hatchenes listed in Appendix table 1. Large arrows
indicate the boundaries between regions. Percentages express the proportion of positlve hatcheries in each coastal region.

- 12 -

URAWA-PATHOBTOLOGY OF PARASITlC PROTOZOANS ON SALMON

and found on wild freshwater fishes in Hokkaido(Nagasawa et al., 1989). There was, however, a slightdifference in the frequency of their occurrence among
the salmonid hosts. These parasites were found mostfrequently on the first-feeding maw salmon, butrarely on juvenile pink salmon. In addition, T. truttae
was found only on chum salmon. These results suggest that host susceptibility to each species of parasite
may differ among salmonid species. Tavolga andNigrelli (1 947) observed in their infection experiment

2°C to 29°C and multiplies most rapidly at about 2425°C. At salmon hatcheries, however, infections
were frequently encountered at temperatures between
1.8"C and 13.6"C, suggesting that I. necator on
salmonids is well adapted to lower temperatures as
pointed out by Robertson (1985).
Ichthyohodo necator and C. piscicola wererecorded from four and three salmonid species,
respectively. These two parasites are known to have awide host range (Hoffman, 1978; Robertson, 1985)

46"
N

140' 142' 144' 146"E

Tsugaru
Strait A

44'
42"
40'
38"

5%

i

40km

- O

Fig. 2.7. The geographical distribution of Chilodonella piscicola at salmon hatcheries in northern Honshu. Solid and open circles indicate the locations of positive and negative hatcheries, respectively. Numerals refer lo hatcheries listed in Appendix table 2. Large
arrows and dotted lines indicate the boundaries between regions and prefectures, respectively. Percentages express the proporlionof positive hatcheries in each coastal region.

- 13

3 1j . S T M % % SCI REP. HOKKAIDO SALMON HATCHERY, NO 50,1996

that different species of fish vary in susceptibility andresistance to I. necator.
It is well recognized that I. necator is a common
pathogen in trout and salmon farms in North America
and Europe (Wood, 1979; Robertson, 1985), whereas
this small flagellate has been overlooked in Japanesesalmon hatcheries until quite recently. The present
intensive survey shows that I. necator is the most epizootic ectoparasite on hatchery-reared salmonids inJapan. The transmission of the parasite by transportof infected live fish was confirmed at one hatchery,
however this probably does not account for the wide
distribution of this parasite since live fish are rarelytransported between hatcheries in the study area. The
parasite is able to survive and multiply in seawater(Urawa and Kusakari, 1990). The expansion of parasite’s distribution may be promoted by this seawater
adaptability, while it seems to be also encouraged byother spread mechanisms.
It is generally considered that ectoparasitic protozoans usually transmit themselves directly fromhost to host. This implies that the occurrence of parasites may be influenced by the type of water supply(spring, well, filtered, or river waters). In the case ofT. truttae, the majority of infections occurred in theponds supplied with river water. A separate observation confirmed that wild salmonids were infectedwith T. truttae (Urawa, unpublished data). Thesefindings indicate that wild fish serve as the main
source of Trichodinu infections in hatcheries. A similar trend was also observed in C. piscicola infections.
Both ciliates occurred frequently along the coast of
Nemuro Strait where about 80% of hatcheries usedriver water, but they were rarely found in Iwate
Prefecture where only 11% of hatcheries used riverwater. Thus, the distribution of these ciliates inhatcheries is affected by type of water supply.
On the other hand, the frequency of Ichthyobodo

infections was most stable among individual types ofwater supply and increased when mixed waters wereused. This increase seems to just reflect the sum ofrisks in each types of water supply. Infections often
occurred in isolated ponds supplied with spring orwell water, in which wild fish were absent. Thus,
besides the direct transmission, there may be anyother mechanisms by which lchthyohodo spreads
among host populations. Htond (1963) described thepresence of I. necator on carp eggs. Some workers
believe that the flagellate forms cysts in unfavorable
conditions which remain in the water as well as on
the host’s body (see Bauer, 1959). However, these
phenomena have not been confirmed by infection
experiment, electron microscopy or other techniques.
Both of I. necator and T. truttae are site-specificto the body surface of their hosts (Urawa, 1992a,
1992b). The observed coexistence of I. necator with
T. truttae was significantly lower than expected, suggesting the presence of interference competition
between these parasites. This assumption was corroborated by the experimental observation that T. truttae
reduced the density of I. necator on the skin of hosts(Urawa, unpublished data).
Trichodina truttae and C. piscicola infections
may be controlled by using only spring or well waters
in which feral fish are absent, whereas this method
does not appear effective for the prevention of I.
necator. Heavy Ichthyobodo infections reduce
markedly the seawater adaptation of anadromous
hosts and consequently cause high marine mortalities(Urawa, 1993). It was confirmed that formalin bathtreatment of infected chum juveniles significantly
increased their return rate as adults (Urawa, 1996).
Thus, it is strongly recommended that anadromous
salmonids should be examined for parasites before
their release from hatcheries.

- 14 -

URAWA- PATHOBIOLOGYOF PARASlTIC PROTOZOANS ON SALMON

3. Ichthyobodo necator
3.1 Morphology, Taxonomy and Seawater Adaptability
Abstract. - Experimental studies revealed that a freshwater ectoparasitic flagellate lchthyobodo
necator could survive and reproduce in seawater after infected chum salmon fry (Oncorhynchus keta)

were transferred directly from fresh water to 33"/00seawater. Minor morphological changes (slight
reduction in body width, loss of twistlike wrinkles on body surface, and reduction in contractile vacuoles) were observed in the attached form of I. necator following transfer to seawater.The field survey
also confirmed that I. necator occurs on chum salmon fry in seawater estuaries (salinity 17-34'/nn) andin freshwater habitats. It was assumed that 1. necator acquired salinity tolerance as a result of adapting
to the migratory behavior of its anadromous host. Two morphologically similar bodonids, I. necatorfrom chum salmon and Ichthyobodo sp. from marine Japanese flounder (Paralichthys olivaceus), weredifferentiated each other by cross infection experiments. Thus, the parasite from marine floundershould be regarded as a separate species from I. necator.

Introduction
The bodonid flagellate Zchthyobodo necator isregarded primarily as a freshwater fish ectoparasitewith a broad host range (Becker, 1977). This protozoan was observed on sea-cultured salmonid smolts
in Scotland (Ellis and Wootten, 1978; Roubal et al.,1987) and Canada (Kent, 1992). It was suspected thatthe parasite can survive on infected fishes transferredfrom fresh water to seawater (Ellis and Wootten,
1978; Needham and Wootten, 1978). The parasite
was found also on several marine flatfish, namely,
wild plaice, Pleuronectes platessa, off the coast ofwest Scotland (Bullock and Robertson, 1982;
Bullock, 1985); winter flounder, Pseudopleuronectes
americanus, off Newfoundland, Canada (Cone andWiles, 1984); and Japanese flounder, Pat-alichthys
olivaceus, reared a t the Hokkaido Institute ofManculture, Hokkaido, Japan (Kusakari et al., 1985).
In these cases, the parasite was identified as I. necator because of morphological similarities. Bullock
and Robertson (1982) and Cone and Wiles (1984)
assumed that the parasite was of freshwater origin
and had adapted to the marine environment.
However, Morrison and Cone (1986) recently reported an Zchthyobodo infection on the gills of haddock,
Melanogrammus aeglejinus, caught in the northwest
Atlantic, 120 km offshore from Nova Scotia, andsuggested that the species found on marine fish
should be a marine variant of I. necator or a separate
species. This interpretation was supported by
Diamant (1987) who reported Zchthyobodo sp. from
common dab, Limcmdu Zimanda, in the North Sea, 30
km off Buckie, northeast Scotland. Thus, it is unclear

whether Zchthyobodo species on marine fishes are of
freshwater origin or a separate marine species. This
confusion is attributable to the lack of experimental
information on the survival of I. necator in seawaterand on its host specificity.
In the present study, survivability of I. necatorin seawater was examined by transferring infectedchum salmon fry, Oncorhynchus keta, from fresh
water to seawater, and the host specificity of I. necator from freshwater chum salmon and Zchthyobodo
sp. from marine Japanese flounder was tested bycross infection experiments.

Materials and Methods
Survivability in seawater
Survivability of I . necator in seawater wasdetermined in the laboratory using 1,000 chumsalmon fry (mean body weight 0.75 g), which had
been maintained in a 40-1 flow-through tank suppliedwith ground water. These uninfected test fish were
exposed to I. necator for 2 weeks by rearing together
with the infected chum salmon fry obtained from
Yoichi Hatchery (western Hokkaido, Japan). Afterconfirming that most test fish were infected with theparasite, 200 of these fish were transferred directly toa 60-1 tank supplied with filtered seawater (0.45 p m
membrane filters; salinity 33.4O/00; mean temperature
11.1 -t 0.3"C). The remaining fish were placed in a30-1 tank supplied with a constant flow of groundwater (salinity o.05°/~~;
mean temperature 10.5 5
0.1 "C). All fish were fed once a day with commercial
dry pellets (3% body weighUday) and held for another 4 weeks.
Ten fish from each group were sampled before- 15 -

2 If - Z -FAbftl SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996w
HOKKAIDO

Chitose River

ILake Shikotsu#

1

Chitose Hatchery

-

0

10

20km

141E

142E

Fig. 3.1.1. Map qhowing the location\ where chum salmon fry \amplea were collected in Hokkaido, northern Japantransfer (0 h) and at 1 h, 1, 2, and 4 weeks post-transfer. Pieces of the skin, fins, and gills were fixed in
1 % glutaraldehyde in 0.1 M phosphate buffer. Afterwashing in the buffer the tissues were dehydrated in
graded ethyl alcohols, and placed in isoamyl acetate.
Subsequent treatment involved critical point drying
and gold coating. The specimens were observed witha scanning electron microscope (SEM; Akashi
Instruments). The parasites were counted under the
SEM within a 0.25 mm quadrate at 113 different sites
on the skin, fins and gills (Fig. 3.1.3) and the parasite
density was expressed as a mean number.
Skin impression smears of infected chumsalmon fry both in fresh water and seawater were prepared by air drying and fixing in absolute methyl
alcohol for 2 min. These smears were stained with
Giemsa stain (buffered at pH 6.4) for 25 min.
Measurements of these specimens were made withthe aid of a camera lucida.
Host specificity
The host specificity of I. necator from chumsalmon and Ichthyobodo sp. from Japanese flounderwas examined by cross infection experiments.
Juvenile Japanese flounder (mean body weight 0.85g) and chum salmon fry (1.22 g), both with no historyof Ichthyobodo infection, were used in 2 experiments.
In the first experiment, 100 juvenile flounderand 100 salmon fry were held together in a 60-1 tankequipped with recirculating seawater, and they
cohabited with 30 caged chum salmon fry infectedwith I. necator. In the second experiment, the same

number of juvenile flounder and salmon fry held inrecirculating seawater cohabited with five caged
Japanese flounder (1 year old, mean body weight
12.5 g) carrying Ichthyobodo sp. The infected chumsalmon and flounder were obtained from Yoichi
Hatchery and Hokkaido Institute of Mariculture,
Shikabe, Hokkaido, respectively. Three weeks after
the exposure, fish of each species in each experimentwere placed in a different tank to avoid accidental
transient infections, and they were held separately for
3 more weeks. Seawater was filtered with 0.45 ,um
membrane filters and maintained at salinity 33.4°/~1(temperature 11.3
0 5 ° C ) . Every fish from eachgroup was sampled at 3 and 6 weeks after exposure,and their gills and fins were examined immediately
under a light microscope (approximately 5 min at X400) for the presence of the parasites.
For t h e morphological observations ofIchthyobodo sp. from the juvenile flounder, light andscanning electron microscope preparations weremade as described in the survival experiments.

*

Field observations
The distribution of I. necator was examined in
the Chitose River (a tributary of the Ishikari River)and its saltwater estuary of Ishikari Bay, western
Hokkaido (Fig. 3.1.1). In March and April 1986
about 30 million chum salmon fry were released into
the Chitose River from Chtose Hatchery. Four hundred fry were collected at 3 sites, Chitose Hatchery,
Maizuru, about 30 km down from the hatchery, andthe estuary (salinity 17.4-34.O0/nn) near the mouth of- 16 -

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

the Ishikari River during March, April and May
1986. Their gills and fins were examined microscopically (approximately 5 min per fish at X 400) to
assess the prevalence of 1. necator on chum salmon
fry both in freshwater and marine environments.

Skin

3000-

b
Q

$:::

G
UY 2000c
0)-0Resultsa,

5 1000-Survivability in seawater
Ichthyobodo necator survived and proliferated
in seawater (salinity 33.4%) after the direct transferof the infected chum salmon fry from fresh water toseawater (Fig. 3.1.2). The number of parasites on theskin and fins increased more slowly in seawater thanin fresh water during the first week, but from week 1
to 2 it increased exponentially to attain the same
abundance as that in fresh water. The parasites wereabundant on the skin and fins in both groups (Fig.
3.1.3), but a difference in the population of the parasites was detected on the gills between the fish heldin fresh water and seawater. Although the parasites
were rarely observed on the gills in fresh water
throughout the experimental period, the numbers ofparasites increased on the gills of fish transferred toseawater (Fig. 3.1.2).
The infected fry began to die in seawater atweek 2 and the mortality exceeded 40% by the end of
the experiment, whereas the total mortality remained
very low (0.8%) in fresh water.

5

a

k

$ 30001

Fins

h

I

I

I

1

2

4

r4

300

B

.2 200UJ

5

Ua,.z 100emaHost specqicity
First experiment. Ichthyobodo necator from
freshwater chum salmon fry was easily transmitted tothe same host species in seawater but not to juvenile
flounder even after 6-week exposure (Table 3.1.1j.
Second experiment. Only a few parasites were
found on the gills and fins of the salmon fry (preva-

0

Weeks after transfer
Fig. 3.1.2. Changes in mean parasite density of Ichthyobodo
necator on chum salmon fry in fresh water (open circles)
and seawater (solid circles) after being held with infectedchum salmon fry in fresh water for 2 weeks.

Table 3.1.1. Attempts to transmil Ichthpbodo necutor and Ichthyobudo sp. to chum salmon fry and Japanese flounder in seawater.
The test fish were exposed to the parasites for the first 3 weeks.
Results*
SourceFish tested

Ichthyobodo necutor

Chum salmon

from chum salmon
Ichthyobodo sp

Japanese flounder
Chum salmon

0 wk0110 (0)0/10 (0)0/10 (0)0/10 (0)3 wk10110 (100)0/10 (0)6 wk
10/10 (100)0/10 (0)6/20 (30)
0/29 (0)5/5 (100)
5/5 (100)*Fractions rcpresent the number of fish infested 10 number of lish examined with prevalences ( 8 )in parentheses at various weeks after
the start of experiment.
from Japanese flounderJapanese flounder- 17 -

2 if . d T-k;wH SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996/: \_________ :---------

2.1 - 3.0
0 >3.0%Fig. 3.1.3. Distributions of Ichthyobodu necator on chum salmon fry 1 week after transferring infected fish to fresh water (A) and seawater (B). The 26,232 and 17,426 parasites were counted on ten chum salmon fry in fresh water and in seawater, respectively.

lence 30%) in seawater at the end of cohabitation
period of 3 weeks with juvenile flounder infectedwith Icktkyobodo sp. However, they disappeared
from the fry within 3 weeks after removing theinfected flounder from the rearing tank. Many
Ickthyohodo sp. were attached to the fins and gills of
all tested flounder (prevalence 100%) at weeks 3 and
6 (Table 3.1.1).
Morphological observations
The attached form of I. necator was pyriform,
and no striking morphological change was apparent
in this parasite when infected fish were transferredfrom fresh water to seawater (Figs. 3.1.4-6).
Twistlike wrinkles were observed on the body surface in fresh water (Fig. 3.1.4), but they became
indistinct when transferred to seawater (Figs. 3.1.5 &6). The free swimming form was almost round withaverage length of 10.1 ,urn and width of 9.6 ,urn infresh water (Fig. 3.1.9), whereas in seawater it was
pyriform with a narrower width (P < 0.001) averaging 7.7 p m (Fig. 3.1.10). Contractile vacuoles were
frequently observed in the parasites in fresh water but
rarely in those in seawater.
Zcktkyobodo sp. from Japanese flounder was
also pyriform when it was attached to the epidermal
cells (Figs. 3.1.7 & 8). Its attached form was similar
in morphology to that of I. necator on chum salmon
except that it lacked wrinkles on its body surface.
The free swimming form was reniform and as for I.
necator there were 2 flagella of unequal length (Fig.
3.1.11). As shown in Table 3.1.2, Zckthyobadu sp.
had an almost same size as I. necator in seawater.

- 18 -

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Figs. 3.1.4-6. Scanning clcctron micrographs of Ichthyobodo necotur on the ventral fins of chum salmon fry. Each scale bar = 5 ,urn.
4. In fresh water. 5. In seawater 1 h after the tranqfer from fresh water. 6. In seawaler 2 weeks after the transfer.

19 -

2 If

5 %A48$$ SCI. REP. HOKKATDO SALMON HATCHERY, NO. 50,1996

figs. 3.1.7 & 8. Scanning clectroii micrographs o l ~ l ~ h / h ) ~ l J h ~'pl d t lon
the dorsal tins oi Japancc lloundcr in wm'atcr. Each wale har = 5:fin. 7. Ic~hrh\~obut/ob p . including a dividing I'orni carrow) 8.
f h h w h d osp. showing rhs I\W 1lqclla.

Figs. 3.1.9-11. Photomicrographs of Ichthyobodo spp. showing nucleus (n) and flagella (0.
Giemsa stain. Each scalebar = 5 pm. 9. Ichthyobodo necator from chum salmon in fresh water. 10. Ichthyohodo necator from chumsalmon in seawater. 11. Ichthyobodo sp. from Japanese flounder in seawater.

- 20

URAWA - PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

h

March

(%I

April

May

(%I

$ 30

30

r
0

Uninfected fish

I" 20

Infected f i s h

.w

8

20

10

10

30

30

ti 20

20

c

c

0

L

al

>

al
v)

0

10

10

30

30

;20

20

z
r
0
m
al

v)

.w
In

6CrJ 10

10

3.2

4.0

4.8

5.6 3.2
4.0
4.8
5.6 3.2
Fork length (crn)

4.0

4.8

5.6

Pig. 3.1.12. Prevalences of lchthyobodo necator and size frequency distnbutions of infected and umnfected chum salmon fry collccted
from Chitose Hatchery, the Chitoae k v e r and estuary of lshikan Bay in 1986. N, number of fish examned, P, prcvalence of inrection
Table 3.1.2. Meawrements* ( p m ) of Ichthjobodo necator trom chum salmon fry in fresh water and seawater, and those oflchthyohodo sp from Japanese flounder in seawaterSpecies
Host
Habitat

I necator
Chum salmon
Frcsh waterin = 60)

I necator
Chum salmon
Seawater(n = 50)

Ichthyvbodo sp
Japanese flounder
Seawater(n = 50)

Body length10.1 f 0.9(8.5-13.0)
9.6
0.9(8.0-11.5)
3.5 k 0.4
(2.9-4.5)

10.1 f 0.7*(9.0-12.0)
7.7 f 1.1
(6.0-9.7)
3.6 f 0.5
(2.5-4.5)

10.8 f 0.9(8.5- 13.0)
7 7 k 0.9
(6.0-10.2)
3.5 t 0.5
(2.8-4.2)

16.5 k 2.2

14.6 f 2.0

17.9 k 3.1(12.0-20.0)
9.7
17(6.0-14.0)(12.0- 19.5)
8.3
1.5(6.0-13.0)(12.0-26.0)
8.8
1.8(6.0-15.0)

Body width
Length of nucleus
Length oflong flagellum
Length ofshort flagellum

*

*

*

*Mean with standard dcviations followed by range in parentheses. Giemsa-stained free-swimming forms were measured- 21 -

5 It . 5 +A$J% SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

Field observations
Prevalences of I. necator and size-frequency distributions of infected and uninfected chum salmon fry
collected at 3 habitats are shown in Figure 3.1.12. At
Chitose Hatchery, 34% of reared salmon fry were
infected with 1. necator in March, but prevalence
decreased to 2% in April as a result of treatmentswith a formalin solution. During March and early
April, hatchery-reared fry were released directly into
the Chitose River, where the prevalences were 20%
and 34% in April and May, respectively. In the
Chitose River, the fry migrated to the sea within 1-4
weeks after the release (Mayama et al., 1982). In theestuary (salinity 17-34O/00) near the mouth of the
Ishikari River, the parasites were still attached to thegills and fins of salmon fry with prevalences of 32%
and 27% in April and May, respectively.

Discussion
The present experimental study shows that the
freshwater ectoparasitic flagellate I. necaror can survive and increase in number under marine conditions
after the direct transfer of infected fish from fresh
water to seawater. Among protozoan fish ectoparasites, I. necator seems to be the only one species that
occurs in both freshwater and marine environments.
A few morphological changes were observed in theattached form of I. necator when transferred from
fresh water to seawater. The parasite in seawater lost
twistlike winkles on its body surface, and had a narrower width and fewer contractile vacuoles than thatin fresh water. These changes may be attributable tothe differences in the surrounding osmotic pressure.
The field survey also demonstrated that I. necator
occurred on chum salmon fry in seawater estuary andfreshwater habitats. There was no apparent differencein the prevalence of parasite infections between chumsalmon fry in the river and seawater estuary. Dartnell(1974) reported that some trichodinids have similar
tolerance to salinity changes as their euryhaline host
(Gasterosteus aculeatus). Although the mechanismof osmoregulation of 1. necator is unknown, the present study suggests that the parasite acquires salinity
tolerance as a result of adapting to the migratory
behavior of its anadromous host.

With an increasing number of reports on theoccurrence of Ichthyobodo on marine fishes, the origin and taxonomic status of this marine form has
been confused. Transmission electron microscopic
studies (Diamant, 1987) showed that marine
Ichthyobodo sp. from wild common dab was ultrastructurally similar to 1. necator from freshwater fishes as described by Schubert (1966) and Joyon andLom (1966, 1969), although Roubal and Bullock(1987) observed morphological differences in theattachment disc and cytostome process between theparasites on salmonids in fresh water and seawater. In
my study with light and scanning electron microscopes, no difference was observed in morphology
between I. necator and Zchthyobodo sp. from chumsalmon in seawater and Japanese flounder, respectively. The experimental study reported herein, however, indicates that Japanese flounder was not susceptible to 1. necator originating from chum salmon.
Similarly, chum salmon was not susceptible toIchthyobodo sp. from Japanese flounder, although a
few parasites were observed on chum salmon as longas the parasites were supplied from infected floundercohabiting in the same tank. Thus, these two morphologically similar flagellates, I. necator andZchthyobodo sp., can be differentiated each other by
their host specificity in cross infection experiments. It
is, therefore, concluded that Zchthyobodo sp. fromJapanese flounder should be regarded as a different
species from I . necator, as proposed by Momson andCone (1986) for Zchthyobodo sp. from haddock.
Zchthyobodo necator infection causes mass mortalities of chum salmon fry in fresh water when combined with stressful stimuli such as crowding andpoor water quality (Urawa, 1987, 1995). During thepresent experiments, about 40% of the infected chumsalmon fry died when the parasite number increased
rapidly in seawater. In a separate experiment, it was
confirmed that the parasite also reduces the seawater
adaptability of seaward-migrating salmon fry becauseof skin epidermal destruction (Urawa, 1993). Thus, it
is supposed that losses of infected salmonids may
occur in marine habitats, even if the fish were readyto smolt. T o avoid this jeopardy, it is necessary to
remove the parasites from infected fish before releaseor transfer to seawater.

22 -URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

3.2 Host-Parasite Interactions
Abstract. - The number of the ectoparasitic flagellate Icktkyobodo necator on chum salmon fry(Oncorkynckusketa) and the host's epidermal responses to infection were monitored for 15 weeks withlight and scanning electron microscopy. Parasite density on the skin peaked at 3 weeks post-infection,at an average of 625 parasites/mm2,and then decreased to near zero by week 10. Alcian blue (AB, pH
2.5)/periodic acid-Schiff s reagent (PAS) staining procedure revealed dramatic changes in both thedegree and histochemistry of epidermal mucus secretion during the course of the parasite infections.
The number of mucus cells declined significantly during the first 4 weeks as parasite density increased,
but thereafter rose to 2,000 cells/mm' by week 8, when mucous cells stained with PAS alone predominated. This increase occurred concomitantly with a reduction in parasite density. In the control fish, theconcentration of epidermal mucous cells was stable at 400-800 cells/rnm2during the same period andacid mucopolysaccharides (PAS-negative, AB-positive) were a major constituent of the mucous cellsecretion. It is suspected that the PAS-positive mucous cells may play an important role in the defense
mechanism against the parasite infection.

