一■■監

第25巻第1号平成9年3月
内
原

容

著

熱帯熱マラリア原虫23kD抗原の疫学的意義
フィリピン・パラワン島での試験調査の結果（英文）・Pilarita Tongol−Rivera，狩野 繁之
Editha C諭ete−Migue1，鈴木
守・・

1 5

フィリピン・東ミンドロ州・サンナルシソ村における腸管
寄生虫感染に関する調査（英文）一千種松田雄一，桐木 雅史，横井一，川合肇，安羅岡一男，Lydia E．Bugawan，BayaniL．Blas覚
7−9マレーシア半島部で採集されたブユ（双翅目：ブユ科）の1新種
S吻％IJ％彫（S伽％1伽卿）ッoηgJ

sp．Nov．

（英文）・・高岡宏行，D．M．Davies

11−16

タイおよびマレーシア半島部で採集されたブユ（双翅目：ブユ科）の1新亜属S吻％1勉挽（D8漉sεll％吻）と1新種S（n）oo％吻の」 （英文）

一……高岡宏行，Peter．H

Adler

17−27

研究ノート

アマゾン日系移住地における眼トキソプラズマ症
・・狩野 繁之，Milton Masato Hida，
宇都宮嘉明，鈴木守一……………
（裏面に続く）

29−32

■■

1

Jpn. J. Tro p. Med. Hyg., Vol. 25, No. 1, 1997, pp. 1 5

A 23kD MOLECULE OF PLASMODIUM FALCIPARUM
BINDS SPECIFIC IgG FROM SPLENOMEGALIC,

PARASITEMIC, BUT ASYMPTOMATIC CHILDRENA PILOT STUDY IN A MALARIOUS COMMUNITYIN PALAWAN, THE PHILIPPINESPILARITA TONGOL‑RIVERA1'2, SHIGEYUKI KANO1, EDITHA CA fETE‑MIGUEL3

AND MAMORU SUZUK11
Received November 13, 1996/Accepted January 16, 1997

Abstract: A pilot sero‑epidemiological study was performed in Palawan, The Philippines, in a community
within which annual malarial transmission takes place. Sera obtained from splenomegalic and asymptomaticchildren with current falciparum infection (Group D were examined using a Western blotting technique todetermine the reactivity to electrophoresed antigenic molecules of Plasmodium falciparum (P.f) . Group I
sera consistently exhibited specific reactivity of lgG to a 23kD molecule, to which sera obtained fromanother group of asymptomatic children with neither parasitemia nor splenomegaly (Group ID exhibited no
distinct reactivity. A 58kD molecule was also exhibited by the Group I sera, while several Group 11 sera
reacted with a 52 kD molecule in association with the 47kD which was reported to be indicative of present
and/or recent past clinical episode of falciparum infection. Determination of the immunological features of
the antigenic molecules of parasites by this type of sero‑epidemiological study will provide a new assay
system for evaluation of immune status in communities endemic for malaria.
Key words: Plasmodium falciparum, malaria, epidemiology, antigenic polypeptides

INTRODUCTION

47kD P.f antigen in a study on sera obtained fromJapanese patients with acute naive falciparum infec‑

Although P.f. infection can be fatal to those without

tions. This molecule also specifically reacted with sera
collected from parasitemic and symptomatic patients inendemic areas in The Sudan. It was thus shown that the
47kD band was indicative of present and/or recent past

immunity, the same parasites cause asymptomatic

P.f. infection with clinical manifestations.

It is generally accepted that responses to malarial

infection vary considerably depending on the immune

status of the affected individual (Marsh, 1992).

malaria in highly irnmune individuals who have experi‑

The present study was carried out in a village

enced repeated infections. Trials to determine theendemic for malarial infection in Palawan, The

immune status of inhabitants of a malarious region havePhilippines, with special focus on splenornegalic children

been made using a Western blotting technique, withexamination of sera obtained from individuals with

who had developed parasitemia but manifested no signif‑
icant clinical signs of malaria. This group of children
was thought to be resistant to malaria, and their seravarious histories of P.f. infection . Thelu et al.(1991)

carried out a longitudinal study in Burkina Faso to
examine the development of immune responses to P.f.

were tested with a Western blotting technique. The

malaria. Three bleedings were carried out before, dur‑ing, and after the seasonal peak of transmission, and theof another group of children who manifested neither
splenomegaly nor parasitemia. Certain immunologic

predominance of reactivities to 115 and 103 kD antigen
molecules was noted. Kano et al. (1990a) specified thefeatures of the defined antigenic molecules and theirusefulness in immuno‑epidemiologic studies of malaria

results were compared with P.f bands exhibited by sera

1

Department of Parasitology, Gunma University School of Medicine, Maebashi, Gunma, 371 Japan2

Department of Parasitology, College of Public Health, University of the Philippines Manila, Manila, The Philippines
Palawan Provincial Hospital, Palawan, The Philippines3

2

are to be discussed.

strain, SGE1, was donated by Professor Ambroise‑
Thomas in 1979, and was maintained in our laboratory

SUBJECTS, MATERIALS AND METHODS
Subjects studied

This study was conducted in the southern part ofPuerto Princesa City, the capital of Palawan province,at the end of the rainy season, December 1992. In aprevious study, it was found that the forest fringe was
the focus of transmission of malaria within the study
area (Rivera et al.. 1993). Both falciparum and vivax
malaria were prevailing in this community. Many chil‑dren residing in this part of the community were found
to exhibit splenomegaly. In the 0‑9 year age group, 19
of 38 children examined had palpable spleens. Interviews
with mothers revealed that splenomegalic children had,suffered repeated febrile and/or malaria attacks; somecases of malaria were confirmed by examination of
peripheral smears. However, there were no cases ofsevere malaria or death due to malaria in previous

by continual in vitro culture with occasional freezing .

The parasite had been cultured in an ordinary CO,
incubator (Miyagami and Waki, 1985) using RPMI 1640
with a 10% volurne of human O type RBC with theaddition of 10% human sera (Trager and Jensen, 1976) .
The high pathogenicity of this strain for Aotus trivir ‑gatus was unchanged, and the parasites, even after
continuation of in vitro culture for more than 10 years,

caused fatal infections when introduced into monkeys.

However, knob formation on infected RBCS had been
10st after the culture.

Western blotting study

Asynchronous cultured parasites, in which were
included no gametocytes, were harvested when par‑asitemia reached >20%, and the infected RBCS Were

hemdlyzed with 0.05% saponin. The lysate wassion, 5ml of blood was collected from each of 11

centrifuged with a refrigerating centrifuge at 3,600 rpm
for 15 minutes, and then washed 3 times with phosphatebuffered saline (PBS). After the final wash, the sedi‑

asymptomatic children (2 ‑ 9 years old) withrrlent was dissolved in the antigen solution (2.3% SDS,

splenomegaly (Group D . Children who resided near theseashore within the study area did not have palpablespleens. As a non‑splenomegalic group (Group ID , 10 ofthese children, 4‑12 years of age, were included in the
study. Giemsa‑stained thin and thick smears were also

5% 2‑ME, I mM PMSF, 0.0625 M Tris‑HCl, pH6.8).

years. After explaining the study and obtaining permis‑

prepared for each donor. The serum from each donor

The resulting material was stored at ‑35'C until use,
and wa adjusted to a concentration of 30pg/20pl whenapplied to one‑dimensional electrophoresis in 10%
acrylamide gels, following the method of Laemmli(1970). The reference markers used were SDS‑PAGE

was separated in a laboratory of the local hospital, andStandards (BioRad, CA). Western blotting was perfor‑

was kept at ‑20 'C. The frozen serum specimens werebrought to Japan for immunofluorescence testing andmed by electrotransfer to polyvinylidene difluoride filter

Western blotting studies.

asitologically negative when no parasites were detectedin any microscopic field of either type of blood smear.

10t (ATTO, Tokyo) with absorbent papers soaked inblotting buffer (0.1M Tris‑HCl, 0.192M Glycine, 20%
methanoD . Non‑specific reactions to the PVDF were
blocked by soaking the membrane in 10% skimmed milk
in PBS at 4'C overnight and then washed three times
with PBS. The blotted PVDF strips were incubated witheach serum sample at 37'C for 2 hours. After washing,

Serological study

goat anti‑human lgG (Dako, Denmark) at 37'C for 1

Parasitological study

Both thin and thick blood filrns were microscopi‑cally studied. The examinees were considered par‑(PVDF) (Clear Bl t‑p, ATTO. Tokyo) using Horizeb‑

the strips were then incubated with peroxidase‑1abelled

An indirect immunofluorescent antibody test

hour. The substrate for the enzyme reaction was from a(IFAT) was performed on all serum specimens using

Konica immunostain kit (Konica, Tokyo, Japan) andthe method reported by Kano et al. (1990b) . The secon‑

the molecular weights (M.W.) of fractionated antigen
bands were determined using the curves obtained with
the Standard markers.

dary antibody employed was an FITC‑conjugated rabbit
immunoglobulins to human lgG (y‑chain specific; Dako,

Denrnark ) . 'Parasites used in the study

An established strain of P.f of Gambian origin andadapted to in vitro culture was used in this study. ThisRESULTSInterviews, spleen examination and parasitologicaland serological testing were performed for the children

3

Table I Profiles of children, with schematic representation of results of electrophoresis, slide examination and IFA T.

Symp‑ Spleno‑ IFA titer

Antig l mQ!

No. Age Sex toms megaly Slide P.f･ P.v. 23 3 l

o

O

o

O

l

9

2
3

6
4

4

2

5

3

6

4

7
8

8
8

9

5

lO

5

ll

8

12

8

13

4

14

11

15

6
6

16
17
18
19

8
12

20

9
9

21

ll

F

MF

‑‑L ++
‑, +

‑ +
M ‑ +
F
‑ +
F
= +
M ‑ +
F
‑ +
M ‑ +
M ‑ +
P

F
F
F
F
F

M

Pf･ &:. P.v.
P , f .
P , f .

4096 64
409 6 64l 024

P , f .

256

P , f ‑

l 024

256
64

64

P , f ,

l 024

64

P. f.

l 024

P . f .

l 024

16
16
16

P , f .

l 024

16

<4

+* ‑

16
16
16

P. v.

F
F

M
M

16

<4
<4

64

16

16

4
256

64
16

64
64

l
I
lI l

l** '

l** l

16
16

16

4
4

256

16

l ‑

I l
l
l
l
l
l
l
l

I** .

1;

S kD

37 47 52 54 58 70 74 83 94 100 102 105 120 160

･ I

l ‑
l

l ‑

I I ‑
I

I

I
I
l

l ‑

I ‑
l ‑

l ‑
I ‑

･ I

･ I I

･ ･ l
･ I l

･ I ‑

I

l l I ‑
l

･ I ‑
I ‑

I ‑
I ‑

* fever and chills, ** faintparticipating in the study. Both parasitological andserological findings, shown in Table 1, demonstrated
predominance of falciparum malaria over vivax malaria

seemed to have had fewer episodes of P.f. infection than

within the study area. All children studied had P.f . titers

higher than P. v. titers by the IFAT, but one who had P.

In the next part of the study, particular parasite
molecules reactive to specific lgG in the collected sera

v. parasites in the blood smear showed P.f･ titer at 1:64
versus P. v. titer at 1:256. This epidemiological findingof the two groups were studied by Western blotting
analysis. A notable difference between sera from thewas also obtained in the previous study performed in thesame area (Rivera et al., 1993). In the present study,
two distinctive groups of children could be differentiat‑

two groups was evident in the blotted maps (Table 1) . A

ed using the lists in Table 1. All children in Group I
exhibited splenomegaly and high P.f titers (1:64 ‑ 1:4096). Nine of these 11 children was parasitemic.
Regardless of apparent infection, however, none of them
manifested clinical signs of malaria. On the other handin Group II, 9 out of 10 children examined manifested

neither splenomegaly nor P.f. parasitemia. One child(No. 16) in this group had contracted vivax infection.
P.f. titers in Group 11 ranged from 1:16 ‑ 1:256, with one

negative case. Again, clinical signs were not found in
this group, except in the case of the girl with vivax
malaria. These findings suggest that the children inGroup I were splenomegalic and resistant to P.f.
malaria and had high serum titers to P.f parasite, andappeared to support the hypothesis that the children inGroup I had been attacked by repeated P.f. infections

and eventually had developed resistance to clinicalmalaria. On the other hand, individuals in Group II

those in Group I, as indicated by lower titers of thespecific antibodies.

