一昌目｝

第25巻第4号平成9年12月
内
原

容

著
モンゴルで採集されたユスリカの研究（英文）

・・佐々

学，鈴木

博

149−189正崇

191−195

タイ国で重症度の異なるデング患者より分離されたデングウイルス4型の構造遺伝子
及び非構造遺伝子NS1領域の遺伝子解析（英文）一Charnchudhi Chanyasanha

197−207

Ro伽iκs餌ol鰍sに感染した丁脚碗oso耀勉ng6〃のサシガメ体内移行経路
およびそれにともなう丁昭ng観の形態変化に関するSEM所見（英文）・・Maria

Carlota

Monroy，古賀

研究ノート

中国産生薬の膣トリコモナスに対する吻o伽oの効果
一張
永生，牧

純，杜

軍，伊藤

洋一

209−213

一………・……・……・…・………・………・…一…・………………・大西健児

215−216

症例報告

無症候群ビルハルツ住血吸虫症：輸入症例としての1例

会報・記録
217−223■■

Co皿igendhm
Jpn．J．Trop．Med．Hyg．，Vol．25，No．4，1997，pp．197−207
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149

Jpn. J. Tro p. Med. Hyg., Vol. 25, No. 4, 1997, pp. 149 189

STUDIES ON THE CHIRONOMIDAE(DIPTERA, INSECTA) COLLECTED

IN MONGOLIA

MANABU SASA* AND HIROSHI SUZUKI'
Received May 28, 1997/Accepted September 16, 1997

Abstract: Collections of adult chironomids were carried out in Mongolia, Central Asia, Iocated about
midway between Europe and Japan, in August 1995 and also in August 1996. The specimens were preservedin 70% ethylalcohol, and individually mounted on slides in gum‑chloral medium after digested in 10% hot
KOH solution and dissected under stereomicroscope. A total of 345 male specimens were studied for species
identification. As the results, a total of 42 species belonging to 23 genera were identified, among which 12
species are considered as belonging to the same one as those already recorded from Europe, and 11 species

among them are also in common with those from Japan, and the rest 29 species are described here as new
species. Three new genara were created in order to accept 4 new species arnong them.
Key words: Chironomid midges, Mongolia, Allergens, Asthma, Nuisances

INTRODUCTION
The insects of the family Chironomidae include
large numbers of species breeding in various types oflakes, rivers and streams, and have recently attracted
special attention as causing a number of problems in theOn the other hand, Iarge numbers of chironomid
species were found to be breeding in various types oflakes, rivers and ditches, and serving as an importantfactor in the spontaneous rernoval of pollutants fromnatural waters. The species of chironomids found innatural waters are quite different according to theof dust containing fragments of certain chironomid

chemical and physical characters of the waters, and they
are recently utilized as the most excellent biologicalindicators of the degree of pollution of rivers and lakes.

species acts as the important allergens causing bron‑

Extensive studies have been carried out in the

chial asthma throughout the world. Such cases wereEuropean, Oriental, Australian and the Nearctic

first reported in 1958 from Sudan, where large amounts
of a chironomid species, Cladotanytarsus lewisi, began toRegions on the taxonomy, biology and ecology of thisgroup of insects, as was reviewed in the monograph ofemerge after a manmade lake was constructed on theRiver Nile, and large numbers of people who moved tober bf species recorded from the British Islands werethe lake‑side started to suffer from bronchial asthma.

about 440 according to Pinder (1978) , and that recorded

However, it was shown in 1985 by Sasa and coworkers
in Japan that many other chironomid species commonly
ernerging from rice paddies, Iakes and rivers are also

from Japan increased from about 160 in 1978 to morethan 800 in 1995, as was reviwed by Sasa and Kikuchi

fields of medical and environmental sciences. For exam‑
ple, as was recently reviewed by Sasa (1989) , inhalation

Chironomidae edited by Wiedelholm (1989). The num‑of the asthmatic patients tested in many rural and urban(1995) , and some 50 new species are being added every
year from this small country. The chironomid fauna ofJapan were extensively studies by Tokunaga (1936 a, b,

areas of Japan were shown to be hypersensitive to thec, 1937, 1940) , and since about 1976 by some 50 presently

chironomid antigens. Furthermore, some chironomids
are serious nuisances to many people residing nearactive entomologists. The chironomid fauna of theeutrophicated lakes and rivers all over the world, andnecessitates the construction of sewage cleaning plants
in many cities, mainly in order to prevent their massive

as by Makarchenko (1987, 1993, 1994) in Siberia, by Ree

acting as causes of allergic diseases, and some one‑third

emergence.
1

2

Oriental Region have also been extensively studied, such

(1981) and Ree and Kim (1981, 1988) in Korea, and by
Wang et al. (1977) and Yan and Ye (1977) in China.
However, the chironomid fauna of the Central Asian

Shofuku Clinic, 1022 Tetsugami, Kurobe‑shi, Toyama 938, Japan
Institute of Tropical Medicine, Nagasaki University, Nagasaki 852, Japan

150

Region have remained almost unknown. Since Mon‑as new species. The morphological descriptions andgolia is located between Europe and Japan, the results offigures are presented also to a part of those considered

the present study carried out for the first time in thisas belonging to the already described species, because
they were found to be morhologically somewhat differ‑ent from the type materials described from Europe orJapan, or in the purpose of supplementing the incom‑

country will contribute a great deal to supplement the
information on the taxonomy and biology of the family

Chironomidae.
This study was conducted as a part of the projectplete original descriptions.

No. 07041156, Grants in Aid of International Scientific

Research, the Ministry of Education of the Japanese
Government, with Prof. Masao Kamiya, University of
Hokkaido, as the Project Leader.

THE SpECIES COLLECTED,AND MORPHOLOGICAL AND TAXSONOMICAL NoTES
1. Camptochironomus mougolabeus sp. nov.
(Figs. I a‑m)

MATERIALS AND METHODSFive males were collected at Bogd (# 1) on August
13, 1996. Holotype: No. 308:73. Paratypes: 308: 74‑77.

Collections of adult chironomids were conducted by
H. Suzuki during the period from July 30 to August 6,
1995, in and around the capital city of Ulaanbaatur, andfrom August 6 to August 22, 1996, at around Bogd, about
700 km southwest of the capital. The adult midges werecollected during the daytime with insect net while rest‑(4.58) mm, WW/WL 0.28‑0.29, ER 0.12‑0.23 (0.18), AR
3.71‑4.05 (3.86), AHR 0.52‑0.57 (0.55), P/H 0.91‑1.11

ing in bushes on the shore of lakes and streams, and also(45.5) , SQ 34‑46 (40.0) , RR 0.33‑0.45 (0.38) , VR 1.06‑1.11

at night with sucking tubes while being attracted onelectric lamps. The specimens were preserved in 70%

0.57‑0.60 (0.59). fTR 0.23‑0.25 (0.24), fBR 1.7‑5.2 (3.7),ethylalcohol, and were mounted individually on slides in

mBR 1.0‑2.3 (1.5), hBR 1.4‑3.1 (2.3).

gum‑chloral medium following roughly to the method
described by Sasa and Kikuchi (1995), but special

WL 3.42 mm, WW/WL 0.26, AR 2.90 (smaller), AHR

devices were made this time by Suzuki for the improve‑ments of the methods of mounting the specimens on
slides for the taxonomic studies of this group of insects.

The methods of descriptions, standard measurements
and preparation of figures mostly followed those de‑scribed by Sasa and Kikuchi (1995), and the technicalterms used in the morphological descriptions mostly
followed those presented by Pinder (1978) or Saether(1980) . The specimens used in the present study, includ‑ing the holotypes and paratypes, are being deposited in
the Institute of Tropical Medicine, Nagasaki University.

RESULTS

Male. In the first 4 specimens with larger body size,
BL 10.08‑10.90 (10.57 in average of 4) mm, WL 4.34‑4.92(1.00) , SO 35‑44 (40.4) , CL 50‑70 (59.0), PN all O, DM 16‑

36 (24.3), DL 30‑50 (30.9). PA 8‑11 (9.0), SC 36‑56(1.09), fLR 1.26‑1.36 (1.31), mLR 0.50‑0.53 (0.52), hLR

In No. 308:77 with smaller body size, BL 9.96 mm,
0.56, ER 0.31, P/H 0.94, SO 32:32, CL 46, PN O, DM 18,
DL 24:26 (smaller), PA 8:8. SC 38, SQ 46:46, RR 0.31, VR
1.05. R/Cu 1.13, fLR 1.57 (larger), mLR 0.53, hLR 0.60,

fTR 0.28, fBR 1.1, mBR 1.2, hBR 1.2.