Introduction
Ichthyohodo necator is an important pathogenicparasite being responsible for host mortalities andlarge economic losses, but the host's defense mechanism has not been clarified yet. Robertson (1979)
reported that numbers of I . necutor markedly
decreased shortly after a peak of infection which wasobserved at 4 weeks after the first feeding in juvenile
rainbow trout (Oncorhynchus mykiss). A similar
decrease i n intensity was observed in juvenile
Atlantic salmon (Salmo salarj by Wootten and Smith(1980) and in carp fry (Cyprinus carpio) by Migala(197 1). It is speculated that immunity develops tosuppress the infection (Robertson et al., 1981).
A remarkable reduction in the number ofmucous cells due to Ichthyohodo infections has been
observed in several fish species (Robertson et al.,
1981; Awakura et al., 1984; Diamant, 1987; Kusakari
and Urawa, 1990; Urawa et al., 1991). Other reports
have described a similar loss of mucous cells associated with other ectoparasites (Hines and Spira, 1974;
Logan and Odense, 1974; Pottinger et al., 1984;
Wells and Cone, 1990). On the other hand, Ellis andWootten (1978) observed an increase in concentration of periodic acid-Schiff s reagent (PAS) -positive
mucous cells in salmonid gills affected by I. necator.
A similar proliferation of PAS-positive mucous cells
was also found in the gills of Japanese flounder(Paralichthys olivaceus) infected with the marine ciliate Cvptocaryon irritans (Kaige and Miyazaki,1985). Pickering and Fletcher (1987) noted that
Ichthyobodo infection induced an increase in thenumber of sacciform cells whose secretion mightarrest the infection and reduce the damage caused by
the parasite. Thus, the epidermal secretions of certain
fish may be important in defence against invading
pathogens.
The present study was designed to monitorchanges in density of I. necator on chum salmon fry(O nc or hy nc hus k e t a ) and the host's epidermal
response in order to understand more fully the function of epidermal mucus secretion.

Materials and Methods
Fish
Chum salmon fry (mean body weight 0.26 g)which had no previous parasite infections were used
for the present experiment. Two groups (each containing 1,400 fish) were held separately in 23-1 tanks.
One group was exposed to I. necator by cohabiting
with 10 infected chum fry obtained from Yoichi
Hatchery in western Hokkaido. The other groupserved as controls. Each tank was supplied with well
water at a constant flow rate of 1.3 l/min. Water temperature was constant at 10.5"C throughout theexperimental period (15 weeks). The fish were fed
twice daily with commercial dry pellets at about 2%
body weight per day.
Light microscope preparations
Five fish were sampled from each group at intervals of 1-3 weeks and preserved in Bouin's solution.

Fixed tissues were processed by standard histological
techniques, embedded in paraffin blocks, and transversely sectioned at 6 p m at six different sites of the
fish body (Sl-6 in Fig. 3.2.2). Sections were rinsed in
3% acetic acid (pH 2.5) for 2 min and stained with23 -

3 If ' 2 TAW% SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

water, the sections were stained with hematoxylin for
10 min, followed by a final 1 5 4 1 1 rinse in runningwater. The sections were dehydrated, cleaned, andthen mounted in Bioleite (Ohken Co.).
Epidermal mucous cells stained either blue (ABpositive, PAS-negative), purple (AB and PAS-positive) or deep red (AB-negative, PAS-positive).
Differential cell counts were made on sections fromeach piece of skin from 10 random quadrates (0.25

1% Alcian blue (AB) in 3% acetic acid at pH 2.5 for
10 min. After a 5-min wash in running water, thestained sections were fixed in 0.3% sodium carbonate
for 30 min. Following a 10 min rinse in runningwater the sections were oxidized for 10 min in 0.5%
periodic acid, washed for a further 10 min, and thenplaced in Schiff's reagent (PAS) for 10 min. The
reaction was stopped with three 2-min baths of 0.05
M sodium bisulphite. After a 10-min rinse in running+ Gills
800
600
a

E
E

400

> 200
v)

!

0

P

2000

w-

0
0

1600

2

1200

800
400

0
1 2 3 4 5 6

8

10

12

15

Time (weeks)
Fig. 3.2.1. Changes in mean density of lchthyobodo necator on the gills, skin, and fins of chum salmon fry Vertical lines indicate SD.

- 24 -

URAWA- PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

s4

0

1.i-2.0

2.1 -3.0
0 > 3.0 %Fig. 3.2.2. Frequency distributions (70)of Irlzthyohodo necafor on chum salmon fry at 1, 3 , and 8 weeks post-infcction. Arrows (S1-6)
indicate thc locations at which thc skin was transvcrscly sectioned for histological obscrvationsmm’), and the concentration and proportion of
mucous cells visible with three different staining
reactions were calculated. Additional histochemical
techniques (see Sano, 1972; Blackstock andPickcring, 1982) were employcd to determine thecontents of mucous cells (Table 3.2.1).

in a scanning electron microscope (Akashi C o . ) .
Parasites were counted on 113 rectangles (0.06 mm’)from the gills, fins and skin (Fig. 3 . 2 . 2 ) , and theresults were expressed as mean number of parasites
per mm’.

Scanning electron microscope preparations
Ten fish were collected from each group atintervals of 1-3 weeks, fixed in 2% phosphatebuffered glutaraldehyde, and dehydrated in gradedethanols. Subsequent treatment involved critical point
drying and gold coating. The specimens were viewed

Changes in parasite densiw
Parasite density on the skin increased markedly
from week 2 post-infection (Fig. 3.2.1). It peaked at
week 3, at an average of 626 parasites/mm2, and thendecreased steadily to 8 parasites/mm2 at week 10. A
minor peak (351 parasites/mm’) occurred at week 12,

Results

25

AM

\ ~ .

AM
AM

1

Fig.325-7. LLght rmcmgaphs of theslon epidcms of churn salmon t?ymfcccctcd mth Ichthyobodo necafor
Alclan blue (AB.pH 2 5) Ipendc
acid-SchifFs reagent (PAS) s m n
Each bar = 30pm 3. Nomul epidermal s k m contammg AB-posth Y e mucous cells (AM) 4. Three
week post-mlccuon Note the paras~tes(P) and camplele dcplerianof epidermal mucous E e l l s ~ 5. Fourwcchs post-mfcctlon Eppldcns
above the basal layer 1s sloughed
off 6. Eighr weeks post-mfcNm
Numerous PAS-posrtlvc mucous
cells (PM) have appeared in lhc
upper layer of epidcmis~ 7. Ten
W c c h post-mlcctlon In addltlon tolhs PAS-positive mucous cells(PM) 111 the upper layer or epldermls, AB-positive mucous cells(AM) have appeared in the basal
laycr- 26 -

URAWA- PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Figs. 3.2.8-11. Scanning clcctron micrographs of the skin cpidemis of chum salmon fry infected with fchthyobudo necutor. 8. One
week post-infection. Note the active secretion or mucus from the epidermal mucous cells in response to the paraGte infections. Bar= 25 pm. 9. Same. The skin surfacc and parasites are covcred with mucus secreted from the mucous cells. Bar = 30 pm. 10.
Three weeks post-infection. The mucous cells have disappeared from the skin surface, and the infected Malpighian cells showed aloss of mcroridges. Bar = 20 pm. 11. Same. The parasite infcctions caused irregular wrinklcs on the cpidemal cell surface. Bar= 8 pm.

27

2 Cj I S,$.bjtJfi SCI REP HOKKAIDO SALMON HATCHERY, NO SO 1996

Figs. 3.2.12 & 13. Scanning eleclron micrographs of the skin surface of chum salmon fry infcctcd with fchthyobodo iiec-amr at week 4
post-infection. 12. Numerous parasites attached to the skin, causing considerable dcstruction. Bar = 15 pin, 13. Large
sloughed off area (SA) of the upper layer of epidermis, on which no parasites appear, in contrast to high infections on the rcinalning epidermal regions. Bar = 70 /rm.

Fig. 3.2.14. Scanning elcctron micrograph of thc skin surface of chum salmon try infcctcd with Ichd~yobodunwutur at week 8 postinfection. The surface of epidermis shows numerous openings of PAS-positive mucous cclls, where the parasltc was rarcly
obycrved. Bar = 25 ,vm.

- 28 -

URAWA- PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

28
n

cu

/

24

E
E 20
\
0
0

Y

--

16

v)

Q)

0

12

0

.........

4 0'

'

\,

I

I

I

I

I

1 2 3 4 5 6

8

10

12

15

I

Time (weeks)
Fig. 3.2.15. Changes in the concentration of skin epidermal mucous cells of Ichthyobodo infected (closed circles) and uninfected (opencircles) chum salmon fry. Vertical lines indicate SD.

followed by a sharp decline to 47 parasites/mm* atweek 15. A similar change in parasite density wasobserved on the fins ( P < 0.001, 12 = 0.796), thoughinfection levels were consistently higher than on theskin (Mann-Whitney U-test; P < 0.05). Infection levels remained low on the gills throughout the experimental period (Fig. 3.2.1).

Within the skin, the parasite was much more abundant in the middle and posterior regions (S3-6 in Fig.
3.2.2) than the anterior regions (S1 and S2). At week
8, when the parasite density decreased, the parasites
showed a minor shift in distribution: high frequency
infections occurred on the caudal fins and the outersurface of the gill cover (S2).

Spatial distribution ofparasites
Figure 3.2.2 illustrates changes in the frequency
distribution of I. necatur on chum salmon fry at 1, 3,
and 8 weeks post-infection. At week 1, when theinfection level was low, the parasites were relatively
abundant on the gills and fins, exclusive of the pectoral and ventral fins, compared with other areas.
When the parasite density sharply increased at week
3, the parasites were found more frequently on thedorsal, ventral, and anal fins than at other sites.

Histopathology
In response to the parasite in the initial stage of
infection, the surface mucous cells actively discharged their contents (Fig. 3.2.8), which occasionally covered the skin surface and the attached parasites(Fig. 3.2.9). By week 3, however, mucous cells wererare at the skin surface (Fig. 3.2.4), and infected
Malpighian cells appeared necrotic, with no surface
microridges (Figs. 3.2.10 & 11). Furthermore, atweek 4 the infected epidermal skin was hyperplastic,

29 -2 L j . 3 -6LnWfl SCI. REP. HOKKAIDO SALMON HATCHERY, NO. SO, 1996

100
80

60
40
20

0

d1
:::.:
8

1

3

4

5
10
6
Time (weeks)

1215Fig. 3.2.16. Changes in the histochemisitry of epidemal mucous cells of uninfected ( A ) and Ichthyobudo infected ( B ) chum salmon
fry. Histochemistry was asscssed by staining with Alcian blue (AB, pH 2.5) and periodic acid-Schiffs reagent (PAS). Vertical bars
indicate SD.

spongiotic, and vacuolated, and was sloughing above
the basal layer (Figs. 3.2.5 & 13). Parasites wereabsent from sloughing tissue, although high infections occurred on intact areas of the epidermis (Fig.
3.2.12). Thereafter, numerous mucous cells appearedin the superficial layer of the skin epidermis (Figs.
3.2.6,7, & 14).
Concentration of mucous cells
In control fish, the average concentration of epi-

dermal mucous cells was stable at 400-800 cells/mm2
over the course of the experiment (Fig. 3.2.15). In
infected fish, it declined to 30 cells/mm2 at week 4,
and thereafter increased to 2,400 cells/mm2 by week
8. The Concentration was reduced between weeks 10
and 12, while again it sharply rose to approximately
2,000 cells/mmLat week 15. A statistically significant
negative correlation existed between the parasite density and the concentration of mucous cells in theinfected epidermis throughout the experimental pen-

- 30 -

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

AB+, PAS -UAB+, PAS

Table 3.2.1. Sizes and histochemical responses of the qecretory contents in the AB-positive (type I) and PAS-positive(type 11) mucous cells in the epidermis of chum salmon fry+ 0 AB -, PAS+20

1 week

16 CilI

infected with Ichthyobodo necator.

0

_____
_____

16

12

0

12

8

8

4

4

0

Staining response
Staining procedure
Alcian blue (pH 1.O)
Alcian blue (pH 2.5)
Alcian yellow (pH 2.5)
Toluidine blue (pH 2.5)
Toluidine blue (pH 7.0)
Periodic acidSchiffs reagent (PAS)
Hematoxylin and eosin

0

- C"

0

cu

3 weeks

E 16 I I

1600
1200

<
E

800
400

Height* (,urn)
Width* ( u m )8 weeks

2E 4 8 1'i; 4 4

I

Q
'c

0

Z

300
200
100

F G

12.4 i 2.2
18.6 i 3.6

1 0 . 8 i 2.7

9.8 i 2.4

0too

S1 S2 S3 S4 S5 S6Red
Unstainedof the fins and gills had less mucus than that of the
body. The parasite infections, however, induced
marked changes in the mucous cell concentration
throughout the body surface (Fig. 3.2.17).

Q

I

Unstained
Unstained

Unstained
Unstained
Unstained
Unstuned
Unstained

.-

u)

0

Type II cells*Values are given as the mean t SD ( n = 30)

o h

~

_____

Type I cells
UnstainedBlue
Yellow
Weak
Weak

0

Fig. 3.2.17. The mucous cell concentrations in the skin (Sl-6
in Fig. 3.2.2), dorsal and anal fins (F), and gills (G) of
Ichthyobodo infcctcd (I, right columns) and uninfected (C,left columns) chum salmon fry at weeks 1 , 3 and 8 postinfection. Closed circles indicate the mean parasite densityof I. necator on the corresponding site. Mucous cell histochemistry was assessed according to the Alcian blue (AB,pH 2.5) /periodic-acid Schiffs reagent (PAS) staining procedure. Vertical bars indicate SD.

od ( P < 0.05, rZ = 0.446).
As shown in Fig. 3.2.17, the mucous cell concentration in uninfected fish was significantly higher
in the anterior regions (S1, 2) of the body than in theposterior regions (S3-6; P < 0.05), and the epidermis- 31 -

Histochemistry
In control fish, the majority of mucous cells
stained exclusively with AB over the experimental
period (Fig. 3.2.16). In the infected fish, cells stainedwith PAS alone predominated during the increase in
mucous cell concentration between weeks 5 and 8(Fig. 3.2.16). In the gills, PAS-positive mucous cells
frequently appeared over the period of the experiment(Fig. 3.2.17). As shown in Table 3.2.1, the PAS-positive mucous cells (type TI) were smaller than the ABpositive cells (type I). Histochemical analysis (Table
3.2.1) suggested that the newly formed mucous cells(type 11) contained neutral mucosubstances that were
AB-negative and PAS-positive, whereas acidmucopolysaccharides (AB-positive, PAS-negative)
were major constituents of the mucous cell (type 1)
secretions in the control fish.

Discussion
The number of I necator on the skin of chumsalmon fry increased during the 3rd week of infection
and then declined to near zero by week 10. A minor
peak followed at week 12. This pattern is similar tothat reported by Robertson (1979) for this parasite on
the gills of farmed rainbow trout and Atlantic salmon

2 I + . 3 -b&.tiff$E SCI REP HOKKAIDO SALMON HATCHERY,NO. 50,1996

fry. A sudden decrease in the number or prevalenceof I. necator was also observed by Migala (197 1) and
Wootten and Smith (1 980). All of these observations
suggest that a defense mechanism is initiated to control the parasite after several weeks of infection.
The density of mucous cells varies greatly,
depending on body region, sex, age, and season(Pickering, 1974, 1977; Wilkins and Jancsar, 1979).
Blackstock and Pickering (1982) noted that the concentration of mucous cells of brown trout (Salmo
trutta) was reduced to half (about 700 cells/mm’) at
the time of yolk-sac resorption and first feeding,which coincided with a period during which theyoung fish are particularly susceptible to skin infections. Pickering (1974) reported that in salmonids thehighest concentrations of mucous cells occurred on
the anterior regions of the body, which is similar toobservations of chum salmon fry in the present study.
Interestingly, the parasite showed the reverse distribution on chum salmon fry during the initial stage of
infection: the highest infections occurred on theregions with less mucus (i.e., the fins, and the skin on
the posterior part of the body), but rarely on the anterior regions. Richards and Pickering (1978) found asimilar distribution pattern of Saprolegnia infection
in salmonids. These observations suggest that mucus
can limit initial settlement of invading pathogens.
The AB (pH 2.5)PAS technique revealed dramatic changes in both the degree and the histochemistry of mucus secretions in the epidermis during thecourse of the parasite infection. The initial skin
response included the explosive discharge of mucus
containing acid mucosubstance (AB-positive, PASnegative), which resulted in complete disappearanceof the mucous cells. Thereafter, there was a marked
increase in the concentration of epidermal mucous
cells (about four times more abundant than in controlfish), together with a histochemical shift in the secretions, which was followed by a distinct decline inparasite density. Blackstock and Pickering (1982)
found that the epidermal mucous cells of brown trout
fry contained acid mucopolysaccharides but rarely
neutral ones (PAS-positive, AB-negative). The present histochemical analysis suggests that the newly
formed PAS-positive mucous cells contain neutral
mucosubstances which exhibit a negative response toAB (pH 2.5) and eosin. Pickering and Fletcher (1987)
noted that the number of acidophilic sacciform cells
increased in the epidermis of brown trout infectedwith fchthyobodo sp. This cell type is easily distinguishable from the present PAS-positive mucous
cells of chum salmon fry by its eosinophilic, PASnegative secretion.
Skin mucus secretions seem to be important natural defences against parasites and other infectious
agents (Ingram, 1980; Fletcher, 1981). The defence
may be mechanical, due to the continuous productionof mucus (Pickering, 1974), whereas the skin mucusof certain fish has been shown to contain moleculeswith possible antibiotic activity (see Pickering andRichards, 1980). The biochemical mechanism
responsible for the protective role of the epidermal
mucous cells against the parasite cannot be elucidated
here, but it is possible that the PAS-positive mucous
cells may contain protective substances that defend
the fish against Ichthyobodo infections. Thesemucous cells concurrently appeared throughout thewhole body of fish in spite in a regional bias in theparasite density. A similar proliferation of PAS-positive mucous cells was induced by the ectoparasitic
ciliate Trichodina truttae (Urawa, 1992b). Thus, theresponse appears to from a part of nonspecific protective mechanisms.

- 32 -

URAWA-PATHOBIOLOGY

OF PARASITIC PROTOZOANS ON SALMON

3.3 Effects on Seawater Survival of Anadromous Fish
Abstract. - Effects of the ectoparasitic flagellate Ichthyobodo necator on growth, survival and seawater adaptation in juvenile chum salmon (Oncorhynchus keta) held in fresh water were evaluated by
an experimental infection. The parasite density on the skin increased 2 weeks after infection, peaked at
week 6 , and then decreased to near zero by week 10. During this 10-week rearing period in fresh
water, the mortality in the infected fish accumulated to 12.4% compared with only 1.9%in the uninfected controls. The heavy infection had significant effects on the feed efficiency, but not on thegrowth of host fish. High mortalities (63-70%) occurred among infected fish in seawater challenge
tests (salinity 3 3 % ~for 48 h) conducted between weeks 4 and 6 when heavy infections caused intensive
erosions and hemorrhages in the epidermis, whereas mortalities were rare (0-3.3%)among the controls
in the test. Serum C I concentration of infected fish was significantly lower in fresh water and higher
in seawater, compared with that of the controls. A previous acclimation in one-third seawater did not
improve the seawater survival of infected fish. These results indicate that Ichthyobodo infection has amarked effect on osmoregulation of juvenile salmon due to destruction of the skm and will consequently reduce marine survival of anadromous hosts

Introduction

Materials and Methods

Many salmonid fishes are pnmarily anadromousand migrate to the ocean, adapting themselves to theincreased osmotic pressure of salt water.
Morphological, physiological, biochemical, andbehavioral changes occur in the course of their parrsmolt transformation and consequently make such
migrations possible (see reviews by Hoar, 1976,
1988; McCormick and Saunders, 1987). For the efficient use of hatchery-reared juveniles in salmonid
enhancement and aquaculture it is important to elucidate factors influencing the quality and survival ofsmolts (Wedemeyer et al., 1980). Several environmental factors are known to affect smoltification andsubsequent marine survival of anadromous salmonids(Wedemeyer et al., 1980; Langdon, 1985). but few
investigations have been conducted to clarify theeffects of pathogenic organisms on seawater adaptation of salmonids.
The flagellate Ichthyohodo necator infects theslun and gills of salmonids and causes severe epidermal destruction (Robertson et al., 1981; Roubal et al.,
1987; Urawa, 1992a). Thus, it was surmised that theparasite infection may disturb the osmoregulation ofhost fish (Robertson, 1979) and affect their seawater
adaptability (Ellis and Wootten, 1978; Wood, 1979).
There have been, however, no experimental works toprovide evidence for such pathogenicity of I. necator.
The present study was conducted to evaluate theinfluence of Zchthynbodo infections on growth, survival, and seawater adaptation in juvenile chumsalmon (Oncorhynchus keta) by using expenmentally-infected fish.

Transmission and fish rearing procedures
Juvenile chum salmon used in the experimentwere obtained from the Chitose Hatchery (42"48'N,
141'29'E) in western Hokkaido, which previously
had no history of parasite infections. Six hundred
juveniles (mean weight 0.74 g, n = 20) were placed ina 23-1 tank, and exposed to I. necator by cohabiting
with 10 infected chum fry obtained from the Yoichi
Hatchery (43" 12'N, 140"47'E) in western Hokkaido.
A control group of 600 fish (mean weight 0.72 g, n =22) was held in another 23-1 tank. Each tank was supplied with well water at a constant flow rate of 1.3
l/min. The water temperature was maintained constant at 10.5"C, and the photoperiod was under natural conditions. Fish were fed daily with commercial
dry pellets at about 2% body weight per day for 10
weeks. Mortalities in each group were recorded daily,
Biometric analysis
Twenty to thirty-five fish were collected weekly
from each group and fixed in a 10% solution of neutrally buffered formalin. The fixed fish were weighed(W, g) and measured (fork length, L, cm) individually. Condition factor (CF) was calculated as C F =100W/L3.Feed conversion efficiency (FE) was calculated from the equation: FE = 100(total weightgained, g)/(total food fed, g).
Light microscopy
Every two weeks, 8-10 fish were formalin-fixedin order to monitor changes in the intensity o f I.
necator using a light microscope at a magnification- 33 -

2 I?

2 TAHPk SCI. REP HOKKALDO SALMON HATCHERY, NO. 50,1996

transferred to 100% seawater (salinity 33"h). SerumC1- concentrations were measured at the end of theacclimation period in 113 seawater, and 24 h and 48 h
after transfer to 100% seawater.

of X 600. The parasite counts were made for eachfish from 18 squares (total 0.68 mm2) on the epidermal skin removed from the left side of the fish body
between the dorsal and ventral fins. The number ofparasites was expressed as a mean density per mm2.
In addition, the gills were removed from fish andexamined for the presence of the parasite.
For histopathological observations, formalinfixed materials were embedded in paraffin wax andsectioned at 6 p m . Sections were stained with hematoxylin and eosin or Giemsa stain.

Statistics
The Mann-Whitney U-test (one way) was used
to analyze differences between the infected and control groups. Correlations were analyzed with
Pearsons correlation matrix.