23kD molecule was consistently reactive with lgGs ofGroup I children who were splenornegalic and par‑asitemic but asymptomatic. The same molecule, how‑ever, was not distinctly reactive with lgGs of Group II

children who were asymptomatic, not parasitemic andhad a non‑palpable spleen. The pattern of the otherbands present varied considerably by serum specimen,

and thus consistent findings concerning the im‑munological features of the corresponding molecules
could not be obtained. However, many more bands were
present for Group I sera than for Group 11 sera. In
particular, bands equal to or higher than 102kD werereactive with several Group I sera. In addition, 4 of the
11 children in Group I exhibited lgG reactivity to a 58kD

molecule, while none of the 10 sera from Group II
exhibited this band. On the other hand, 4 of 5 sera
reactive to the 47kD molecule in Group 11 also exhibiteda 52kD molecule.

4

DISCUSSION
Using a Western blotting+ technique, we have shownthat the 23kD P.f. molecule binds pecific IgGs fromsplenomegalic but asymptomatic childr' n with par‑asitemia. The children of this group (Group D showed

immunogenic relationship between these two molecules.
The other antigenic molecules detected in this study
require further investigation to determine their.immuno‑
epidem̲iological significance.
The objective of the present pilot study is to provide

higher IFAT titers than those of non‑spleh.omegalic anda new malariometric index using defined tnolecules.
Using presently available methods, it is diificult tonon‑parasitemic children (Group ID. Indeed, manydifferentiate those who are susceptible and those who

more bands were exhibited by sera from Group I than by
the sera from Group II, vhich had lower IFAT titers. It
might be suggested that the 23kD molecule was exhibit‑
ed by sera because antibodies to the parasite compo‑

gram, especially f,o,r children who are the major target

nents were simply present at high concentrations inof the control operation. In addition, this kind of study

Group I. However, this possibility could be ruled out,
sinqe s9me of the sera in Group 11 also exhibited highwill provide suggestions for determihation of parasite
molecules as candidates for vaccine development using

IFAT titers (eg. N0.14=1:64, N0.18=1:64, N0.21=1:

an immuno‑epidemiological approach.

are pr( tected from clinical disease irrespective of appar‑

ent parasitemia. Determination of these criteria is indis‑

pensable for establishment of a rational malaria pro‑256), while the reactivities of these sera to the 23kD
band were null Qr faint. In corltrast, some antigen bands,

such as the 47kD and 52kD bands, were exhibited by low‑titer (1:16) sera in Group 11 and not by high‑titer sera

in Group I. These findings support the view thatreactivity to the 23kD molecule is specific toasymptomatic children with splenomegaly and par‑asiternia, and suggest that reactivity of serum to the
23kD molecule is a reflection of resistance to sympto‑
matic malaria. Further work should still be required todetermine whether the reactivity was a unique featureof the strain used or whether this phenomenon could besubstantiated with other isolates and sera taken fromother geographical locations.
One of the present authors previously reported thatthe 47kD molecule was strongly exhibited by sera of non‑
immune Japanese patients who had contracted falcipar‑
urn malaria (Kano et al., 1990a). This was also true of
Sudanese children who lived in an endemic locality anddeveloped fever and parasitemia. It therefore appearedthat presentation of the 47kD molecule indicated apresent or recent symptomatic episode of malaria devel‑
oped in suscept,ible individuals. This finding is consistent

with those of the present study. In Group II, the 47kD

molecule was exhibited by sera of 5 of 10 non‑splenomegalic children who were considered less resis‑
tant to clinical malaria. In fact, results of a question‑

naire obtained for those 5 children supported theirrecent history of clinical malarial infection. On. the other

hand, in Group I, only 3 children exhibited the 47kD
band, suggesting that the 47kD molecule may be moreclosely related to the presence of severe symptoms inpatients. Although the number of sera studied was small,association of the 47kD with the 52kD bands was also
noted in the Group II, suggesting the presence of an

Financial sipport: This study was supported by WHO‑TDR Research Tr =ining Grant o. 900729, a Monbusho
Research Trainirig" Fellowship, Deutsche Gesselshaft fur

Technische Zusammenarbeit‑College of Public Health,
University of The Philippines, Manila Institution Stren‑

gthening Project, Grant‑in‑Aid Cooperation Research(B 06454197) from the Ministry of Education, Science,Sports and Culture of Japan, and Grant‑in‑Aid for
Scientific Research on Priority Areas (08281203) from
the Ministry of Education, Science, Sports and Cultureof Japan.

REFERENCES
1 ) Kano, S., E1 Gaddal, A.A. and Suzuki, M. (1990a) :Clinical and epidemiological studies on a 47 kD Plas‑
modium falciparum antigen. Jpn. J. Trop. Med. Hyg., 18,
317‑3242 ) Kano, S., Waki, S., Igarashi. I., Nakazawa, S., Masuda,
G. and Suzuki, M. (1990b): Retrospective malaria diag‑nosis by indirect fluorescent antibody titration on
Japanese Patients. Jpn. J. Parasitol., 39, 475‑481

3 ) Laemmli, U.K. (1970): Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.
Nature, 227, 680‑6854 ) Marsh, K. (1992): Malaria ‑a neglected disease? Par‑
asitol., 104, S53‑S69

5 ) Miyagami, T. and Waki, S. (1985): In vitro cultivationof Plasmodium falciparum under aerobic atmosphere ina C02 incubator. J. Parasitol., 71, 262‑263
6 ) Rivera, P.T., Kano, S., Miguel. E., Tongol, P. and Suzuki,
M. (1993) : Application of seroepidemiology in identifica‑tion of local foci in a malarious community in Palawan,
the Philippines. Am. J. Trop. Med. Hyg., 49, 608‑612
7 ) Thelu, J., Sheick‑Zakiuddin, I.. Boudin, C., Peyron, F.,

Picot, S. and Ambroise‑Thomas,. (1991): Develop‑

5ment of natural immunity in PZαs窺04伽吻血lo吻鰯郷malaria：Study of antibody response by Westem blot−
ting．」．Clin．MicrobioL，29，510−5188）Trager，W．and
ParasitesinJensen，JB．（1976）：Humancontinuous

malaria

culture、Science，193，673−675

7Jpn. J. Trop. Med. Hyg., Vol. 25, No. 1, 1997, pp. 7 9

A SURVEY OF INTESTINAL PARASITIC
INFECTIONS IN SAN NARCISO, VICTORIA,
ORIENTAL MINDORO, PHILIPPlNESYUICHI CHIGUSAl, MASASHI KIRlNOKll, HAJIME YOKOll,

SATORU KAWAll, HAJIME MATSUDAl, KAZUO YASURAOKAl,
LYDIA E. BUGAWAN2 and BAYANl L. BLAS2
Received November 29, 1996/Accepted January 30, 1997

Key words Parasitological survey, San Narciso, Victoria, Oriental Mmdoro Philippmes

dition in rural populations in developing countries. Intes‑

6%)two different parasites, 42 (17.4%) three parasites,
12 (5.0%) four parasites, 5 (2.1%) five parasites and 2

tinal parasitic infections are not unusual in these popula‑(0.8%) six parasites.

tions. Here we describe the prevalence of intestinal
helminth and prt)tozoan infections among the inhabit‑

are Ascaris lumbricoides. Trichuris trichiura, hookworm,

Parasitism is usually considered as a common con‑ants in San Narciso, Victoria, Oriental Mindoro,
Philippines where malaria and schistosomiasis areendemic (Saniel, 1951).
The study area San Narciso, Victoria is on Mindoro
Island, the seventh largest island of the Philippines. The

mountains lie at almost full length of the island from thenorth to the south, and the island is divided into two
provinces; Oriental Mindoro, the east of the mountainridge and Occidental Mindoro, the west of the mountainridge. The economy of this island is based on agricul‑
ture, especially rice farming. The barrio, San Narciso issituated at the west shore of Lake Naujan in Oriental

Mindoro.
In determing the prevalence of intestinal parasitic
infections, the samples of 242 individuals aged I to 88(142 males and 100 females) screened in the barrio wereallocated proportionately in the age‑stratified popula‑tion. The sample included approximately 24% of the
population. In August 1994, each inhabitant was given astool container and instructed to return it the following

day with stool specimen. Instructions were given to
ensure procurement of freshly passed stool samples intheir local dialect. Stool examination was made withformalin ‑ ether concentration technique.

Of 242 persons examined, 190 (78.5%) were foundinfected with one or more intestinal parasites (Table 1) .

Seventy two (29.8%) had one parasite infection, 57(23.

The eggs of helminths and cysts of protozoa foundSchistosoma japonicum. Fasciola sp., Entamoeba his ‑tolytica. E. coli. Endolimax nana. Giardia lamblia andIodamoeba buetschlii. T. trichiura (48.8%) was the
predominant helminth in the barrio, followed by A.
lumbricoides (43.8%). Hookworm prevalence was rela‑
tively low(11.6%).Eggs of S. japonicum and Fasciolasp. were found in 5.4% and 0.8%,respectively. No tape‑worm eggs were detected throughout the survey. The
most prevalent protozoan was E. coli (20.7%) in thisstudy area, followed by E. nana (12.0%) and G. Iamblia(10.3%). Both E. histolytica and I. buetschlii weredetected in 5.4%. No attempt was made to identify the
species of hookworm although previous studies suggest‑ed that all infections were with Necator americanus(Carney et al., 1981). The prevalence of hookworm
infection was significantly higher in males than in
fernales (p<0.01)while Ascaris and Trichuris infection
were not different between sexes. The protozoan preva‑
lence showed no clear difference between sexes.

Age‑prevalence profiles for three major soil‑transmitted helminths, Ascaris. Trichuris and hook‑worm infections are shown in Fig. 1. Trichuris infec‑
tion attalned an early peak of over 60% in the age‑group0‑10 years, then it shbwed a definite downward trendwith increasing age. A similar profile was observed in
Ascaris infection. Studies of the relationship between
age and the prevalence of ascariasis in a range of1

Department of Medical Zoology Dokkyo University School of Medicine, Mibu, Tochigi 321‑02, Japan2

Department of Health, Republic of the Philippines, Manila, Republic of the Philippines

880

Table I Prevalence of intestinal protozoan and hel‑minthic infections by sex in San Narciso, Victo‑

ria, Oriental Mindoro, Philippines in August
1994.

60

No. of examined

PARASITE

142

q'
o 40

=
o5
>

100

242

Protozoan

La,

20
O

MALE FEMALE TOTAL

Entamoeba histolytica

7t( 4.9)6( 6.0)13( 5.4)

E . coli
Eudolimax nana

29 (20 . 4)

21 (21 . O)

50 (20 . 7)

17 (12 . O)

12 (12 . O)

29 (12 . O)

Giardia lamblia

11( 7.7)

14 (14 . O)

25 (10 . 3)

Iodamoeba buetschlii7( 4.9)6( 6.0)13( 5.4)

HelminthO I O 20 30 40 50 ̲

Ascaris lumbricoides (fer.)

48 (33 . 8)

35 (35 . O)

83 (34 . 3)

Age in years

A . Iumbricoides (unf . )10( 7.0)

13 (13 . O)23( 9.5)

Trichuris trichiura

71 (50 . O)

47 (47 . O)

118 (48 .8)

Hookworm

23 (16 . 2)5( 5.0)28 (11 . 6)

Schistosoma japoleicum9( 6.3)4( 4.0)13( 5.4)

Fasciola sp .

1( 0.7)1( 1.0)2( 0.8)Figure I Age prevalence profiles for A. Iumbricoides. T.
trichiura and hookworm infections in San Nar‑ciso. Oriental Mindoro, Philippines in August
1994

LEGEND : ‑F ; Ascaris lumbricoides‑; Trichuris trichiu ra

HI‑ ; Hookworm

LEGEND: t : No. of positives
fer. =fertilized egg
unf . = unfertilized egg

Number in parentheses indicates the percentage

communities reveals the existence of two patterns(Crompton, 1989).In one the prevalence value remains
high regardless of the age of the subjects and in theother the value declines once the subjects have attained
adulthood as seen in Fig. 1. It remains unclear as to why

the prevalence declines markedly with age in somecommunities and not with others. Obvious explanations
suggest that some degree of immunity may have devel‑
oped (Jones, 1977) or that behavioural changes that

accornpany adulthood result in reduced exposure to
infective eggs. There is, however, still no solid evidence

for either of these propositions (Crompton, 1989).
In contrast, the prevalence of hookworm infection in the

age‑group 0‑10 years was less than 10%, then reached apeak at age of 20 and remained to be plateau of 12‑19%
throughout adulthood. Hookworm infection is acquired
farther from the immediate vicinity of the house and themates, being more active, run a greater risk of acquiring
the infection than do young children (Pesigan et al.,1958). Due to the low prevalence of S. japonicum andFasciola sp. infections, the sample of infected cases

within each age class was too small for meaningful
age‑stratified analysis.