Ground color of scutum yellow, stripes brown,
scutellum yellow, postnotum dark brown; femora andtibiae entirely yellow, tarsi I and 11 Iargely yellow andapical portion brown, tarsi 11 to V brown; abdominal
tergites almost entirely brownish yellow, hypopygium
brown. Head in Fig. I a. Eyes bare, antenna with 13
flagellar segments. Frontal tubercles (Fig. I b) promi‑
nent, almost cylindrical, 43 pm long, 18 pm in diameter,and 45 pm apart from each other. Antepronotum (Fig.
1 c) united in the middle, without lateral seta.Wing bare,

As the results of these collections, a total of 345

venation in Fig. I d, typical as a member of the Chir ‑adult male chironomid specimens were collected andmounted on slides, and they were classified into 42
species belonging to 23 genera, as shown in Table 1.
Twelve among them are considered as belonging to thesame species as recorded from Europe, and 11 among
them are also in common with those recorded fromonomus complex. Tip of front tibia (Fig. I e) with abroad and rounded terminal process. Tips of middle andJapan, indicating that they are widely distributing in the

Hypopygium in Fig. I h, (dorsal, Ieft half) and Fig. I i(ventral view, right half) . Ninth tergite with a longholarctic region. The number of species in common
with those recorded from Japan but not from Europe is
only one. The rest 29 species are described in this paper

hind tibiae (Fig. I f, g) with two comb scales, both witha short spur. Pulvilli well developed, brush‑1ike, claws

are simple and with pointed apex.

Abdominal tergites with numerous short setae.

posterior process flanking anal point, its posterior
margin only slightly concave (not deeply concave as in151ole e oo ee')
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Camptochironomus mongolabeus sp. nov. a: head, b: frontal tubercles; c: antepronotum; d : wing; e: tip offront tibia; f: tip of middle tibia; g: tip of hind tibia; h: hypopygium, dorsal view, Ieft half; i: hypopygium, ventral
view, right half; j: anal point, Iateral view; k: dorsal appendage, ventral view; m: dorsal appendage, dorsal view.

Fig. 2 Camptochironomus mongolbeceus sp. nov. a: frontal tubercles; b: hypopygium, dorsal view; c:hypopygium, ventral view; d: anal point and dorsal appendages.

152

the previously known species) . Ninth tergite with a pairof low, broad and rounded lobes laterally (Fig. I h) .
Anal point constricted at base and apically rounded in
dorsal view (Fig. I h) , and somewhat sickle‑shaped inlateral view (Fig. I j). Dorsal appendage (DA in Fig. 1h; also in Figs. I m, dorsal view, and k, ventral view,
both enlarged) elongate oval, smaller and shorter thanventral appendage, with 8 setae on inner margin andplex, SQ 22:24, RR 0.37, VR 1.06, R/Cu 1.16. fLR 1.28
(relatively small), mLR 0.50, hLR 0.59, fTR 0.23, fBR
1.4, mBR 1.7, hBR 1.9. Pulvilli large and brush‑1ike.
Hypopygium in Figs. 2 b (dorsal) and 2 c (ventralview) . Anal point flanked by a V‑shaped lobe reaching
just to tip of anal point, the posterior margin of ninth
tergite not concave but convex. Dorsal appendage (Fig.
2 d) much smaller than ventral appendage, highly con‑dorsal side is entirely clothed with microtrichia. Ven‑tral appendage (VA) very long and finger‑like, the tip

stricted at base and triangularly expanded, clothed withreaching near tip of gonostylus, with numerous shortextremely long, finger‑like, and clothed with numerous
short setae and microtrichia, as in the former species.
Gonostylus also very wide at base and gradually taper‑ing towards rather pointed apex, also like in the former
species, 2.37 times as long as wide, with short setae
along inner margin.
Remarks. This specimen also belongs to the genus

setae on dorsal side and thickly clothed with microtri‑chia on both sides. Gonostylus relatively short and very
stout, 2.5 times as long as wide, widest near base andapically rounded, with short setae on inner margin.

Remarks. These specimens are considered as
belonging to the genus Camptochironomus Kieffer, 1918,

numerous setae and microtrichia. Ventral appendage

since anal point is flanked by a process of ninth tergite,

Camptochironomus, since the shape and structure of

dorsal appendage is rather small, elongate oval andventral appendage and gonostylus are quite characteris‑tic and similar to the above species, and ninth tergite
seems to have median posterior lobe flanking anal point.
It differs from C. mongolabeus in that the posterior lobeof ninth tergite is just as long as the 'anal point andreaches to the tip of it without showing concave poste‑rior margin, and dorsal appendage is quite different inshape, strongly constricted at base and expanded to abroad and triangular lobe bearing numerous long setae
and microtrichia. The present species has distinct dark

clothed with numerous short setae and microtrichia, andventral appendage is very long, finger‑1ike and alrnost
entirely clothed with numerous short setae and microtri‑chia (ref. Cranston et al., 1989a). They are closer to C.
pallidivittatus (Malloch) than to C. tentans (Fabricius)among the two European species, in that gonostylus is
more robust, but in the present specimens gonostylus isfurther stouter and shorter (3.0 times as long as wide intentans according to the figure of Pinder, 1978, Fig. 142
B, about 2.5 times in the present specimens) . In theabove European species, anal point is much more slen‑der, and posterior process flanking anal point is deeply
divided into two lobes posterior margin only slightly
concave in the present species.

2. Camptochironomus mongolbeceus sp. nov.

and pale rings on tibiae and tarsi, while femora andtibiae are entirely yellow in the former species.

3. Chironomus mongolcedeus sp. nov. (Figs. 3 a‑d)
A male was collected at Bogd (# 21), 1,500 m highfrom sea level, on August 13, 1996. Holotype: No. 308:
51.

(Figs. 2 a‑d)Male. BL 6.06 mm, WL 3.46 mm, WW/WL 0.26.

A male was collected at Bogd on August 13, 1996.
Holotype: No. 308:79.

Ground color of scutum dark brown, stripes, scutellum
and postnotum black, abdominal tergites almost uni‑
formly dark brown, VI, VII and Vlll with a narrow paleband along caudal margin, Ieg segments almost uniform‑ly brownish yellow, excepting tibiae which have short

Male. BL 9.64 mm, WL 4.46 mm, WW/WL 0.28.
Ground color of scutum yellow, stripes dark brown,
scutellum yellow, postnotum black; femora entirely
yellow, tibiae with a conspicuous dark brown apicalring, basal 2/3 of front tarsus I and basal 1/2 of middle

and hind tarsi I yellow and their distal portions darkbrown, tarsi 111, IV and V of all legs entirely dark brown.

Frontal tubercles (Fig. 2 a) Iarge, cylindrical. Eyes
bare, ER 0.37. Antenna with 13 flagella segments, ARbasal and apical dark rings. Frontal tubercles (Fig. 3 a)relatively small, 24 pm long, 12 pm in diameter, and 50
pm apart from each other. Eyes bare, ER 0.35. Antenna
with 11 flagellar segments, AR 3.88, AHR 0.71. Palp
10ng, P/H 1.24. SO 26:26, CL 25. Antepronotum black,very narrowly united in the middle, PN O:O. DM 18, DL

4.28 (larger than in the former species) , AHR 0.69. Pl

27:28, PA 6:6, SC 28. Wing bare, bluish, anal lobe

H 1.00. Antepronotum united in the middle, without
lateral seta. DM 16, DL 32:32, PA 9:10, SC 21. Wing
venation typical as a member of the Chironomus com‑

rectangular, SQ 18:18, RR 0.30, VR 1.05, R/Cu 1.16. fLR

1.41, mLR 0.59, hLR 0.71, fTR 0.21, fBR 2.2, mBR 2.0,
hBR 2.8. Pulvilli well developed, brush‑like.

153

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Fig. 3

Chironomus mongolcedeus sp. nov. a: frontal tubercles; b: hypopygium, dorsal view; c: Ieft dorsal appendage;d: right ventral appendage. Fig. 4 Chironomus mongoldeeus sp. nov. a: frontal tubercles; b: abdominal tergites
I‑VI, showing dark areas; c: hypopygium; d: Ieft dorsal appedage; Ieft ventral appendage. Fig. 5 Chironomus
mongolefeus sp. nov. a: frontal tubercles; b: hypopygium; c: anal point (1ateral view) , and dorsal appendages;
d: Ieft dorsal appendage; e: right dorsal appendage.

154

Hypopygium in Fig. 3 b. Anal point stout, parallel‑
sided and apically rounded. Dorsal appendage (Fig. 3 c)boot‑shaped, distal lobe short, stout and rectangularly
curved, widest at about middle, and basal portion low,
wide and flat, with 5 Iong inner setae. Ventral append‑age (Fig. 3 d) finger‑1ike, parallel‑sided and with 24

recurved setae and 4 caudally directed short setae.
Gonostylus widest at about middle and not constricted
near apex.