Results

Seuwater challenge tests
To determine effects of the parasite infection on
the seawater adaptability of juvenile chum salmon,
the following seawater challenge test was carried out
every two weeks. A sample of 30 fish was taken fromeach group, and held in a 20-1 tank supplied withoxygenated seawater (salinity 33% and temperature
about 1lOC) for 48 h, during which time mortalities
were recorded.
The ion regulation of salmon juveniles was estimated by measuring serum C1- concentration. Blood
samples were taken with a heparinized capillary tube
from each live fish ( n = 5-7) before and after the seawater test. In order to monitor successive changes i nserum C1- concentration of the fish transferred to seawater at 6 weeks after infection, blood was also taken
at 0, 1, 3, 12, 24, 48, and 96 h post-transfer from theinfected and control fish. After centrifugation at
10,000 rpm for 5 min, the serum C1- concentration
was immediately measured using a chloride counter(Hiranuma Co.).
In addition, the effects of a two-stage acclimation in seawater were tested at week 6. Forty fishfrom each group were kept each in a 23-1 tank of onethird seawater (salinity ll01~o)for 12 h, and thenParasite density
The mean density of I . necator on the skinincreased after 2 weeks of infection, reached a peakof 690 parasites per mmz at week 6 post-infection,and then decreased gradually to 90 parasites per mm2
by week 10 (Fig. 3.3.1). The parasite rarely appearedon the gills throughout the experimental period.
Freshwater survival
The heavily infected fish had many small patches of hemorrhages on the body surface and showed aslight reduction in appetite. Except for these effects,
infected fish exhibited no apparent symptoms. A
moderate mortality started at week 2 among theinfected fish reared in fresh water and it persisted forthe following 4 weeks (Fig. 3.3.1). The highest weekly mortalities (3.2-3.5%) occurred between weeks 4
and 5 when the parasite density abruptly increased on
the skin. The cumulative mortality was 12.4% in theinfected fish, but only 1.9% in the control group during the 10-week rearing (Table 3.3.1).
Growth and condition factor
The infected fish were smaller than the controls
after 4 weeks of infection, but there were no signifi-

Table 3.3.1. Final weight, weight gain, feed conversion efficiency, and cumulativc mortality of juvenile chum salmon (initial averagc
weight 0.72 g, n = 20) infected with Ichthyobodo necator for 10 weeks and comparison with control fish (initial average weight
0.74 g, n = 22). Thirty-five fish in each group were measured at thc end of the experiment.

Group

Final
weight

Total
gain

2.15

Feed
conversion
efficiencv*' (96'0)
83.8

(e)

(e'0)

Infected

2.88 f 0.93*'

Control

3.02* 1.02

factor*'0.770 f 0.080

12.4

2.29

99.2

0.769 f 0.053

1.9*'Feed conversion efficiency = 100 (total weight gained, g) / (total food fed, g).
*'Condition factor = 100 (weight, g) /(fork length, cm)).
SD.
*'Mean*34

Condition

Cumulative
mortality(%)

URAWA~

PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

cant differences in body size or condition factor
between two groups over the experimental period(Table 3.3.1 and Fig. 3.3.1). Feed conversion efficiency was lower in the infected fish than in the controls at the end of the experiment (83.8 and 99.2%,
respectively, Table 3.3. I). This reduced feed conversion rate was most prominent between weeks 4 and 6
when infections were heavy (76.5 and 108.9% in theinfected and control groups, respectively).
When the condition factor of individual fish wascompared with parasite density on the skin (Fig.
3.3.2), statistically significant negative correlations
existed from week 4 to week 8 ( P < 0.05), but not atweeks 2 and 10 when infection levels were low.
There was no distinct relationship between the parasite density and length or weight of individual hosts
over the experimental period.

Epidermal pathology
Severe epidermal destruction including hyper-

r

$

0
4

Y

0

0

m

0LI
0

I

I

I

I

I

2

4
6
Weeks

8

10

Fig. 3.3.1. Changes in the mean density of lchthyobodo necator on the skin, weekly percentage mortality, and mean body weight in
thc infected and uninfected control groups of juvenile chum salmon held in fresh water. Vertical bars indicate SD.

35 -

"

ま 仁ふ 所報 ScI ． REp

ぬ MONMTcmRY． HOKMLOS， NO

50 ， l996

800
]0
400
5

三

o

あ

500

2wk@

0

「

O
0
2

=

Q.
あ

c

7

o
lア
o
t
9ぬ
0f

o0

n
o

QLQ

0巾
co

0
0
0
2

00

十の

0

8H

4wk¥
R=
0.825

250

0

@a
(1)

0
0
l

P < 0.01

4ム

O
O

丁

0.7

0.8

0.9

1.0

COndition factor
Fig. 3.3.2. Relationships between the density of Ichthyobodo necator On the skin and condition factor (CF) tn individual chum salmon
juveniles a@ 2, 4, 6, 8, and I0 weeks afterinfection. CF=

100 (weight tn g) / (fork length in cm)3.

O

O

丁

の一

き
巳 o‑
oLの0 ユの[二@ノ‑ナ
0卜 0004﹂
[
QQ
T 0

エ

「

@ infectedo control

●

、、
●

50
O

……‥

0‑‑‑C""

0

O ………

6

4

2

"O

………

"O

L

8

]0

Weeks
Fig

Changes in the frequency (%) 0r juvenile chum salmon with skin erosion caused by lchthyohodoinfections

plasia, spongiosis, vacuolation, and hemorrhage
Occurred fn the skin 0f juvenile chum salmon heavily
infected with /. necator, as previously reported by
Urawa (1992a). MOst infected fish (80‑90%) sufferedfrom skin erosion (sloughing 0f the entire hyperplas‑―

tic epidermis above the basal layer) between weeks 4
and 6, but thereafter the frequency 0f affected fish
decreased t0 zero by week I0 (Fig. 3.3.3).

36―

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

In fresh watero control.......

..........

gc 100Q)*
0

In seawater

170r

0

E
1303

tj

v,

110 -

0

I

I

2

4

I

6
Weeks

I

I

8

10

Fig. 3.3.4. Biweekly changes in the serum C1- concentration in fresh water and seawater after 48 h of exposure, and the mortality inseawater within 48 h in the Ichthyohodo-infected and control groups of juvenile chum salmon. Vertical bars indicate SD. *, P <0.05; **, P < 0.01.

37 -2 If - 3 Tbdjf& SCI REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996= 200-**
infectedo control

0

'
C

Q,

.... ......4........

140-..............

......................

3

z 100

v)

I

;; 3

I

6

I

I

12

24
Hours

1

1

48

96

Fig. 3.3.5. Changes in the serum C1- concentration of Ichrhyobodo-infected and control chum salmon juveniles held in fresh water following exposure to seawater at week 6. Vertical bars indicate SD. **, P < 0.01.

180

.-5
c,

C

160

==140
0 0

p,€
-0 - 120
0

L ..................
1/3SW

sw

12

36
Hours

..................
..................

060

Fig. 3.3.6. Changes in the serum C1- concentration in juvenile chum salmon acclimated in one-third yeawatcr for 12 h following exposure to 100% seawater. Ichrhyobodo-infected fish 6 weeks after infection and corresponding controls were compared. Vcrtical bars
indicatc SD. **, P < 0.01.

ma1 erosion, while mortalities were quite low (0-Seawater survival
In the seawater challenge tests (salinity 33Oh for48 h), high mortalities (63.70%) occurred among theinfected fish between weeks 4 and 6 during which

3.3%) in the controls throughout the experiment (Fig.
3.3.4).
When high mortalities were recorded in infectedfish between weeks 4 and 8, serum C1- concentrations

period the parasite infections caused severe epider-

38 -URAWA- PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

of infected fish were significantly lower in fresh
water and higher in seawater 48 h after transfer, thanthose of the controls ( P < 0.05, Fig. 3.3.4), showing
significant negative correlations in fresh water andseawater ( P c 0.01). Compared with the controls,
whose serum C1- concentration gradually reachedseawater adaptive levels (about 130 meq/l) within 48
h after the transfer to seawater, the mean concentration of serum C1- in the infected fish suddenly
increased to 180 meq/l within 3 h after the seawatertransfer and continued at much higher levels over the
next 4 days (Fig. 3.3.5). This initial sharp rise inserum C1- levels was followed soon afterwards bydeath in many infected fish.
In the two-stage acclimation test, no mortalities
were observed in the first stage (1/3 seawater for 12h), but the infected fish showed a high mortality(50%) in the second stage (100% seawater) of acclimation. The survivors still had significantly higher
serum CI- concentrations in seawater than the controls (Fig. 3.3.6).

Discussion
The present experiments clearly indicated that
Zchthyobodo necator infection has a marked effect on
the seawater adaptation of juvenile chum salmon.
Heavy infections caused high mortalities (63-70%) of
host fish just after transfer to seawater and the serumC1- concentration of the infected fish was much higher than that of the uninfected controls. It has been
well documented that Ichthyobodo infections insalmonids cause necrosis of epidermal cells and as aconsequence extensive epidermal destruction of theskin (Robertson et al., 1981; Urawa, 1992a). The present study also confirmed that most infected fish suffered severe skin erosion when infections wereheavy. There was a significant positive correlation
between the frequency of fish with skin erosion andtheir mortality or serum C1- levels in seawater ( P <0.01). No pathological changes, however. wereobserved in the gills throughout the experiment.
Thus, the high mortalities of infected fish during seawater adaptation were attributable to epidermal
destruction of the skin caused by Ichthyohodn infections. A sudden rise in serum C1- concentration
observed immediately after the transfer of infectedfish to seawater suggests that acute dehydration

occurred due to an osmoregulatory disorder of the
injured skin.
In contrast, moderate mortalities continued
among infected fish reared in fresh water. Bi-weekly
comparisons between the parasite density and condition factor in individual hosts (Fig. 3.3.2) suggest thatthe initial parasite infections break out indiscriminately among host fish but subsequent increases ofparasite number may emaciate and kill weakened
fish. Because the infected fish had a lower concentration of serum C1- in fresh water than did the controls,
the major cause of fish deaths in fresh water may also
be osmoregulatory breakdown, followed by hemodilution and circulatory failure. Similar osmoregulatory
disturbances in salmonids leading to mortalities ordifficulty in seawater adaptation have also been
demonstrated in fungus (Richards and Pickering,
1979; Uno, 1990) and cestode infections (Boyce andClarke, 1983).
Juvenile chum salmon can acquire salinity tolerance and migrate to sea soon after emergence fromthe gravel beds (Black, 1951; Houston, 1961;
Weisbart, 1968; Hasegawa et al., 1987). Iwata andKomatsu (1984) found that juvenile chum salmon
released in rivers remain temporarily in brackish
water before migrating to seawater. They confirmedthat acclimation to one-third seawater for 12 henhanced hypo-osmoregulatory ability of juvenile
chum salmon. In the present experiment, however,
one-third seawater acclimation could not improve theseawater survival of Zchthyobodo-infected chumsalmon juveniles. This indicates that high mortalities
will occur among infected fish when they migrate toseawater estuaries. Since the parasite can survive andmultiply on host fish after direct transfer to seawater(Urawa and Kusakari, 1990), further fish mortalities
may break out during their subsequent marine migrations.
Zchthyobodo infections are very frequently
encountered in salmonid hatcheries (Urawa, 1 9 9 2 ~ ) .
Thus, it is strongly recommended that hatcheryreared anadromous salmonids should be examined
for the presence of I. necator before being transferred
into rivers or seawater net pens. A treatment of
infected fish with formalin bath several weeks prior
to release can improve their marine survival (Urawa,1996).

- 39 -

2 bt ' d TA;.aff%
SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

3.4 Effects of Rearing Conditions
Abstract. - Chum salmon (Oncorhynchus keta) juveniles, experimentally infected with the bodonid
flagellate Ichthyobodo necator (Henneguy, 1883), were reared in 18-1tanks at three densities (300, 600
and 1,200 fish) for 5 weeks. Water inflow rates were adjusted to give five combinations of crowding
and flow rate. The parasite density on the fish body surface increased to 2,700-4,100 parasites/mm2
over the course of the experiment, and did not differ significantly among the groups. Total mortality
was 76-90% in infected groups kept under the most unfavorable conditions (crowded or low watersupply), but only 11-15% in the other infected groups. In the uninfected control groups, the mortality
was low (0.7-2.6%) and the degree of crowding or water supply had no significant effect on growth or
mortality. These results indicate that Ichthyobodo infections have marked effects on both host growthand survival when infections are combined with environmental stress induced by overcrowding and/or
inadequate water supply, although the parasite density is not affected by these factors.

Introduction
Disease is a result of complex interactions
between host, pathogen, and environment (Snieszko,1974). It is well accepted that environmental stress
reduces host resistance against invading pathogens(Wedemeyer, 1970; Hoffman, 1976; Schreck, 1981;
Wedemeyer and McLeay, 1981; Shepherd, 1988).
Hatchery practices such as high rearing density andpoor water quality can affect the physiological statusof anadromous salmonids (Eddy, 1981; Fagerlund etal., 1981; Redding and Schreck, 1983; Schreck et al.,
1985; Patino et al., 1986; Boeuf and Gaignon, 1989)
and also their return rate (Hosmer et al., 1979). Thus
it is important to define safe levels of water quality
and crowding in the course of hatchery practices.
However, no single aspect of water quality should beconsidered in isolation from the influence of otherstress factors. Smart (1 98 1) has pointed out that nutritional deficiencies and protozoan infections may
reduce the ability of fish to withstand any deterioration in water quality.
The bodonid flagellate Ichthyobodo necator is a
widespread ectoparasite of various fishes and causes
a serious disease in freshwater and marine aquacultures (Robertson, 1985). Although it is generally
believed that the disease occurs only under poor reari n g conditions (Bauer, 1959; Wedemeyer andMcLeay, 1981), heavy infections are frequently
observed among hatchery-reared salmonids evenunder optimum conditions (Urawa, 1 9 9 2 ~ )There.
isno experimental evidence to confirm the relationshipbetween outbreaks of this parasitic disease and rearing conditions.
The purpose of the present study was to investigate the effects of fish rearing density and water

inflow rate on the abundance of I. necator and thedegree of its pathogenicity to juvenile chum salmon(Oncorhynchus keta).

Materials and Methods
Experimental design
Parasite-free chum salmon juveniles (mean bodyweight 0.45 g, n = 120) were used for this experiment. A stock of 6,600 fish was divided equally into
two 40-1 tanks, each supplied with a constant flow of
10.5"C well water. One group was kept for 1 weekwith 20 caged juvenile chum salmon infected with I.
necator derived from Yoichi Hatchery, and the othergroup served as an uninfected control. Both groups
were fed commercial dry pellets at about 2% of bodyweight per day for 1 week.
After exposure to the infected fish, each group
was divided into 5 groups consisting of 1 high density (HD, 1,200fish), 3 medium density (MD, 600 fish)and 1 low density (LD, 300 fish) groups. MD groups
were supplied with well water at 3 different flow
rates, 370 (low inflow, LI), 750 (medium inflow, MI)or 1,500 ml/min (high inflow, HI). H D and L D
groups were supplied water at MI rate. The contorol
fish were also divided into the same number ofgroups as the infected fish and subjected to the same
conditions of stocking density and water flow. Eachgroup was kept in an 18-1 tank and fed as previously
for the experimental period of 5 weeks. Water temperature and dissolved oxygen (DO) concentration
were monitored in each tank outlet at 2-3 day intervals with a dissolved-oxygen meter (YSI model 57),
and ammonia concentration was measured at about
1-week intervals using the indophenol blue method.
Water temperatures were kept at 10.5"C in all tanks
throughout the experimental period.

- 40 -

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

centrifuged at 12,000 rpm for 5 min, and serum C1concentrations were measured by chloride counter(Hiranuma Co.).

Fish sampling and parasite counts
Fish ( n = 20-30) were sampled from each tank at
0, 1, 2, 3, and 5 weeks after the start of the experiment. The fish were anaesthetized in MS222 (about
100 mg/l), immediately weighed and measured (forklength), and fixed with 10% neutral buffered formalinfor parasite counts. These treatments were confirmed
to have no effects on the number of I. necator on hostfish. The parasites were counted on the dorsal andventral fins, and the body surface between these finswith a scanning electron microscope (SEM), as
described by Urawa (1992a). Condition factor (CF)was calculated from the formula: CF = lOOw / l',where w is the weight of each fish and 1 is corresponding fork length. Food conversion ratio (FCR)
was calculated from the equation: FCR = 100 (total
weight gain, g) / (total food fed, 8).

Statistical analysis
The Mann-Whitney and Kruskall-Wallis tests(one way) were used to assess the significance of differences among groups. The significant level was set
at P < 0.05 for all comparisons.

Results
Water chemistry
Rearing density, water inflow rate, dissolved
oxygen and NH,-N concentrations for the five different combinations of crowding and inflow rate
throughout the experimental period of 5 weeks arepresented in Table 3.4.1. Dissolved oxygen levels
were reduced to lower than 7 mg/l for the first week
in the tanks of HD-MI and MD-LI in both infected
and control groups. NH4-N concentrations were also
highest (12-35 p g / l ) in these tanks, but these were
within safe levels for salmonids (Smart, 1981).

Seawater challenge tests
For the evaluation of seawater adaptability, seawater challenge tests were carried out on 30 fish fromeach group at week 3 in 20-1 tanks supplied with aerated seawater (salinity 33% ; temperature 11"C) for
48 h. For serum chloride (C1-) measurements justbefore and after seawater challenge tests, fish werebled from the caudal vessels into heparinized hematocrit centrifuge tubes. The tubes were immediately

Parasite density
Initial parasite density averaged 2.2 and 6.6 parasites/mm2 on the skin and fins, respectively. By theend of experiment (week 5 ) , the parasite density

Table 3.4.1. Fish density, water inflow rate, dissolved oxygcn (DO), and N H - N concentrations in tanks with different degrees of
crowding and water inflow rate.
DO (mgfl)

Fish density (kg/m')

croup*'

Week0 Week 1 Week3 Week5

Infected group
MD-LI
MD-MI
MD-HI
HI-MI
LD-MI

17.9
18.2
17.4
34.7
8.9

Control group
19 1
MD-LI
MD-MI
19.6
19.5
MD-HI
HD-MI
38.3
LD-MI
8.7

Weeks 0-1

*
*

21.1
21.3
23.6
42.8
10.3

18.9
28.2
28.2
36.9
14.5

3.9
24.9
21.7
23.6
13.2

6.0 0.7*2
7.8 0.4
9.0 zk 0.4
5.7 k 0.5
8.8 0.5

22.2
23.7
21.6

30.2
31.9
31.1

32.8
38.6
36.1

6.5 0.5
7.8 f 0.5
8.9 k 0.4

49.2

61.3

70.2

5.6 f 0.5

11.1

15.2

17.7

8.9

*

*

* 0.4

NH-N
(mg/l)
Weeks 0-5

Weeks 1-3

Weeks 3-5

7.3 f 1.0
7.8 f 0.3
8.9 i 0.2
8.0 0.8
9.0 0.2

9.2 0.2
8.0 0.3
9.0 f 0.3
7.5 0.4
9.0 f 0.2

0.012 f 0.007
0.014 f 0.001
0.007
0.002
0.028 f 0.002
0.005
0.001

5.5 t 0.4
7.1 f 0.4
8.6 f 0.3

0.035 f 0.001
0.018 f 0.007
0.004 f 0.003

5.5 f 0.6

0.031 k 0.005

8.7

0.001

*

*

* 0.3
* 0.3
* 0.3
5.7 * 0.3
8.7 * 0.3
5.8
7.1
8.7

*

*
*

* 0.3

*
*

* 0.002*'HD, high rearing density (1,200 fish); MD, medium rearing density (600 fish); LD, low rearing density (300 fish); LI, lowwater inflow (370 d m i n ) ; MI, medium water inflow (750mUmin); HI, high water inflow (1,500 m lh in ) .
*'Mean
SD.

*

- 41

3 1j . 3 T&2%&SCI. REP HOKKAIDO SALMON HATCHERY, NO. 50,1996

Skin

4

A

Skin

)

0 low density

Bohigh inflow
medium inflow
low inflow

0 medium density

3
n
N

E

€ 2

zP

-

::I
C

Q

tn

r
0'0

30

Fins

'

Fins

I

I

I

0

3
Weeks

5

-

0

3
Weeks

5

Fig. 3.4.1. Effccts of rearing density (A) and water inflow rate (B) on the mean density of Ichfhyobodo necator on the skin and fins ofjuvenile chum salmon. Water inflow rate and rearing density was adjusted at medium levels in the groups of A and B, respectively.

increased to 2,700-4, 100 and 6,200-7,200 parasites/mm2 on the slun and fins, respectively, with nosignificant differences among groups (Fig. 3.4.1).
Control fish remained uninfected throughout theexperiment.
Mortality
The weekly mortality rate increased rapidly in
HD-MI and MD-LI groups from week 3, cumulating
at 76% and 90%, respectively, by the end of theexperiment (Fig. 3.4.2). The total mortality was 11.414.5% in the other infected groups and only 0.7-2.6%
in the controls.

GrowthThe mean body weights of infected fish in the
HD-MI and MD-LI groups were significantly lower
than that in the corresponding controls at week 3 (P <0.01, Fig. 3.4.3). Significant differences were also
observed in the HD, MD, MI and HI groups at week
5. The condition factor of fish in the HD and LI
groups was significantly lower in infected fish than in
the controls at weeks 3 and 5 , except for the LI groupat week 5 (Fig. 3.4.4). Among the infected groups,
there was a negative relation between condition factor and rearing density at weeks 3 and 5, and a positive relation between condition factor and inflow rate
at week 3. In the controls, the condition factor was- 42 -

URAWA --PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

100

-

- -

0 low density

n

x

80

inflow- .mediumlow inflow

high density>rB
0 high inflow

0 medium density

-

-s

-

A

w

.
I

(0

60

P

/D

E

.-> 40
Q)

Y

(0

i 20
3
I

0

L

I

I

0

1

2
3
Weeks

1

I

J

I

I

I

4

5

0

1

2
3
Weeks

I

I

I

4

5

Fig. 3.4.2. Effects of rearing density (A) and water inflow rate (B) on the cumulative mortality of juvenile chum salmon infected with
Zchthyobodo necator. Water inflow rare and rearing density was adjusted at medium levels in the groups of A and B, respectively.

not influenced by fish density nor inflow rate over theexperimental period. Compared with the controls, thefood conversion rate was apparently lower in theinfected HD and LI groups at week 3, and in all
infected groups except for LI at week 5 (Fig. 3.4.5).
Seawater survival

Seawater challenge tests (48 h) produced highmortalities (80-100%)in all the infected groups (Fig.
3.4.6). In particular, all test fish died in the infected
HD and LI groups. The serum CI concentration of
infected fish was significantly higher (over 160
meqll) in seawater and lower (under 110 meqll) in
fresh water compared with that of normal fish.
Seawater mortalities were rare (0.67%) in the control groups, while the serum C f concentrations of the
HD and LI groups were significantly higher in seawater and lower in fresh water than those of the othercontrol groups.

Discussion
The present experiment reveals that the pathological potential of Ichthyabodo tiecator is strongly
intensified by unfavorable rearing conditions,although the density of I. necntor was not influenced

by fish density or inflow rate. High rearing density(HD) and inadequate low inflow rate (LI) resulted in
reduced survival, growth, and food conversion efficiency of infected fish, while these conditions did notaffect the uninfected controls. The unfavorable rearing conditions induced low dissolved oxygen (5-6mg/l) in both the infected and control groups. Theselow dissolved oxygen levels may be more stressful to
Ichtkyobodo-infected fish than to normal uninfected
fish. Itazawa (1971) suggested that the minimum
level of dissolved oxygen required to maintain rnaximum feeding, growth and efficiency of food conversion is 4-4.5 mg/l for rainbow trout (Oncorhynchus
mykiss) at about 10.5"C. Alabaster and Lloyd (1982)
considered that the minimum sustained dissolved
oxygen necessary for the normal life cycle ofsalmonids is 5 mg/l under favorable conditions. The
present study suggests that these minimum oxygen
levels are dangerous f o r Ichthyobodo-infectedsalmonids.
It was reported that Ichthyobodo infcctions
strongly reduce the seawater survival of juvenile
chum salmon because of skin destruction (Urawa,1993). The present seawater challenge tests also
showed that mass mortality (SO-100%)occurred in all
the infected groups, with the highest mortality- 43 -

2 Ij . d TZltrll X I . REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

A

B

A

o.6w
m
0.8 r

0 weeksr

0.7

0.4

06

1 week,

1 O r

0.5

08

0.8
L

0 0.7
c

06

-s
0

m 0.6
05

0.4

--

1.4

.- 0 8
.-

m 12

x

I

U

10

C

07

.? 0 8

0 06

0.6

05

0.4

0.9

2.0

0.8

%

U

r

18

0.7

1.6

0.6

14

0 5

5 weeks

r

HD

12
1.0

nc

HD

MD

LD

LI

MI

MD

LD

LI

MI

HI

Fig. 3.4.4. Effects of rearing density (A) and water inflow
rate (B) on the condition factor of juvenile chumsalmon. Open and shaded columns represent the uninfected control and Zchthyobodo-infected groups, respectively. Bars represent SD. HD, high rearing density;
MD, medium rearing density; LD, low rearing density;
LI, low water inflow; MI, medium water inflow; HI,high water inflow; *, P < 0.05; **, P < 0 01

08
V."

B

08

HI

Fig. 3.4.3. Effects of juvenile rearing density (A) and water
inflow rate (B) on the growth of juvenile chum salmon.
Open and shaded columns represent the uninfected control and Zchth?iobodo-infected groups, respectively. Bars
represent SD. HD, high rearing density; MD, medium
rearing density; LD, low rearing denslty; LI, low water
inflow; MI, medium watcr inflow; HI, high water inflow;**. P < 0.01.