The prevalence of E. coli and E. nana infections

was not remarkably age‑dependent while G. Iamblia
prevalence was already 19.4% in the youngest age class(0‑10 years) and then decreased gradually with age.

In 1978, Carney et al. (1981) exarnined fecal sam‑ples in San Narciso and found that Ascaris (78.5%)
infection was commonest and being followed by Tri ‑churis (76.1%), hookworm (27.8%) and Schistosoma (15.
9%) infections. The most prevalent protozoan was E.
coli (17.1%),being followed by E. nana (3.9%), G.
lamblia (3.2%) and E. histolytica (2.2%).

One year later, in 1979, Cabrera and Cruz (1983)
examined 364 fecal samples for geohelminths in San
Narciso and found that Trichuris infection was mostprevalent(84.9%),being followed by Ascaris (74.7%)
and hookworm(14.6%).Although data of their studies
cannot be directly compared with those of our studies, it

seems likely that in San Narciso, Victoria, Oriental
Mindoro, geohelminth infections have decreased to someextent in the last one and a half decades, while proto‑

zoan infections have remained almost unchanged or
showed an upward trend. At present time, no reasonable
explanation on the cause of this phenomenon is possible.

ACKNOWLEDGEMENTS
We wish to express our deepest indebtedness toMses. Victoria B. Tuazon, Evangeline A. Orallo, Ray‑rose L. Terrado, and Florida L. Octavo and the staffs
involved in Schistosomiasis Control Team, Oriental
Mindoro for their painstaking skillful technical assis‑

9tance and cordial cooperation.

The authors wish to thank the health workers of
San Narciso for their painstaking activity of collecting

samples and to Ms. Mayumi Ohshita and Ms. Junko
Taya, Dokkyo Univ. School of Medicine, for theirtechnical assistance. Finally, but not least, we areindebted to Sasagawa Memorial Health Foundation for
their financial support.

(APCO) ,Tokyo
2 ) Carney, W. P., Banzon, T., Veyra, V. D., Papasin, M. C.
and Cross. J. H.(1981):Intestinal parasites of man in
Oriental Mindoro, Philippines, with emphasis on schis‑tosomiasis. Southeast Asian J. Trop. Med. Pub. Hlth.,
12, 13‑183 ) Crompton, D. W. T. (1989):Ascariasis and its prevention
and control, eds., Crorhpton, D.W. T. et al., 9‑44, Taylor& Francis, London, New York and Philadelphia4 ) Jones, H. I. (1977) :Hemagglutination tests in the study ofAscaris EPIDEMIOLOGY. Ann. Trop. Med. Parasitol.,

REFERENCES
1 ) Cabrera. B. D. and Cruz, A. C. (1983) :Collected papers on

the control of soil‑transmitted helminthiases by the
APCO research group, Vol. 11 ,eds., Yokogawa, M. et al.,266‑287, The Asian Parasite Control Organization

71, 219‑226
5 ) Pesigan, T. P., Farooq, M., Hairston, G., Jauregui, J. J.,

Garcia, E. G., Santos, A. T., Santos, B. C. and Besa, A.
A. (1958) :Studies on Schistosoma japonicum infection in

the Philippines, 345‑578, Bull. WHO, Geneva

11Jpn. J. Trop. Med. Hyg., Vol. 25, No. 1, 1997, pp. 11‑16

SIMULIUM (SIMULIUM) YONGI SP. NOV.
(DIPTERA= SIMULIIDAE) FROMPENlNSULAR MALAYSIA
Hiroyuki TAKAOKAI AND Douglas M. DAVIES2
Received November 27, 1996/Accepted December 19, 1996

Abstract: Simulium (Simulium) yongi sp. nov. is described based on reared females, reared males, pupae
and mature larvae collected from Peninsular Malaysia. The immatures of this species were previously
misassociated with an adult female holotype of S. (S.) pahangense Takaoka and Davies. This new species
is characterized by the combination of the following features: scutum unpatterned, tarsal claws simple andanterior gonapophysis enlarged and widely bare along posterior margin in the female; ventral plate trans‑verse without toothed margins and style with a prominent basal protuberance pointed dorsally in the male;and gill with six filaments per side and cocoon wall‑pocket‑shaped, with very thin and almost transparent
wall in the pupa. This species seems to be related to S. (S.) rudnicki Takaoka and Davies from Langkawi
Island, Peninsular Malaysia, S. (S.) ufengense Takaoka from Taiwan, S. (S.) fuzhouense Zhang and Wang
from Fujian, south China, and S. (S.) taipokauense Takaoka, Davies and Dudgeon from Hong Kong, fromwhich it differs readily by the darker legs.

Key words: black fly, Simuliidae, Simulium, Malaysia, new species

Our recent survey of black flies in Peninsular

iridescent pruinosity, moderately covered with dark

Malaysia has found a new black fly species belonging tostout hairs. Antenna composed of 2 + 9 segments,

the subgenus Simulium s. str. The pupal and larval

reared females, reared males, pupae and mature larvae.

brownish black except scape, pedicel and base of Ist
flagellar segment yellow when viewed from dorsally, or
brownish black except scape, pedicel and a few basal
flagellar segments yellow to dark yellow when viewed
from front; each flagellar segment with a distinct pit
distally on each lateral surface, gradually becoming

Holotype and most paratype specimens will bedeposited at the Natural History Museum (BMNH),

smaller in size toward apical tip, except those on seg‑ment 9 Iocated medially (Figs. 2 & 3) ; each pit provided

London, U.K.

densely with minute sensilla (Fig. 4); each flagellar

stages of this species are similar to those misassociatedwith the adult female holotype of Simulium (Simulium)

pahangense Takaoka and Davies, 1995.
This new species is here described based on thesegment also with a small pit distally on dorsal surface,[Simulium (Simulium ) pahangense Takaoka and Davies,

except that on segment 9 Iocated medially. Maxillary
palp brownish black, with 5 segments, proportional
lengths of 3rd, 4th and 5th segments 1.0:1.2:2.9; 3rd

1995: part (pupa and larva misassociated with adultsegment not enlarged; sensory vesicle of moderate size,

holotype) lelliptical, with rugged surface, 0.3 x length of 3rd

Simulium (Simulium) yongi sp. nov.

segment, with a moderate round opening distally.
Head. Narrower than width of thorax. Frons brownish

Maxillary lacinia with 13 inner and 13 outer teeth.
Mandible with ca. 30 inner and 11 or 12 outer teeth.

black, shiny, not pruinose, with several dark stout hairs

Cibarium (Fig. 7) with a cluster of ca. 50 conical tuber‑along lateral margins; frontal ratio 1.2:1.0:1.1; frons‑cles medially. Thorax. Scutum brownish black, shiny,
thinly whitish grey pruinose, moderately covered withyellow pubescences as well as dark brown ones, inter‑

DESCRIPTION. Female. Body length 3.0‑3.5 mm.

head ratio 1.0:4.2. Fronto‑ocular area (Fig. 1) welldeveloped. Clypeus brownish black, shiny, with silvery

1

2

Division of Medical Zoology, Oita Medical University, Hasama, Oita, 879‑55, Japan
Department of Biology, McMaster University, Ontario, L8S 4K1, Canada

12

spersed with long, upright dark hairs on prescutellar
area. Scutellum brownish black, with long dark hairs as

wide, rounded posterolaterally, strongly sclerotized andwell as short hairs. Postscutellum brownish black,shiny, whitish grey pruinose, and bare. Pleural mem‑

proct slightly produced ventrally along medial margin,
covered with numerous short hairs laterally and ventral‑brane bare. Katepisternum longer than deep, and bare.
Legs. Foreleg: coxa pale yellow; trochanter yellowish
brown; femur dark brown; tibia largely white except

covered with numerous short hairs. Spermatheca globu‑

distal 1/5 brownish black and inner surface of distal 1/2

small adjacent area of spermatheca unsclerotized.

largel concave anteromedially in ventral view; para‑ly. Cercus rounded posteriorly, ca. 0.5 x as long as wide,lar, well sclerotized, with minute internal setae; tube andbrown to brownish black, with a large white sheen onouter surface in light; tarsus brownish black; basitarsus

Male. Body length 3.5 mm. Head. Slightly wider thandilated, ca. 4.8 x as long as its greatest width. Midleg:thorax. Upper eye consisting of large facets in 20 hori‑coxa dark brown; trochanter dark brown with basezontal and 20 vertical rows. Clypeus black, white

yellow; femur dark brown except outer surface of basesomewhat yellowish; tibia white except distal 1/5 darkbrown, with a large white sheen on posterior surface inpruinose, iridescent when illuminated, sparsely covered

light; tarsus brownish black except basal 1/2 of basitar‑sus or a little more whitish. Hind leg: coxa dark brown;
trochanter pale yellow; femur brown to dark brown withbase pale yellow; tibia (Fig. 8) brown to brownish blackwith basal 1/5 and posterior surface of basal 3/4 white,with dark brown hairs. Antenna composed of 2+9 seg‑ments, dark brown except base of Ist flagellar segment
pale; Ist flagellomere somewhat elongate, 1.6 >< as longas 2nd flagellomere; flagellar segments 1‑8 each with 1
or 2 small pits distally on each lateral surface (Figs. 5 &6) , which are not so developed as compared to those of. Maxillary palp composed of 5 segments, propor‑

and with a large white sheen on posterior surface inlight; tarsus brownish black except a little more thantional lengths of 3rd, 4th and 5th segments 1.0:1.2:2.6; 3rdbasal 1/2 of basitarsus white and basal 1/2 of 2nd tarsal

respectively; calcipala short, ca. 0.7 >< as long as its

cal sensory vesicle, 0.2 x length of 3rd segment, andwith a small opening distally. Thorax. Scutum brownish
black (3 narrow longitudinal black vittae visible inalcoholic specimen) , with silvery iridescent patternbasal width; pedisulcus distinct at basal 1/3 of 2nd tarsal

differing with angles of light: when illuminated anterior‑segment yellow; basitarsus parallel‑sided (W:L = 1.0:5.3),

ca. 0.64 x and 0.74 x as wide as hind tibia and femur

segment of normal size, with a small, globular or ellipti‑

segment. Tarsal claws simple without any tooth. Wing.

ly and viewed dorsally scutum shows subanteriorly a

Length ca. 2.6 mm; costa with spinules and hairs; subcos‑

transverse pair of narrow silvery iridescent spots widely

ta fully haired; basal section of vein R bare except distal

spaced in middle (distance between spots ca. 1/3 x that

1/4 to 2/3 with hairs; hair tuft at base of stem vein darkof scutum) ; when illuminated laterally or posterolateral‑brown; basal cell absent. Abdomen. Basal scale brown‑
ish black with a fringe of dark hairs; 2nd segment dark

fade and are replaced by an anterior pair of narrowbrown except basal 1/2 pale, with a dorsolateral pair ofsilvery iridescent spots broadly connected in middle; allother tergites dark brown to brownish black, with darkhairs, tergites 6‑8 shiny. Genitalia (Figs. 10‑13).
Ventral surface of abdominal segrnent 7 with a weakly
developed sternal plate medially. Sternite 8 well sclerot‑

ized, bare medially, with ca. 6 pale short setae sub‑medially near posterior border, and with ca. 14 dark
10ng hairs laterally on each side; anterior gonapophyses

triangular in shape, membranous, expanded ventrally,each covered with ca. 16 short setae as well as numerous

microsetae except narrow portion along inner margin
and rather wide portion along posterior margin bare andtransparent; inner borders narrowly separated fromeach other. Genital fork of inverted‑Y form, with wellsclerotized stem; arms rather wide, each with strongly
sclerotized distal ridge having a distinct projection
directed anterodorsally. Paraproct nearly as long as

ly and viewed dorsolaterally subanterior pair of spots

iridescent spots on shoulders which extend posteriorly
along lateral margins in a wide band and connect to alarge transverse posterior spot on prescutellar area;
when illuminated anterolaterally and viewed dorsolater‑
ally scutum on each shoulder has a large anterior irides‑

cent spot including anterior and subanterior narrow
spots mentioned above, which is also contiguous to abroad lateral iridescent band along lateral border;
scutum uniformly covered with dark brown recumbent
pubescence, interspersed with long upright hairs onprescutellar area. Scutellum brownish black, silvery
iridescent when illuminated, with several upright darkhairs as well as dark short hairs. Postscutellum brown‑
ish black, silvery iridescent when illuminated, and bare.