Remarks. This species belongs the genus Chir ‑onomus with S‑type (shoe form) of dorsal appendage
after Strenzke (1959), and most species of this group

have been found breeding in rather polluted sewage

fTR 0.21‑0.25 (0.23). Beard ratio of tarsomeres I
highly variable, fBR 1.9‑5.0 (3.1), mBR 2.4‑5.3 (3.4),
hBR 3.1‑6.0 (4.2). Pulvilli well developed.
Hypopygium in Fig. 4 c. Anal point stout, widest atbase, and sickle‑shaped in lateral view. Dorsal append‑age (Fig. 4 d) composed of a low and wide basal portion
bearing 4 or 5 inner setae, and a distal horn, which isbare, darkly pigmented, Iong, slender, smoothly curved
and apically pointed. Ventral appendage (Fig. 4 e) Iong,straight, widest at base and tapering towards rounded
apex, bearing some 24 recurved setae arising from the
distal half. Gonostylus slender, inner margin slightly
concave, and lateral margin abruptly constricted nearwaters at least in Japan. However, almost all of themshow peculiar pale and dark marks on abdominal ter‑apex.

gites, and anal point is narrow and constricted basally,

this genus collected in Mongol all belong to the groupwith the E‑type (elephant‑horn form) of dorsal append‑
age in the classification of Strenzke (1959). The speci‑

and those found in sulfuric hot spring waters in Japanare all black in body coloration and with narrow anal
point (Sasa and Kikuchi, 1995). This species is quite
unusual in that abdominal tergites are almost uniformly
brown, and anal point is very wide and stout.

4. Chirouomus mongoldeeus sp. nov. (Figs. 4 a‑d)Twenty one males were collected at Karakorum (#32) on August 13, 1996. Holotype: No. 308:52. Par‑

Remarks. This and the four following species ofmens of this species are highly variable in most measure‑ment data, including body and wing length, AR, LR, BR,
and the numbers of setae on head and thorax, but can be
identified and differentiated from other species of thisgenus in the peculiar coloration of abdominal tergites
and the peculiar shape of anal point, dorsal and ventral
appendages, and gonostylus.

atypes: No. 308:53‑57, 310:64‑78.

Male. Six specimens collected at the same time at
Karakorum (# 32) were measured. BL 7.28‑11.12 (8.52

5. Chironomus mongolefeus sp. nov. (Figs. 5 a‑e)in average of 6) mm, WL 3.38‑4.81 (3.86) mm, bothThree males were collected at Karakorum (# 24) on
August 18, 1996. Holotype: No. 308:58. Paratypes: No.

highly variable, WW/WL 0.27‑0.29 (0.28). Ground color

308:59, 60.

of scutum yellow, stripes brown, scutellum yellow, post‑

Male. BL 7.60, 7.32, 7.64 mm, WL 3.36, 3.40, 3.35

notum dark brown, Iegs almost uniformly yellowish

mm, WW/WL 0.28, 0.29, 0.27. Ground color of scutum
yellow, stripes largely dark brown and marginal por‑tions black, scutellum yellow, postnotum black, femora
largely yellow and distal portion brown; tibiae brown
for basal 1/3 and distal portion, the middle portion
yellow, tarsi I yellow for basal half and brown for
distal half, other tarsal segments brown; abdominal

brown. Abdominal tergites 11 to Vffl each with a large

brown oval area in the middle surrounded by yellow
zones along lateral and posterior margins (Fig. 4 b) .
Frontal tubercles (Fig. 4 a) widest at about middle andspindle‑shaped, 22 pm long, 12 pm in diameter, and 23
pm apart from each other in the holotype. Eyes bare,
ER 0.09‑0.29. Antenna with 11 flagellar segments, AR
3.48‑5.00 (mean, 4.08), AHR 0.53‑0.62 (0.58). Palp long,P/H 1.18‑1.44 (1.31). SO 32‑61 (45.6), CL 13‑52 (38.3).
Antepronotum united in the middle, without lateral seta.
DM 17‑28 (22.5: DL 26‑51 (34.6), PA 6‑8 (7.1), SC 18‑46(33.3) , all highly variable.

tergites largely brown, 11 to Vlll with yellow band alongcaudal margin.
Frontal tubercles (Fig. 5 a) prominent, cylindrical.
Eyes baie, ER 0.34, 0.30, 0.27. Antenna with 11 flagellar
segments, AR 3.61, 3.94, 3.65, AHR 0.67, 0.56, 0.67. Palp
10ng, P/H 1.18, 1.20, 1.13. SO 36:37, 44:46, 34:34, CL 38,

Wing bare, membrane slightly bluish, R‑M darkly

40, 20. Antepronotum narrowly united in the middle,

pigmented. Costa does not extend beyond tip of R4 + 5,

without lateral seta. DM 18, 14, 22, DL 22:25, 34:31, 30:

which is distal to tip of Cul, R/Cu 1.09‑1.14 (1.12). R2+

26, PA 7:7, 6:7, 9:9, SC 25, 34, 30.

3 ending closer to tip of R1 than to tip of R4 + 5, RR
0.23‑0.45 (0.31). R‑M slightly proximal to FCu, VR 1.01‑

with 26:26 fringe hairs in the holotype. RR 0.16, 0.17,

1.04 (1.03) . Anal lobe slightly produced inwards. fLR 1.32‑

0.25, VR 1.01, 0.99, 1.03, R/Cu 1.13, 1.13, 1.12. fLR 1.37,

1.49 (1.44), mLR 0.56‑0.62 (0.60), hLR 0.70‑0.77 (0.74),

1.48, 1.39, mLR 0.53, 0.55, 0.53. hLR 0.70, 0.71, 0.71, fTR

Wing bare, bluish, R‑M darkly pigmented. Squama

155

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Chironomus mongolfegeus sp. nov. a: frontal tubercles; b: hypopygium; c: Ieft dorsal appendage; d: Ieft Ventral

appendage. Fig. 7 Chironomus mongolgeheus sp. nov. a: frontal tubercles; b: hypopygium; c: Ieft dorsal
appendage; d: Ieft ventral appendage. Fig. 8 Chironomus mongolheius sp. nov. a: frontal tubercles; b:hypopygium; c: Ieft dorsal appendage; d: Ieft ventral appendage.

156

0.23, 0.24, 0.21, fBR 1.5, 1.4, 2.1, mBR 2.0, 1.8, 5.3, hBR

structure of hypopygium, as shown in the key. It is

2.4, 1.8, 4.0, (BR all highly variable) .

somewhat related to C. salinarius Kieffer, a cosmopoli‑tan species breeding in brackish swamps, in that anal
point is constricted in the middle and dorsal appendage
is smoothly curved, but differs from the latter in thatbody coloration is paler, anal point is stout, dorsal
appendage is wider, and gonostylus is strongly constrict‑ed near apex (ref. Sasa and Kikuchi, 1995).

Hypopygium in Fig. 5 b. Anal point very stout,
sickle‑shaped in lateral view (Fig. 5 c). Base of dorsal
appendage low and broad, with 4 or 5 basal setae, distal
horn stout, parallel‑sided, inner margin nearly straight

but apically hooked inwards (Fig. 5 d, e) . Ventral
appendage (Fig. 5 f) widest at base, with 18 recurved
and 4 caudally directed short setae. Gonostylus slender,widest at about basal 1/3, Iateral margin convex and not

7. Chironomus mongolgeheus sp. nov. (Figs. 7 a‑d)

apically narrowed.
Remarks. This species is somewhat related in the
shape of stout anal point to C. anthracinus Zetterstedt
among the European species of this genus, but differs
essentially in the shape of dorsal appendage, which islong, slender and strongly curved in the latter (cf.
Pinder, 1978) . Three among the Japanese species of thisAugust 13, 1996. Holotype: No. 308:61. Paratypes:

brown, scutellum yellow, abdominal tergites almost

genus have the stout anal point, C. nipponensis To‑

uniformly brownish yellow; femora and tibiae uniformly

kunaga, C. fujitertius Sasa, and C. echizensis Sasa, butthey also differ from the present species in the shape ofyellow, tarsi I , 11 and 111 Iargely yellow and each withdorsal appendage and gonostylus (cf. Sasa and Kikuchi,
1995) .

Thirty seven males were collected at Bogd (# 1) onother 36 males, No. 308:62; 310:O1‑35.

Male. BL 9.96‑11.12 (10.35 in average of 8) mm,
WL 4.80‑5.02 (mean 4.87) mm (both larger than in mostother species of Chironomus) , WW/WL 0.26‑0.29 (0.27) .
Ground color of scutum yellow, stripes and postnotum

an apical dark ring, IV yellow for basal half and brown
for distal half, V brown.
Frontal tubercles (Fig. 7 a) prominent, 68 pm long,

28 pm wide and 40 pm apart from each other in the
6. Chironomus mongolfegeus sp. nov. (Figs. 6 a‑d)
A male was collected in a tent, in Gobi Desert, on
August 10, 1995. Holotype: No. 306:70.

Male. BL 7.44 mm, WL 3.40mm, WW/WL 0.29.
Ground color of scutum yellow, stripes brown, scutellum
yellow, postnotum brown, Iegs almost uniformly brown‑

ish yellow, abdominal tergites also largely brownish
yellow and caudal margins of II, 111, IV, Vll and Wll are

yellow. Frontal tubercles (Fig. 6 a) prominent, almost
cylindrical, 40 pm long, 16 pm in diameter, and 39 pm

apart from each other. Eyes bare, ER 0.36. Antenna
with 11 flagellar segrnents, AR 3.83, AHR 0.57. Palp
long, P/H 1.15. SO 28:30, CL 22. Antepronoturn narrow‑holotype. Eyes bare, ER 0.15‑0.29 (0.21). Antenna with
11 flagellar segments, AR 4.45‑5.41 (4.74, very high) ,AHR 0.56‑0.63 (0.60). Palp relatively short, P/H 0.93‑
0.98 (0.96). SO 38‑58 (44.3), CL 36‑50 (41.8). Ante‑pronotum narrowly united in the middle, without lateral
setae. DM 18‑24 (20.7), DL 36‑42 (40.3), PA 5‑8 (6.8),
SC 36‑44 (41.2). Wing bare, SQ 14‑28 (22.3), RR 0.36‑
0.50 (0.44), VR 1.05‑1.11 (1.08), R/Cu 1.12‑1.17 (1.15).
fLR 1.26‑1.37 (1.32, relatively small), mLR 0.57‑0.59(0.58), hLR 0.68‑0.71 (0.70), fTR 0.20‑0.22 (0.21), fBR
4.3‑7.3 (5.8, very high), mBR 2.7‑4.0 (3.2), hBR 3.0‑4.9(3.9). Pulvilli well developed.