A

2.0 r

3 weeks

B

1.6
1.2
0.8

(100%)in the unfavorable rearing groups (HD and
LI). Direct transfer to seawater may impose a considerable stress, even on healthy anadromous fish. Whileno significant differences in the seawater mortality
were detected among the controls, the serum C1- levels of the HD and LI groups were significantly lower
in fresh water and higher in seawater than that ofother control groups. This suggests that stressinduced by unfavorable rearing conditions disturbs
the osmoregulation of anadromous fish, as reported
by several workers (Schreck et al., 1985; Boeuf andGaignon, 1989).
It is generally believed that crowding of fish is
conductive to the development and spread of ectoparasitic diseases (Hoffman, 1976). In the present experiment, however, the number of I. necatur increased
rapidly on host fish regardless of rearing conditions
such as fish rearing density and inflow rate. Thisimplies that I . necatur can multiply and spread

-44-

0.4

0
2.0

5 weeks

r

r

1.6
12

0.8

0.4

-

n

HD

MD

LD

LI

MI

HI

Fig. 3.4.5. Effects of rearing density (A) and water inflow
rate (B) on the food conversion ratio of juvenile chumsalmon. Open and shaded columns represent the uninfected control and Ichthyobodo-infected groups, respectively. HD, high rearing density; MD, medium rcmng
density; LD, low rearing density; L1, low water inflow;

MI, medium waler inflow; HI, high water inflow.

among hatchery-reared fish independent of host condition once the parasite has successfully settled onhost fish. This is evidenced by the fact that heavy

(JRAWA‑PATHOBIOLOGY

0F PARASITIC

PROTOZOANS

0N

SALMON

Infected fish

100

190
て

あ

4

70

二

7

50

130 三

茸

110
山の

0

HD

MD

LI

LD

0

）ポ

ム

十十

4
Lo

=

十

50

十

十中

十

4Q卜
90 @

小

M@

HI

十

十

110 さ

十

1

]70
100
Fig, 3.4.6.

COmparison.*:

COntrol fishの190

of seawater mortality (salinity33o/00for 48 h; shaded columns), and serum Ct' concentrations tn fresh water(solid circles)and seawater (open circles)after48 h Of transfer between Ichthyobod0‑iafecled juvenile chum salmon and the unin‑

fccted controls at weeklow

water inflow; M1,

3. Bars indicate SD. HD, high rearing density; MD, medium rearing density; LD, low rearing density; L1,
mediumwater inflow; H1, highwater inflow

Ichthyobodo infections occur very frequently inhatchery salmonids reared even under optimum con‑ditions (Urawa, 1992c).
In conclusion, Ichthyobodo
infections havemarked effects on both host growth and surviva1,

especially when the infections are combinedwithenvironmental
stress inducedby overcrowdingorinadequate water supply, although the parasite densi‑
ty ts not influenced by these factors.

―

45

―

3b j .

$A$f$ii SCI. REP. HOKKAIDO SALMON HATCHERY, NO SO. 1996

3.5 Controls
Abstract. - Seasonal changes in the density of the ectoparasitic flagellate fchthyobndo necator on
juvenile chum salmon (Oncorhynchus keta) and the host's seawater adaptability were monitored at
Yoichi Hatchery along the Yoichi River in western Hokkaido. The parasites appeared on the body surface of salmon alevins just before the completion of yolk sac absorption. The parasite density
increased rapidly during the first-feeding period, resulting in the reduced seawater adaptability of hostfish. A formalin bath (250 ppm for 1 h) controlled the attaching parasites, and improved the host's seawater survival within one month post-treatment. The number of adult salmon returns significantly
increased in the Yoichi River after the control of parasite infections.

Introduction
The bodonid flagellate Ichthyobodo necator isan epizootic ectoparasite in salmonid hatcheries(Wood, 1979; Urawa, 1992~).The parasite can survive and multiply in both freshwater and marine
environments (Urawa and Kusakari, 1990). Infections
cause the severe epidermal destruction of salmonids(Robertson et al., 1981; Urawa, 1992a) and reduce
the seawater adaptability of anadromous hosts(Urawa, 1993). Thus it is assumed that Ichthyobodo
infections strongly affect the early marine survival of
salmon, resulting in the low levels of adult returns.
The purpose of the present study was to investigate seasonal occurrence of I. necator on hatcheryreared juvenile chum salmon (Oncorhynchus keta)
and to improve their marine survival by control ofparasite infections.

Materials and Methods
Hatchely
Yoichi Hatchery is located in western Hokkaido(43"12", 140"47'E), 4 km upstream from the estuary of the Yoichi River draining into the Sea of Japan(Fig. 3.5.1). This salmon hatchery has been operated
since 1970 by the Yoichi Cooperative Fisheries
Association, and annually released several million
chum salmon juveniles directly into the Yoichi Riverafter 2-3 months of feeding. Most of these chumsalmon may migrate seaward soon after their releasebecause the estuary is close to the hatchery. Most
returning adult chum salmon are caught by a trap
completely blocking up the river near the hatchery
between September and November. Thus, wild chumsalmon are rarely present in the Yoichi River.
Fish and rearing conditions
In the spring of 1987, about 8.8 million juvenile
chum salmon were reared in 4 indoor and 2 outdoor

ponds each supplied with well water mixed withwater from the Yoich River. Fish were fed commercial dry pellets during the period of February-April,and released directly into the Yoichi River in middle
April. The water temperature ranged from 6°C to 9"
C in ponds during the rearing period.
Treatments
A formalin bath (250 ppm for 1 h) was conducted in all hatchery ponds on March 12, I3 or 21, about
one month before the fish releases (Table 3.5.1).
Fish sampling and parasite counts
Fish samplings were carried out in 4 indoor
ponds (A-D in Table 3.5.1; each stocked with about
1.1-1.2 million fry) at 2-week intervals between late
January and middle April, 1987. Sixty fish were collected from each ponds and immediately fixed in
10% neutral buffered formalin. The fixed fish were
measured (fork length and weight) and then the parasite number was counted on the dorsal and anal fins(see Urawa, 1992a) with a light microscope.
Seawater challenge tests
To evaluate the seawater adaptability of juvenile
chum salmon, seawater challenge tests were carriedout at 2-week intervals. Fifty fish from each ponds
were held in 20-1 tanks with seawater (salinity 3 3 O / ~ )for 48 h, during which fish mortality was recorded. In
addition, serum CI- concentrations were measured for
10 fish before and after the test, as in Urawa (1993).

Results
Parasite density
In all rearing ponds examined, Ichthyobodo
necator infected chum salmon alevins with a lowmean density (0.6-8.6 parasites/mm*) at the first sampling, and then increased its number after the yolksacs were completely absorbed (Fig. 3.5.2). In pond

- 46 -

URAWA

46'

140"

142"

~

PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

144'

146"E

N

44

42

40

Fig. 3.5.1. Maps showing the location of Yoichi Hatchery and the arrangement of indoor ponds (A-D in the right figure) examined in
the present study. Arrows in the pm&sindicate a linc of inflow water.
Table 3.5.1. History of juvenile chum salmon stocked in the indoor ponds A-D of Yoichi Hatchery in 1987.
Date of
Pond

A
B
C

D

Initialfeeding
FEB. 1
FEB. 3
FEB. 13
FEB. 13

Formalin
beatment
MAR. 12MAR. 12MAR. 13MAR. 21Fishrelease
APR.7
APR.7
APR. 16
APR. 16A, the parasite density averaged 603 parasites/mm2 2
weeks after the first feeding of fry, but decreased tonear zero after a formalin bath treatment. The parasite
was also disinfected and remained in low infectionlevels by a formalin treatment in other ponds, except
for pond B where the parasite density increased again
after the treatment (Fig. 3.5.2).
Seawater survival

The seawater mortality of juvenile chum salmon
was less than 10% when the yolk sac was completely
absorbed, but thereafter it decreased gradually withincreasing parasite density (Fig. 3.5.2). The serumC1- concentration of infected fish was lower in fresh

Initialnumberof fishstocked
1,104,000
1,137,000
1,125,000
1,184,000

Numberoffish
released
1,082,000
1,113,000
1,103,000
1,171,000

water and hgher in seawater than that of normal fish
(Table 3.5.2). Within 4 weeks after the formalin treatment, however, disinfected fish completely recovered
their seawater adaptability (Table 3.5.2).
Adult returns
Number of juvenile chum salmon released from
Yoichi Hatchery increased gradually every year, andmore than 7 million fish have been annually released
from 1983 (Fig. 3.5.3). Adult salmon returns
remained in very low levels (less than 4,000 fish) for
19 years (1970-1 988) after the hatchery operation
started, whereas they increased up to ten thousand
fish between 1989-1991 (Fig. 3.5.3).

47 -

A

0

I

V

0

P

Parasite density
per rnm*

o

g

o

o
2

0
o

2

Body weight (gl

m
0

0

I

0

I

0

-I

Ib-

A

0
m

A

0

A

0

u l o
0

A

8

Body weight (9)

0

0

g

Survival (%)
in seawater

1
-

u l o

Survival (%)
in seawater

Parasite density
per mm2

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

70

75

80

85

90

Year
Fig. 3.5.3. Annual changes in the number of juvenile chum salmon released (open circles) and adult returns (shaded bars) in the Yoichi
River between 1970 and 1991.

Discussion
It was reported that yolk-sac salmonid fry aresometimes infected lightly with I. necator (Wood,
1979; Castillo et al., 1991). The present study also
confirmed that the parasite attached to the body surface of chum salmon alevins with a low density. The
parasite density rapidly increased after their complete
yolk-sac absorption. It is speculated that mucous cells
play an important role in the defense mechanisms
against fchthyobodo infections (Urawa, 1992a).
Blackstock and Pickering (1982) noted that the density of mucous cells of salmonids was reduced to half
at the time of yolk-sac resorption and first feeding.
This period corresponds to a time during which thedensity of I. necator increased rapidly on chumsalmon fry.
A formalin bath (250 ppm for 1 h) was very
effective for the control of parasite infections at
Yoichi Hatchery, as reported by Wood (1979). The
parasite density remained in very low levels after thetreatment, except in pond B, in which it increased

again. This exceptional increase is probably due tothe fact that the formalin treatment was delayed onlyin pond D (Table 3.5.1), from which the parasite
could transmit into the lower pond B. Thus it is probable that the recovering host can acquire an effective
immunity to the parasite if the huge source of infection is absent, as known in other protozoans (seereviews by Woo, 1987; Houghton et al., 1988).
T h e infection experiment (Urawa, 1993)
revealed that lchthyobodo infections drasticallyreduced the seawater adaptability of juvenile chumsalmon due to the skin destruction. This is corroborated by the present investigation in Yoichi Hatchery,
where the seawater survival of young salmon reducedwith an increase in the parasite density. A formalinbath improved the seawater adaptability of juvenile
chum salmon at 3-4 weeks post-treatment when they
were released from the hatchery.
Annual survey (1986-1990) indicated that
Ichthyobodo infections occurred every spring in
Yoichi Hatchery (Urawa, 1 9 9 2 ~ )Salmon.
fry werereared every year under almost same hatchery condi-- 49 -

2 Cf. 3 T - W l SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50, 1996

tion with no control of parasite infections. Thus, theparasite diseases might have broken out every year
since 1970, resulting in mass mortality of infectedfish when they migrated to salt water.
Although number of juvenile chum salmon
released from the hatchery increased gradually year
by year, adult returns had been very scanty until
1988. However, adult returns significantly increased
between 1989-1991. More than 95% of chum salmon
spend two- to four-year marine life (age 0.2-0.4)
before their return to the natal river (Salo, 1991).
Thus these increased chum salmon returns in 19891991 are largely a consequence of the improved earlymarine survival of juvenile salmon due to the controlof Ichthyobodo infections.
Ichthyobodo necator has widely spread insalmon hatcheries (Urawa, 1 9 9 2 ~ )Consequently,.
hatchery-reared salmonids should be examined forthe parasite infections, and infected fish should betreated by one month prior to their releases. There
have been various treatment methods for external
protozoan parasites (Hara, 1972; Hoffman andMeyer, 1974; Wood, 1979; Griffin, 1989), among
which formalin may be the most widely used therapeutic agent in hatcheries. In Japan, however, its use
for parasite control in food fish has not been permitted by the Pharmaceutical Law. While standard formalin treatment (167-250 ppm for 1 h) does notaffect growth, survival and seawater adaptability ofsalmonids (Sumari et al., 1979; Smith et al., 1987),
formalin occasionally causes pathological changes injuvenile fishes (Wedemeyer, 1971). In addition, there
are reports in Scandinavia that a formalin-resistantstrain of Ichthyobodo has been produced as a result of
regular prophylactic treatment (Robertson, 1985).
Therefore new methods should be found out to control Ichthyobudu infections.

50 -UUAWA- PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

4. Trichodirza truttae
4.1 Morphology and Taxonomy
Abstract. - The parasitic ciliate Trichodina truttae Mueller, 1937 is redescribed from silverimpregnated specimens collected from the body surface of chum salmon (Oncorhynchus kefa) fry
reared in five hatcheries of Hokkaido and northern Honshu, Japan. The description is supplemented byscanning electron microscopic observations. The distinguishing morphological features include its
large size (body diameter 115-178pm), high number (10-23) of radial pins per denticle, and the presence of two markedly different lengths of cilia withm the adoral ciliary spiral. Geographical variation
was observed in the body size and number of denticles.This is the first record of T. truttae from Japan.

from the gills of hatchery-reared cutthroat trout(Oncorhynchus clarki) in Oregon, and has been subsequently reported from various salmonids in western
North America and the USSR (Davis, 1947, 1953;
Bogdanova, 1963, 1967, 1977; Bogdanova andShtein, 1963; Arthur and Margolis, 1984). Because
this finding constitutes the first record of T. truttae
from Japan, the parasite is redescribed according toKlein's (1958) dry silver-impregnation technique andscanning electron microscopy (SEM), and some
aspects of its geographical variability are noted.

Introduction
Ciliates belonging to the family Trichodinidae
are common and occasionally pathogenic parasites offreshwater and marine fishes (Lorn and Hoffman,
1964; Lorn and Laird, 1969; Hoffman, 1978). In
Japan, tnchodinid infections frequently occur on cultured freshwater and marine fishes, but taxonomic
and pathological studies of this group have been veryfew (Egusa, 1978). Although Ariake (1929) andSuzuki (1950) reported several new species of trichodinids from Japan, these descriptions were consideredby Albaladejo and Arthur (1989) to be too inadequate
to permit positive reidentification. The only othercon firmed i den t i f i c a t io n s of T r i c h o din u fromJapanese fishes are those of Ahmed (1976, 1977),
who reported T. reticulafa Hirschmann and Partsch,1955 on goldfish (Curassius aurutus), crucian carp
(C. aurutus) and color carp (Cyprinus carpio).
In 1968, high mortalities occurred among chumsalmon (Oncorhynchus keta) fry reared in Ichani
Hatchery, eastern Hokkaido, due to trichodinid infections (Takeda et al.. 1969). Although the parasite wasreported as T. domerguei (Wallengren, 1897), its
identification is suspect, since it was not based on
detailed morphological studies (Nagasawa et al.,1987). Several other papers also reported unidentified
trichodinid ciliates from farmed salmonids in Honshu(see Nagasawa et al., 1987). Recently, many cases ofTrichodinu infections have been encountered among
hatchery-reared salmon fry.
The present study was conducted to clarify the
species of trichodinid ciliate occurring on the skin ofchum salmon fry in northern Japan, and consequently
the ciliate was identified as Trichodina truttae
Mueller, 1937. This species was originally described

Materials and Methods
Trichodinids were collected from the body surface of chum salmon fry from five hatcheries in
Hokkaido and northern Honshu between 1981 and1990 (Fig. 4.1.1). Air-dried smears of living ciliatestaken from the skin were prepared for light microscopical observations. Smears were immersed in a
2% solution of silver nitrate (AgNOJ for 10 min, andexposed to ultraviolet light for 15-20 min (Klein's dry
silver-impregnation technique). h addition, some formalin preserved specimens were impregnated withsilver according to Wellborn (1967). For SEM, ciliates were fixed by immersion of whole living chumsalmon fry in 1% glutaraldehyde in 0.1 M phosphatebuffer. Parasites were centrifuged and dehydrated
through a graded alcohol series. The alcohol was
replaced with isoamyl acetone, and the samples weredried in a Hitachi critical point drying unit, gold-coated in a sputter-coater (Eiko), and then observed using
an Akashi scanning electron microscope. Terminology and methods of measurement followed theuniform specific characteristics proposed by Lorn
(1958) and Arthur and Lom (1984).

51

2 bf . 5 j-mW$$ SCI. REP. HOKKAIDO SALMON HATCHERY, NO. SO, 1996

142E
I

Asahi Hatchery has been deposited in the National
Science Museum, Tokyo (NSMT-Pr. 165).

144E

I
Sea of
1 Okhotsk

Description
The following description is based mainly on
silver-impregnated ciliates collected from Asahi and
Chitose Hatcheries (Table 4.1. l), supplemented by
SEM observations.
A relatively large trichodinid with a disc-shaped
body (Fig. 4.1.6), 114.7-177.5 ,urn in diameter.
Adhesive disc cup-shaped, 81.1-125.0 pm in diameter, surrounded by a wide border membrane, 6.5-12.0
p m in width. Adoral ciliary complex consisting of
three distinct ciliary bands: basal ciliary ring, locomotor ciliary wreath, and marginal ciliary ring (Figs.
4.1.6-8). Center of adhesive disc dark and uniformly
granular in silver-impregnated specimens.
Denticulate ring 45.3-75.0 pm in diameter, consisting of 24-31 denticles (Figs. 4.1.2 & 3). Span of denticle 24.5-35.5 ,urn; length 12.8-20.0 pm. Blade of
denticle acutely falciform with highly concave anterior border and sharply angled posterior margin (Fig.
4.1.4). Blade joining central portion of denticle posterior to median axis of thorn. Length of blade 8.8-14.0
pm. Central part thick, 4.5-8.5 ,urn in width, its
external portion cylindncal. Thorn thickened at base,tapering gradually to a sharp point. Length of thorn
10.4-16.0 I'm. Number of radial pins per denticle 1023 (Figs. 4.1.4 & 9). Adoral ciliature forming a spiralof about 450" (Fig. 4.1.5). The majority of cilia comprising the adoral ciliary spiral short (Fig. 4. I .7), only
final area near buccal cavity comprised of long cilia(Fig. 4.1.8). Macronucleus U-shaped, about 80 ,urnin
diameter.

42N

Pacific
Ocean

38N-

Fig. 4.1.1. Map of collection localities for Trichodina truttae infecting chum salmon fry in northern Japan.
Numbers refer to hatcheries given in the legend of Fig.
4.1.10.

Results
Trichodina truttae Mueller, 1937
(Figs. 4.1.2-9)
Host: Chum salmon fry (Oncorhynchus keta).
Site of infection: Body surface.
Locality of samples: Asahi Hatchery, Rishiri Island,
northern Hokkaido; Chitose Hatchery, western
Hokkaido; Ichani Hatchery, eastern Hokkaido;
Ohkawa Hatchery, Miyagi Prefecture, Pacific coastof northern Honshu; Gakko Hatchery, Yamagata
Prefecture, Japan Sea coast of northern Honshu.
Deposition of specimens: One slide including silverimpregnated specimens from chum salmon fry from

Geographical comparison
Figure 4.1.10 compares variation in parasite size
(denticulate ring) and number of denticles among T.
truttae populations collected from five hatcheries innorthern Japan and one in Canada. The ciliates from
Asahi (location 1) and Ichani (location 2) Hatcheriesin Hokkaido are significantly larger than those fromother hatcheries ( P < 0.01). The number of denticles
appears to increase with decreasing latitude in northern Japan except for specimens from Chitose
Hatchery (location 3) where the denticle number isvery small (24-26).

- 52 -UUAWA- PATHOBIOLOGY OF PARASITICPROTOZOANS ON SALMON

Figs. 4.1.2-5. Photomicrographs of Trichodinu truttue from chum salmon fry. 2. Adhesive disc of silver-imprzgnaled specimen collected from Asahi Hatchery. Bar = 20 pm. 3. Adhesive disc of silver-impregnated specimen from Chitose Hatchery. Bar = 25
I'm. 4. Detail of adhesive disc in Fig. 4.1.3,showing radial pins, which appear as pairs in this specimen. Bar = 10 ,urn. 5. Oralview of fresh specimen from Chitose Hatchery, showing adoral ciliary spiral. Bar = 40 p m . LAC, long adoral cilia; SAC, shortadoral cilia.