Pleural membrane bare. Katepisternum longer thandeep, and bare. Legs. Foreleg: coxa pale yellow; tro‑

chanter and femur dark brown; tibia brownish black
except median large portion on outer surface white and

13

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Figures 1−20．Adult female and male of S吻％1勉窺（S枷％1劾窺）ッo％g歪sp．nov．1，fronto−ocular areaof♀；2＆3，rightoutsideantennaview；3，inside

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（ventral view）；12＆13，paraproct and cercus（12，ventral view；13，lateral view）；14，coxite

and

style

prominent

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aprominent

protuberance（endbasalview）；17＆18，ventral

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sclerite（endprotuberance（lateralview）；16，coxite，＄tyle

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andview）；15，styleventralplate

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Figures 21‑27. Pupa and larva of Simulium (Simulium) yongi sp. nov. 21, pupal thoracic integument
near gill base showing a small area densely covered with tubercles (lateral view; scale 0.1mm) ; 22, anterior 1/2 of pupal thoracic integument with branched trichomes and gill fila‑
ments (lateral view; scale 0.2 mm) ; 23, basal portion of a ventral pair of gill filaments
showing inner filament with narrowed base (ventral view; scale 0.1 mm) ; 24 & 25, cocoon (24,dorsal view; 25, Iateral view; scale I mm) ; 26, apex of larval mandible; 27, Iarval head capsuleshowing hypostomiurn (hypostomal setae omitted) and postgenal cleft (ventral view)

with a white sheen in light; tarsus brownish black;basitarsus somewhat dilated, ca. 5.6 x as long as its

greatest width. Midleg: coxa, trochanter and femur
brownish black; tibia brown to brownish black except
posterior surface of basal 3/5 white and with a white
sheen in light; tarsus dark brown to brownish black
except basal 1/2 or a little less yellowish, though its
border not well defined. Hind leg: coxa brownish black;dark hairs; segments 2, 5‑7, each with a pair of silvery

iridescent spots dorsolaterally, those on segment 2
broadly connected in middle. Genitalia (Figs. 14‑20).
Coxite in ventral view nearly quadrate, a little longer
than its width. Style narrow and elongate, ca. 1.8 x as
long as coxite, spatulate ventrodorsally, with a subter‑

minal spine, and with a prominent basal protuberance
which is pointed dorsally and furnished with 8‑12 c. oni‑

trochanter yellow; femur and tibia brownish black

cal spines on anterior surface. Ventral plate in ventral

except base yellow; base of tibia whitish sheeny whenilluminated; tarsus brownish black except a little lessview transverse, much wider than its length, rounded
posterolaterally, with posterior margin untoothed andthan basal 1/2 of basitarsus and basal 1/3 of 2nd tarsal

somewhat concave medially, and covered with finesegment yellowish; basitarsus (Fig. 9) enlarged (W:L =1.0:4.4), ca. 0.78 x as wide as hind tibia and femur;
calcipala small, W : L = 1.3 : 1.0; pedisulcus distinct.
Wing. Length ca. 2.4 mm; other features as in except
subcosta and basal portion of radius completely bare.

Abdomeu. Basal scale blackish with a fringe of longdark hairs. Terga dark brown to brownish black withappressed setae centrally; basal arms long, curved out‑

wardly and forwardly. Parameres wide basally, eachwith numerous parameral hooks. Median sclerite nar‑row, slightly widened toward apex.

Pupa Body length (excluding gill filaments)

3.0‑3.5mm. Head . Integument yellowish brown, bare, with 1

15

facial and 2 frontal pairs of long, branched trichomes(split into 2‑4); antennal sheath bare. Thorax. Integu‑
ment yellowish brown, bare on anterior 1/2 except small
area at base of gill densely covered with tubercles ofirregular shapes (Fig. 21), moderately covered withsmall, cone‑shaped tubercles on posterior 1/2; thorax
anteriorly with 3 dorsal and 2 Iateral pairs of long,number, median tooth and each corner tooth longer thanothers; Iateral margins moderately serrate apically; 8 or

9 hypostomal bristles diverging posteriorly from lateralborder on each side. Postgenal cleft (Fig. 27) medium in

depth, 1.8‑2.0 X as long as postgenal bridge; Iateral
margins on posterior l/2 nearly parallel‑sided or slight‑

branched trichomes (split into 3‑6), posteriorly with 1

ly converged a ,base. Thoracic cuticle almost bare.
Abdominal c ticle, bare except last segment moderately

lateral pair of branched trichomes (split into 2 or 3) . Gillcovered with short, colorless setae on each side of anal(Fig. 22) with 6 filaments arranged in sessile pairs; outersclerite. Rectal papilla of 3 Iobes, each with 15‑17 finger‑

filaments of each pair brown, subequal in length (1.5‑2.0likmm) and thickness, acutely bent at basal 1/5; inner
filament of each pair somewhat paler and shorter thanouter ones, subequal in thickness to outer filament
except that of lower pair much narrower basally, ca.

broadened anterior arms ca. 0.65 x as long as posterior

secondary lobules. Anal sclerite X‑shaped, withones. Ventral papillae absent. Posterior circlet with 154‑

170 rows of hooklets with up to 22 hooklets per row.

0.6 >< as wide as outer filament of the same pair; all

TYPE SPECIMENS. Holotype

filaments gradually tapered toward apical tip except
inner filament of lower pair increasing its width frombase to basal 1/5 of its length, then tapered towardapical tip (Fig. 23), with annular ridges and furrows
throughout their length except near base, densely cov‑
ered with minute tubercles. Abdomeu. Tergum I yellow‑

mounted, reared from pupa collected from a cascading
stream, crossing the Hulu Langat‑Semenyih Road, near
Sungai Tekala, 18th mile from Kuala Lumpur, Selangor
State, Malaysia, 25.III.1996, by H.Takaoka and Y.Hoi‑

ish, with I bifid slender hair on each side. Tergum 2 onholotype; 6each side with 1 Iong bifid seta, I short spinous seta, andmature larvae, collected from a cascading stream, cross‑4 short simple hooked spines of equal size. Terga 3 and4 each with 4 hooked spines along posterior margin on

ing the road from Tapah to Tanah Rata, 33 km to Tana
Rata, Tapah, Perak State, 13.III.1996, by H.Takaoka; 4pupae, I pupal exuvia and 4 mature larvae, collectedfrom a tributary of Selangor R., at farm, W Fraser's
Hill, Pahang State, 2.XII.1975, by D.M.Davies, J.J.S.
Burton and P.Tan; I pupa and I mature larva, 21 Km

each side. Tergum 8 with a transverse row of spine‑
combs on each side. Terga 6, 7 and 9 each lacking spine‑

combs, with a transverse row of comb‑1ike groups ofminute spines on each side. Tergum 9 Iacking terminal
hooks. Sternum 4 with I simple hook and a few shortsetae on each side. Sterna 5‑7 each with a pair of simple

hooks on each side. Grapnel‑1ike hooklets absent.
Cocoon. Wall‑pocket‑shaped, very' thin, transparent
film‑like, anterior margin irregular in form, usually
deeply concave dorsoposteriorly as shown in Fig. 24;
anterolateral portions rather high up to anterior end,
appearing as a flap when viewed from side (Fig. 25);
cocoon usually somewhat extending ventrolaterally.

Sen. Paratypes: 4(BMNH), slide‑, 2 c71 , reared from pupae, 5 pupae, 5mature larvae (BMNH), in alcohol, same data as, I c?1 , reared from pupae, 5 pupae, 5NE of Kuala Lumpur, 1.1 Km N of Gombak Hospital,
Selangor State, 26.XI.1975, by D.M.Davies and P.Tan; 1pupal exuvia and 2 mature larvae, tributary of Kenyoi
R., 24 Km SE of Bentong, Pahang State, 4.XII.1975, by

D.M.Davies, J.J.S.Burton and P.Tan.

ECOLOGICAL NOTES. Pupae and larvae of thisspecies were found on rock suface of jetting water ofcascading streams, 1.5‑5.0 m wide, exposed to the sun orpartially shaded. A few pupae were also collected on

Mature larva. Body length 6.5‑8.0 mm. Body color

trailing grasses in the fast flowing water. Water temper‑

greyish. Cephalic apotome pale yellow with a small dark

ature ranged from 20 to 26'C. Altitude varied from 230

area in middle along posterior border, with faint positive

to 1,010 m.

head spots. Antenna composed of 3 segrnents and apicalsensillum, much longer than stem of labral fan; Iength
ratio of segments (from base to tip) 1.0:1.4:0.5. Labral
fan with 50‑54 main rays. Mandible (Fig. 26) with usual
mandibular serration of I medium tooth and I small one(sometimes posterior small tooth forked apically) ,This species was collected together with S. (S.)
pahangense and S. (S.) grosslfilum Takaoka and Davies

without supernumerary serrations; comb‑teeth de‑ovaries was seen beyond stage II, suggesting anautogeny

creased in length from Ist to 3rd. Hypostomal teeth 9 inin ovarian development.

In the stream of Tapah, 8 of 74 Iarvae of this species

examined were infected with mermithid larva (e) .

Three reared female flies were kept alive withsugar solution for five days but no development of

16

ETYMOLOGY. The specific name, yongi, is given infrom Hong Kong (Takaoka et al, 1995) shows somehonor of Prof. Yong Hoi‑Sen, Department of Zoology,
University of Malaya.

similarities to this new species but differs by having the

DISTRIBUTION. Peninsular Malaysia.
REMARKS. This new species is characterized by the
combination of the following features: antenna with awell developed pit on each lateral surface of flagellar
segments 1‑9, scutum unpatterned, tarsal claws simple,

anterior gonapophysis enlarged and widely bare andtransparent in the female; ventral plate transverse,
without toothed margins, and style with a prominentmuch longer basal protuberance of the style and thesaddle‑shaped ventral plate with a nipple‑1ike medianprocess.

ACKNOWLEDGEMENTS
We are grateful to Dr. R.W. Crosskey, the Natural
History Museum, London, U.K. for valuable suggestions.
The senior author thanks Prof. Yong Hoi‑Sen, Depart‑
ment of Zoology, University of Malaya, Kuala Lumpur,

Malaysia, who kindly provided hirn with laboratory

basal protuberance in the male; and gill with six fila‑

facilities and advice, took him on collectiong trips, andments per side, and cocoon wall‑pocket‑shaped withfrom Langkawi Island, Kedah State, Peninsulargave comments on the manuscript. Our appreciation
goes to Mr. Lee Han Lim, Head, Division of Medical
Entomology, Institute for Medical Research, Kuala
Lumpur, Malaysia, for his advice. This study was
supported by a Grant‑in‑Aid to HT from the Japan

Malaysia (Takaoka and Davies, 1995), of which theSociety for the Promotion of Science.

very thin, transparent wall in the pupa.
The female of this species resembles that of S. (S. )rudnicki described based on female specimens collectedscutum and the genitalia. are very similar to those of S.

(S.) yongi, although leg colorings are different fromeach other. The femora of S. (S.) rudnicki are mostly
yellowish.