Hypopygium in Fig. 7 b. Anal point long, narrow,ly united in the middle, PN O:O. The numbers of setae on

slightly constricted in the middle, and darkly pigmented,

scutum and scutellum are DM 18, DL 30:30, PA 8:8, andSC 30. Wing bare, SQ 18, RR 0.27, VR 1.01, R/Cu 1.14.
Front tarsi both lost, mLR 0.61, hLR 0.71, mBR 3.1, hBRor narrow and sickle‑shaped in lateral view. Dorsal
appendage (Fig. 7 c) composed of a long and low basebearing 6‑8 inner setae, and a narrow, straight andparallel‑sided distal horn, which is slightly curved
inwards at distal 1/5. Ventral appendage (Fig. 7 d)4.2. Pulvilli well developed.

Hypopygium in Fig. 6 b. Anal point more stout thanin the next species, widest at base, and slightly constrict‑

10ng, narrow, slightly curved and bearing some 30 recur‑

ed in the middle. Dorsal appendage (Fig. 6 c) wider andmore strongly curved, ventral appendage (Fig. 6 d) also
wider than in the next species, widest at base and nearlystraight, with 28 recurved setae and 5 short caudally
directed setae. Gonostylus slender, inner margin con‑cave, Iateral margin abruptly constricted near apex.
Rernarks. This species is similar in basic structure
to the next species, but can be differentiated by theved setae on distal 1/4. Gonostylus narrow, inner
margin concave, and lateral margin constricted neara pex.

Remarks. This species is somewhat similar among
the European species to C. annularis (Degeer) in that
anal point is slender, frontal tubercles well developed,
tarsi with long beards, and body coloration is not black
but largely brown, but differs from the latter in that

157

drosal appendage is stout and more strongly curved,
ventral appendage is curved, and gonostylus is wider
and constricted near apex. It is most closely related
among the Japanese species of this genus to C. fuji ‑primus Sasa, since anal point is narrow, antepronotum
without lateral setae, dorsal appendage is horn‑like,according to the key of Pinder (1978) , in that anal point

scutal stripes and abdominal tergites almost uniform incolor, and frontal tubercles are well developed, but inthe latter body coloration is largely greenish yellow, AR

is narrow and constricted at base, dorsal appendage is
not shoe‑form, tarsi with long beards, and body is notentirely black, but the latter is a very large species
breeding in lakes throughout the world, abdomen withconspicuous marks, and dorsal appendage is band‑form.
It is most closely related among the Japanese species
also to C. fujiprimus Sasa, but also differs from thelatter in body coloration and in the shape of dorsal

is smaller (3.2‑3.9) , fLR Iarger (1.41‑1.53) , fBR smaller

appendage and gonostylus.

(1.9‑2.4), anal point is constricted in the middle, andgonostylus is narrower and not constricted near apex

9. Dicrotendipes nervosus (Staeger, 1893)(Figs. 9 a‑e)(ref. Sasa and Kikuchi, 1995) .

Twelve males were collected at Bogd on August 13,

8. Chironomus mongolheius sp. nov. (Figs. 8 a‑d)Thirty six males were collected at Bogd (# 4) , 1,500

1996 (# 7), at about 1,500 m high from sea level. No. 308:
81, 82, 310:81‑85, 311:39‑43. In the first two specimens

m high from sea level, on August 13, 1996. Holotype:
No. 308:63. Paratypes: No. 308:64‑71, 310:38‑63.
Male. BL 6.28‑9.86 (8.32 in average of 6) mm, WL .
2.88‑4.22 (3.63) mm, WW/WL 0.26‑0.28 (0.27). Groundcolor of scutum yellow, stripes brown (dark brown insome specimens) , scutellum yellow, postnotum darkbrown, Ieg segments almost uniformly brownish yellow(tarsi V darker) and without dark rings, abdominal

measured, BL 5.22, 5.10 mm, WL 2.64, 2.46 mm, WW/
WL 0.27, 0.28. Body almost entirely yellow, scutaltergites almost uniformly brown (tergites Vll and Vlll withfTR 0.23, 0.24, fBR 2.7, 2.8, mBR 3.7, 3.2, hBR 5.9, 3.5.
Frontal tubercles in Fig. 9 a. Antepronotum (Fig. 9 b)

pale area along caudal margin) . Frontal tubercles (Fig.

8 a) prominent, 38 pm long, 11 pm ip diameter, and 34

pm apart from each other. Eyes bare, ER 0.13‑0.29(0.23). Antenna with 11 flagellar segments, AR 3.98‑
4.75 (4.35), AHR 0.56‑0.67 (0.60). Palp relatively short,
P/H 0.83‑1.00 (0.92). SO 42‑64 (48.8), CL 36‑70 (47.5).
DM 24‑44 (34.2) , DL 24‑46 (32.0) , PA 8‑16 (10.7) , SC 32‑
52 (40.0).

Wing bare, R‑M area dark, SQ 18‑24 (20.7, relative‑ly smalD , RR 0.28‑0.40 (0.33) , VR 1.03‑1.07 (1.05) , R/Cu

stripes, postnotum, distal half of front tibia, all tarsal

segments and hypopygium slighly brownish. ER 0.24,
0.23, AR 3.00, 2.95, AR 0.67, 0.63. P/H 1.26, 1.36, PN all

O, DM 21, 18, DL 14:16, 15:16, PA 6:7, 5:6, SC 13,16. Wingbare, SQ 18:18, 11:13, RR 0.21, 0.20, VR 1.02, 1.07, R/Cu
1.12, 1.11. fLR 1.58, 1.64, mLR 0.52, 0.53, hLR 0.67, 0.68,

narrowly united in the middle, without lateral setae.
Hypopygium in Fig. 9 c. Both dorsal appendage (Fig. 9d) and ventral appendage (Fig. 9 e) extremely long,gonostylus also very long, narrow and srnoothly curved.

Remarks. The above measurement data and thestructure are almost coincident with those obtained withspecimens of D. nervosus collected in Europe (Pinder,
1978, Fig. 157 D) and Japan (Sasa and Kikuchi, 1995) ,but the shape of appendages and gonostylus seeims to be

1.13‑1.16 (1.14). fLR 1.25‑1.46 (1.37), mLR 0.49‑0.57(0.54), hLR 0.62‑0.68 (0.65), fTR 0.23‑0.28 (0.25), fBR

slightly different from those of the above reports.

3.3‑7.8 (5.3), mBR 2. ‑3.8 (3.3), hBR 3.4‑4.8 (4.2).

10. Cryptotendipes mongolijeus sp. nov.

Hypopygium in Fig. 8 b. Anal point relatively short,(Figs. 10 a‑j)narrow and constricted in the middle. Dorsal appendage(Fig. 8 c) composed of a high base with rounded marginand bearing 6‑8 inner setae, and a distal horn which islargely straight and abruptly hooked apically. Ventral

Three males were collected at Bogd on August 13,
1996 (# 9). Holotype: No. 310:87. Paratypes: No. 310:

appendage (Fig. 8 d) relatively short, straight andalmost parallel‑sided, bearing 32 short, recurved setae

and 6 caudally directed short setae in the holotype.

Gonostylus with nearly straight inner margin andsmoothly rounded lateral margin, not constricted neara pex.

88, 89.

Male. BL 4.88, 4.70, 3.92 mm, WL 2.14, 2.16, 1.80mm, WW/WL 0.31, 0.31, 0.29. Ground color of scutum
pale, stripes dark brown, scutellum pale, postnotum
dark brown, Iegs and abdominal tergites brownish yel‑low. Head in Fig. 10 a. Frontal tubercles absent. Eyes
bare, ER 0.44, 0.32, 0.33. Antenna with 13 flagellar
segments, AR 2.97, 2.71, 2.29. Palp nearly as long as the

Remarks. This species is most closely related

width of head, P/H 0.91, 0.96, 1.05. SO 10:10, 12:12, 12:12,

among the European species to C. plumosus (Linnaeus)

CL 14, 16, 12. Antepronotum (Fig. 10 b) united in the158

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appendage; e: Ieft ventral appendage. Fig. 10 Cryptotendipes mongolijeus sp. nov. a: head; b: ante pronotum;
c: scutum and scutellum; d: wing; e: tip of front tibia; f: tip of middle tibia; g: tip of hind tibia; h hypopygium;i: anal point; j : dorsal appendages.