- 53 -

d l j . 2 -d Mlf$fiSCI. REP. HOKKAIDO SALMON HATCHERY, NO 50,1996

Figs. 4.1.6-9. SEM photomicrographs of Trzchodina truttae from chum salmon fty reared in Chitose Hatchev. 6 . Oral view of entirc
specimen. Bar = 35 pm. 7. Enlargement of oral surface showing short adoral cilia. Bar = 7 pm. 8. Enlargement of oral surface
showing long adoral cilia and buccal cavity. Bar = 8 pm. 9. Adhesive disc showing iadial pins and denticles. Note the low number of radial pins per denticle (about 12) indicative of a recently divided specimen. Bar = 5 pm. ACS, adoral ciliary spiral; BC,
buccal cavity; BM, border membrane; D, denticle; LAC, long adoral cilia; LCW, locomotor ciliary wreath; MCR, marginal ciliary
ring; RP, radial pins; SAC, short adoral cilia.

radial pins per denticle (14-26) is the highest number
reported for any member of the genus (Arthur andMargolis, 1984). In addition, their SEM observations,confirmed by the present study. note that the presenceof two markedly different lengths of cilia within theadoral ciliary spiral may also be unique to T. truttue.
Morphological features of our specimens are compa-

Discussion
Arthur and Margolis (1984) stated that the diagnostic features of T. truttue are its large size and large
number of radial pins per denticle. This species is thelargest ciliate among over 100 species of Trichodirzu
described from freshwater fishes, and the number of54

URAWA - PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Table 4.1.1. Comparison of morphometries of Trichodim truttae from salmonids in Japan and Canada. Measurements are given in
micrometers, and are based on silver-impregnated specimens.
~~

SourceHost
Site
Locality

Present study

adhesive discdenticulate ringNumber of:denticles
radial pins per denticle
Dimensions of denticles:
length
blade
thorn

*

114.7-143.9(125.5 9.4)
81.1-104.8(90.0 6.9)
45.3-62.1(51.0 f 4.4)*

114.0-170.5
12.2)(148.095.0-136.8(121.1 10.1)
45.6-69.4(58.5 5 6)

26-31
(27.2 t- 0.9)
12-23

24-26
(24.8 0.7)
10-20

*

27-3 1(29.5 1.0)
14-22

14.3-19.0(16.5 i 1.4)
11.0-14.0(12.1 f 0.9)
12.3-16.0
1.0)
(14.3
4.5-6.2(5.5 0.5)
28.5-35.5(32.0 1.8)*

12.8-20.0(15.4 2.2)
8.8-11.2(9.7 0.9)
10.4-15.2
1.6)
(13.4
6.1-8.5(7.0 0.6)
24.5-32.2(28.5 2.0)

*
*
*
*
*

14.2-22.8(19.6 1.7)
8.6-14.7(11.7 k 1.4)
10.4-18.0(14.5 k 1.9)
4.9-8.6(6.8 1.0)
24.7-37.0(32.9 2.7)

6.5-10.0
1.0)
(8.4

*

7.2-12.0
1.7)
(9.4

*

7.6-12.4(10.4 i 1.4)

about 450"

about 450'

440-450"

*

*

span

Width of bordei
membrane* SD

125.0-177.5(1447 f 14.2*)
100.0-125.0(108.8 7.2)
56.5-75.0(63.7 f 5.2)*

center*Mean( n = 21)

Arthur & Margolis (1984)
0. kisutch
Body surface
Quinsam Hatchery,
Vancouver Island,
B.C., Canada( n = 25)

Body surface
Asahi Hatchery,
Rishiri Island,
Hokkaido, Japan(n = 25)-

Diameter ofbodyAdoral ciliature

Present study
0. keta
Body surface
Chitose Hatchery,
Hokkaido, Japan

Oncorhynchus keta-

rable with those of T. truttae as given by Arthur andMargolis (1984) (Table 4.1.1).
Many of Japanese specimens have low numbers(8-12) of radial pins per denticle (Fig. 4.1.9). These
individuals are smaller than ciliates with high denticle number (18-23). These smaller ciliates haverecently undergone mitosis. They have completed
production of a new ring of denticles but have not yet
replicated the number of radial pins.
Trichodina truttue shows geographical variabili-

*
*
*
*

*

*
*

ty in body size and number of denticles within northem Japan (Fig. 4.1.10). The ciliates had a larger bodysize in Asahi and Ichani Hatcheries than in the otherthree hatcheries. When the host fish were collected,
the rearing water temperature was about 5,C in theformer two hatcheries, but between 8,C and 10,C in
the other hatcheries. Then, these differences in the
body size may be due to differences in environment(e.g., water temperature) and/or differences in thenumber of recently divided ciliates among the popu-

55 -2 Lf . d T&Ef$#,SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50, 1996

I+
0lations. Kazubski and Pilecka-Rapacz (1981) similarly reported seasonal and interpopulational variability
in the body dimensions of T. nigra Lom, 1961, andBasson and Van As (1989) found intraspecific variation in dimensions and shape of denticles of
Hemitrichodina robusta Basson & Van As, 1989.
Takeda et al. (1969) recorded T. domerguei fromchum salmon fry reared in Ichani Hatchery without
providing any morphological description. Takeda(1971) also reported a heavy infection of Cyclochaeta(= Trichodina) domerguei on chum salmon alevins in
Nemuro Hatchery, eastern Hokkaido, and listed
Sakhalin taimen (Hucho perryi) and masu salmon(Oncorhynchus masou) as experimental hosts.
Although T. domerguei has, on one occasion (seeBogdanova and Shtein, 1963), been reported fromchum salmon fry, this species is primarily a parasiteof gasterosteid fishes (see Lorn and Stein, 1966). The
present finding of only T. truttae from the same
hatcheries as those from which T. domerguei wasreported by Takeda et al. (1969) and Takeda (1971)
and the lack of supporting evidence to validate theiridentifications leads me to believe that these reports
are probably referable to T. truttae.

Fig. 4.1.10. Geographical variation in the diameter of the denticulate ring and number of denticles for Trichodina trurtue. Each open circle represents the mean, the vertical bar
represents the range. The number above or below eachpoint is the sampling locality. 1, Asahi Hatchery (n = 25);
2, lchani Hatchery ( n = 20); 3, Chitose Hatchery ( n = 21);4, Ohkawa Hatcheq (n = 20); 5 , Gakko Hatchery (n = 20);
6, Quinsam Hatchery (49"30'N, 124"ZO'W) on Vancouver
Island, B.C. (n = 25; from Arthur and Margolis, 1984).

56 -URAWA - PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

4.2 Pathogenicity and Host-Parasite Interactions
Abstract. - A transmission experiment was carried out to evaluate the effects of Trichodinu truttae
infection on the growth and survival of juvenile chum salmon (Oncorhynchus keta) and the host's
defense responses. The intensity of infection rose sharply to reach a peak of an average of 5,730 (range
1,770 - 12,600)parasites per fish at week 3 post-infection,and then declined markedly to near zero by
the end of the experiment (week 6). The parasite was site-specific to the skin of host fish and induced
epidermal hyperplasia. Heavy infections caused intensive flashing among infected fish, which was
accompanied by mass host mortalities with a cumulative loss of 56%. The growth and seawater adaptation of juveniles were, however, not significantly affected. In the course of infections, drasticchanges occurred in the degree and histochemistry of the skin epidermal mucus secretion. The densityof mucous cells containing acid mucopolysaccharides (Alcian blue-positive, PAS-negative) decreased
with increasing parasite intensity, while the parasite infections induced a marked increase of PAS-positive mucous cells, in turn, which may have caused a distinct reduction in parasite intensity. These
results suggest that the increased production of PAS-positive mucous cells is an effective defense
mechanism against trichodinid infections

aquaria supplied with well water at a flow rate of 3.6
I/min. One group was held for one week together
with five chum juveniles infected with Trichodinu
truttae derived from Asahi Hatchery on Rishiri
Island, northern Hokkaido (see Urawa and Arthur,1991). The other group was held as an uninfected
control. These two groups were reared for 6 weeks
with commercial dry pellets fed at approximately 2%
body weight per day. Daily monitoring included
checking fish mortalities, water temperature and dissolved oxygen in each aquarium. Water temperatures
were stable at 10.5"C, and dissolved oxygen concentrations were maintained above 9 mg/l throughout theexperiment.

Introduction
Trichodinids are probably the most commonly
encountered protozoan parasites on wild and cultured
fishes in marine as well as freshwater environments.
Hoffman (1978) listed 74 trichodinid species, ofwhich at least 19 were believed to cause considerable
damage to their hosts under certain conditions. There
have been, however, no investigations to confirm thepathogenicity and host-parasite interactions underlaboratory conditions.
Trichodina truttae Mueller, 1937 is specific tosalmonid fishes and widely distributed on the North
Pacific coasts of North America, Sakhalin Island, andnorthern Japan (Mueller, 1937; Davis, 1947, 1953;
Bogdanova, 1963, 1967, 1977; Bogdanova andShtein, 1963; Arthur and Margolis, 1984; Urawa and
Arthur, 1991). This large ciliate has often been
observed on the skin (accidentally on the gills) of
juvenile salmonids reared in hatcheries, where heavy
infections have occasionally been associated withhost mortalities (Bogdanova and Shtein, 1963;
Takeda et al., 1969; Takeda, 1971; Hoskins et al.,1976).
The purpose of this infection experiment is toclarify the effects of T. truttae on the growth and survival of juvenile chum salmon (Uncorhynchus ketu)
and to investigate the host's defense mechanisms.

Sampling and biometric procedures
Twenty to 30 fish were collected at weeklyintervals from each group and immediately fixed in
10% neutral buffered formalin. Infected fish were
preserved separately in individual bottles. The fixed
fish were measured and weighed individually, andcondition factor (CF) was calculated using the formula: CF = 100W/L3,where W is the weight (g) of theindividual fish, and L is the corresponding fork length(cm).
Detection of parasites
Since the parasites were removed from the host's
skin when fixed in formalin, they were collected bylight centrifugation (1,500 rpm, 5 min) of sediment ineach bottle, and counted with a stereo microscope. In
a separate study to determine the spatial distributionof the parasites on the host, the attachment site ofMaterials and Methods
Fish-rearing and transmission procedures
Two groups, each containing 380 juvenile chumsalmon (average weight 0.78 g), were placed in 25-1

57

2 If

$-FAW$$ SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

individual parasites was recorded on the skin andgills of lightly infected fresh fish ( n = 20) under the
microscope. The parasitological terms used here arein accordance with those of Margolis et al. (1982).

tions, a blood sample was taken with a heparinizedcapillary tube from the caudal artery of each live fish(n = 10) just before and after the seawater challenge
test. After centrifugation at 10,000 rpm for 5 min, theserum C1- concentration was immediately measured
using a chloride counter (Hiranuma Co.).

Histological procedures
Five fixed fish from each group were examined
weekly throughout the six week period of the experiment to determine changes in the density and histochemistry of epidermal mucous cells due to parasitic
infection. The staining method employed was aslightly modified version of the Alcian blue
(AB)/periodic acid-Schiff s reagent (PAS) techniquedescribed by Blackstock and Pickering (1982). The
skin between the dorsal and ventral fins was removed
from one side of the fixed fish and washed in runningwater. The thin sheet of tissue was rinsed in 3%
acetic acid at pH 2.5 for 2 min, and stained with 1%
Alcian blue in 3% acetic acid at pH 2.5 for 15 min.
After a 10-min wash in running water, the stained tissue was immersed for 30 min in 0.3% sodium carbonate. Following a 10-min rinse in running water
the tissue was oxidized in 0.5% periodic acid for 5-10
min, washed for a further 10 min and then placed in
Schiff s reagent for 30 min. The reaction was stopped
with three 2-min baths of 0.05 M sodium bisulphite
followed by a final 10-min rinse in running water.
Specimens were dehydrated, cleared and mounted in
Bioleite (Ohken Co.). Superficial mucous cells in theskin epidermis were stained either blue (AB-positive,
PAS-negative), purple (AB-positive, PAS-positive)
or deep red (AB-negative, PAS-positive). Differential
counts were made for each fish from 10 random
quadrates (total 0.38 mm2), and the concentrations
and proportions of mucous cells displaying the threedifferent staining reactions were calculated. In addition, the fixed fish were embedded in paraffin wax,
sectioned at 6 pm, and the sections were stained withhematoxylin and eosin, Giemsa's stain or AB (pH2.5)/PAS, as given in Urawa (1992a).

Statistical analysis
Levels of statistical significance among thegroups were obtained using the Mann-Whitney U-test(one way). Correlations were analyzed with Pearsons
correlation matrix.

Results
Parasite intensity
All fish of the test group were infected with T
truttae at one week post-infection, while no fish were
infected in the control group throughout the experiment. The parasite intensity increased sharply during

0 Control

Seawater challenge tests
In order to evaluate the effects of parasite infection on the seawater survival of juvenile chumsalmon, seawater challenge tests were carried out at 4
and 6 weeks post-infection. Thirty fish from eachgroup were held in 20-1 tanks supplied with oxygenated seawater (salinity 33O/m) for 48 h, and theirmortalities were recorded. To estimate ionoregulationof fish by measuring serum chloride (C1-) concentra-

0

1

fish

2

3
4
Weeks

5

6

Fig. 4.2.1. Weekly changes in the mean intensity of
Trichodina truttae and the host mortality in juvenile
chum salmon. Bars indicate SD.

58

UFL4WA- PATHOBIOLOGYOF PARASITIC PROTOZOANS ON SALMON..

..... ...... ......

.......... .........

........... .............................

..

I 1.7sFig. 4.2.2. Spatial locations of Trichudinu truttue on juvenile chum salmon. A total of 235 parasites was counted on 20 fish at low levels of infection (mean intensity = 11.8). Closed and open circles represent the facing and reverse sides of attachment site, respectively.

the next two weeks, reaching a peak at week 3 withan average of 5,730 (range of 1,770 to 12,600) parasites per fish, and then declined markedly to an average of 85 (range 18-444) parasites by the end of theexperiment (week 6) (Fig. 4.2.1).
Spatial distribution ojthe parasite
The trichodinid occurred widely on the bodysurface and fins of host fish (Fig. 4.2.2), but not on
the gills. The frequency distributions of the parasite
varied markedly from site to site: the parasite numbers were relatively high on the head region anterior
to the gill operculum (28.1%) and on the caudal fin(26.8%), but low on the pectoral and dorsal fins (1.3
and 1.7%, respectively).
Mortality
Fish started to flash two weeks after parasite
infection. Severe flashing and jumping out of thewater occurred in the following two weeks, which
was accompanied by rapid mortalities. Weekly mortality in the infected fish reached a maximum(32.1%) at week 4, one week after the peak of parasite intensity, and then decreased to zero at week 6(Fig. 4.2.1). Overall mortality at the end of six weeks
was 56.0% in the infected fish, as compared to only
0.6% in the controls.
GrowthInfected fish had reduced growth rates during

the first two weeks of infection, but at the end of theexperiment, there were no significant differences inmean length or weight between the infected and control groups (Fig. 4.2.3). The condition factor of theinfected fish was lower at the end of the first week,
but higher during the last two weeks of the experiment, compared with that in the controls (Fig. 4.2.3).
No discernible relationships were evident between
parasite intensity and weight or length of individual
host fish throughout the experimental period ( P >0.05). When the condition factor of these fish wascompared with parasite intensity (Fig. 4.2.4), however, there were significant negative correlations atweeks 4 and 5 ( P < 0.05), and a positive correlation
at week 1 ( P < 0.05).
Seawater survival
Mortality in seawater after 48 h was less than
7% in both the infected and control groups (Table
4.2.1). There were no significant differences in theserum C1- concentrations between the groups, neither
in fresh water nor seawater (Table 4.2.1).
Histopathology
AB-positive mucous cells are common in theepidermis of uninfected skin (Figs. 4.2.5 & 9), but
most of these disappeared during the first two weeks
of infection (Figs. 4.2.6 & 10). In contrast, numerous
PAS-positive mucous cells appeared in the upper
layer of epidermis at week 4 (Figs. 4.2.7 & 1 l), after

59 -

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Weeks
Fig. 4.2.3. Weekly changes in the mean folk length, weight, and condition factor 0f Trichodina truttae infected and uninfectedchum

salmon juveniles. Bars indicate sD. *, P < 0.05; **, P < 0.01.

which AB‑positive cells reappeared abundantly underthem (Figs. 4.2.8 & 12). MOre apparent changes
included marked epidermal hyperplasia (Fig. 4.2.7)
While the protruding edges 0f the border membrane
0f the tnchodinid parasite seemed t0 injure the host's
epidermal cells (Fig. 4.2.13), severe skin erosions
were

not

Mucouscell concentration
The concentration of AB‑positive

mucouscells

significantly reduced with increasing parasite
intensity during the first3 weeks, while ft increased
sharply during the last 2 weeks when the parasite
intensity decreased (Fig. 4.2.14). In contrast, PAS‑
positive mucous
cells appeared at week 3, their con‑centration rapidly rising t0 approximately 2,300 cells
wasobserved

60

OF PARASITIC PROTOZOANS ON SALMONURAWA-PATHOBIOLOGY

300

10000
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150

0
2000
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Condition factor
Fig. 4.2.4. Weekly comparisons between the intensity of Trichodina truttae and condition factor in individual chum salmon juveniles.
Table 4.2.1. Mortality and serum CI- concentration in Trichodina truttne infected and uninfected juvenile chum salmon held for 48 hin seawater (salinity 33'/w) at weeks 4 and 6 post-infection. Thirty fish from each group were placed in seawater, and 10 fish were
examined for serum CI- concentration.

Group

Weeks
postinfection

Mortality (8)in seawater
within 48 h

in fresh water

i n seawater"'

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131.3

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InfectedControl

6~~~*I48 h after the transfer
SD*'Mean

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61

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%If

+ tb@T$IlSCI RBF' HOKKAIDO SALMON HATCHERY, NO 50.1996

Figs. 4.2.5-8. Light micrographs of the skin of juvenile churn salmon infected with Tnchodrm truulfae A h i n blue (AB. pH 2 5)lPA.S
stam Each bar = M) prn 5. Normal cprderrms conmrung several AB-posinvc mucous cells (dycd bluc)~ 6. Two weeks alkrm€.zccmn Nore h e disappearance oI mucous cells h o r n the e p z d e n s 7. Four w e b after infection A large nurnbcr of PASpositive mucous cells (mows, dycd deep red) appcvcd m the upper layer of epidermis 8. Six weeks dler intecnon In addilton
10 PAS-pomnue mucous cells (mows).AB-pomnue ~ U C O U Scells revwed in rhe epidermis

URAWA-PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

'3

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63

$el Z f M M SCI REP HOKKAlDOSALMONHATCHERY,NO 50,1996

13

Fig. 4.2.13. Light micrograph of the skm of J U Y ~ chum
~ C
salmon mu1 Tnchodmo mtwe on the cpidems NoLC possiblc I " J U ' ~ ofepidermal cclls caused by pmtmding edges of the tnchodlrud border membrane (mow) Gtemsa's stain Bar = 50 fim

2-aControl fish

ln
0

ln-

a%

1 -

-

URAWA -PATHOBTOLOGY OF PARASITIC PROTOZOANS ON SALMON

per mm2at week 4, and being maintained at high levels until week 6. In the controls, the number of ABpositive mucous cells remained almost constant (8001,300 cells per mm2),and PAS-positive mucous cells
were rarely found over the course of the experiment(Fig. 4.2.14).

Discussion
This infection experiment demonstrates that
Trichodina truttae can cause mass mortality of juvenile chum salmon in fresh water when the mean
intensity increases up to 5,000 parasites per fish.
Infections, however, had no significant effects on theosmoregulation or seawater adaptation of the anadromous host fish, although the ectoparasitic flagellate
Ichthyobodo necator is known to reduce seawater
survival of infected chum salmon due to severe epidermal destruction (Urawa, 1993).
In general, trichodinids are believed to feed ondebris of host cells on the surface (Lom, 1973;
Ahmed, 1977). L o m (1973) observed that
Trichodinella epizootica injured host cells by theaction of its border membrane and by pulling theCells into its vaulted disc. The present histologicalobservations also confirm that Trichodina truttae
causes sloughing of the epidermal cells as well as
epidermal hyperplasia. Epidermal damage such as
necrosis and desquamation, however, was not sosevere as seen in Ichthyobodo infections (Robertson
et al., 1981; Urawa, 1992a). This was corroborated
by the fact that T. truttae had n o influence on
osmoregulatory ability or seawater adaptation of hostfish. On the other hand, heavy trichodinid infections
caused extreme flashing of juveniles without anyother apparent symptoms. Thus, acute mortalities infresh water are probably attributable to stress arisingfrom intense irritation caused by numerous parasites
attaching to the entire body surface.
The intensity of T. truttae rose rapidly during
the 3rd and 4th weeks of infection, and then declined
sharply to near zero during the last 2 weeks.

- 65

Responding to the parasite infections, drastic changes
occurred in the epidermal mucus secretion. The density of AB-positive mucous cells containing acidmucopolysaccharides decreased with an increase ofparasite intensity, while the infections induced amarked increase in PAS-positive mucous cell density,
resulting in a sharp reduction of parasite intensity. A
similar epidermal response, including the mass occurrence of PAS-positive mucous cells was a l s oobserved in the skin of salmon fry infected with I.
necator (Urawa, 1992a). Epidermal mucous secretions seem to be an important natural defense mechanism against pathogenic organisms. The defense may
be both mechanical and due to the antibiotic properties of the mucus secretions (Pickering and kchards,
1980). PAS-positive mucous cells have been suspected to contain some protective substances against
Ichthyobodo infections (Urawa, 1992a). Increased
production of PAS-positive mucous cells may be anon-specific defense mechanism against ectoparasitic
infections, although the biochemical mechanism of
this protection has not been clearly elucidated.
The growth of fish appear to be not influenced
by T. truttae infections, while statistically significant
negative correlations existed between the parasite
intensity and condition factor of individual hosts 4
and 5 weeks after infection. It has been suspected thattrichodinid numbers increase when host fish are
under unfavorable conditions (Lom, 1970; Egusa,
1978; Wood, 1979). This is not supported by the present case, but it is possible that the parasite intensity
may be affected by host condition associated withpossible defense abilities.
The present study confirmed that T. truttae
becomes a serious pathogen capable of causing highmortalities in hatchery-reared salmonids. Further
studies are needed to elucidate host-parasite interactions, especially the role that epidermal mucus secretions play in the defense against invading parasites,
and the exact mechanism of host deaths caused by trichodinid infections.

2 1t . 2

SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

5. Chilodonella piscicolu
5.1 Morphology and Pathogenicity
Abstract. - An ectoparasitic ciliate, Chilodonella piscicola (= C. cyprini), became epizootic andcaused chronic mortalities in juvenile masu (Oncorhynchus masou) and pink (0.gorbuscha) salmon
reared at Nemuro Hatchery, eastern Hokkaido, in the spring of 1988. Cumulative mortalities amounted
to 20% in masu salmon over 5 months, and to 10%in pink salmon over 2 months. Transmission experiments in maw salmon fry confirmed that C. piscicola can cause severe proliferation of the gill epithelium in the absence of other stressors. The infected fish exhibited reduced growth and chronic mortalities after the mean intensity had reached a peak (619 parasites per fish) at week 4 post-infection. The
marked epithelial hyperplasia was followed by intense fusion of adjacent gill lamellae and filaments.
Respiratory failure due to gill epithelial hyperplasia may have been a primary cause of the deaths.

Introduction
Chilodonella piscicola (Zacharias, 1894) (= C.
cyprini, according to Shul'man, 1984) is a ciliatedprotozoan widely dispersed i n the Northern
Hemisphere (Hoffman, 1978) and also in t h eSouthern Hemisphere (Ashburner and Ehl, 1973).
This species is commonly found on the gills and skinof various freshwater fishes, and often associatedwith moderate to severe mortalities of hatcheryeared juvenile salmonids and cyprinids (Schaperclaus, 1935;Davis, 1953;Bauer, 1959). Anotherparasitic ciliate, C. hexasticha (Kiernik, 1909), hasbeen found on diseased warmwater fishes (Hoffman
et al., 1979; Paperna and Van As, 1983; Imai et al.,
1985; Ogawa et al., 1985). Lorn and Nigrelli (1970)
recorded the Chilodonella-like parasite (Brooklynella
h o st i l i s) from diseased marine fishes. T h epathogenicity of these ciliates, however, is still a matter of debate. Lom and Nigrelli (1970) noted that theciliates produce disease only in hosts that have been
debilitated by other factors. Egusa (1983) suggested
that, in some cases of host mortaliy, the massive
numbers of C. piscicola found on affected areas may
merely reflect other increased stress factors.
Surprisingly, however, no experimental infections
have been carried out to evaluate pathogenicity of C.
piscicola and related species.
Moderate but sustained mortalities occurredamong juvenile masu (Oncorhynchus masou) andpink ( 0 .gorhuscha) salmon reared at Nemuro

and C. piscicola. Also, I have elucidated the parasite's
pathogenicity by transmission experiments.

Materials and Methods
Hatchery-reared fish
At Nemuro Hatchery in eastern Hokkaido,
approximately 1,050,000 masu salmon fry (meanweight, 0.19 g ) were reared from January 11, 1988 in
an indoor concrete raceway of 85 m' with water levels of 0.1-0.3 m. In addition, about 3,510,000 pinksalmon fry (mean weight, 0.24 g) were stocked in adifferent indoor raceway of 242 m2with a water levelof 0.1 m. Each raceway was supplied with a mixtureof river and well waters (water flow ranged 54-1,010
l/min, depending upon fish growth; pH 7.0). Fishwere fed commercial dry pellets, and mortalities wererecorded. Flow rates of water, levels of dissolved
oxygen, and water temperatures were monitored at
10-day intervals with a dissolved-oxygen meter (YSTmodel 57). Neither pathogenic bacteria nor viruses
were detected in affected fish throughout the rearing
period.

Hatchery, eastern Hokkaido, in the spring of 1988.

Light microscopy
Fish were fixed in 10% neutral buffered formalin or Bouin's solution and processed into paraffinblocks with standard histological techniques.
Sections were cut 5 p m thick and stained with hematoxylin and eosin (H & E) or Giemsa's solution. In
addition, smears were made from the affected gills
and stained with Giemsa's solution or impregnatedwith AgNO, according to Klein's (1958) technique.