This species is closely related to S. (S.) ufengensedescribed from Taiwan (Takaoka, 1979), and S. (S.)
fuzhouense from Fujian, south China (Zhang and Wang,
1991) , by having similar male genitalia. However, there

are differences in the leg colorings, the arrangement of
the pupal gill filaments, and the cocoon. In the latter twos ecies, the femora of adult legs are largely yellowish,
four of six pupal gill filaments are distinctly slenderer

than the remaining two filaments, and the cocoon has athick anterodorsal margin.
S. (S.) taipokauense (only the male adult is known)

REFERENCES
1 ) Takaoka, H. (1979): The blackflies of Taiwan (Diptera:
Simuliidae) . Pac. Ins., 20, 365‑403

2 ) Takaoka, H. and Davies, D.M. (1995): The black flies(Diptera: Simuliidae) of West Malaysia. Kyushu Univer‑sity Press, Fukuoka, viii + 175p

3 ) Takaoka, H.. Davies, D.M. and Dudgeon, D. (1995):
Black flies (Diptera: Simuliidae) from Hong Kong:
Taxonornic notes with descriptions of two new species.
Jpn. J. Trop. Med. Hyg., 23, 189‑196

4 ) Zhang, T. and Wang, D.Q. (1991): A new species ofSimulium from Fujian, China (Diptera: Simuliidae) .
Acta Zootax. Sin., 16, 109‑113

17

J pn. J. Trop. Med. Hyg., Vol. 25, No. 1, 1997, pp. 17 27

A NEW SUBGENUS, SIMULIUM (DA VIESELLUM) ,AND A NEW SPECIES, S. (D.) COURTNEYI, (DIPTERA=SIMULIIDAE) FROM THAILAND AND
PENlNSULAR MALAYSIAHIROYUKI TAKAOKA* AND PETER H. ADLER2
Received December 27, 1996/Accepted January 22, 1997

Abstract. A new subgenus, Daviesellum, in the genus Simulium is established to accommodate two species
from Thailand and Peninsular Malaysia. One of these species is Simulium pahangense, for which the maleand larval silk‑gland chromosornes are described for the first time. A new species, S. courtneyi, is illustrated

and described from larvae, pupae and pharate males and females. The immature stages of both species
inhabit torrential habitats, and larvae of both species contain the trichomycete fungus Harpella melusinae,
representing the first record of this species from Thailand. Simulium (Daviesellum) is related perhaps mostclosely to the subgenera Gomphostilbia and Morops. Sulcicnephia unidens is synonymized with S. (D.)
pahangense .

Keywords: Simulium, black fly, Simuliidae, Daviesellum. Thailand, Malaysia, new subgenus

INTRODUCTION
In a study of Simuliidae from Peninsular Malaysia,

Takaoka and Davies (1995) described Simulium(Simulium) pahangense from a single adult female
swept from over a stream in Fraser's Hill. In the samenew subgenus is described within the genus Simulium .

MATERIALS AND METHODS
Taxonomic procedures used are the same as thosedescribed by Takaoka (1983). Morphological terminol‑

were collected from a rock surface in jetting water andone of these two species was tentatively associated withthis species and the other was described as Sulcicleephia

ogy follows that of Crosskey (1969) . One pharate maleand female of the new species each was dissected par‑tially from the pupal exuviae, dried with hexamethyldi‑
silazane (Brown, 1993), and pinned. Peritrophic mern‑unidens (Takaoka and Davies, 1995). However, ourbranes from larval midguts were examined for tri‑

recent collections made in Thailand and Malaysia showthat the female of S. pahangense and pupa and larva of
Su. unidens belong to the same species, and the pupa
and larva originally associated with S. pahangense con‑chomycetes, according to the procedure of Adler et al.
(1996) . The holotype and some paratypes are depositedstream, pupae and larvae of two undescribed species

sist of a distinct new species ( = S. (S.) yongi Takaoka
and Davies, 1997) .

in the Natural History Museum (BMNH) , London, U.K.
Additional paratypes and slides of trichomycetes aredeposited in the Clemson University Arthyopod Collec‑
tion (CUAO , Clemson, South Carolina, U.S.A.

In this paper, S. pahangense is held as a valid nameSix larvae of S. pahangense were preserved in acetic

and Su. unidens is sunk as a junior synonym of theformer species; and the male and larval silk‑glandchromosomes of S. pahangense are described for theethanol (1 : 3) and their silk‑gland chromosomes were
stained according to the procedures of Rothfels andDunbar (1953). Chromosornal terminology follows thatfirst time. Further, one new species related to S. pahan ‑of Rothfels et al. (1978) . All photographic negatives andgense is described from pharate females, pharate males,

prints of chromosomes are housed in the Clemson

pupae and mature larvae collected from northern

University Arthropod Collection.

Thailand. To accommodate these two rare species, a1

2

Division of Medical Zoology, Oita Medical University, Hasama. Oita 879‑55, Japan
Department of Entomology, Clemson University, Clemson, SC 29634‑0365, U.S.A.

18 'Subgenus Daviesellum sgen. nov.

PHYLOGENETIC RELATIONSHIPS AND TAX‑ONOMIC REMARKS. Although the sister species S.
Type‑species: Simulium (Daviesellum) pahangense Ta‑
kaoka and Davies, 1995

DIAGNOSIS: Female and Male. Antenna composed ofscape, pedicel and 9 flagellar segments. Basal section ofradius bare. Pleural membrane and katepisternum bare.
Female. Frons shiny, with several hairs along lateral

margins. Cibarium with numerous minute tubercles.
Scutum white pruinose but not patterned. Tarsal claw
with a small subbasal tooth. Abdomen with dorsolateralpair of white pruinose spots on segment 2, and tergitesof segments 6‑8 shiny. Anterior gonapophysis produced
posteriorly, tongue‑like. Genital fork with well develop‑

ed wide arms. Paraproct of unique form, with a clusterof numerous dark, stout spines. Spermatheca with inter‑
nal setae. Male. Scutum white pruinose, with 3 faintlongitudinal vittae. Abdomen with dorsolateral pair ofwhite pruinose spots on segments 2, 4‑8. Coxite longer
than wide, gradually narrowed posteriorly. Style elon‑
gate, 1.5‑1.7 x length of coxite, slender, gradually taper‑

ed toward apex, with an apical spine. Ventral plate inform of transverse, haired plate, with a narrow longitu‑

dinal median process. Pararnere narrow with numerous
parameral hooks. Median sclerite wide, plate‑like.
Pupa. Head with 3 pairs of trichomes. Gill composed of
6 filaments in pairs. Tergum 2 on each side with 4 stout

pahangense and S. courtneyi sp. nov. might represent
highly derived members of an existing subgenus, themorphological gap separating them from all other sub‑genera is substantial. We, therefore, establish a newsubgenus, Daviesellum, to accommodate these species.
Subgenus Daviesellum shares preimaginal features
undoubtedly convergent
with those of othersubgenera, such as the Palearctic subgenus Obuchovia
and the New World subgenera Hemicnetha. Hearlea, andShewellomyia. These features include a densely woven,boot‑shaped cocoon with a high neck; short, rather
compact pupal gills with fairly stout filaments; an elon‑

gate, gradually expanded larval abdomen; an enormous
number of hooklets in the posterior circlet and on theprothoracic proleg; Iabral fans with short stems andstout primary rays; and a hypostomium with smoothly
curved anterolateral margins and dense rows of thickbristles. Nearly all of these characters are typical of
simuliids that aggregate in clusters or mats in torrential

habitats. Chromosomal similarities between Daviesellumand Hemicnetha, such as an expanded centromere regionin chromosome I and a splayed end in 111S, are judged tobe convergent or plesiotypic.

Male genitalic features of Daviesellum suggest a
relationship with some species‑groups of the subgenera

hooks, equivalent in size to those on terga 3 and 4. Terga

Gomphostilbia (e.g. baisasae. batoense, ceylonicum
groups) and Morops (e.g. banauense and sherwoodi

5‑9 without spine‑combs, and terminal hooks absent.

groups) . All these share an elongate, gradually tapered

Sterna 5‑7 each with pair of simple, stout hooks on eachstyle bearing one apical spine and a ventral plate that, inside. Cocoon boot‑shaped. Larva. Cephalic apotome
wide with 13 apical teeth composed of I prominent

lateral view, has a large, strongly produced keel. The
parameral spines in Daviesellum are smaller and more
subequal in size than those typically found in Gompho‑

median tooth accompanied at its base by 2 small teeth

stilbia and Morops.

on each side, and 4 small individual teeth on each side;
lateral teeth absent. Postgenal cleft deep. Mandible withto the Australasian and Oriental Regions (Takaoka

widest just before posterior margin. Hypostomium very

well developed comb‑teeth, mandibular serration com‑posed of a single tooth, and supernumerary serrations
absent. Rectal papilla of 3 Iobes, each with numerous
secondary lobules. Proleg with sclerotized rod on eachside. Anal sclerite of usual X shape, posterior arms

much longer than anterior ones, somewhat widened

Both Gomphostilbia and Morops are nearly endemic
1996), further suggesting a relationship with Daviesel‑lum. Gomphostilbia has been found in Thailand andPeninsular Malaysia; however, the presence of Morops
in these two areas is unconfirmed.

This new subgenus is readily distinguished frommedially when viewed posteriorly. Posterior circlet withother subgenera by the following unique characters: inthe female, paraproct with a cluster of spines; in theover 400 rows of up to over 40 hooklets per row. Lastmale, elongate coxite and style; in the pupa, stout hooks

abdominal segment remarkably bulged laterally oron the second tergum; and in the larva, proleg with asomewhat ventrolaterally, forming a large and a small
papillae, which are visible when viewed ventrally.

sclerotized rod on each side, postgenal cleft with anter‑

ETYMOLOGY. This subgenus is named in honor ofsimuliid authority Douglas M. Davies.

olateral extensions, hypostomium very wide with 13
apical teeth, mandibular serration (sensillum) cornposedof a single tooth, and posterior circlet with over 400
rows of up to over 40 hooklets per row.

19

Simulium (Daviesellum) pahangense
Takaoka and Davies, 1995
Simulium (Simulium) pahangense Takaoka and Davies,
1995: 150‑155 (holotype female only and not pupa
and larva) .

Sulcicnephia unidens Takaoka and Davies, 1995: 10‑13(pupa and larva) . NEW SYNONYMY.

plate. Sternite 8 well sclerotized, with anterior expan‑
sion medially, bare medially but with several hairs andseveral short setae along posterior border on each side;

anterior gonapophysis tongue‑shaped, protruding ventro‑
posteriorly, thin, membranous, covered with ca. 10 shortsetae as well as numerous microsetae. Genital fork of
inverted‑Y form; stem slender and well sclerotized;
arms each with large, strongly sclerotized distal ridge.
Paraproct of unique shape, well sclerotized, widely bare2 + 9 segments, brownish black except scape, pedicel andventrolaterally, expanded anteriorly in form of roundand compressed lobe with cluster of short, stout spines
along ventral inner margin, with numerous stout hairson setose area near posterior margin. Cercus nearly
semicircular, covered with numerous stout hairs. Sper‑matheca nearly ovoid, well sclerotized except tube andsmall area of tubal base transparent, with reticulate
pattern near tubal base; tube somewhat enlarged basal‑

Ist flagellar segment yellowish brown. Maxillary palply; minute internal setae present.

DESCRIPTION. Female. Body length ca. 2.8 mm.
Head. Narrower than width of thorax. Frons black,shiny, thinly grayish pruinose, with several dark, stout
hairs along lateral and lower margins; frontal ratio 1:1:
1; frons‑head ratio 1.0:3.3. Fronto‑ocular area (Fig. 1)

well developed. Clypeus black, shiny, thinly grayish
pruinose, with dark stout hairs. Antenna composed ofcomposed of 5 segments, proportional lengths of 3rd, 4th

and 5th segments 1.0:0.8:1.7; 3rd segment not enlarged;
sensory vesicle (Fig. 2) of medium size, oblong, withrugged surface, 0.2 x length of 3rd segment, with large
round opening distally. Maxillary lacinia with 16 innerand 18 outer teeth. Mandible with ca. 42 inner and 22
outer teeth. Cibarium (Fig. 3) with numerous minutetubercles along posterior margin. Thorax. Scutum

Male. Body length 2.8 mm. Head. Slightly wider thanthorak. Clypeus black, thinly whitish gray pruinose, withsparse dark hairs. Upper eye consisting of large facets in

19 horizontal rows and in 18 vertical columns. Antenna

cornposed of 2 + 9 segments, brownish black except
scape, pedicel and base of Ist flagellar segment yellow
to dark yellow; Ist flagellomere elongate, 1.4 x as long

black, shiny, whitish gray pruinose, moderately covered

as 2nd flagellomere. Maxillary palp with 5 segments,

with short, dark, recumbent pubescence, interspersed

proportional lengths of 3rd, 4th and 5th segments 1.0:1.0:

with long, erect, dark hairs on prescutellar area. Scute.1‑

2.5; sensory vesicle small, oblong, 0.19 x length of 3rd

lum dark brown with short, dark pubescence and long,dark hairs. Postscutellum dark brown, shiny, whitish
gray pruinose and iridescent when illuminated, bare.

segment. Thorax. Scutum black, white pruinose in

Pleural membrane bare. Katepisternum longer thantinct against dark brown ground color of scutum) ,densely covered with dark pubescence, intermingledwith dark, Iong, upright hairs on prescutellar area.

deep, bare. Legs. Almost blackish except fore coxa,base of mid basitarsus and base of hind 2nd tarsornere
pale brown, and basal 1/2 of hind basitarsus (Fig. 4)
whitish. Base of mid and hind tibiae with slight whitish
sheen when illuminated. Fore basitarsus somewhat dilat‑

certain angles of light, with 3 indistinct narrow longitu‑

dinal vittae (in alcohol these 3 Iongitudinal vittae dis‑Scutellum black, covered with dark, Iong, upright mar‑
ginal hairs as well as dark, short hairs. Postscutellum

parallel‑sided, width : Iength= 1:5; calcipala and pedisul‑

black, bare. Pleural membrane' bare. Katepisternum
longer than deep, bare. Wing. Length not measurable;
other features as in, except subcosta bare. Legs.
Coloration nearly as in. Fore basitarsus somewhat

cus distinct; all tarsal claws with a small subbasal tooth

dilated, 5.3 x as long as its greatest width. Hind basitar‑(Fig. 5). Wing. Length 2.9 mm; costa with spinules andhairs; subcosta bare; basal section of vein R bare; hairssus (Fig. 11) gradually widened toward distal tip, 4.4 x
as long as its greatest width, which is 0.67 x and 0.79 x
as wide as hind tibia and femur, respectively; calcipalaed, 5.0 X as long as its greatest width. Hind tibia slightly

narrower than femur; hind basitarsus (Fig. 4) nearlyat base of stem vein dark brown; basal cell absent.
Abdomen. Basal scale black with fringe of dark hairs;
2nd segment black, with pair of large, dorsolateralsilvery iridescent spots broadly connected to each othermedially; tergites 3‑5 small, black; tergites 6‑8 Iarge,
black, shiny, with dark hairs. Genitalia (Figs. 6‑10).