159

middle, with 5:5, 4:4, 2:2 Iateral setae. Distribution of

genus in that setae on dorsal appendage are arising fromsetae on scutum and scutellum in Fig. 10 c. DM 10, 8, 4,
arising from some distance from the anterior margin of
scutum. DL 10:10, 12:12, 8:8. SC 6, 6, 10.
Wing bare, finely granular, venation as in Fig. 10 d.

not only the apical portion, but are distributed to the
distal half of the inner margin.

SQ 21:22, 20:20, 24:24. Anal lobe slightly produced

A new genus belongipg to the Harnischia complexof the tribe Chironomini. Antenna with 11 flagellar
segmets. Antepronotum with lateral setae (a character

inwards. RR 0.37, 0.36, 0.35, VR 1.11, 1.20, 1.11, R/Cu
1.07, 1.06, 1.04. Tip of front tibia (Fig. 10 e) with a broad

Mongolchironomus gen. nov.

and rounded scale. Tips of middle and hind tibiae (Figs.

in common to members of the Harnischia complex).

10 f, g) with two comb scales, both with a spur. fLR

Gonostylus fused with gonocoxite, Iong, slender, expand‑

1.36, 1.34, 1.44, mLR 0.45, 0.49, 0.45, hLR 0.69, 0.65, 0.62,

ed medially near the base, and inner margin is concavefTR 0.25, 0.25, 0.2, fBR 3.4, 3.6, mBR 3.5, 3.2, hBR 3.6,

towards rounded apex. Ventral appendage absent.
Dorsal appendage situated in the basal portion of

3.6. Pulvilli well developed, brush‑1ike.

Hypopygium in Fig. 10 h. Ninth tergite with two

gonocoxite, thumb‑like, parallel‑sided and apicallyshort setae at the base of anal point, otherwise without
setae in the median portion. Anal point (also in Fig. 10

rounded, bearing 3 to 5 setae and clothed with microtri‑chia except on the lateral portion, and directed laterally

i, ventral view) constricted in the middle and apically(not medially like in most other species of this com‑plex). Containing only the following two species col‑rounded, with some 16 short setae arising from theventral and lateral portions. Dorsal appendages (also in
Fig. 10 j) rather small, horn‑like, narrow and straight,
with V‑shaped base and 4 or 5 setae on inner margin andapex. Ventral appendage absent. Gonostylus fused withgonocoxite, inner margin strongly produced inwards

near the base and conspicuously concave towards
rounded apex, with 12‑14 short setae along inner mar‑gin.

Remarks. This species is a typical mernber of thelected this time in Mongolia.
The species of this genus are similar in the structureof hypopygium to Microchironomus Kieffer, 1918, in that
dorsal appendage of gonocoxite is slender, rod‑1ike andbearing a few setae, ventral appendage is absent, andgonostylus is expanded basally, but differs from it inthat dorsal appendage is broad, covered by microtrichia
and directed laterally, and gonostylus without apicalhook. Genotype: M. mongoljekeus sp. nov.

Harnischia complex of the tribe Chinonomini, sinceantenna with 11･ flagellar segments, apex of middle andhind tibiae with two comb scales each with a spur,
dorsal appendage is reduced and ventral appendage is

11. Mongolchironomus mongoljekeus sp. nov.
(Figs. 11 a‑m)absent, and seems to be belonging to the genus

Altogether 14 males were collected at Bogd on
August 13, 1996. Holotype: No. 308:84. Paratypes:

Cmptotendipes Lenz, 1941, as its dorsal appendage is rod‑

other 13 males, No. 308:85, 310:91‑97, 311:44‑48.

like, with a few short setae but without microtrichia.
Reviews of this genus were made by Saether (1977) and

Male. BL 4.10‑4.66 (4.30 in average of 8) mm, WL
1.70‑1.96 (1.80 in average of 14 )mm, WW/WL 0.28‑0.31

Cranston et al. (1989a) , and the present species seems to(0.29). Median and lateral stripes dark brown andbe closest to a Nearctic species, C. emorosus (Townes,
1945) , in that ninth tergite without dorsal hump, inner
margin of gonostylus with conspicuous median projec‑

clearly differentiated from the large yellow humeral andthe median posterior areas of scutum; scutellum yellow,

tion that forms distinctive concavity in the apical half,largely yellow. Head in Fig. 11 a. Eyes bare, ER 0.28‑
0.42 (0.35). Palp long, P/H 1.02‑1.15 (1.08). Frontal
tubercles (Fig. 11 b) very small, semicircular, 13 pm
wide, 11 pm high and 90 pm apart from each other. AR
2.26‑2.66 (2.41 in average of 14), AHR 0.55‑0.65 (0.60).
Palp relatively long, P/H 1.02‑1.15 (1.08). SO 12‑20

anal point is well developed, and dorsal appendage
without microtrichia. However, in the original descrip‑tion of this species by Townes (1945, p. 161, Fig. 185) by

the generic name of Harnischia, anal point is not con‑stricted at base, and gonostylus is much longer andnarrower, and seems to be quite different from thepresent species. The present species also differs fromthe two species of this genus recorded from Japan, C.
oyabeprimus Sasa, Kawai et Ueno, 1988 and C. tamacutus
Sasa, 1983, in the shape of gonostylus and anal point.
The present species is also unusual as a member of thispostnotum dark brown; Iegs and abdominal tergites(14.6), CL 10‑24 (14.1). Antepronoturn (Fig. 11 c)
united in the middle, with 3‑5 (4.0) Iateral setae, and thebridge connecting the two lobes without processes suchas seen in the next species. DM 3‑6 (4.7), DL all 8, PA
3‑6 (4.1), Sd 6‑9 (8.0).

Wing in Fig. 11 d. Squama with 12‑18 (14.7) fringe

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Mongolchironomus mongoljekeus sp. nov. a: head; b: frontal tubercles; c: antepronotum; d: wing; e: tip offront tibia; f: tip of middle tibia; g: tip of hind tibia; h: front tarsus V; i: hypopygium; j: anal point, dorsal view;

k: anal point, ventral view; m: dorsal appendages. Fig. 12 Mongolchironomus mongolkeleus sp. nov. a: head;b: antepronotum; c: wing; d: tip of front tibia; e: tip of middle tibia; f: tip of hind tibia; g: hypopygium; h:anal point, dorsal view; i: anal point, ventral view; j: dorsal appendages.

161

hairs, anal lobe obtuse. RR 0.32‑0.42 (0.37). VR 1.06‑
1.15 (1.12), R/Cu 1.06‑1.09 (1.07). Tip of front tibia

VR 1.08, 1.13, 1.11, R/Cu 1.05, 1.08, 1.06. Tip of front(Fig. 11 e) with a broad and somewhat sickle‑shaped

pointed one, a broad and rounded one, and a long,somewhat quadrangular one. Tips of middle and hindprocess. Tips of middle and hind tibiae (Figs. 11 f, g)

with two comb scales, both with a short spur. fLR 1.32‑
1.44 (1.38), mLR 0.45‑0.51 (0.47; very smalD, hLR 0.63‑
0.67 (0.65), fTR 0.21‑0.24 (0.23), fBR 3.3‑6.4 (5.1), mBR

tibia (Fig. 12 d) with 3 processes, a narrow and sharply

tibiae (Figs. 12 e, f) with a narrow and a broad comb
scales, both with a spur. fLR 1.41, 1.45, 1.44, mLR 0.53,
0.53. 0.51, hLR 0.68, 0.68, 0.66, fTR 0.26, 0.27, 0.25, fBR

2.4‑3.2 (2.9), hBR 4.0‑7.0 (5.4). Tips of tarsi V with a

6.3, 4.2, 5.2, mBR 5.3, 5.3, 5.1, hBR 5.7, 4.9, 4.3. Pulvilli

pair of simple claws, ari ernpodium, and a pair of brush‑
like pulvilli (Fig. 11 h, front tarsus V).

pad‑1ike and covered with brush.
Hypopygium in Fig. 12 g. Anal point (Fig. 12 h,dorsal; Fig. 12 i, ventral view) is more conspicuously

Hypopygium in Fig. 11 i. Bands of ninth tergite
separated in the middle. Anal point (Fig. 11 j, dorsal;
Fig. 11 k, ventral view) slightly constricted at base, withsome 16 short setae at its base on dorsal side, and amedian V‑shaped groove with straight edge bearing 3
pairs of setae on ventral side. Gonocoxite each with afinger‑like, Iaterally directed dorsal appendage (Fig. 11m) bearing a long and curved apical seta and 3 or 4 innerconstricted at base than in the preceding species, the V‑
shaped groove on the ventral side (Fig. 12 i) has a pairof rounded processes bearing 6 strong setae. Dorsal
appendages (Fig. 12 j) are shorter and slightly expand‑ed, not conical as in the preceding species, and･ bear 4

setae on inner margin but without apical seta. Gonos‑
tylus fused with gonocoxite, widest at base, abruptly

ly curved inwards, bearing 6 setae on the basal expanded

curved and constricted in the middle, the setae on inner
margin are distributed more widely than in the preced‑
ing species, not concentrated on the basal convex por‑portion of inner margin.

tion.

setae, and clothed with microtrichia. Gonostylus widest
at base and constricted in the middle, distal half abrupt‑

Remarks. This species can be distinguished fromthe previously known species of the Harnischia complex
by the characters stated in the definition of this new
genus.