The diseased fishes were heavily infected with C. piscicolu and displayed severe hyperplasia of the gill filaments. In this paper I report light and scanning electron microscopical observations of the diseased fishes

Scanning electron microscopy
The infected gills were removed and fixed in 2%
phosphate-buffered glutaraldehyde (pH 7.2; total

66 -URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

osmolality, about 21 0 milliosmoles). The tissues
were dehydrated in a graded ethanol series. The
ethanol was replaced by isoamyl acetate, and then thetissues were critical-point dried. The dried tissues
were gold-coated and examined with a scanning electron microscope (SEM).
Experimental infection
The pathogenicity of C. piscicola was experimentally examined with masu salmon fry (meanweight, 0.3 g) obtained from Chitose Hatchery, western Hokkaido. Two groups of 300 uninfected fish
were each kept in 18-1 aquaria. One group was reared
together with five caged masu salmon that were
infected with C. piscicola derived from Nemuro
Hatchery. The other group, which served as the control, was reared with five parasite-free masu salmon.
Test fish in both groups were fed commercial dry pellets at a feeding rate of 3% body weight per day for 8
weeks. Each tank was supplied with well water*(mean temperature & SD, 10.5
0.1"Cj at a constant flow rate of 4 Vmin. Dissolved oxygen ranged
from 10.0 to 10.8 mg/l, and unionized ammonia
ranged from 0.000 to 0.001 mg/l in the lower end ofboth tanks during the experiment. Neither of the factors were harmful to the fish in t h s experiment. Fromeach group, 21-33 fish were collected at 0, 4, and 8
weeks post-exposure. Each fish was separately fixedin 10% neutral buffered formalin, measured for fork
length, and weighed. The gills were removed andmounted in water under a cover slip. The slides weremicroscopically examined to determine histopathological changes and number of parasites on the gills.
Additional counts were made of the parasites that
detached from the host when the host was fixed in theformalin solution. The terms prevalence and mean
intensity follow the definitions proposed by Margolis
et al. (1982). The condition factor (CF) was calculated from the equation: CF = lOOwlP, where w is the
body weight (gj of individual fish, and 1 is the corre-

Figs. 5.1.1 & 2. Ventral views of Chilodonella piscicolu from masu salmon. Each bar = 15 pm. 1. Giemsa's stain. 2. Silverimpregnated specimen showing 12 (6 preoral) kineties in the right band of ciliature and 12 in left band. CT, cytopharyngeal tube;
LK, left band of kineties, MN, macronucleus; PK, preoral kineties; RK, right band of kineties.

- 67 -

3 If . 3 T.LM% SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

sponding fork length in cm. The Mann-Whitney Utest (one way) was used to test for differences
between the infected and control groups.

Results
Parasite
The parasite found on the gills and occasionallyon the fins of masu and pink salmon juveniles was
identified as Chilodonella piscicola. It was dorsoventrally flattened, and oval or cordate in shape with aposterior notch (Figs. 5.1.1-3), measuring 50-77 p mlong and 42-74 p m wide. Macronucleus was more orless oval (Fig. 5.1.1), 19-32 pm long, and 10-21 p m
wide. Micronucleus was not detected. Dorsal bodysurface was ornamented with numerous narrowridges parallel to the body axis and a prominent porelike opening (Fig. 5.1.3). This porelike dorsal structure was previously observed by Wiles et a]. (1985),
but its function is unknown. A short ciliary row with
8-10 cilia was on the anterior dorsal side of the body.
Ventral surface was covered with thick cilia (Fig.
5.1.5). The left band of ciliature had 12-16 hneties
and the right band had 11-13 kineties (Fig. 5.1.2). Of
the kineties in the right band of ciliature, 5-7 werepreoral, extending from the front of anterior end of
the left band (Fig. 5.1.2). The ventral pellicle wasslightly concave between the two bands of ciliature(Fig. 5.1.5), and a rod-shaped cytopharyngeal tube
protruded on the anterior end of this concave (Figs.
5.1.1 & 6). The left anterior margin formed a fistshaped apparatus ornamented with several stout setae(Fig.. 5.1.6). It is possible that this apparatus twists
itself around the cytopharyngeal tube during feedingon host tissues. The posterior edge of the body was
V-shaped with a stout shoehorn-shaped border (Fig.
5.1.5), which appeared to insert its tip into degenerated host epithelial cells (Figs. 5.1.3 & 4). Although
Wiles et al. (1985) found rows of short cuticular protuberances on ventrolateral margins of C. piscrcola
from goldfish (Carassius auratus) in eastern Canada,
such accessories were not observed in my specimens.
The light microscopical features of the parasite wereconsistent with those of C. piscicola as described by
Kazubski and Migala (1 974) (Table 5.1.1).

Gross observations
Masu salmon. Mortality peaked in the end of
February, 1988, about 2 months after the start of rearing, and it gradually declined between March andMay (Fig. 5.1.7). About 20% of the original population died during the 5-month rearing period. The diseased juveniles were found at the lower end of the
raceway, where they showed listlessness, loss ofappetite, extended gill covers, and darkened bodycolor. In the rearing raceway, mean water temperature remained at 6-7°C from January to April androse to 8.5"C in late May (Fig. 5.1.7). Dissolved oxygen gradually decreased to its lowest level (6.5 mg/l)
in late April (Fig. 5.1.7), and then it was back to its
initial level (9.0 mg/l) by mid-May.
Pink salmon. Approximately 352,000 juveniles(10%) of the original stock died over 2 months.

Table 5.1.1. Morphological comparison of Chilodonella from masu and pink salmon with Chilodonella piscicola and C. hemstichafrom common carp (Cyprinus carpio).

Pink salmon
Gills(n = 30)

Chilodonella
piscicola*'
Common calp
Skin, gills( n = 35)

Chilodonella
hexasticha*'
Common caIp
Skin, gills(n = 65)64 (52-77)*'
57 (43-74)

61 (50-73)
49 (42-64)

60 (47-80)
42 (35-46)

58 (48-70)
46 (35-61)

13.4 (12-14)
11.6 (11-13)

13.9 (13-16)
12.1 (11.13)

12.8 (11-15)

8.4 (6-10)

11.8 (10-13)

6.1 (5-8)

Parasite

Present species

Present species

Host
Site of infection

Maw salmon
Gills(n = 30)

Body dimensions
Length ( Pm)
Width (,urn)

No. of kineties
Left side
Right side*'After Kazubski and Migala (1974).
*'Mean with range in parentheses.

- 68 -

URAWA-PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Figs. 5.1.3-6. Scanning electron micrographs of Chilodonellu piscicola attaching to the gills of maw salmon. 3. Dorsal view of twoparasites, showing prominent porelike openings (arrows). Bar = 20 pn. 4. Lateral view of the parasite. Host epithelial cells
seem to bc sloughed off by the parasite's posterior margin. Bar = 10 pm. 5. Ventral view of the parasite, showing thick cilia andrigid posterior margin (arrow). Bar = 10 pm. 6. Enlargement of anterior part of ventral body, showing a protmded cytopharyngeal tube (CT) and a fist-shapcd apparatus (FA). Bar = 5 pm.

- 69 -

2 If. 3+AW% SCI. REP. HOKKAlDO SALMON HATCHERY, NO. 50,1996

Fig. 5.1.7. (Top) Changes in the water temperature (closed squares) and dissolved oxygen (open circles) at Nemuro Hatchery.
(Bottom) Cumulative mortality (closed circles) of masu salmon fry at Nemuro Hatchery and mortality at 10-day intervals (stippledcolumns, expressed in units of ten thousand fish).

sionally present (Fig. 5.1.13).

Clinical signs were similar to those seen in masu
salmon.
Microscopical observations
Affected fishes were characterized histologicallyby a proliferative reaction of the gill epithelium.
Massive epithelial hyperplasia resulted in extensive
fusion of adjacent lamellae and obliteration of theinterlamellar space (Figs. 5.1.11 & 12). Severe fusionof the gill filaments also occurred in areas where theparasites were scattered (Figs. 5.1.%lo),whereas few
parasites were found on the normal gill filaments.
The hyperplastic tissues were edematous (Fig.
5.1.12), revealing cellular degeneration, mild erosion,and inflammatory response. Other responses included
marked swellings at the tips of filaments where
thrombus and extravasation of blood cells were occa-- 70

Experimental infection
All fish examined were found infected following
the exposure to C. pucicolu. The mean intensity was
619 and 167 parasites at weeks 4 and 8 post-infection, respectively (Fig. 5.1.14A). A moderate mortality started at week 5 among the infected fish, and thetotal mortality reached 6% by week 8 (Fig. 5.1.14B).
No parasites were found in the control group, and its
total mortality was less than 1%.Although the mean
fork length and weight of the infected fish were lower
than those of the controls, the differences were notsignificant ( P > 0.05; Fig. 5.1.14C). On the other
hand, the condition factor of the infected group was
significantly lower than that in the control group (P<0.05; Fig. 5.1.14D). When the condition factor andURAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Figs. 5.1.8-10. Scanning electron micrographs of the gills of maw salmon infected with Chilodonella piscicola. 8. Entire view of anaffected gill. Fusion of adjacent gill filaments occurred at random. Bar = 0.5 nun. 9 & 10. Enlargements of the affected gill,showing severe fusion of adjacent filaments where the parasites (arrows) appear. Each bar = 0.2 nun.

- 71 -

S If . 6T.LW8 SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

Figs. 5.1.11-13. Histological sections of the gills of masu and pink salmon infected with Chilodonella piscicoh. 11 & 12. Infectedgill filaments of pink salmon showing epithelial hyperplasia and extensive lamellar fusion caused by the parasites (arrows).
Giemsa's stain. Each bar = 70 pm. 13. The affected gills of masu salmon showing severe hyperplasia, swelling and fusion of thefilaments. H & E. Bar = 100 pm.

72 -URAWA-PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

0-q'

5n

E0

v

50

4-

&

3-

1.4-

C

*

D

*

C

.-

0

*: .

........

c
,

L

2

0.8

i

I

I

0
4
8
Weeks post-infection

I

i

I

0
4
8
Weeks posthfection

Fig. 5.1.14. Changes in (A) mean intensity of Chilodonellapiscicola, (B) percent cumulative mortality, (Cj mean fork length, and (D)mean condition factor in the experimenlally infected maw salmon fry (closed circles) and the controls (open circles). Bars indicate
SD. *, P i 0.05 compared with the control.

Table 5.1.2. Occurrence of hyperplasia of gill epithelium and adhesion of gill filaments III masu salmon fry experimentally infectedwith Chilodonella piscicola.
Number of fish (%)
Group

Weeks
post-

With hyperplasia

With adhesion

infection

Examinedof gill epithelium

lnfccted

0
4
8

21
28
30

4 (19)
28 (100)
30 (100)of gill filaments
0 (0)21 (75)
1 1 (37)

Control04823Prevalenceof
infections
(%)

~~

0
100
100

32
30
~

parasite intensity were compared in the infectedgroup (Fig. .5.1.15), they showed no relationship atweek 4, but revealed a negative correlation (Y =-0.467; P < 0.02) at week 8.
Hyperplasia of the gill epithelium was found in
all infected fish and some control fish (Table 5.1.2).

Adhesion of the gill filaments also occurred in 75%
and 37% of the infected fish at weeks 4 and 8,
respectively. No fusion of filaments was encounteredin the control group.

- 73 -

5 If . ?CAW+SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50, 1996

4 weeks
post infectionn= 28
r -0.302
P> 0.1

-

v)

Q)

U

1500

0

v)

-x
E

1000

0

a
f 500- l
6

o

\

"

f

1500

cn
Q)

.-u)
+d

E 1000
(P

P

6

0

\

I \
0.6

8 weeks
post infection
n=30
r =-0.467
P c 0.02

-

0

0

0.8

1.0

1.2

1.4

1.6

Condition factor
Fig. 5.1.15. Comparisons between the parasite intensity and thecondition factor of masu salmon fry 4 and 8 weeks after
exposure to Chilodonella piscicola.

Discussion
Hyperplasia of the salmonid gill epithelium is
produced by various direct or indirect factors, such ascontinuous exposure to high levels of unionizedammonia (Burrows, 1964; Larmoyeux and Piper,
1973; Smith and Piper, 1975; Klontz et al., 1985;
Lang et al., 1987), bacterial gill disease (Kudo andKmura, 1983; Ferguson, 1989), high population density, low dissolved oxygen levels, and water temperature changes (Snieszko, 1974; Smith and Pier, 1975;
Wedemeyer et al., 1976; Wood, 1979; Roberts andShepherd, 1986). Because Chilodonella spp. areoccasionally found in weak fish affected by thesecomplex stressors, there has been some doubt about
the parasite's pathogenicity.
The present transmission experiment clearly
indicates that severe hyperplasia of the gill epitheli-- 14 -

um of masu salmon fry is caused by C. piscicola
infections in absence of other stressors. The infectedmasu salmon fry also exhibited reduced growth andchronic mortalities, which occurred after parasite
intensity peaked at week 4. The infected fish showed
an inverse correlation between the intensity of infection and host condition factor at week 8 post-infection, but not at week 4. These results suggest thatchronic infections affect the growth and survival of
host fish. The marked epithelial hyperplasia was followed by extreme fusion of adjacent gill lamellae andfilaments. The cause of death may be respiratory failure due to branchial hyperplasia produced by C. piscicola. The gill epithelial proliferation is probably
attributable to continuous stimulation by the thickventral cilia and by the feeding activity of the parasite. Thus, chilodonelliasis of salmonids is characterized as a chronic proliferative gill disease.
The distribution of C. piscicola on the gills was
distinctive: most parasites were found only on hyperplastic areas. This distribution suggests that thehyperplastic tissues attract additional parasites andpromote increased parasite reproduction. These proliferative cells may be easily utilized as food by theparasite, as pointed out by Bauer (1959), who also
noted that the parasite sucks out the contents of individual host cells by protruding the cytopharynx. My
histological and SEM observations suggest that thecytopharynx may not stick into the epithelium, while
the anterior fist-shaped apparatus seems to facilitate
grazing on host tissues. In addition, weak host tissue
cells appear to be sloughed off by the parasite's rigid
posterior margin (Figs. 5.1.4 & 5). The SEM observations suggest that this posterior margin may also
act as an attachment organelle, whereas Wiles et al.
(1985) supposed that chilodonellids somehow use
their ventral cilia or cytopharynx (or both) to attach
to hosts' cells. A transmission micrograph(MacMillan, 1991) implied that the posterior margin
becomes embedded in the gill epithelium. Further
studies that include transmission electron microscopy
are needed to clarify the feeding and attaching mechanisms of the parasite.
As the infection experiment revealed, C. piscicola infection was an exclusive cause of the salmonid
gill disease at Nemuro Hatchery. Overall mortality of
10-20%was significantly higher than average mortality in healthy hatchery-reared fish. Hoffman andMeyer (1974) reported that formalin is effective fortreating Chilodonella-infected fish. However, despite

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

repeated 1 hour treatments with a 15,000solution offormalin at Nemuro Hatchery, the health of theaffected fish was not improved nor was the parasite
completely eliminated. Affected pink salmon fry
were released into a river, and more may have died in
the estuary, because branchial hyperplasia reduces
the host's ability to adapt to seawater.

In recent years, infections by C. piscicola havealso occurred among masu and chum (0. keta)
salmon fry in other hatcheries of northern Japan(Urawa, 1992~).There is thus a need for more workon the life cycle of C. piscicola, the host's defense
mechanisms against the parasite, and control methods.

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SCI REP HOKKAIDO SALMON HATCHERY. NO. 50.1996

6. Concluding Remarks
The present survey shows that the three protozoan species, Ichthyohodo necator, Trichodinu truttae and Chilodonella piscicola, commonly appear on
the external surface of hatchery-reared salmonids. If
their pathogenic potentials are low, the infections canbe ignored in the course of hatchery practices. Thus,an essential object of this study is to evaluate thepathogenic virulence of each parasite species against
host fishes by infection experiments.
The pathogenic consequences vary from species
to species, depending on attaching method, parasite
intensity, host condition and environments. Both of I.
necator and T. truttae are parasitic on the body surface of salmonids, but their mode of attachment andfeeding distinctly differs each other. Ichthyohodo
penetrates to epidermal cells by an adhesive discfrom which a cytostome process extends into the hostcytoplasm (Joyon and Lom, 1966, 1969; Schubert,
1966, 19681, and cause severe epidermal destruction,
which leads to host deaths due to osmoregulatory
breakdown. In contrast, T. truttue does not penetrate
to the host tissues, but causes acute mortalities among
hosts maybe due to extreme stimulation by feeding
activity with sick cilia and adhesive disc. In the caseof C. piscicola infections, the major cause of chronicmortalities is respiratory obstacle attributable to thegill epithelial hyperplasia.
Since host mortalities usually occur when thenumber of parasites has remarkably increased, it isfundamental for control of parasite infections tounderstand the biological principles of population
dynamics. In most cases of infections, interestingly,
the parasite number increased during the first several
weeks of infection, but thereafter decreased to nearzero with no treatments. The present study indicatesthat the host's mucus secretion is one of the importantfactors controlling the number of external parasites. It
is confirmed that the skin epidermal mucous cells
dramatically change their density and contents in
response to Ichthyohodo or Trichodina infections.
The proliferation of PAS-positive mucous cells,
which occurred after normal mucous cells (AB-positive, PAS-negative) had vanished, should be an effective defense against ectoparasite infections. Although
the exact protective mechanism is unknown yet, thesePAS-positive cells may contain protective substances.
Whereas epidermal hyperplasia and mucus

secretions are host's defense responses against external parasites, in some cases, the parasites seem toturn these responses to possible advantage for their
growth and reproduction. It i s possible that
Trichodina consciously causes mucus secretions fromthe host and feeds them. If this is true, the reductionof normal mucous cells may cause nutritional starvation to the parasites. Chilodonella also seems to cause
the gill epithelial hyperplasia in order to feed sloughing cells or bacteria multiplying on the weakened filamental epithelium.
The host-parasite relationship is a dynamic association controlled by host, protozoan, and environmental factors. Most of fish parasites seem to havelittle effect on the host because parasite survival isdependent to some extent upon the host survival.
However, this perception may not be applied in intensive fish-rearing conditions. The present study
reveals that the pathogenic impact of I. necatorincreases especially when the infections are combined with unfavorable environment such as highrearing density and poor water quality. Thus, the highreproductive ability of external protozoans makes
them very dangerous to host fish under stressful condition in hatcheries.
Various natural factors reduce the abundance offish populations. In wild environment, it is difficult toquantify the influence of protozoan infections on fish
population, although there are several methods for
estimating natural mortality due to parasites (Moller
and Anders, 1986; Sindermann, 1987). The present
infection experiments provide indirect evidence of
natural host mortality caused by I. necator. Amongectoparasitic protozoans, I. necator should be themost dangerous pathogen for anadromous salmonid
survivals. This ectoparasite can reproduce in both
fresh water and seawater, possibly as a result of its
coevolutional adaptation to the anadromous hosts.
Furthermore, the heavy infections reduce the seawater adaptability of anadromous fish due to skindestruction, resulting in mass fish mortalities during
the early marine life. This is corroborated by the fieldsurvey that the control of parasite infections prior tosalmon releases significantly improves their marine
survival. In consequence, diagnosis and control of I.
necator in hatcheries are indispensable for a successof anadromous salmonid enhancement.
It is concluded that the three protozoan species
are harmful parasites capable of causing large losses
in the course of artificial salmon ranching. Their

76 -URAWA - PATHOBIOLOGY OF PARASITICPROTOZOANS ON SALMON

pathogenic potential is affected by attachmg method,parasite intensity, host condition, and environments.
The ideal goal should be to prevent parasite infections in hatcheries on the basis of these biologicalinformation. Future subjects will be to clarify themode of parasite infections and the exact defence
mechanism of host fish, and to establish safety methods for the control of parasite infections.

71 -

S 1f

d f,Lh.wf@ SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

Summary
Urawa, S. 1996. The pathobiology of ectoparasitic
protozoans on hatchery-reared Pacific salmon. (Ph.D.
thesis submitted to Hiroshima University in July
1992.) Sci. Rep. Hokkaido Salmon Hatchery, (50), 199.
The recent development of Pacific salmon
enhancement increases the risk of disease outbreaks
in hatcheries and also possibly in natural waters. The
present study was conducted to determine the occurrence and geographical distribution of external protozoan parasites on Pacific salmon (chum, pink, masu
and sockeye salmon) reared at 204 hatcheries innorthern Japan, and to clarify their pathogenic potentials and host-parasite interactions by infection experiments.
A flagellate Ichthyobodo necator and two ciliates Trichodina truttae and Chilodonella piscicola
were widespread in salmon hatcheries at water temperatures between 2" and 15°C. The percentage of
positive hatcheries was 37.3% for I. necator, 15.2%
for T. truttae, and 8.8% for C. piscicola. Comparisons between their occurrence and type of watersupply in hatcheries suggest that wild fish serve as
the reservoirs of infections with T. truttae and C. piscicoh, but other transmission mechanisms exist in
Ichthyobodo infections.
Transmission experiments reveal that theectoparasitic bodonid I. necator can survive andreproduce in both fresh water and seawater. The fieldsurvey also confirmed that the parasite frequently
infected chum salmon fry in saltwater estuaries aswell as in freshwater habitats. It is assumed that theparasite acquired salinity tolerance as a result of
adapting to the migratory behavior of its anadromous
host. Two morphologically similar bodonids, I. necator from salmon and Ichthyobodo sp. from marine
flounder were differentiated each other by crossinfection experiments.
The density of I. necator on the skin of chumsalmon fry peaked at 3 weeks after infection and thendecreased to near zero by week 10. Alcian blue (AB,pH 2S)/periodic acid-Schiff s reagent (PAS) staining
procedure revealed dramatic changes in both thedegree and histochemistry of epidermal mucus secretion during the course of the parasite infections.
Normal epidermal mucous cells (AB-positive, PASnegative) vanished in response to the initial parasite- 78 -

infection, but thereafter the extensive proliferation of
PAS-positive mucous cells occurred in the infected
epidermis, which was followed by the reduction of
the parasite density. Thus, the PAS-positive mucous
cells may play an important role in the defense mechanism against Ichthyobodo infections.
Fish mortalities accumulated to 12.4% in the
Ichthyobodo-infected group while only 1.9% died in
the control group during the 10-week experimental
period in fresh water. In seawater challenge tests(salinity 33%0 for 48 h), high mortalities (63.70%)
occurred among infected salmon fry when the heavy
infections caused intensive erosions and hemorrhages
in the skin epidermis. Serum C1- concentration of
infected fish was significantly lower in fresh waterand higher in seawater, compared with that of thecontrols. These results indicate that Ichthyobodo
infections disturb the osmoregulation of juvenile
salmon due to skin destruction and will consequently
reduce marine survival of anadromous fish.
Ichthyobodo-infected chum salmon juveniles
were stocked in 18-1 tanks at three different rearing
densities (300, 600 and 1,200 fish) and water inflow
rates (370, 750 and 1,500 d m i n ) for 5 weeks. The
parasite density on the skin increased regardless ofrearing density or inflow rate. The total mortality was
76-90% in the infected groups kept under the mostunfavorable conditions (crowded or low water supply), but only 1 1 - 15% in the other infected groups. In
the uninfected control groups, the mortality was low(0.7-2.6%), being not affected by the degree ofcrowding or water supply. The results suggest thatthe pathogenic potential of I. necator is strongly
intensified when the infections are combined withenvironmental stress induced by over-crowding
and/or inadequate water supply, although the parasite
density is not affected by these factors.
Seasonal changes in the density of I. necator onchum salmon fry and the host's seawater adaptability
were investigated at Yoichi Hatchery along the
Yoichi E v e r , western Hokkaido. The parasite density
increased rapidly during the first-feeding period,
resulting in the reduced seawater adaptability of hostfish. A formalin bath (250 ppm for 1 h) could control
the attaching parasites, and consequently improved
the seawater survival of host fish within one month
post-treatment. The number of adult salmon returning
to the Yoichi River significantly increased after thepractice of parasite controls. These results corroborate the hypothesis that heavy Ichthyobodo infections

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

cause high mortalities among anadromous salmonids
during their marine life. Thus, diagnosis and controlof I. necatur in hatcheries are essential for a successof anadromous salmonid enhancement.
The intensity of Trichodina truttae increased
sharply to reach a peak of an average of 5,730 parasites per fish at week 3 post-infection, and thendeclined markedly to near zero by the end of theinfection experiment (week 6). The parasite was sitespecific to the body surface of host fish and induced
epidermal hyperplasia. Heavy trichodinid infections
caused extreme flashing among infected fish, which
was accompanied by mass host mortalities with acumulative loss of 56%. These acute mortalities areprobably attributable to extreme stimulation by feeding activity of the parasite with sick cilia and adhesive disc. Drastic changes occurred in both the quality and quantity of the skin epidermal mucus secretionof the host fish during the course of Trichodina infections, just like in lchthyobodo infections. These
results suggest that increased production of PAS-positive mucous cells may be a non-specific defense
mechanism against external parasites.
The holotrichous ciliate Chilodonella piscicola
became epizootic and caused chronic mortalities injuvenile masu and pink salmon reared at Nemuro
Hatchery, eastern Hokkaido. Cumulative mortalities
amounted to 20% in masu salmon over 5 months, andto 10% in pink salmon over 2 months. Transmission
experiments in juvenile maw salmon confirmed that
C. piscicola can cause severe proliferation of the gillepithelium in the absence of other stressors. The
experimentally infected fish exhibited reduced
growth and chronic mortalities after the mean intensity had reached a peak (619 parasites per fish) at week
4 post-infection. The marked epithelial hyperplasia
was followed by intense fusion of the adjacent gilllamellae and filaments. Respiratory failure due to gillepithelial hyperplasia may be a primary cause of thedeaths.
It is concluded that the three external protozoan
parasites are epidemic pathogens capable of causinglarge economic losses both in hatchery and wild conditions during the course of artificial salmon ranching. Their virulence is affected by the mode of attaching and feeding, parasite intensity, host condition,and environments. Thus, parasite infections should becontrolled in hatcheries with due regard to these factors.