Ventral surface of abdominal segment 7 Iacking sternaland pedisulcus well developed. Abdomen. Basal scale
black, with fringe of long, dark hairs. Dorsal surface ofabdominal segments brownish black to black except
anterior 1/2 yellow, with dorsolateral pair of whitepruinose spots on segments 2 and 4‑8, though those on
segments 7 and 8 thinly pruinose; those on segments 2

20
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S'(D'
Figures 1‑16. Female and(D.)
and malemaleofofS.
) pahangense. 1, fronto‑ocular area; 2. 3rd maxillary palpal
segment; 3, cibariurn;
hind tarsus; 5,
Ci ariurn; 4,4,tarsal claw; 6‑10,
genitalia
6, 8th sternite.

b hindanterior gonapophyses, and genital fork in situ (ventral view) ; 7 & 8, paraproct and cercus (7, ventral
view; 8, Iateral view) ; 9, paraproct (medial view); 10, spermatheca; 11, c l hind basitarsus; 12‑16, c lgenitalia
12, coxite, style and ventral plate in situ (1eft coxite and style omitted, ventral view) ;13, coxite and style (1ateral view) ; 14, ventral plate (lateral view) ; 15, ventrar plate (end view) ; 16.
ventral plate, parameres and median sclerite in situ (dorsal view) . Scale bar 0.05 mm ( only applying
to figs. 6‑10 & 12‑16)

21

and 6 Iarge and connected widely to each other in middle

isolated from postocciput. Antenna (Fig. 25) composed

and those on segments 4 and 5 small and probablyof 3 articles and apical sensillum, a little longer thanstem of labral fan; Iength ratio of articles (from base tooverlooked. Genitalia (Figs. 12‑16). Coxite much lon‑
ger than wide. Style elongate, ca. 1.5 x as long as
coxite, gradually tapered toward apex, curved dorsallyand with I apical spine. Ventral plate in ventral viewtransverse, shorter than wide, with posterior marginrounded and somewhat produced posteriorly like eaves,with median longitudinal process produced ventrally,and fully covered with minute setae. Paramere slender,
with numerous hooks apically. Median sclerite plate‑like, wide basally, somewhat narrowed medially andagain widened apically.
Pupa. Body length (excluding gill filaments) ca. 3.5 mm.

Head. Integument pale yellow, face covered denselytip) 1.0:1.3:0.4. Labral fan with ca. 52 main rays, ofwhich pectination as in Figs. 26 & 27. Mandible (Fig. 28)

characterized by well developed comb‑teeth and inner
teeth, with I mandibular serration (sensillum), without

supernumerary serrations. Hypostomium (Fig. 29) very
wide, Iacking lateral serrations; anterior teeth (Fig. 30)

13 (12 in I specimen) , with median tooth much longer
and thicker than others; 11 or 12 hypostomal bristles
lying close in row, diverging posteriorly from lateralborder on each side. Postgenal cleft (Fig. 29) deep,
anterior margin except middle portion extending for‑ward, reaching posterior border of hypostomium, Ieav‑
ing incomplete postgenal bridge medially. Proleg with

with small tubercles, frons almost bare; I facial and 2

an elongate sclerite rod on each side (Fig. 31) . Thoracic

frontal pairs of simple, short trichomes (Fig. 17).

cuticle bare. Abdominal cuticle bare except dorsolateralThorax. Integument pale yellow, bare except both sides
and posterodorsal surface densely covered with small

uncolored setae. Rectal papilla of 3 Iobes, each with 18‑

tubercles; 2 dorsal and 3 Iateral pairs of simple, shorttrichomes, all subequal in length. Gill (Fig. 18) with 6
pale or yellowish, short filaments in pairs, almost sessile

except dorsal pair short‑stalked, tapering apically; allfilaments subequal in length (1.0‑1.4 mm) and thickness,

their maximal diameter as thick as or a little thicker
than interspiracular trunk; cuticle of filaments withnumerous, sharp, transverse ridges forming well‑definedareas on last segment sparsely covered with minute,25 finger‑like secondary lobules. Anal sclerite X‑
shaped, with short, slender anterior arms ca. 0.3 ><length of posterior ones, which are narrow in dorsal
view (Fig. 32) but somewhat widened medially in end
view (Fig. 33). Posterior circlet with ca. 460 rows ofhooklets with up to 53 hooklets per row. Last abdominal
segment remarkably bulged ventrolaterally, forming
double papillae, which are discernible when viewed

reticulate patterns, uniformly covered with minutetubercles. Abdomen. Tergum I pale, densely covered

ventrally (Fig. 34).

with minute tubercles, with I simple seta on each side.
Tergum 2 pale, with 4 hooked spines (Fig. 19) directed
forward and 2 simple, short setae of different length, oneach side. Terga 3 and 4 each with 4 hooked spines (Fig.

Larval silk‑gland chromosomes (Fig. 35). 6 Iarvae (220) and I simple, short seta on each side. Terga 5‑9
without spine‑combs. Tergum 9 without terminal hooks.
Sternum 5 with pair of simple, stout hooks (Fig. 21)
situated close together submedially on each side. Sterna
6 and 7 each with pair of simple, stout hooks similar to

those on sternum 5, but widely spaced on each side.
Grapnel‑Iike hooklets absent. Cocoon (Fig. 22). Boot‑
shaped, with high neck, thickly woven, individual
threads undetectable; anterior rim not thickened; sur‑face smooth but with many bumps of silk in 2 Thailands pecimens.

cfl and 4 ) examined. Haploid number = 3. Arm
associations standard. Homologues tightly paired.
Chromocenter absent. Centromere bands thick, darkly
staining. Centromere region of chromosome 1 Iarge,
expanded; slightly expanded in chromosome II; notexpanded in chromosome 111. Nucleolar organizer in
middle of 111S. IIL with symmetrical marker subbasal,
followed by DNA puff; parabalbiani in approximately
middle third of arm (diffuse edge distaD, followed bygray band. IIS with trapezoidal marker basal, followedby Ring of Balbiani; bulge subterminal. 111S with end
splayed; blister subterminal with two heavy bands moreterminal. 111L with basal marker in basal third of arm.
One c l with heterozygous inversion in base of IS.

Mature larva. Body length ca. 6.5 mm. Body color

SPECIMENS EXAMINED. Holotype

reddish brown. Cephalic apotome (Fig. 24) widest some‑what before posterior border, pale yellowish, somewhat
darkened medially just before posterior border, withswept while flying over foam of a stream, one of tribu‑
taries of Selangor R., at farm, EIO1'44'20"/N3'40', W

faint positive head spots; cervical sclerites discrete and(BMNH),

Fraser's Hill, Pahang State, MALAYSIA, 2.XII.1975, by
D.M. Davies, J.J.S. Burton & P. Tan. I pupal exuvia

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Figures 17‑34. Pupa and larva of S. (D.) pahangense. 17‑22, pupa

17, frons; 18, interspiracular trunk (a)and gill filaments showing enlagement of part of filament (b) ; 19‑21, spinous hook (19, on 2nd tergurn;20, on 3rd tergum; 21, on 5th sternite) ; 22, cocoon (lateral view) ; 23‑34, Iarva
23, whole body(lateral view) ; 24, head capsule (dorsal view) ; 25, antenna; 26, innermost ray of labral fan; 27, part ofone of major rays of labral fan) ; 28, apex of mandible; 29, head capsule showing hypostomium andpostgenal cleft (vental view) ; 30, anterior teeth of hypostomium (ventral view) ; 31, proleg showing alateral sclerotized rod; 32 & 33, anal sclerite (32, dorsal view; 33, end view) ; 34, posterior tip of larval

abdomen showing ventrolateral papilla on each side (vental view) . Scale bars 0.03 mm for figs. 19, 20,
21, 26 & 27; 0.05 mm for figs. 18b & 25; 0.2 mm for fig. 18; 1.0 mm for figs. 22 & 23.

23

with cocoon (holotype of former Sulcicnephia unidens) ,ECOLOGICAL NOTES. At the type locality (ca. 1,000

2 pupae, 2 mature larval head capsules, and 3 premature

m in aititude) of Fraser's Hill, pupae and larvae werelarvae (BMNH), same data as holotype; I c;collected from a rock surface in jetting water of a
stream 34 m wide, exposed to the sun. Water tempera‑ture was 21 'C. Collected with this species were S.

rearedfrom pupa, 2 mature larvae (BMNH), a cascading
stream, crossing Hulu Langat‑Semenyih Road, near
Sungai Tekala, Selangor State. MALAYSIA, 25.III.1996,
by H. Takaoka. A. Takaoka & H.S. Yong; I pharate,(Simulium) hirti

l pupa, and 5 immature larvae (3 examined

March 1996 to recoll, ect this species at the type locality

chromosomally, CUAO , and photographic negatives of

was disappointing. Neither simuliids nor other aquatic
insects were breeding in this stream, which was muddy
due ta a golf course under construction upstream. In
Selangor, Malaysia, a few immatures of this species

larval silkgland chromosomes (CUAO , Suai Mai
waterfall, E98"27'/N9"20', Phangna Province,Tl"‑rAILAND, 14.X.1994, by G.W. Courtney; 3 immaturelarvae (examined chromosomally, CUAO , Huai Tom
Than Lod, 244 m in altitude, E99'17'/Nl4"39', Kan‑
chanaburi Province, THAIL. AND, 30.X.1994, by G.W.
Courtney; 5 immature larvae (CUAO , Mai Yai water‑fall. E98'30'/N8 53', Surat Thani Province,
THAILAND, 15.X.1994, by G.W. Courtney.

!ervis. S. (S.) tani. S. (S.) bishopi, S.

(S:) yongi, and S. (Gomphostilbia) sp. Our trial made in

were coliected from a rock surface in a jetting, cascad‑ing stream 2‑5 m wide, in a lowland area (ca. 230 m inaltitude) . Water temperature was 26 'C. Two otherspecies, i.e., S. (S.) grosszfi!um and S. (S.) yongi, werealso collected from the rock surface in the same stream.

Collections in Thailand were made from wellshad=ed waterfalls and torrential areas of streams, 18‑20 'C,

Figures 35. Total chromosomal complement from larval silk glands of S. (D.) pahangens, e (male larva fromThailand, Phangna Province. Suai Mai waterfall, 14.X.1994) , showing majar landmarks. Chrorno‑some arms are labeled IS, IL, IIS, IIL. 111S, and 111L. The end of chromosome arul IIS overlaps the
middle portion of chromosome arm 111L. CI. CII, CI11 = centromeres of chromosomes I, II, and 111,

respectively. Bl = blister, Bm = basal marker, Bu = bulge. D =DNA puff, gB = gray band, NO =nucleolar organizer, P = parabalbiani, S = symmetrical group, si =heterozygous inversion, * =ring of Balbiani

24

flowing over bedrock. One stream (in Kanchanaburi
Province) had heavy deposits of marl. None of thelargely concave anterolaterally when viewed ventrally.
Spermatheca nearly globular in shape.

larvae in our collections had patent infections of para‑sites or pathogens. However, the guts of all six larvae of

Pharate male. Body length 3.0 mm. Head. As in c ! of S.