12. Mongolchirouomus mongolkeleus sp. nov.
(Figs. 12 a‑j)

Three males were collected at Bogd (# 9) on AugustRemarks. These specimens are quite similar in thebasic structure to the preceding species, and were con‑sidered first as its larger form, but is described as adifferent species belonging to the same new genus, since
BL, WL, and AR are much larger, palpi are shorter than
the width of head and P/H is conspicuously smaller than
1.0, and the numbers of scutellar setae and the values ofLR are larger. The shape of dorsal appendage is differ‑88.

ent between the two groups, the base is U‑shaped andexpanded towards middle in this species (Fig. 12 j),Male. BL 5.46, 5.36, 5.44 mm, WL 2.40, 2.18, 2.10 mm(all larger than in the preceding species) , WW/WL 0.28,

while in the former the base is only slightly concave andtapering towards apex (Fig. 11 m) . Anal point is longer,

13, 1996. Holotype: No. 308:86. Paratypes: No. 308:87,

0.30, 0.30. Scutal stripes black and clearly differentiatednarrower, constricted at base and with lateral ridges infrom the large yellow humeral and median posterior
areas, scutellum yellow, postnotum black, Iegs andthis species (Fig. 12 h) , while it is stouter, parallel‑sided

abdominal tergites brownish yellow. Head in Fig. 12 a.
ER 0.31, 0.26, 0.32. Frontal tubercles very small, cres‑cent‑shaped, 10 pm wide and 8 pm high. AR 3.11, 3.03,
3.00 (larger than in the preceding species), AHR 0.65,

groove on the ventral side of the base of anal point bears

0.70, 0.62. Palp shorter than in the preceding species, Pla pair of large dark processes in this species (Fig. 12 b) ,H 0.81, 0.79, 1.00. SO 16‑20 (17.7; Iarger), CL 11, 10, 10.

while they are absent in the former (Fig. 11 c) .

and without lateral ridge in the former (Fig. 11 j). Thea pair of rounded process bearing 5 or 6 setae in thisspecies (Fig. 12 i), while they are absent in the former(Fig. 11 k) . The bridge connecting antepronotum bears

Antepronotum (Fig. 12 b) connected in the middle, with
2:2, 4:4, 4:4 Iateral setae, the bridge connecting the two

10bes bears a pair of long, darkly pigmented processes
which are absent in the preceding species. DM only 3, 3,
O, DL 12‑15 (13.8), PA 5 or 6 (5.5), SC 14‑17 (15.7; muchlarger) .

13. Polypedilum mongollemeus sp. nov.
(Figs. 17 a‑k)

A male was collected at River Orkhon (# 35) , 1,775
m high, on August 18, 1996. Holotype: 308:89.

Male. BL 4.08 mm, WL 2.32 mm, WW/WL 0.30.

Wing bare, venation in Fig. 12 c. Squama with 12‑

Ground color of scutum brown, stripes dark brown,

18 (15.0) fringe hairs in the holotype. RR 0.42, 0.33, 0.36,

scutellum brown, Ieg segments uniformly brown, abdom‑

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Micropsectra junci (Meigen, 1818) a: head; b: frontal tubercles; c: antepronotum; d: scutum and scutellum;
e: wing; f: tip of front tibia; g: tip of middle tibia; h: tip of hind tibia; i: hypopygium; j: anal point; k: dorsal

Fig. 14

appendage, dorsal view; m: dorsal appendage and digitus, ventral view; n: median and ventral appendages.
Micropsectra mongolmeneus sp. nov. a: frontal tubercles; b: antepronotum; c: wing; d: tip of front tibia; e:
tip of middle tibia; f: tip of hind tibia ; g: hypopygium; h: anal point; i: dorsal appendage, dorsal view; j: dorsal

appendage and' digitus, ventral view; k: median and ventral appendages.

163

inal tergites almost uniformly brown, tergites VII and
VIII each with a narrow pale area along caudal margin.
Head in Fig. 17 a. Frontal tubercles absent. Eyes bare,
ER 0.45. Antenna with 13 flagellar segments, AR 1.63,

AHR 0.56. Palp long, P/H 1.27. SO 9:10, CL 12.
Antepronotum (Fig. 17 b) separated in the middle witha V‑shaped groove, without lateral setae. Distributionof setae on scutum and scutellum in Fig. 17 c. DM 16,
DL 15:15, PA 5:5, SC 12.
Wing membrane bare, bluish and finely granular,
venation in Fig. 17 d. SQ 21:22, RR 0.17, VR 1.10, R/Cu
1.11. Anal lobe nearly rectangular. Tip of front tibia

3.82, 3.10 mm, WL 2.32, 2.26, 2.36 mm, WW/WL 0.28,
0.27, 0.29. Scutal stripes and postnotum brown, otherbody portions largely yellow. Head in Fig. 13 a. Eyes
bare, ER 0.39, 0.28, 0.30. Frontal tubercles (Fig. 13 b)very small, 4 pm wide and 5 pm high, 22 pm apart fromeach other. Antenna with 13 flagellar segments, AR 1.
14, 1.14, 1.11, AHR 0.51, 0.48, 0.48. Palp very long, P/H

1.41, 1.36, 1.37. SO 12:12, 12:14, 14:14, CL 16, 18, 18.

Antepronoturn (Fig. 13 c) widely separated in the
middle, PN all O. Distribution of setae on scutum andscutellum in Fig. 13 d. DM 13, 12, 23, DL 11:12, 11:11, 11:

13, SC 12 (scutellum is lost in 2 specimens) .

(Fig. 17 e) with a rounded scale bearing a long and stout

Wing (Fig. 13 e) with rather small numbers ofseta. Tip of middle and hind tibiae (.Figs. 17 f, g) withmacrotrichia on the distal half and on the principal

a narrow and a broad cornb scales, the former with along spur and the latter without spur. fLR 1.23 (varyveins. SQ all O, RR 0.36, 0.35, VR 1.14, 1.11, 1.14, R/Cu

Hypopygium in Fig. 17 h. Anal point long, slenderand almost parallel‑sided. Ninth tergite with 6 Iong

1.10, 1.10. Terminal process of front tibia (Fig. 13 f)
short, narrow and sharply pointed. Terminal comb scaleof middle and hind tibiae (Figs. 13 g, h) contiguous andwithout spur. fLR 1.45, 1.54, mLR 0.54, 0.69, fTR 0.24,
0.22, fBR 4.2, mBR 5.8 (hind tarsi all lost) . Pulvilli

setae in the middle portion, and 6 setae on both sides ofabsent.

smalD , mLR 0.58, fTR 0.23, fBR 3.0, mBR 3.5 (hind tarsi10st) . Pulvilli well developed, brush‑1ike.

posterior margin flanking anal point. Dorsal appendage(Fig. 17 i, right) roughly C‑shaped, inner margin ofbasal portion very high and strongly concave, with 3
inner setae, and looks quite differently when seen fromthe frontal angle (Fig. 17 j, Ieft) . Ventral appendage(Fig. 17 k) Iong, slender and parallel‑sided, with 8

Hypopygium in Fig. 13 i. Anal point (also in Fig. 13j) small, narrow and parallel‑sided, with a U‑shaped
basal ridge, and a small tubercle bearing 4 short setae on

its base. Dorsal appendage (Fig. 13 k) semicircular, 34

pm high and 40 pm wide in the holotype, Iength/width
ratio 0.85, 0.90, 1.00, with 10 short setae on dorsal siderecurved setae and a long apical seta. Gonostylus slight‑

and a long basal seta arising from a large tubercle.

ly expanded in the middle, with an apical seta and 12
10ng setae along inner margin.
Remarks. This species is a typical member of theDigitus and ventral aspect of dorsal appendage in Fig. 13

nubeculosum group of subgenus Polypedilum s. str., sirice

dorsal appendage composed of a broad base bearing
inner setae, and a distal horn bearing a long lateral seta.

In the key to this group compiled by Sasa and Kikuchi(1995), it is closest to P. kunl amiense Sasa et Hase‑
gawa, 1988, in that body with brown and yellow colora‑
tions, antepronotum without setae, abdominal tergites
are almost uniformly brown, AR is larger than 1.4 andm, Di/DA ratio 0.75, 0.74, 0.78. Median and ventral
appendages in Fig. 13 n. The former relatively long,narrow and nearly straight, bearing some 10 simple
setae on basal half and some 20 short spoon‑Iike setaeon distal half, MA/VA Iength ratio 0.91, 0.96, 0.93.
Ventral appendage finger‑1ike, bearing some 20 short,recurved setae on dorsal side and 4 caudally directed
setfae on ventral side of the distal portion. Gonostylus
fused with gonocoxite, slightly expanded in the middle,
with 12 short setae in two rows on inner margin.

smaller thari 1.7, frontal tubercles are absent, and lateral

Remarks. This species is structurally a typicalseta on dorsal appendage arises from about middle ofmember of the genus Micropsectra Kieffer, 1909, and isprovisionally identified as M. junci (Meigen, 1818),
since the structures are almost coincident with thosethe distal horn, but differs essentially in the shape of
dorsal appendage (basal portion is strongly elevated anddistal horn is more strongly curved in the present
species) , and fLR is much smaller (1.85‑1.93 in P.
kunigamiense, after Sasa and Hasegawa, 1988).
14. Micropsectra junci (Meigen, 1818)(Figs. 13 a‑n)Three males were collected at the side of River
Orkhon on August 18, 1996 (No. 308:91‑93). BL 4.10,

described by European workers, especially by Pinder(1978, p. 144, Fig. 175A) , in that combs of middle andhind tibiae are fused and without spur, scutal stripes are
distinct, AR is about 1.1, anal point is narrow and tri‑

angular, and median appendage is relatively long andbearing spoon‑shaped setae. This species has also beenrecorded from Lake Towada, northern Honshu, by Sasa
(1991, p. 69).