79 -

b
j.5

SCI. REP HOKKAIDO SALMON HATCHERY, NO. 50,1996

Acknowledgments

References

I wish to express my sincerest gratitude toProfessor Kiyokuni Muroga of Hiroshima University
for constant guidance in the course of t h s study. My
deep appreciation is expressed to Drs. Leo Margolis
and Susan M. Bower of Pacific Biological Station,
Canadian Department of Fisheries and Oceans, and
Dr. J. Richard Arthur of Maurice Lamontagne
Institute, Canadian Department of Fisheries andOceans: they kindly gave valuable suggestions on
preliminary manuscripts. I acknowledge my grateful
thanks to Dr. Teruhiko Awakura of Hokkaido Fish
Hatchery, Dr. Kazuya Nagasawa of National Instituteof Far Seas Fisheries, Dr. Kazuo Ogawa of the
University of Tokyo, and Drs. Shin-ichi Uye andToshihiro Nakai of Hiroshima University, for theirinterest, encouragement and criticism.
The present study was carried out as a research
program of Hokkaido Salmon Hatchery, Fisheries
Agency of Japan. My special thanks are due to thepersonnel of Hokkaido Salmon Hatchery for generous consideration and support in this study. I am alsoindebted to Dr. Muneharu Kusakari of Hokkaido
Fisheries Experimental Station and Mr. Noriyuki
Uelu of Okayama Prefectural Fisheries Experimental
Station for their kind cooperation in the experimentswith marine fish. My thanks are extended to Mr.
Yoshitaka Yoshida of Aomori Prefectural Fish
Culture Center, Mr. Minoru Ube of Iwate Prefectural
Southern Sea Farming Center, Dr. Akira Kumagai of
Kesennuma Miyagi Prefectural Fisheries
Experimental Station, Mr. Hiroshi Konaka of Akita
Prefectural Freshwater Fisheries Experimental
Station, and Mr. Yutaka Kasahara of Yamagata
Prefectural Sakata Fisheries Office, for providing thesamples in Honshu.
Finally, I acknowledge the support of my farnly, Mutsuko and Yuka, who encouraged me throughout the study.
The thesis includes eight original papers thathave been individually published in scientific journals (Urawa and Kusakari, 1990; Urawa and Arthur,
1991; Urawa and Yamao, 1992; Urawa, 1992a,
1992b, 1992c, 1993, 1995 in references). The published papers are reproduced with kmd permission
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Becker, C. D. (1977): Flagellate parasites of fish. In
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Wedemeyer, G. A., and D. J . McLeay (1981):
Methods for determining the tolerance of fishes toenvironmental stressors. In Stress and Fish (editedby A. D. Pickering). Academic Press, London. pp.
247-275.
Wedemeyer, G. A., F. P. Meyer, and L. Smith(1976): Diseases of fishes. Book 5. Environmental
stress and fish diseases. T. F. H. Publications Inc.,
Neptune, New Jersey. 192 p.
Wedemeyer, G. A., R. L. Saunders, and W. C. Clarke(1980): Environmental factors affecting smoltification and early marine survival of anadromous
salmonids. Mar. Fish. Rev., 42, 1-14.

Weisbart, M. (1968): Osmotic and ionic regulation inembryos, alevins, and fry of the five species ofPacific salmon. Can. J. Zool., 46,385397.
Wellborn, T. L. (1967): Trichodina (Ciliata:
Urceolariidae) of freshwater fishes of the southeastern United States. J . Protozool., 14,399-412.
Wells, P. R., and D. K. Cone (1990): Experimental
studies on the effect of Gyrodactylus colemanensis
and G. salmonis (Monogenea) on density of
mucous cells in the epidermis of fry of
Oncorhynchus mykiss. J. Fish Biol., 37,599-603.
Wiles, M., D. K. Cone, and P. H. Odense (1985):
Studies on Chilodonella cyprini and C. hexasticha(Protozoa, Ciliata) by scanning electron
microscopy. Can. J. Zool., 63,2483-2487.
Wilkins, N. P., and S. Jancsar (1979): Temporal variations in the skin of Atlantic salmon Salmo salar
L. J. Fish Biol.,15,299-307.
Woo, P. T. K. (1987): Immune response of fish toparasitic protozoa. Parasitology Today, 3, 186188.
Wood, J. W. (1979): Diseases of Pacific salmon; theirprevention and treatment. 3rd edition. Washington
State Department of Fisheries, Olympia,
Washington. 82 p.
Wootten, R., and J. W. Smith (1980): Studies on theparasite fauna of juvenile Atlantic salmon, Salmosalar L., cultured in fresh water in eastern
Scotland. Z. Parasitenkunde, 63,221 -231.

87 -

3If

d TAR% SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50, 1996

APPENDIXESEXPLANATION OF APPENDIXES

Region and hatchery.- Hatcheries are mapped by number in Figs. 2.1, 2,4-7.
Fish species: Alevins are shown by "A"in parentheses following the fish name.

No. offish examined.- Asterisks indicate that fish were treated with forrnalin before the parasitologicalexamination.
Mean length and weight.- ND means data not available.
Parasites.- Numerals show the prevalence (%) of Zchthyobodo necator (ICH). The absence or presence ofthe ciliate parasites, Trichodina truttae (TRI) and Chilodonella piscicola (CHI), is expressed by "+" or"-",respectively.
Type of water supply.- S, spring water; W, well water; I, infiltrate water; R, river water; L, lake water.
Temperature of water supply.- Water temperature ("C)in a rearing pond where fish were sampled is indicated in parentheses.

88 -URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Appendix table 1. The occurrence of Ichthyobodo necator (ICH), Trichodina fruttae (TRI), and Chilodonella piscicola(CHI) on hatchery-rearedjuvenile salmonids in Hokkaido.
RegionFish
No. Hatchery

Date

Number

Mean

Mean

of

of fishlengthWeightcollection

examined

(cm)

(g)

Parasites
ICH

~

TRI

-

Water supply
CHI

Type (temp.)

I. Nemuro Strait
Chum

MAY-25-89

30

4.9

1.18

63

R

Maw

MAR- 10-89

20

2.8

0.23

100

R

Pink

MAY-10-89

61

3.3

0.24

0

R

2 Hamanaka

Chum

APR-29-89

40*

4.2

0.73

0

3 Nizibetsu

ChumMAR-26-82

8

0.30

25

Chum

MAR-15-89

10

3.5
41

0

SockeyeMAR-15-89

30

2.5

0.64
0.12

Chum (A)MAR-15-89

10*

3.3

0.33

0

1 Bettoga

4 Nishihetsu

W+R (6.0)

+
+

S

+
+

R (4.10)0s (8.5)S (9.0)Chum

MAY-11-89

30*

62

2.59

0

5 Honbetsu

Pink

MAR-13-91

30

3.5

0.37

0

S (7.0)6 Tokotan

ChumMAR-20-89

40*

3.5

0.38

0

S+R

Chum

APR-lI-S9
MAR-14-91

30*

4.0

0.69

0

S+R

Chum

30

4.0

0.62

0

S+R (4.0)

7 Shunbetsu

Pink

APR-21-89

30

3.2

0.18

0

S (5.0)8 Kenebetsu

ChumMAR-23-82

7

3.5

0.40

0

R

Chum

MAR- 15-89

3.7

0.48

0

Chum

APR-I 8-89

10
51

4.5

1.03

0

+

R (7.0)9 Nakashibetsu

Chum

APR-21-89

60

4.5

0.98

0

+

S+R (6.5)

10 NemuroPink

MAY -2 1-87

50

4.0

0.60

0

S+R

Pink

MAY-16-88

10

4.2

0.42

0

Masu

MAY-2 1-87

50

4.1

0

S+R
S+R

Masu

APR-07-88

10

4.6

0.91
0.83

0

S+R

Masu

MAR-0491

10

3.1

ND

- 0

Chum

MAR-25-82

7

3.3

0.30

57

Chum

MAR-16-89

30*

39

0.52

0

Maw

MAY-22-87

1 0*

3.6

0.29

0

Chum

FEB-06-82

10

3.8

0.35

S+W+R

Chum

FEB-20-82

10

3.8

0.37

100
70

Chum

MAR-02-82

5

4.0

0.42

100

S+W+R

Chum

MAR-25-82

5'

4.1

0.69

0

S+W+R

Chum

MAR-16-82

10

ND

ND

0

Chum

APR-07-88

60

3.4

0.35

0

+

S+R

14 Motosakimui

ChumMAR-30-88

10

4.1

0.68

10

+

15 Mosekmbetsu

Chum

MAT-24-82

6

3.6

ND

50

S+R
S+R

11 Ichani

12 Tyurui

13 Kunbetsu

R (15.0)

R (6.0)

StR

+
+

W+R
W+R (5.0)
W
S+W+R

S+R

Chum

MAY-20-88

60

4.1

1.06

0

S+R

16 Uebetsu

ChumMAR- 19-88

30

4.2

0.79

100

W+R

17 Rikushibetsu

Chum

KIN- 10-88

60

5.2

1.37

0

S (7.1)18 Rausu

Chum

MAR-25-82

4

3.6

0.41

R

Chum

MAY-26-88

60

4.8

1.15

0
0

19 Yunosawa

Chum

MAY 18-88

60

5.1

1.33

0

R

20 Sashim

Pink

APR-30-88

10

3.5

0.29

R

21 RusaChum

MAY-30-88

60

4.9

1 24

0
0

- 89 -R

S+W

2 If $-VA@f%SCI REP. HOKKAIDO SALMON HATCHERY, NO. 50, 1996

Appendix table 1 (continued).
~

RegionFish
No Hatchery

Date

Number

Mean

Mean

of

of tishlengthWeightcollection

examined

(cm)

(g)

~

Parasites
ICH

TRI

-

Water supply
CHI

Type (temp )II. Sea of Okhotsk
Chum

APR-11-89

10

3.7

0.38

0

R

23 Hamawaohetw Pink

22 lwaobetsu

APR-11-89

10

3.3

0.31

0

24 Onnebetsu

Pink (A)APR-11-89

10

3.2

0.29

0

R
R

25 Okushihetsu

Pink (A)Plnk

MAR-29-88

30

3.1

0.24

3

R (4.8)APR-11-89

10

3.3

0.28

0

R

Chum

MAR-29-88

10

4.5

0.92

0

S

Chum

APR-01-89

10

5.0

1.34

0

S

Plnk

APR 11-89

10

3.3

0.22

0

S

Chum

MAR-24-82

17

3.8

0.44

0

S

Chum

MAR- 16-89

10

4.0

0.58

S

Chum

APR-12-89

10

4.5

0.90

0
0

Plnk

MAR-24-82

10

3.4

0.21

0

26 Raiun

27 ShanS

Masu

MAY-12-84

5

4.2

0.64

0

S
S

Masu

JAN-22-87

10

3.1

0 20

100

S

Masu

JAN-27-87

10

2.8

0.13

100

S

Maw

JAN-28-87

20*

3.0

0.15

0

S

Masu

APR- 12-89

10

3.2

0.35

20

S

28 Akinokawa

Chum

APR-11-89

10

4.1

0.69

0

S

29 Yanbetsu

ChumMAR-29-88

10%

3.9

0.63

0

S+R

Chum

APR-12-89

10*

3.9

0.58

0

S+R

30 Mokoto

Chum

APR- 12-89

10

3.9

0.52

0

S

31 Aioi

Chum

APR- 12-89

10

4.0

0.62

0

S+R

32 Kaminobato
33 Abashiri

Chum

APR- 12-89

10

3.6

MAR-29-88

20

3.3

0
100

S

Chum (A)

0.38
0.31

Chum

APR-12-89

10

3.9

0.57

100

s+L

Chum

MAR-30-88

10

3.6

0.46

0

S

Chum

APR-07-89

10

3.6

0

S

35 Kitami

Chum

MAR-16-89

20

3.5

0

W

36 Tokoro

Pink

APR-13-89

10'

3.3

0 36
0.40
0.26

0

S+R

37 Maruseppu

ChumMAR-30-88

30

4.0

0 60

0

S+R

Chum

APR-07-89

10

4.1

0.67

0

S+R

Chum

APR-18-85

10

4.4

0.59

10

W

Plnk

APR- 18-85

10

37

0.30

10

W

Pink

APR-17-X9

10*

4.6

0.98

0

w (9 0)W

34 Oketo

38 Yubetsu

S+L(1.8)

Chum

MAR-30-88

20

4.3

0.77

0

Chum

AFR-07-89

10

46

0.97

0

W

Chum

MAR-28-86

20

4.0

0.67

0

w (7.9)Chum

MAR-30-88

10

0

W

APR-13-89

10

1.45

0

w (8.0)Chum (A)
Pink

MAR-31-88

10

5.1
5.0
3.4

1.45

Chum

0.39

0

R

APR-13-89

101'

3.2

0.28

0

R

42 Horonaiminami Chum

MAR-3 1-88

10

4.3

0.76

40

W

43 Horonai

ChumMAR-24-82

5

3.3

0.23

30

W

Chum (A)

JAN-09-87

29

3.5

0.33

W

Chum

MAR-31-88

10

4.3

0 84

0
100

Chum

APR-13-89

10

5.9

2.20

100

w (5 5 )39 Yubetsugosen
40 Shokotsu

41 Okoppe- 90 -W

URAWA - PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Appendix table 1 (continued).
RegionDate

Number

Mean

Mean

Fish

of

offishlengthWeightcollection

examined

(cm)

(p)

ICH

Chum

MAR-23-82

11

3.9

0.52

9

Chum

J A N 3 1-89

10

3.6

0.41

0

w (5.4)Chum

MAR-20-89

10

4.3

0.83

0

W (4.0)No. Hatchery
44 Tohshibetsu

Parasites
TRI

Water supply
CHI

TvDe (temo.)W

Masu

MAR-23-82

5

2.8

0.19

100

Masu

JAN-31-89

10

2.9

0.23

0

w (5.4)Masu

MAR-20-89

10

3.4

0.40

0

W (4.0)Pink

MAR-23-82

10

3.2

0.21

20

W

Chum

MAR-27-86

20

4.1

0.76

0

w (2.0)Chum

APR-05- 88

10

4.5

0.98

0

W (3.1)Chum

FEB-03-89

10

3.3

0.39

0

W (3.6)

Pink

APR-05-88

10

3.6

0.38

0

W (3.1)

Masu

MAY-3 1-88

10

3.6

0.53

100

W (6.5)Masu (A)

FEB-07-89

10

2.8

0.21

0

W (3.6)

Chum

MA€-27-86

19

4.6

0.94

Chum

MAY-07-88

10

4.2

0.79

0
0

W (6.5)W

47 Onishibetsu

Pink

MAR-31-88

50

3.2

0.22

0

48 Souya

Chum

APR-08-88

60

4.1

0.75

0

S+R
R

49 Kafukai

Pink

APR-17-90

10

3.0

0.23

0

50 Higashirishin

Chum

MAY-08-88

61

4.5

0.91

0

5 1 Asahi

ChumMAR-26-86

6

3.6

Chum

MAY-08-88

23

4.5

0
0

Chum
Chum

APR-27-88

45
10

5.3
3.7

Chum

MAR-23-82

4.4

Chum

MAR-30-88

Chum

FEB-06-89

8
30
10

33

0.30
0.93
1.27
0.35
ND
0.79
0.29

Chum

MAR-29-89

60

4.7

Chum

MAR-24-86

41

Chum

APR-20-87

Chum
Chum

45 Utanobori

46 Tonbetsu

W

111. Sea of Japan

52 Rishin
53 Teshio++RR (5.8)R (5.4)
R

0

W

0

W

0

W

0

s+w

0

s (9.5)

0.93

0

W

3.4

0.33

0

35

3.8

0.49

74

w (3.3)w (3.5)MAR-20-88

10

4.9

1.17

0

W

JAN-30-89

10

3.4

0.34

0

W (6.6)Chum

APR-0 1-89

10

4.5

0.94

40

W (6.0)Masu

APR- 10-89

10

35

0.37

100

W (6.3)

Masu

MAY-31-89

20

4.1

0.62

10

W+R (8.7)

APR-06-87

35

3.6

0.40

56 Mashke

Chum
Chum

APR-09-87

10

4.9

1.23

0
20

I
R

57 Hamamashu

Chum

MAR-09-88

62

57

1.91

58 Atsuta

Chum

APR-11-88

53

46

0.99

0
0

W+R
S +R

59 Shlkotsuko

Kokanee

APR-16-87

20

2.4

0.08

0

S

54 Nakagawa

55 Enbetsu

FEB-21-82

4.3

91

3 If . 3 th.6fff$
SCI. REP H O W 0 SALMON HATCHERY, NO. 50,1996

Appendix table 1 (continued).
~

RegionFish
No. Hatchery

Date

Number

Mean

Mean

of

of fishlengthWeight61 Yoichi

62 Fumbira

Water supply

collection

examined

ICH

JAN-1 1-82

40

( 4
ND

(n)

Chum (A)

ND

0

Chum

FEB-09-82

11

ND

FEB- 16-82

15

ND

ND
ND

100

Chum

100

+
+

Chum

FEB-24-82

20

ND

ND

100

+

Chum

MAR-02-82

10

ND

60

Chum

MAR-09-82

15

ND

ND
ND

Chum

FEB-06-86

60

ND

ND

0

Chum

MAR-27-86

40

4.6

0.78

48

Chum

MAR-23-87

25

4.3

0.56

20

Chum

MAR- 16-88

40

4.1

0.69

18

Chum

MAR-22-88

35

4.2

0.47

17

Chum

MAR-08-89

24

4.1

0.54

21

S+R

Masu

APR-26-88

17

5.0

1.14

76

S+R

Masu

MAR-09-89

10*

4.2

0.87

Maw

FEB-20-89

10

3.8

0.43

90
40

W+R
W+R

~

60 Chitose

Parasites

TRI

CHI
~

80

~

Type (temp.)
S
S+R
S+R

+
+
+

+

S+R
S+R
S+R
S
S+R
S+R
S+R
S+R

S+R

Sockeye

APR-12-88

8

11 3

13.23

38

SockeyeDEC-13-90

7

12.5

18.28

43

Chum

MAR-19.86

10

3.5

0.26

100

Chum

MAR-1 1-87

90

35

0.39

100

Chum

MAR-09-88

15

3.7

0.30

47

+
+

Chum

APR-08-88

10

4.5

0.64

100

+

Chum

FEB-01-89

10

3.6

0.28

100

Chum

FEB-22-89

25

3.5

0.39

100

Chum

JAN-19-90

10

3.4

3.17

100

Chum

MAR-05-84

3.7

0.26

5

W+R (8.0)
W+R

Chum (A)JAN-30.86

3.4

0.25

35

W+R

Chum

JAN-30-86

19
20
20

3.9

0.36

100

W+R

Chum

MAR-0 1-88

30

4.6

1.oo

63

W+R

S+R

+

W+R
W+R
W+R (6.8)++

W+R (7.7)
W+R
W+R

+

Chum

MAR-24-89

15

4.4

0.80

40

W+R (6 6)

63 Bikuni

Chum (A)JAN-27-87

60

2.8

0.23

0

S+R

64 Kamoenai

Chum

MAR-0 1-87

53

0.8

0.49

0

S+W (7.0)

65 Iwanai

Chum

MAR-01-87

60

4.2

0.81

0

R (7.8)66 Shiribetsu

Chum

MAR-02-87

60

3.4

0.33

0

S+W+R (6.0)

Maw

MAR-02-87

30

3.3

0.39

67

S+W+R (6.0)

67 Suttsu

Chum

MAR-02-87

55

3.7

0.30

0

W+R (5 3)

68 Shimamaki

Chum

MAR-02-87

60

3.4

0.30

0

W+R (6.5)

69 SetanaChum

MAR-03-87

60

4.2

0 74

0

S+R (5.3)

70 Toshibetsu

Chum

MAR-02-87

60

4.3

0.94

0

S+W (7.0)

71 Kumaishi

ChumMAR-03-87

60

3.9

0.52

0

W+R (6.5)
W+R (6.5)

Masu

MAR-03-87

10

3.8

0 63

0

72 Ainumana

Chum

MAR-03-87

65

3.6

0.43

0

S (5.8)73 Toppu

Chum (A)

MAR-04-87

52

3.4

0.41

0

W+R (5.0)

Masu

MAR-04-87

101

3.5

0.57

30

74 Assabu

Chum

MAR-04-87

45

4.0

0.66

0

+

75 Kaminokuni

Chum

MAR-04-87

34

4.6

0.93

0

+

76 Kiyobe

Chum

MAR-04-87

60

3.8

0.51

0

+

7 1 Oyobe

Chum

MAR-30-87

30

4.0

0.57

100

- 92

+

W+R (5.0)+

W+R (3.0)
W+R (6.0)
R (7.0)S (7.5)URAWA- PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Appendix table 1 (continued).
RegionFish
No Hatchery

Date

Number

Mean

Mean

of
collection

offishlengthWeightexamined(cm)

MAR-04-87
MAR-05-87

60

4.0

0.61

0

34

MAR-05-87
MAR-05-87

42

5.1
4 .O

1.35
0.37

4.2

0.77
0.6 I

0
0
0
0
0
100

-

Parasites

Water supply

-

TRI

-

CHI

Type (temp.)