S. pahangense that we examined from Thailand (Phan‑
gna and Kanchanaburi Provinces) contained numerous
thalli and trichospores of the trichomycete fungus

pahangense except the following characters: Upper eye

Harpella melusinae L6ger and Duboscq. This tri‑chomycete has been recorded previously from theflagellar segments completely fused becoming elongate
Ist segment, and 3rd and 4th ones incompletely separat‑
ed in right antenna, and Ist and 2nd flagellar segments
and 7th and 8th ones incompletely separated respective‑
ly in left antenna. Maxillary palp with 5 segments,

Oriental Region (Adler et al., 1996), but this is the firstrecord from Thailand.

consisting of large facets in 17 horiz'ontal rows and in 16

vertical columns. In I c71 antennae abnormal, Ist and 2nd

DISTRIBUTION. Peninsular Malaysia and Thailand

proportional lengths of 3rd, 4th and 5th segments 1.0 : 1.0 :(new record) .

2.3; sensory vesicle small, oblong, ca. 0.16 x length of

3rd segment. Thorax. As in c ! of S. pahangense except

Simulium (Daviesellum) courtlreyi sp. nov.

DESCRIPTION. Pharate fernale. Body ,1ength 3.0 mm.
Head. As inof S. pahangense except the following

scutum dark brown to blackish brown, and scutellum
not pigmented. Wing. Length not measurable; otherfeatures as in, except subcosta bare. Legs. Coloration

nearly as in. Fore basitarsus somewhat dilated, ca. 6.

characters: Frontal ratio 1.1:1.0:0.9; frons‑head ratio 1.0:

2 x as long as its greatest width. Hind basitarsus

3.8. Antenna dark brown except scape and pedicel yel‑low, and Ist flagellar segrnent yellow entirely whenviewed ventrally but only basally when viewed dorsally.

gradually widened toward distal tip (length‑width ratio
not measurable) . Calcipala and pedisulcus well devel‑
oped. Abdomeu. As in c71 of S. pahangense except" dorsal

Proportional lengths of 3rd, 4th and 5th maxillary palpal

surface dark brown. Genitalia (Figs. 41‑45). Coxite

segrnents 1.0:0.9:2.0; sensory vesicle 0.25 x length of 3rd

10nger than wide. Style elongate, ca. 1.8 x as long as
coxite, curved dorsally, gradually tapered toward apex,

segment. Maxillary lacinia with 14 or 15 inner and 16
outer teeth. Mandible with ca. 40 inner and 22 outerteeth. Thorax. As in of S. pahangense except scutum
dark brown to blackish brown, and scutellum not pig‑
mented. Legs. Coloration still incomplete appearing asfollows: All coxae and trochanters brown to dark brown
except fore coxa dark yellow. All femora pale except
distal 1/5 or 1/6 blackish brown. All tibiae blackish
brown with a large pale portion medially, and with baseyellow. Fore tarsus blackish brown; mid and hind tarsi
grayish brown except basal l/2 of mid basitarsus gray‑ish and basal 1/2 of hind basitarsus and 1/3 of hind 2nd

with I apical spine. Ventral plate in ventral view trans‑verse, Ionger than wide, gradually narrowed posteriorly,

with median longitudinal process produced ventrally,
covered densely with minute setae, with arms slightly
diverging from each other; ventral plate in end view
much produced ventrally along posterior margin, withlateral margins gently rounded and apically convergent,and fully covered with minute setae. Paramere slender,
with numerous hooks. Median sclerite plate‑1ike, nar‑row in basal 1/3, gradually widened toward apex, androunded apically.

tarsomere yellowish white. Fore basitarsus somewhat
dilated, 5.1 x as long as its greatest width. Calcipala,Pupa. As in S.pahangense except the following charac‑pedisulcus and tarsal claw as in of S. pahangense.
Wing. Length not measurable; other characters as inof S. pahangense. Abdomen. As inof S. pahangense
except 6th to 8th tergites shiny grayish. Genitalia(Figs. 36‑40). Nearly as inof S. pahangense except
the following characters: Sternite 8 furnished with 12

ters: Body length (excluding gill filaments) ca. 3.0 mm.

10ng, stout hairs and a few short setae along posterior
margin on each side. Anterior gonapophysis with ca. 20

silk.

Head. Face and frons covered densely with small tuber‑cles (Fig. 46). Thorax. Gill filaments (Fig. 47) brown,

subequal in length (0.6‑1.0 mm) and thickness, theirmaximal diarneter a little thinner than interspiraculartrunk. Cocoon. Surface smooth, shiny, without bumps ofshort setae as well as numerous microsetae. Stem of

Mature larva. As in S. pahangense except the following

genital fork slender with dilated anterior tip; each arm
with large, strongly sclerotized distal ridge bearing acharacters: Body length 9.0‑9.5 mm. Body color darkgrayish brown. Antenna much longer than stem of labral

distinct projection directed anterodorsally. Paraproctfan; Iength ratio of articles (from base to tip) 1.00:1.57:

25

0.45. Labral fan with ca. 55 main rays. Hypostornium

and posterior arms much narrower than that of S.

(Fig. 49) with 9 bristles on each side. Postgenal cleft

pahangense. Posterior circlet with ca. 420 rows of hook‑(Fig. 49) deep but not reaching posterior border oflets with up to 44 hooklets per row.

hypostomium; anterior margin irregularly defined, usu‑
ally with anterolateral extensions. Rectal papilla with 8

TYPE SPECIMENS. Holotype pharate

or 9 finger‑1ike secondary lobules on each lobe. Anal

exuvia and cocoon (BMNH) , Montathom waterfall, 730
m in altitude, E98'55'/N18'49', Chiang Mai Province,sclerite (Fig. 52) with basal portion connecting anterior

with pupallli l;:":' t=:; i a'}{' ̲' ' ̲'= "'S

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('; ']lU"II"//////// }Jl ' // J ll"! 'Lk¥ 'lti li"I' lrl"I'l l"'f'!'J"i'f"I "*¥ll)IJ i:: ;i;,1';ff j' !;ii"/]//////;; /;/iy;;;",i jl;:" 'i';/ ;:/';i;;i;:1';', i
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iif/////'/'i/jrr/
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i:iLi; {i;iF l/'{/ /'/f" i ;j /:/"7// l)/:,;'///'i/:; ;.;; ; ;" :r";{ :l lrff //{'{/{ ‑.̲̲}:: i" ̲

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LL; '1:: 'i:l; *:;{: ;,)Figures 36‑45. Female and male genitalia of S. (D.) courtneyi sp. nov. 36‑40,

45

44

genitalia
36, 8th sternite
and anterior gonapophysis (right half only, ventral view) ; 37, genital fork; 38 & 39, paraproct andcercus (38, ventral view; 39, Iateral view) ; 40, spermatheca; 41‑45, c l genitalia
41, coxite, styleand ventral plate (left coxite and style omitted, ventral view) ; 42 & 43, ventral plate (42, end view;
43, Iateral view) ; 44, paramere (right side only, dorsal view) ; 45, median sclerite. Scale bar 0.05 mm(applying to all figures)

26

THAILAND, 4. XII. 1995, by M. Burgett & M.
Titayavan. Paratypes: I pharate

DISTRIBUTION. Thailand.

, I pharate c l , dried

and pinned, 5 pharate c ! (2 partially damaged) , 6 pupae,ETYMOLOGY. This species is named in honor of

11 pupal exuviae (7 partially damaged) , 2 mature larvae

Gregory W. Courtney who provided us with the material
frorn Thailand.

and 6 immature larvae (CUAO , all in alcohol, samedata as holotype; 3 pharate c?1 (1 with abnormal anten‑nae) , 2 pupae and 2 pupal exuviae (BMNH), all in

TAXONOMIC REMARKS. This new species shows

alcohol, same data as holotype except date 29.XII.1995;

close similarities to S. pahangense in all stages. How‑ever, it differs frorn the latter species chiefly by thefollowing features: the shape of the genital fork andparaproct in the female; the shape of the ventral plateand median sclerite in the male; the size and coloring of

1 immature larva (CUAO , same data as holotype
except date 3. XI. 1994 and collector G.W. Courtney; 8

immature larvae (CUAO , Mai Yai waterfall, E98'30'/
N8'53', Surat Thani Province, THAILAND, 15. X. 1994,
by G.W. Courtney.

the gill filaments, and the integument of the frons dense‑ly covered with tubercles in the pupa; and the shape of

ECOLOGICAL NOTES. This species 'vas taken fromtwo heavily shaded streams, 16‑20 'C and 5‑8 m wide,
flowing over a series of bedrock cascades. Simulium
courtneyi was collected with S. pahangense at only one
site, Mai Yai waterfall in Surat Thani Province (15October 1994). Although we examined the gut of only
one larva of S. courtneyi (from Chiang Mai Province) , itcontained thalli of the trichomycete H. melusinae.

the postgenal cleft, as well as the length and coloring of

the body in the larva.

ACKNOWLEGEMEN TS
We are grateful to Dr. D. M. Davies, Professor
Emeritus, McMaster University, Ontario, Canada, and
Dr. R.W. Crosskey, the Natural History Museum, Lon‑.

48 *¥'
47

l

l

l

J '

l

:

l

¥:{{ f'; t i .. : " t ' .."" . ;"'‑"';:'s;;;;' I : ' " ¥/ ･. ‑""*' 't.
"" !s:2'･.･'::'.* 'i'. ". ..** '::l" :: .=1

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.

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I =*'= .*=s;'=' *SSS,=::;i!*'*::;. #‑ " f' 'l*,'''* I't':"': ' ' 'j;･;: S:; i{:'i j"=: ' =49
Figures 46‑52. Pupa and larva of S. (D.) courtneyi sp. nov. 46 & 47, pupa 46, frons (right half only) ; 47,gill filaments (1eft side, Iateral view) ; 48‑52, Iarva 48, whole body (lateral view) ; 49, head capsuleshowing hypostomium and postgenal cleft (ventral view) ; 50, anterior teeth of hypostomium (dorsalview) ; 51, apex of mandible; 52, anal sclerite (end view) . Scale bars 0.2 mm for fig. 47; 1.0 mm for fig.

48

27

don, U.K., for their valuable suggestions. Thanks are
due to Prof. Yong Hoi‑Sen, University of Malaya, Kuala

Simuliidae (Diptera) of Africa and its islands. Bull. Br.
Mus. Natur. Hist. (Entomol.) suppl., 14, 195p.

Lumpur, Malaysia, who helped HT in various ways

4) Rothfels, K. H. and Dunbar, R. W. (1953): The salivary
gland chromosomes of the black fly Simulium vittatum

during collection trips in Peninsular Malaysia. We thankDr. G.W. Courtney, Grand Valley State University,
Michigan, U.S.A., for providing material and habitat
information, and Dr. D. C. Currie, Royal Ontario Museurn,

Toronto, Canada, for reviewing the phylogenetic portionof the paper. This study was in part supported by aGrant‑in‑Aid to HT from the Japan Society for the
Promotion of Science.

REFERENCES
1 ) Adler, P. H., Wahg, Z. and Beard, C. E. (1996) : First

records of natural enemies from Chinese blackflies(Diptera: Simuliidae) . Med. Entomol. Zool., 47, 291‑292
2 ) Brown, B. V. (1993): A further chemical alternative to
critical‑point‑drying for preparing srnall (or large)
flies. Fly Times 11, 103 ) Crosskey, R. W. (1969) : A re‑classification of theZett. Can. J. Zool., 31, 226‑241

5) Rothfels, K.. Feraday, R. and Kaneps, A. (1978): A
cytological description of sibling species of Simulium
venustum and S. verecundum with standard maps for thesubgenus Simulium Davies (Diptera). Can. J. Zool., 56,
1110‑1128
6) Takaoka, H. (1983): The blackflies (Diptera: Simulii‑dae) of the Philippines. Japan Society for the Promotionof Science, Tokyo, xi + 199p.
7) Takaoka, H. (1996): The geographical distribution of
the genus Simulium Latreille in the Oriental and Aus‑
tralasian Regions. Jpn. J. Trop. Med. Hyg., 24, 113‑124
8) Takaoka, H. and Davies, D. M. (1995): The black flies(Diptera: Simuliidae) of West Malaysia. Kyushu Univer‑sity Press, Fukuoka, viii + 175p.