164

15. Micropsectra mongolmeneus sp. nov.
(Figs. 14 a‑k)

A male was collected at the side of the River
Orkhon on August 18, 1996. Holotype: 308:94.

Male. BL 5.80 mm, WL 3.18 mm, WW/WL 0.26
(very narrow) . Body almost entirely black. Frontal
tubercles (Fig. 14 a) small, cup‑Iike, 9 pm wide, 5 pm
high, and 77 pm apart from each other. Eyes bare, ER
0.39. Antenna with 13 flagellar segments, AR 2.69, AHR
0.66. Palp long, P/H 1.37. SO 16:16, CL 20. Ante‑pronotum (Fig. 14 b) widely separated in the middle,
without lateral setae. DM 6, DL 12:14, PA 3:4, SC 10.
Wing (Fig. 14 c) with only about 10 macrotrichia inthe extreme tip area (quite unusual character as amember of this genus) . Squama bare, anal lobe nearlyflat. RR 0.24, VR 1.01, R/Cu 1.07. Tip of front tibia(Fig. 14 d) with a short, narrow and apically pointed
spur. Terminal comb scales of middle tibia (Fig. 14 e)contiguous, very low and without spur. Terminal comb
scales of hind tibia (Fig. 14 f) also very low and contigu‑

ous, but with one short spur. fLR 1.15 (very smalD,
mLR 0.46 (unusually small), hLR 0.62, fTR 0.20, fBR
6.5, mBR 4.0, hBR ,3.7. Pulvilli absent.

Hypopygium in Fig. 14 g. Bands of ninth tergite
separated. Anal point (Fig. 16 h) very small, widest atbase and apically rounded, with a few short setae nearits base. Dorsal appendage roughly semicircular, 62 pm
wide and 122 pm long, and with 10 short setae on dorsalside, basal seta absent (Figs. 14 i,j). Digitus short, 32

dorsal appendage are narrower, median appendage isshorter, ventral appendage is parallel‑sided and not
expanded in the middle. Among the species recorded
from Japan, it is most closely related to M. yunoprima
Sasa, 1984, collected in large numbers from the moun‑tain lake of Yunoko, Nikko National Park, but thelatter is still larger in body size and AR, wing with more

numerous macrotrichia on larger area, ventral append‑

tge is not medially expanded, median appendage isshorter and with smaller numbers of longer and nair‑rower spoon‑1ike setae (Sasa, 1984).

16. Tanytarsus mongolneous sp. nov. (Fig. 15 a‑k)Three males were collected at Bogd on August 13,
1996. Holotype: No. 308:96. Paratypes: 308:97, 98.
Male. BL 2.72, 2.66, 2.68 mm. WL 1.40, 1.32, 1.26mm, WW/WL 0.28, 0.27, 0.29 mm. Ground color of
scutum yellow, stripes, scutellum, postnotum andabdominal tergites brown, Iegs brownish yellow. Headin Fig. 15 a. Eyes bare, inner margin concave but
without dorsomedial extension, ER 1.32, 1.26, 1.50.
Frontal tubercles (Fig. 15 b) prominent, widest at baseand apically rounded, 16 pm long, 7 pm in diameter, and32 pm apart from each other. Antenna with 13 flagellar
segments, AR 1.06, 1.04, 1.05, AHR 0.53, 0.53, 0.51. Palp
about as long as the width of head, P/H 1.08, 0.98, 0.82.
SO 9:9, 10:10, 10:10, CL 8, 10, 14. Antepronotum (Fig. 15c) widely separated in the middle, without lateral setae.
DM 8, 7, 8. DL 6:7, 8:8, 6:6, PA all 1, SC 6, 5, 6.

pm long and 8 pm wide, Di/DA 0.52. Median andWing with relatively small nurnbers of macrotrichia

ventral appendages in Fig. 14 k. The former 92 pm long,
composed of a straight shaft bearing 6 or 8 setae, and aon the distal portion and on the principal veins, venation

distal expanded portion bearing 30 short, spoon‑likesetae. Ventral appendage 184 pm long (MA/VA 0.50),
conspicuously expanded at about middle, bearing 20

0.41, 0.50, 0.49, VR 1.14, 1.19, 1.15, R/Cu 1.06, 1.07, 1.09.

recurved setae on inner side of distal half. Gonostylus
long, parallel‑sided, bearing 8 Iong setae on inner mar‑g) with two narrow comb scales, both with a spur. fLR

gin of apical portion.

fTR 0.28, 0.26, 0.28, fBR 4.2, 4.3, mBR 5.2, 4.1, hBR 8.4,

Remarks. This specimen was collected at the samein Fig. 15 d. Squama bare, anal lobe nearly flat. RR
Tip of front tibia (Fig. 15 e) with a short, narrow andpointed spur. Tips of middle and hind tibiae (Figs. 15 f,
1.54, 1.61, 1.55, mLR 0.54, 0.51, 0.50, hLR 0.63, 0.62, 0.63,

7.6. Pulvilli absent.

site as in the preceding species, and also belongs to the

Hypopygium in Fig. 15 h. Bands of ninth tergite

genus Micropsectra, but obviously represents a different
species, since body is much larger, entirely black, wing

separated in the middle. Anal point (also in Fig. 15 i)
triangular, sharply pointed apically, with a pair of lat‑with only a few macrotrichia restricted to the tip area,

eral ridges and 4 spine clusters in two rows, with 12
short basal setae and 4 pairs of lateral setae. Dorsal
appendage (Fig. 15 j) somewhat sickle‑shaped, inner
margin concave, with 6 short setae on dorsal surface,
and with 3 basal setae arising on prominent tubercles.

AR is much larger, VR is nearly 1.0 and smaller, fLR

and mLR is much smaller, and median appendage ismuch shorter. Among the European species of thisgenus, it is somewhat related to M. notescens (Walker)

in that AR is high, body is large and entirely black,
median appendage is relatively short and with spoon‑

Digitus long and slender, entirely exposed on inner side

like setae, but the latter differs from the present species

Fig. 15 k; the former very long and slender, Ionger thanthe latter, MA/VA 1.17, 1.39, 1.19, with both simple andespecially in that mLR is larger than 0.5, anal point andof dorsal appendage. Median and ventral appendages in

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Tanytarsus mongolneous sp. nov. a: head; b: frontal tubercles; c: antepronotum; d: wing; e: tip of front tibia;
f: tip of middle tibia; g: tip of hind tibia; h: hypopygium; i: anal point; j: dorsal appendage and digitus; k: medianand ventral appendages. Fig. 16 Tanytarsus mongolopeus sp. nov. a: head; b: antepronotum; c: scutum andscutellum; d: wing; e: tip of front tibia; f: tip of middle tibia; g: tip of hind tibia; h: hypopygium; i: anal point;

j: dorsal appendage and digitus; k: median and ventral appendages.

166

curved, and highly branched long and narrow setae
arising on the distal portion, and some 10 simple setae
arising on the median portion. Ventral appendage also
10ng, slightly tapering towards rounded apex, with 18‑20recurved setae arising in the distal portion. Gonostylus

with inner margin nearly straight, widest at about
middle, and with 8 short setae on inner margin.

the latter but bearing very long and simple setae whosetips extending beyond tip of ventral sppendage. Ventral
appendage finger‑like but curved inwards apically, with

only 8 recurved setae. Gonostylus widest at about
middle and inner meargin nearly straight, with 8 setae
on inner margin.

Remarks. This specimen also belongs to the

Relnarks. This species is a member of theyunosecundus group of the genus Tanytarsus. since anal

yunosecundus group of the genus Tanytarsus van derpoint with lateral ridges and spine clusters, digitus long,

Wulp, 1874, since anal point with lateral ridges and spine

ly pointed apically, dorsal appendage with a large basal

and median appendage is very long and longer than theventral appendage, but differs from the above species
and also from all the previously known species of thisgroup in that distal setae of median appendage are allvery long and simple, anal point with as many as 12
spine clusters in two rows, and dorsal appendage isnarrow and sickle‑shaped (Sasa and Kikuchi, 1995).

tubercle bearing 3 setae, and ventral appendage bears
nume‑rously branched apical setae.