IV. Tsugaru Strait
78 Fukushima

Chum

79 S h u c h

Chum

80 Moheji
81 Kamnso
82 Shodoman
83 H a a h
84 S W s h n a

Chum
Chum
Chum
Chum
Chum

MAR-05-87
MAR-05-87
MAR-05-87

60
10
10
10

4.1
4.4
4.0

0.88
0.62

s (7.4)
W+R (6.7)
W (8.0)W (6.0)W (5.8)
W+R (2.6)

R (6.0)V. Pacific Ocean (west)
85 Yajiri
86 OofunaMAR-30-87

30
10

4.2
3.6
3.7
4.7
4.1
3.9

0.64
0.41
0.41

88 Ojironai

Chum
Chum
Chum
Chum
Chum
Chum

89 Yurrapu

ChumMA€-30-87
MAR-30-87
MAR-03-87

4.1

0.64
0.70
0 54
0.71

90 Yakumo
91 Oshamanbe

Chum

APR-01-87

29

3.8

0.50

Chum
Chum

MAR-06-87
MAR-30-87

10
20

0.44

92 Nukihetsu#l

Chum

MAR-06-87

15

3.7
4.0
4.4

93 NukibetsuM

Chum
Chum

MAR-06-87

15
10

4.6

0.77

MAR-06-87

4.4

0.61
0.38
0.42
0.46
0.75
1.15
1.62
1.94
0.70
0.30

87 Shikahe

94 Keqen#l
95 Kesen#2
96 Chimaibetsu
97 Nohoribetsu
98 Ayoro
99 ShLkiU

Chum
Chum
Chum
Chum
Chum
Chum
Chum

100 Shiraoi
101 Niikappu
102 Shizunai

Chum
Chum
Chum (A)103 Mitsush

Chum
Chum
Chum

MAR-05-87
MAR-30-87
MAR-25-86

MAR-06-87

10
10
10
10
30

0.56
0.57

0
40
50
70
100
10
93
0
0
0
100
100
0
55
100
0

JAN-14-87

20
10
35
60
60
60
10
20
30

JAN-07-87

35

6.4
4.2
3.7
3.3

APR-26-87
JAN-14-87

10

5.4

0.32
I14

0
100

40
60
20

3.9

0.34

0

3.7
4.7

0.43

0

65

30*

4.6

1.19
0.86

MAR-30-87

MAR-06-87
MAR-I 1-87
MAR-11-87
APR-24-87
MAY-09-89
APR-24-87

3.8
3.7
4.3
4.3
4.9
5.5

0

0
0
67
90
0

S+R
S+R (4.5)

R
R (5.5)
R
S+R

+

S+R
s+w
S (10.4)

S
S+W+R (7.2)
S (8.0)s (10.0)
S+R (3.5)
S+R
S+R (5.0)
S+R
W+R
I+R
1+R
I+R
W

W
W
W+I (8.4)
W+I
I(8.0)
W+R

104 Motourdkawa

Chum

105 Erimo

Chum

APR-15-88
APR-25-87
APR-25-87

VI. Pacific Ocean (east)
Chum
106 Huoo
Chum
Chum

MAR-24-84
APR-25-85
FEB-28-87

3
10
5

4.0
4.0
4.0

0.38

100

0.38
0.60

90

100

S +R
S +R
S+R (6.0)

Chum

APR-19-88

20*

53

1S O

15

R

Chum

10
10
40
10

4.2
4.1
4.7
4.4

0.67
0.42

100
0

6

ND

1.06
0.88
ND

0
0
50

S+I (5.5)
IS
s (7.9)I

107 Rakko
I08 Komonbetsu

Chum
Chum

MAR-I 3-89
APR-25-85
APR-13-88

109 Taiki

Chum
Chum

APR-26-89
MAR-24-84

93 -

13

2 It . 2 TA6Ja SCI. REP. HOKKAIDO SALMON HATCHERY, NO. 50,1996

Appendix table 1 (continued).
RegionDate

NumberFishofof fishcollection

examined

Chum

APR-25-85

Chum
Chum
1 I 1 Tokachi # I

112 Satsunai

Mean
lengthMean
Weight

in

4.0

0.44

50

Type (temp.)
R

AF'R- 13-88

15

4.2

0.71

100

I

APR-26-89

53

4.6

1.03

0

I(6.4)Chum

APR- 18-88

n

4.9

1.16

0

W+R

Chum

MAR-23-84

6

4.4

0.81

17

W+R

Chum

MAR-14.89

10

4.7

1.06

0

W (7.0)Chum (A)MAR-24-84

16

3.8

0.55

0

W+R

Chum

APR-I 8-88

27

4.9

1.16

0

W

No. Hatchery
110 Kousei

113 Tokachi #2

Parasites

TRI

Water supply
CHI

Chum

MAR- 13-89

60

5.2

1.40

80

W+R (6.8)

Chum

MAR-24-84

6

4.6

0.98

100

S+R

Chum

MAR-13-89

10

1.28

0

S+R (6.9)

Chum

MAR-27-82

15

5.0
4.5

0.66

0

W+R

Chum (A)MAR-23-84

36

3.3

0.42

0

W+R

Chum

MAR-14-89

10

4.3

0.54

90

W+R (6 6)

I16 Urahoro

Chum

APR-18-89

47

3.9

0.51

0

R (6.4)

117 Oubetsu

Chum

APR-26-88

44

4.6

0.93

0

W

118Tyaro

Chum

APR-26-88

10

4.0

0 61

90

W +R

119 Shoro

Chum (A)MAR-24-89

10

3.6

0.41

R (3.0)Chum

MAR-14-90

78

3.7

0.43

Chum

MAR-23-87

10

4.3

0.77

Chum

APR-25-89

10

3.9

0.61

Chum

MAR-14-89

10

4.0

0.59

0
0
0
0
0

Chum

APR-25-89

10

4.8

1.18

0

W+R

Chum
Chum

MAR-23-87

10

0.43

0

W+R

MAR-26-82

7

3.7
3.7

0.52

14

S

Chum (A)MAR-21-84

15

4.0

0

S

Chum

MAY-20-88

20*

4.0

ND
0.51

0

S

Chum

MAR-14-89

30

3.9

0.52

0

S (7.0)Masu

AUG- 12-88

16

5.2

1.63

100

S

124 Tyorobetsu

Chum

APR-26-88

20

4.7

1.02

25

W+R

125 Chyanbetsu

ChumMAR-22-84

6

3.8

0

I+R

Chum

APR-25-88

40*

4.5

ND
0.88

n

I+R

126 Biwase

Chum

APR-03-89

58

3.6

0.38

0

S+R (5.5)

114 Ootsu
115 Makubetsu

120 Turui #1
121 Turui #2
122 Ashibetsu
123 Kushiro

R
W
S

W+R

127 Shinkawa

Chum (A)APR-25-88

38

3.3

0.29

0

R

128 Horoto

Chum

APR-19-89

10

3.6

0.4 I

0

R (7.0)129 Ochislu

Chum

MAY-1 1-89

40*

4.1

0.72

0

R(11.9)- 94 -

URAWA- PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Appendix table 2. The occurrence of Ichthyobodo necator (ICH), Trichodina truttae (TRI), and Chilodonella piscicoZa(CHI) on hatchery-reared juvenile salmonids in northern Honshu.
RegionDate

Number

Mean

Mean

of

offishlengthWeight-collection

examined

-

(cm)

(g)

ICH

30
20

3.9
37
4.4

0.55
0.50
0.74

0
25
0

3.6
4.2
39

0.40

0

0.66
0.47
0.40
0.40

1no
27

0.81
1.07
0.94

0
87
I00

1.35

0
100
47
0

Fish
No. Hatchery

Parasites

Water supply
~

TRI

CHI

Type (temp.)~

VIl. Tsugaru Strait and Mutsu Bay
1 Masukawa
2 Kanita

Chum
Chum

3 Nouchi
4 Shimizu

Chum
Chum

5 Noheii #I

Chum
Chum

APR-02-87
MAR-01 -88

Chum
Chum
Chum

MAR-07-89
MAR-WS~
MAR-07-89

8 Mutsu
9 Kawauchi

Chum
Chum
Chum

10 Oohata

Chum
Chum

MAR-22-89
MAR-23-89
MAR-02-88
MAR-23-89

11 Noushi

6 Noheji #2
7 Tanabu

MAY-I 7-88
MAR-08-88
MAR-06-89
MAR-07-89

46
30
31
30
60
20
30
30
30
40

3.7
3.4
4.4
5.0
4.8
5.1
4.4

0.80
0.58

MAR-01-88

30
30

Chum
Chum

MAR-22-89
MAR-16-88

30
46

3.9
3.8
3.7

Chum

MAR-23-89

30

3.7

0 29

APR-09-87

30

5.2

APR-27-87

30

1.31
1.20

MAY-11-88
MAR-10-89
MAR-24-89
APR-28-87
MAR-20-89
APR- 15-87
FEB-29-88

30
30
30
32
30

5.0
4.8
3.9
4.2
5.5
4.2

VIII. Pacific Ocean
Chum
12 Oippe
Chum
Chum
Chum
Chum
13 Rokkasho
Chum
14 Oirase
Chum
I5 Mabuchi
Chum
Chum
Chum
Chum
16 Niita
Chum
Chum
17 UgeChum
18 Takage
Chum
19 TakiChum
20 Kokuji
Chum
21 Noda
22 Akka
Chum
Chum
23 FudaiChum
24 Akedo
Chum
25 KomotoChum
26 Settai
Chum
27 Tarou
28 Matsuyama
Chum
Chum
29 Tugaruishi
Chum
30 OmoeChum
31 Oosawa
32 Sekiguchi
Chum

MAR-09-89
APR-20-87~m-n9-89
APR-19-90

30
60
30
29
30

5.0
4.9
5.7
5.0

0.45
0.42

1.02
0.47
0.62
1.64
0.63
1.26
1.18
1.78
1.26

5.1

1.37
0.68
0.55
1.12

30

4.2

APR-19-9030

4.0

APR-09-90

30
30

5.0
4.8

30
30

4.9
4.1
4.7

0 67

4.6
4.5
5.3

0.98
0.86
1.36

MAR-07-90
MAY-01-9 1
MAY-0 1-91
APR-24-91
APR-23-91
APR-09-90
APR-23-91

30
40
30
30

1.13
I14
0.90

W (7.0)W (9.1)
W+R (9.4)
W+I(11.7)

w (12.2)
W+R (8.1)
W+R (7.7)
W (12.6)
W (13.6)
W+I (8.5)
W+R (4.2)
S+W (7.8)
S+W (8.4)

0

W (7.2)0

W (9.8)

3

W+I

43
0
100
0
0
0
17
0
0
0

W+I

100
0
0
0
0
0

0
100

n
0
100

I(9.0)
W+I (9.8)

w (10.0)
W
R (7.6)
W+R
W+R (5.8)
S+R (5.5)

S
S (8.4)
W+R (7.9)
R (6.6)S (8.0)l(8.3)
l(8.4)
I(8.4)
I(l3.3)w (12.0)
W (10.9)W (9.1)0

I(8.4)0w (7.4)W (9.8)

1.64

n
n
n

1.31

0

W (9.8)APR-24-91
APR-10-90
APR-10-90
APR-20-90

30
30
30
30

6.0
4.5
58

2.09
0.86
1.76

4.7

0.93

APR-10-9030

MAR-08-90

30

5.7
5.3

- 95 -

05

R (13.6)
W+R (8.6)

I(7.4)
I+R (12.4)

2 I f . d TAW#. SCI REP. HOKKAIDO SALMON HATCHERY, NO 50,1996

Appendix table 2 (continued)~~

RegionDate

Number

Mean

Mean

Fish

of
collection

offishlengthWeightexamined

(cm)

(9)

Chum
Chum

MAR-08-90
MAR-08-90
MAR-08-90
MAR-13-90
MAR-13-90
MAY-02-91
APR-10-90
APR-20-90
MAR-12-90
MAR-12-90
MAR-12-90
FEB-14-90
JAN-20.91
FEB-16-90
FEB-16-90
FEB-16-90
FEB-16-90

20
30
30
30
30
30
30
30
30
30
30
30
20
10
10
30
30

5.1
5.2
5.2
4.6
5.1
5.3
5.7
4.3
4.4
4.2
4.8
4.9
4.3
4.4
4.3
4.5
5.1

113
158
1 40
0 89
131
145
172
0 77
0 78
0 63
113
0 97
0 64
0 83
0 69
0 84
121

45
0
0
0
0
0
37
0
0
0
0
0
20
100
100
0
0

20
20
30
60
30
30
36
20
29
30
60
30
30
20
30
16
33
20
34
20
23
20
29
20
60
20

3.4
4.0
4.4
4.0
4.9
4.5
3.9
3.4
4.1
4.5
4.3
3.3
4.2
4.2
3.3
4.8
4.1
4.4
4.5
4.8
5.1
4.6
4.0
4.7
4.6
4.1

0 32
0 58
0 83
0 63
1 05
0 96
0 46
0 38
0 66
0 87
0 68
0 30
0 74
0 68
0 28
0 94
0 70
0 15
0 95
105
1 25
0 94
0 61
107
0 83
0 53

50
100
100

Chum
Chum
Chum
Chum
Chum

MAR -08-88
APR-06-88
MAR-13-89
MAR-14-88
MAR-06-89
MAR-07-88
MAR-16.88
JAN- 17-91
APR-16-87
MAR-15-88
APR-07-88
FEB-28-89
APR-15-87
MAR- 15-88
FEB-28-89
APR-11-88
APR-11-89
APR-12-88
APR-11-89
APR-11-88
APR-11-89
APR-11-88
APR-04-89
APR-08-88
MAR-30-89
APR-08-88

Chum
Chum
Chum
Chum
Chum

APR-06-89
APR-08-88
APR-06-89
APR-08-88
APR-06-89

10

21
23
20
20

4.0
4.2
4.2
5.1
4.6

0 52
0 74
0 71
1 06
103

100
0
0
0
100

No. Hatchery

Parasites[CH

nu

Water supply
CHI

Type (temp.)~

33 Orikasa
34 ohzuchl
35 Kozuchi
36 Unozumai
37 Kashi
38 Katagishi
39 Kumano
40 Yoshhama
41 Urahama
42 Morikawa
43 Takada
44 Ohkawa
45 Koizumj
46 Yawata
47 Mizujiri
48 Mitobe

Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum

W (10 8)w (12.2)
w (10.2)l(6.0)W (8 5)I(8.8)
l(8.1)+++I ( 9 2)I(9.7)W (10.5)I(7.4)
I+R (10.9)
S +R
I(7.2)
I+R (6.0)
I (1 0.4)
W (11.0)

IX.Sea of Japan
49 Isomatsu

50 lwaki

51 Akaishi

52 Oirase

53 Sasanai
54 Ohmine
55 Mase56 Fujikoto
57 Ani58 Nomura
59 Omono
60 Kimigano
61 Koromo
62 Ishizawa

Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum (A)
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum
Chum

96

0
0
0

+

+
+

0

55
100
0
0
3
0
100
0
100
100
0
0
0
100
0
0
100
0
I00

+

+

G+R (2.7)
G+R
R (5.4)W ( I 1.7)
W (12.3)
w (11.0)
W (10.8)
W (11.0)
W+R
W+R (5.7)
W+R (8.0)
W+R (7.7)
S
S (7.1)R (7.0)
W+R (4.0-12.0)
W+R (4.0-12.0)
S (7.5-8.5)
S (7.5-8.5)
W (7.0-14.0)
W+R (7.0-8.6)
W+R (6.5-11.0)
W+R (6.0-11.0)
W+R (5.0-12.1)
W (8.5-10.9)
S+R (4.5-10.0)
W (8.0-8.9)
W+R (5.0-10.0)
W+R (5.0-10.0)
S+R (6.5-10.0)
I+R (6.5-10.0)

URAWA -PATHOBIOLOGY OF PARASITIC PROTOZOANS ON SALMON

Appendix table 2 (continued).
RegionDate

Number

Mean

Mean

Fish

of

offishlengthWeightcollection

examined

(cm)

(e)

ICH

Chum

APR-08-88

17

4.0

0.65

100

Type (temp.)
W+R (4.0-12.0)

Chum

APR-06-89

16

3.8

0.55

0

W+R (4.0-12.0)

Chum

AE'R-08-88

16

3.9

0.56

94

W+R (5.0-10.0)

Chum

APR-05-89

41

4.2

0

W (5.0-10.0)

Chum

APR-08-88

20

4.9

Chum

MAR-28-89

20

5.3

0.64
0.94
1.49

Chum

MAR-31-88

27

6.3

Chum

APR-09-88

20

5.3

Chum

MAR-28-89

23

Chum

APR-05-89

Chum

No. Hatchery

Parasites

TRI

Water supply
CHI~

63 Ayu64 Nishime
65 Akishi0

W+R (S.0-12.0)

0

W+R (8.9-13.1)

2.43

0

S (8.5-11.0)

1.40

0

S (8.5-11.0)

5.4

1.74

0

S

21

5.1

1.23

0

S (8.6-9.8)

MAR- 18-88

20

4.7

1.03

5

W+R (6.0-12.0)

Chum

APR-05-89

25

4.1

0.65

100

W+R (6 0- 12.0)

Chum

APR-08-88

21

5.2

1.30

0

Chum

APR-05-89

42

4.6

0.99

0

69 Minowa

Chum

MAR-23-90

65

5.5

1.59

0

70 Masu

Chum

MAR-28-90

60

5.3

1.36

60

71 Gakko

Chum

MAR-28-90

59

5.7

1.79

+

72 Takase

Chum

MAR-28-90

60

6.5

2.52

0
0

73 Nikko

Chum

MAR-20-90

30

50

1.15

0

+

74 AkaChum

MAR-12-90

60

5.9

1.99

0

W (11.7)

75 Sanze

Chum

MAR-30.90

30

7.4

3.73

0

W

66 Kawabukuro

67 Naso
68 Zougata- 97 -

S (9.5-10.0)

s (10.0)+

S+R (10.5)
S+W (9.0)
S+R
W+R (13.0)+

W+R (12.2)

SCI

MP

・

まつぶ所報

・

・

さけ

ぬ MONHATCmRY

HOKMIDOS要， NO． 50 ， l996

旨たが．この間に
体表粘液細胞は質量共に大きな変化を示した．無感染対照魚の体表における粘液細胞数は400‑800

サケ 属 魚類に外部寄生する 原生動物の病
理生物学

細胞/mm' で，酸性粘液多糖類(アルシアン青陽性，PAS陰件) が主たる構成成分であった．

―方 ，感染魚でば，体表粘液細胞数が寄生虫数の増加と共に減少したが，その後PAS 陽性の粘液細胞が急激に増加して8週間後には

浦和 茂彦

2， onn 細胞ノ mm，以上となり，この増加に反比例して寄生太平洋沿岸各国においてサケ属伍類の増殖が近年盛ん

虫数が減少した．また，宿主 卜 における寄生虫の分布もになったが，同時にふ化場や自然界において魚病の発生

粘液細胞の少ない部位が中心となる傾向が認められたする危険性が増大している．飼育
魚に外部寄生性原生動以上のことから， PAS 陽性粘液細胞の増加が宿主の有効物がしばしば認められることが知られているが，これらな防御因子となっていることが示唆された・

・

原虫の病理生物学的研究は枝めて少なく，対処方法も
確(4) サケ稚魚の成長，生残および海水適応能力に対する魚類に出現する
外部寄生虫の種類，地理的分布および
宿

7 npcnrnr

主範囲を明らかにすると共に，優占的な
原生動物3種の病

ける淡水中での累棺死亡率は・対照区で僅かI． w% であっ・

立していない．木研究では ふ化場で飼育されたサケ属の影響を感染実験により検討した．W0 週間にお害性を感染突駿により検討し， 更には駆除実験により病たのに対し，感染区では12 ． 4% であった．感染により
餌料

害防除の基礎となる知見を得た

効率は低下したが，稚魚の成長に対する有意な影響はみ□北日本の204 か所のふ化場で，サケ属4種 (サケ，カ

週間毎に行ったところ，寄生数の増加により表皮層の激

ラフトマス．サクラマス，べ二ザケ) の 幼稚魚におけるしい剥離が起きた
感染後4 6週間日に，海水中での死亡率

・られなかった．海水移行試験(塩分33 。/" ， 4S 時間) を 2・

外部寄生虫の分布調査を行った．その結果・鞭毛虫のが 63‑70% に増加し，感染魚の血清中塩素イオン
温度は対lchthyobodonecator

照照よりも淡水中で低く，海水中で高い値を示した．以t，"ftae(同 15 刈(出現率37 幻 ．繊毛虫のTrjChon.inaとChi.1n如れ e@lapisc だ。la(同 9幻の原生上の結果から， 7． necator

感染は宿主の体表を破壊して浸動物3穐が優占種となってることがわかった /． nfrnTnr

透圧調節に障害を与え，降悔したサケ稚魚の
海洋主残率

は 4 伍種すべてより記録され， C ・が ，。，。ola はべ二ザケをを苦しく低下させると推定された．

・除く 3伊種， 1.truttae はサケ椎佳のみにみられた．繊毛虫
2種は河川水を用いたふ化場で頻繁に寄生がみられたこと(5) /． n，。ntnr の寄生数と宿主(サケ稚魚) の成長と生残から，天然魚が主な感染源と判断された．

に対する飼育環境の影響を検討するため，飼育密度と注

しかしf． ncCo・

to 「は 用水の種類に関係なく発生することから， 魚から魚、水量をそれぞれ
3段階に設定して感染魚と対照焦、 を 5週間への直接感染以外の伝播機構の存在する可能件が示唆さ

飼育した．寄生数にはグループ間に有意差が生じなかっ

れた・

たが．累積死亡率は， 最も不適な環境ド (高密度あるい76‑90% に達し， 他の感は低注水量) で飼育した感染区で染E ではn‑15% であった．無感染対照区における累積死
亡率は0． ?‑2.6%で ，成長や死亡に対する飼育環境の影響は水中でも宿主上で生残し繁殖できることが幸内実験によみられなかった．以上のことから， f． n。，nfnr

り証明された．
本種は宿主焦、の際海性に適応して海水適ものは飼育環境の影響を受けないが，飼育環境が悪化し応能方を獲得したと考えられた．近年ヒラメなど
海産魚、

明らかにな
た場合は感染魚の大量死亡をもたらすことが類からも形態的に極めて類似したIrh 祈 y。 わ "七 が報告されった・・(2) f． necator は本来淡水性外部寄生虫であるが、海洋に移動したサケ稚魚にも感染していることが確認され・海の寄生そのているが，交互感染
突験を行ったところ，宿主感受
作は両極問に明らかな 差がみられ，海産角、に寄生する
ル h@hwbodo(6)

毎年 /． neca@ 。，・の発生する北海道西部の余市ふ化場

で， 1987 年1‑4月に飼育サケ
仔椎仔における木虫の出現状は f． "。。"ぬ 「とは別種と判断された．

況と宿主の海水適応能力への影響を定期的に調べると共(3)

サケ稚魚の体表に寄生する f． n。。fit。 「と宿主の相互に，駆虫処理した
場合のサケの上残率への影響を検討し関係を感染実験により検討した．平均寄生数は感染3週間た ．本由は仔魚期に感染し，その寄生数は椎焦 (摂餌開後にビークとなり(625 虫体/mm始) 期になると急激に増加し，それと共に稚魚の海水適り，その後者しく
減少し

98

URAWA‑PATHOBIOLOGY

OF PARASITIC

PROTOZOANSONSALMON

応能力ば低下した．飼育
魚すべてに対してホルマリン薬

PAS 陽性粘液細胞の増加は外部寄生虫に対する非特異的浴をI回行ったと三ろ， ほほ完全に駆虫され，サケ稚魚はな防御反応と考えられた．

薬浴後t か月以内に海水適応能力が回復し・その後4 月中
親魚回帰
旬までに余市川へ放流された．余市川へのサケ(8) 北海道東部のふ化場で飼育されたサクラマ
ス および

尾数は，これら薬浴放流群の回帰年 (I989‑91年) にそれ

カラフトマス
稚魚における繊毛虫 Chilo 血nellan@s。か。伍以前より3倍以上増加した．以上のことから・
f． "。cutf)rの発生状況を明らかにすると共に，感染実験により
本虫感染が海洋におけるサケの大きな死亡要因の―つになっの病害件を検討した．
本種は宿主の主に鯛表面に寄生し，ていることが確認されると共に，放流の約Iか月前までに

感染魚の慢性的死亡を引き起こして，累積死亡率はサク

寄生虫検査を行い．必要に応じて駆虫することによってラマスで20% ，カラフトマスで10% に及んだ．本種は単独

放流魚の生残率が増加することが実証された．

で 鰹上皮細胞の肥厚と鯛薄板や鯛弁の癒首を起こすこと
が感染実験により確認され，こオ L ら 肥厚による呼吸障害(?@

サケ稚魚の成長， 牛残に対する寄生性繊毛虫が死亡の原因と判断された．

Trichod@ntitruttaeの影響および宿主の防御反応を
感染実
験により検討した．
本椀は宿主の体表に寄生し・寄生数

以上のように，外部寄生性原生動物
3種は明らかな
病害

は感染2週間後より急増し， 3週間日に平均
5， 700 中体にな性を持ち，サケ属魚類の増殖過程で大きな減耗を引き起った後，減少した．感染
魚は異常な行動を小し，6週間のこす可能性のあることが感染実験などにより示された．

累積死亡率は56% に達したが，寄生を受けた表皮層は昔

病害の程度は，寄生虫の種類によって異なり，寄生様式．

十肥厚する程度で，稚魚の成長や海水適応能力は有意な寄生数，宿主の状態および環境によって影響を受けるこ

影響を受けなかった．大量寄生した虫体の繊毛運動と摂とがわかった． これら寄生虫による減耗を効率的に防止

餌行動による過剰な刺激が稚魚の死亡に結びつくと推定するには・原因種と発生状況を正確に把握し．各寄生虫された．感染魚の体表 卜 皮における粘液細胞は， /． n" ，d‑の病害性と生物学的特性を考慮した上で感染防除を図る

巾 r 感染の場合と同様の変化を示し，宿主上皮表面にPASことが重要である陽性細胞が大量に出現して寄生数が減少した．従って．

― 99・