9) Takaoka, H. and Davies, D. M. (1997): Simulium(Simulium) yongi sp. nov. (Diptera: Simuliidae) from
Peninsular Malaysia. Jpn. J. Trop. Med. Hyg., 25 (inpress)

Jpn. J. Trop. Med. Hyg., Vol. 25, No. 1, 1997, pp. 29‑32

29Research Note:

OCULAR TOXOPLASMOSIS IN AN

AMAZONIAN JAPANESE SETTLEMENTSHIGEYUKI KANO1, MILTON MASATO HIDA2, YOSHIAKI UTSUNOMIYA3
and MAMORU SUZUKI
Received December 18, 1996/Accepted February 12, 1997

Abstracts: Infection with Toxoplasma gondii is a frequent cause of retinal disease, and relatively highprevalence of the disease has been reported in Brazil. We experienced acquired and congenital forms ofocular toxoplasmosis in an Amazonian Japanese settlement, Tom6‑A u. Fundus photographs of the patients
representing typical lesions of chronic retinitis are shown.

INTRODUCTION

water networks by Amazon branches, makes highly

try to country. In Brazil, Lamb and Feldman (1968)

vegetated flat area at 11‑30 m above sea level. The
Japanese immigrants have been settling there since
1962, and now 70 families with about 350 people aresupporting themselves mostly by agriculture. The num‑

reported that the prevalence of the infection was 52%,
and particularly in the Amazonian area, it was as highber of Japanese Brazilian inhabitants subjected to thepresent study was 102, aged from 5 to 85.

The geographic distribution of toxoplasmosis iscosmopolitan, however the incidence varies from coun‑as 56%. We also reported the distribution of anti‑
Toxoplasma antibodies for those living in an Amazonian
Japanese settlement, Tom6‑Acu (Sato et al., 1989), in
which we showed the overall prevalence of 75,2% with‑out age or sex dependency. Our epidemiological study
also revealed that some individuals were suffering from

Serologic and Ocular Examination
Antibodies against Toxoplasma gondii were assayedby micro‑titer method with Latex agglutination test
(Toxo‑test MT, Eiken KK, Japan) . Titers equal to or

impaired vision. Thus in 1991, we tried to determine theOphthalmic examinations were conducted for those
who complained of impaired vision, in August, 1991.
After dilation, individuals were screened with direct

prevalence of ocular toxoplasmosis in the same area byophthalmic examinations and found out two cases withretinochoroidal lesions, which are described in the pres‑ent report together with some epidemiological discus‑sion.

SUBJECTS, MATERIALS AND METHODS
Study Area
The study area, Tom6‑A u (1atitude 2'31' S, Iongi‑
tude 48"22' W) , is located 125 km to the south of Bel m,whose average temperature is 21‑33'C and the rain fallin a year amounts 2,635 mm. The place, with natural

higher than I : 32 were considered positive.

ophthalmoscopy for the presence of posterior uveitis by
the identification of inflammatory retinal or retinochor‑

oidal lesions. Fundus photography was performed byusing portable retinal camera and Ektachrome RD135
film (Kodak).

RESULTS
Case 1The patient was a 42‑year‑old male who immigrat‑
ed into Tom ‑A u in 1972. He was fond of eating raw

1 Department of Parasitology, Gunma University School of Medicine, 3‑39‑22 Showa‑machi, Maebashi, Gunma, 371 Japan
2 Department of Ophthalmology, Universidade Estadual Paulista, Faculdade de Medicina de Botucatu, 18618‑000 Rubiao Junior,Botucatu, Sao Paulo, Brasil
3 Department of Internal Medicine, Institute of Tropical Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki, 852 Japan
Address for correspondence:
Shigeyuki Kano, Department of Parasitology, Gunma University School of Medicine, 3‑39‑22 Showa‑machi, Maebashi 371 , Japan30

Photo 1. Fundus photograph of the left eye of the Case 1.
Superior to the macula is a yellowish‑white atro‑
phic lesion with partially pigmented indistinct
margin.

Photo 2A. Fundus phot(̲)graph of the right eyc of the Case 2,
in which an atrophic lesi(̲)n, whitish‑gray'"ithpigmentation, Iies in thc macular region

vith avell‑delineated borc]er.

meat and used to eat sliced pork uncooked. The last
episode of eating raw pork was with his neighboring
friend, one year before the onset of the disease. In 1974,

he began to complain the impairment of vision in theinferior nasal field in the left eye, and was treated witha anti‑malarial drug. A fundus photograph taken in1991 is shown as Photo 1. An unilateral yellowish‑
white atrophic lesion with partially pigmented indistinct

margin, was observed located superior to the macula in
the left eye. Antibody titer against Toxoplasma was l:l024 in 1991̲ As a matter of fact, the friend of his who

ate the raw meat together had become symptomatic of
ocular toxoplasmosis 9 months prior to the Case 1's
onset, and went back to Japan to be treated. (The detailof the history is unknown) . Thus, the route of trans‑mission to the Case I was considered to be from con‑sumption of cyst‑infected raw meat in the diet.

Case 2The patient was a 13‑year‑old boy who had been
blind from birth in the left eye, and was complaining of
the defect of the superior nasal field of vision in the righteye. Strabismus was also observed in the left eye.
Fundus photographs are shown as Photo. 2A and 2B.

Photo 2B. Fundus photograph of the left eye of the Case 2.
The same findings as in the right eye are noted.

31

Table I Anti‑Toxoplasrna antibody titers of theCase 2's family

Year(Age ) 1988 1989 1990 1991

Mother(35)

FatherCase 2(37)(13)

SisterBrother

2,048** 4,096

settlers.

Toxoplasmic retinochoroiditis is usually seen as alate manifestation of quiescent congenital forms of
toxoplasmosis. Based on the various epidemiological

2,048

studies, Stagno (1980) anticipated that approximately 632,7682,048 2,048(11)

32,768

8,192

of every 1,000 pregnant women will acquire toxoplas‑
mosis during the 9 months of gestation. And the aver‑age incidence of congenital toxoplasmosis was reported(9)32,7682,048

t0, be 1:1000 Iive birth and 10% showed ophthalmic

512

gray with pigmentation, were observed in the macular

involvement (Kean et al., 1991). The Case 2, unfortu‑
nately, suffered from this congenital ocular toxoplas‑
mosis, manifesting central and bilateral lesions as areusually the case with congenital ones. Subsequent sib‑lings of the Case 2 were thought to be protected byregions with well‑delineated borders. Thus, the patient

their mother's acquired immunologic defense mecha‑

was thought to have been suffering from congenital

nism, as one may theoretically expect them to be, and in

ocular toxoplasmosis. Antibody titers against Toxoplas‑ma are shown in Table 1. At the time of our survey, noother clinical manifestations of congenital toxoplas‑
mosis were recognized on this patient.

fact, no ocular lesion was recognized on them. However,the considerably high antibody titers were detected forthe family, so that their long‑term and intense exposure
to the organism was thought to be actually taking place.
Their close contact with cats will be the most probablecause of their repetitive infections, although the survey*Ages in parentheses are those in August, 1991.
**Antibody titers are represented in reciprocal.

In both photographs, bilateral atrophic lesions, whitish‑

Case 2's Family
Antibodies to Toxoplasma for the Case 2's parents
and 2 siblings were also measured, and the titers wereshown in Table 1. His mother manifested considerablyhigh titers at I : 2048 (1988 and 1990) and I : 4096
(1989), and also his father showed positive titer at 1:
512 (1988). Although his sister and brother showed thetiters as high as I : 32,768 in 1989, ocular toxoplas‑
mosis was not recognized on either of them. The family
was keeping several cats in the house and the children
were making close contact with the cats, playing oftenwith them or even sleeping with them.

DISCUSSION
Acquired toxoplasmic chorioretinitis was once
thought to be uncommon, however in Brazil, ocular
toxoplasmosis has been reported to comprise approxi‑
mately 50% of all uveitis, and incidence of acquired
subclinical toxoplasmic infection that causes retinochor‑

oiditis is considered much more frequent than previously
believed (Silveira et al., 1988; Glasner et al., 1992).

of the oocysts produced from the cats have not been
conducted.
Our previous serological study on the prevalence of
toxoplasmosis in the same area (Sato et al.. 1989) alsosuggested the significant health hazard caused by thedisease. Dedicated efforts have to be made for thecontrol of toxoplasmosis in Tom6‑A u.

ACKNOWLEDGMENTS
This study was supported in part by grants fromJapan International Cooperation Agency. We express
sincere gratitude to Mr. Tsugio Shishido, Secretary
General, and other staff of Benefic ncia Nipo‑Brasileirada Amaz6nia, for providing us with local facilities. We

also thank Dr. Akio Kobayashi, Professor Emeritus of
Jikei University School of Medicine, Professor Koichi
Shimizu and Dr. Tomohiro lida of Department of Oph‑thalmology, Gunrna University School of Medicine, for
helpful discussion.

Besides, the brazilian are usually fond of eating beefwell charcoal‑grilled so called "churrasco" and rarely

REFERENCES

eat raw meat, however, Japanese Brazilians have a1 ) Glasner, P. D., Silveira, C., Kruszon‑Moran, D., Martins,preference for eating them raw, Iike "sashimi" or "shabu

M. C.. Burnier. Jr., M., Silveira, S., Camargo, M. E.,
Nussenblatt, R. B., Kaslow, R. A. and Belfort; Jr.. R.

shabu", as the Case I practiced. This custom of eating
may increase their chance to contract ocular toxoplas‑
mosis. In the context of the control of the disease,particular attention has to be paid among the Japanese(1992) : An unusually high prevalence of ocular toxoplas‑

mosis in southern Brazil. Am. J. Ophtalmol., 114, 136‑
144

32

2) Kean, B. H., Sun, T. and E1lisworth, R. M. (1991): Color
Atlas / Text of Ophthalmic Parasitology. 9‑21, Igaku‑

Japanese Settlement. Jpn. J. Trop. Med. Hyg., 4, 339‑
346

Shoin, Tokyo
3) Lamb, G.A. and Feldman, H. A. (1968): A nationwide5 ) Silveira, C., Belfort, Jr., R., Burnier. Jr.. M. and Nussenb‑

serum survey of Brazilian military recruits, 1964. 111.
Toxoplasma dye test antibodies. Am. J. Epidemiol., 87,
323‑3284) Sato, S., Kano, S., Hida, M. M., Yanagisawa, H., Og‑
awara, H. and Suzuki. M. (1989) : High antibody positive
rates of ascariasis and toxoplasrnosis in an Amazonianlatt, R. (1988) : Acquired toxoplasrnic infection as thecause of toxoplasmic retinochoroiditis in families. Am.
J. Ophtalmol., 106, 362‑364
6 ) Stagno, S. (1980): Congenital toxoplasmosis. Arn. J. Dis.
Child., 134, 635‑637

March1997

VOL．25NO．1

CONTENTS
Original

ArticleTongo1 Rivera，P．，Kano，S．，C諭ete−Migue1，E．，and Suzuki，M．
A23kD

from
A

MoleculeofPlαs郷04伽n血lo吻捌窺Binds

Splenomegalic，Parasitemic，but
Pilot

Study

in

a

Malarious

Specific

AsymptomaticCommunity

in

IgG

Children

Palawan，The

Philippines

・1−5

Chigusa，Y．，Kirinoki，M．，Yokoi，H、，Kawai，S、，MatsudaナHりYasuraoka，K．，Bugawan，L．E．andBlas，B．L．

ASurveyoflntestinal，ParasiticInfectioninSanNarciso，Victoria，
Oriental・7−9Mindoro，Philippines…

Takaoka，H．and Davies，D．M．
S吻％1勉吻（S初％1勉窺）夕o銘g づsp．Nov．（Diptera：Simuliidae）from

Peninsular

Takaoka，H．andA

New

・11−16

Malaysia
Adler，P．H．

Subgenus，S加％1勿御（P卿歪6s811％吻），and（Diptera：Simuliidae）from

Research

Thailand

and

a

New

Peninsular

Species，S（n）oo％7孟nのノ4

Malaysia……一・17−27

Note

Kano，S．，Hida，M．M．，Utsmomiya，Y．，Suzuki，M．

OcularToxoplasmosisinan

AmazonianJapanese

Settlement

・・29−32