18. Cricotopus (Cricotopus) annulator Goetghebuer,

clusters, digitus present and median appendage is longer

than the ventral appendage (ref. Sasa and Kikuchi,
1995). However, it is quite unusual as a member of thisgroup and differs remarkably from the previously
known species in that anal point is triangular and sharp‑

1927

17. Tanytarsus mongolopeus sp. nov. (Figs. 16 a‑k)
A male was collected at Karakorum on August 18,
1996. Holotype: No. 308:99.

Male. BL 3.08 mm, WL 1.40 mm, WW/WL 0.31.

(Figs. 18 a‑i)

Thirteen males were collected, 10 at Karakorum(# 28) on August 18, 1996 (No. 309:12, 18, 24‑28; 311:66‑71), and 3 at River Orkhon (# 38) on August 18, 1996
(No. 311:83‑85) .

postnotum dark brown, Iegs and abdominal tergites

Male. Eight males were measured. BL 2.86‑3.86(3.38 in average) mm, WL 1.58‑2.00 (1.82) mm, WW/brownish yellow. Head in Fig. 16 a. Eyes bare, renifor‑

WL 0.31‑0.33 (0.32). Ground color of scutum yellowish

m, ER 1.55. Frontal tubercles absent, a remarkabledifference from the above species. Antenna with 13
flagellar segments, AR 0.81 (smalD, AHR 0.49. Palp
10ng, P/H 1.08. SO 10:10. CL 14. Antepronotum (Fig.

brown, stripes brown, humeral areas yellow; scutellum16 b) widely separated, without lateral setae. Distribu‑

as in Fig. 18 g ; tergite I entirely white, oral half of IItion of setae on scutum and scutellum in Fig. 16 c; DM
only 4, DL 8:10, PA 1:1, SC 6.
Wing with very small numbers of macrotrichia only

white and its distal half brown, 111 Iargely brown andwith narrow pale areas along oral and caudal margins,
IV with a pale area on caudal 1/3, V to VII with a palearea along caudal margin, VIII entirely brown, IX andhypopygium largely white.
Head in Fig. 18 a. Eyes pubescent, ER 0.59‑0.84(0.71). Antenna with 13 flagellar segments, AR 1.21‑

Ground color of scutum yellow, stripes, scutellum andin the distal portion, venation in Fig. 16 d. Squama bare,anal lobe nearly flat. RR 0.49, VR 1.30 (larger) , R/Cu

1.05. Tip of front tibia (Fig. 16 e) with a long andnarrow spur. Tip of middle tibia (Fig. 16 f) with only

and postnotum dark brown, femora and tarsi largely
brown, tibiae largely yellow and with short brown rings
on both ends; dark and pale areas on abdominal tergitesg) with two narrow comb scales, both with a spur. fLR

1.40 (1.32), AHR 0.52‑0.56 (0.54). P/H 0.97‑1.09 (1.03).
SO 8‑10 (8.8), CL 8‑13 (11.0). Antepronotum (Fig. 18 b)

one comb scale bearing a spur, tip of hind tibia (Fig. 16
1.86 (larger) , mLR 0.55, hind tarsi lost. fTR 0.33. Tarsi

united in the middle, with 3‑6 (4.0) Iateral setae.

V slender, pulvilli absent (Fig. 16 h, front tarsus V) .

Distribution of setae on scutum and scutellum in Fig. 18

Hypopygium in Fig. 16 h. Bands of ninth tergite
separated. Anal point (Fig. 18 i) widest at base andc. DM 16‑24 (20.4), all minute. DL 18‑26 (22.2), allcontricted near apex, with lateral ridges and 12 spine

minute, decumbent and arising from very srnall pits. PA
3‑6 (4.0), SC 6‑8 (6.8), both well developed. SQ 6‑10

clusters in two rows, Ivith 3 pairs of lateral setae and 8(8.2) , RR 0.45‑0.49 (0.47) , VR 1.08‑1.13 (1.11) , R/Cu 1.04‑

short basal setae. Dorsal appendage (Fig. 16 j) Ionger
than wide and sickle‑shaped, with 5 dorsal and 3 basal

1.10 (1.07). fLR 0.66‑0.69 (0.67), mLR 0.49‑0.51 (0.50),
hLR 0.57‑0.61 (0.59), fTR 0.12‑0.13, fBR 0.18‑0.23 (0.21),

inner setae. Digitus long, entirely exposed on inner sidemBR 2.1‑2.3 (2.2), hBR 2.2‑2.4 (2.3). Pulvilli absent.

of dorsal appendage. Median and ventral appendages inDistribution of setae on abdominal tergites in Fig.

Fig. 16 k. The shaft of the former about half as long as

18 g (No. 309:18), 8 on I, 14 on II, 16 on 111, 18 on IV, 20

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o'05mmO:' 18g1:+cFig. 17 Polypedilum mongollemeus sp. nov. a: head; b: antepronotum; c: scutum and scutellum; d: wing; e: tip offront tibia; f: tip of middle tibia; g: tip of hind tibia; h: hypopygium; i: dorsl appendage, dorsal view; j: dorsal

appendage, ventral view; k: ventral appendage. Fig. 18 Cricotopus annulator Goetghebuer, 1927 a: head; b:
antepronoturn; c: scutum and scutellum; d: tip of front tibia; e: tip of middle tibia; f: tip of hind tibia; g: abdominal

tergites; h: hypopygium; i: inner lobes of gonocovite.

168

on V, 22 on VI, 24 on VII, and 20 on VIII, and those on
II to VI are arranged roughly into the lateral and theposterior groups, and with a few median setae on IV to
VI. Hypopygium in Fig. 18 h. Anal point absent, ninth
tergite with about 10 short setae in the median portion.
Inner lobe of gonocoxite (Fig. 18 i) composed of three
processes, the basal one longest, rounded and clothedwith stout and pointed setae, the middle one rectangularand also with strong setae, the distal one small androunded. Gonostylus simple, not expanded in the middle
and without preapical tooth, apical spur prominent.
Remarks. The above stated body coloration, espe‑
cially the distribution of pale areas on abdominal ter‑gites and legs, is almost coincident with that of C.

annulator described by Hirvenoja (1973) and Pinder(1987) with the European specimens. The structure ofhypopygium, especially that of inner lobes of gonocox‑
ite, is also quite similar to the figures given in thesereferences, but the presence of the third small process
found in the present specirnens are not drawn with theEuropean specimens, and if it is really absent, the pres‑dark brown, Ieg segments and abdominal tergites almost
entirely brown. Head in Fig. 19‑a. Eyes pubescent, eachwith a dorsomedial projection, ER 0.84, 1.18, 1.00.
Antenna with 13 flagellar segments, AR 2.03, 2.11, 1.83,
AHR 0.55, 0.54, 0.55. Palp short, P/H 0.69. 0.76, 0.69. SO

1+4:2 4, 1 4:2+4, 1+4:1+4. CL all 6. Antepronotum(Fig. 19 b) united in the middle, with 4:5, 4:4, 6:6 Iateral

setae. Distribution of setae on scutum and scutellum in
Fig. 19 c; DM 20, 20, 24, all minute; DL 28:28, 24:24,
24:24, all minute, decumbent and arising on small pits.
PA all 4, SC 8, 10, 8, all stout and long.

Wing bare, very finely granular, venation in Fig. 19
d. Costa does not extend beyond tip of R4 + 5, which isdistal to tip of Cul, R/Cu 1.06, 1.04, 1.06. VR 1.14, 1.14,

1.15, RR 0.53, 0.52, 0.54. Cu2 almost straight. Tip offront tibia (Fig. 19 e) with a long apur, 62 pm and 1.6
times as long as the diameter of front tibia at the tip.
Tip of middle tibia (Fig. 19 f) with two short spurs. Tipof hind tibia (Fig. 19 g) with a long spur (60 pm) , a

short spur (12 pm) , and a comb composed of 14 free
simple spurs 30‑63 pm long. fLR 0.53, 0.52, 0.54, mLR

ent specimens could be a different new species. The
present specimens are also closely related to C. to ‑0.44, 0.42. 0.43, hLR 0.52, 0.49, 0.49, all very small. fTR all

kunagai Hirvenoja, 1993, which was originally recorded
from Sakhalin by Tokunaga (1940) with the name of C.

Pulvilli absent.

0.13, fBR 2.3, 2.5, 2.0, mBR 2.4, 2.7, 2.5, hBR 2.3, 3.0, 2.7.

inner lobes of gonocoxite, and the identity of the latter

Distribution of setae on abdominal tergites in Fig.
19 h, those on 11 to VI are arranged into the median andthe lateral groups, the nurnbers are 42 on I, 38 on II, 36

species with C. annulator needs to be studied in future.

on 111, IV, VI, VII and VIII, and 40 on V in No. 309:03.

19. Cricotopus (Cricotopus) bicinctus (Meigen, 1818)
Nine males were collected at Karakorum (# 27) on
August 18, 1996. No. 309:17, 21‑23; 311:61‑65. This
species was originally described from Europe, and also
recorded from a number of localities in Japan. It isespecially characteristic in body coloration, scutum,

tergite with 10 short setae in the median portion. Inner

scutellum and postnotum almost evenly dark brown,

cially characterised in that legs and abdominal tergites

femora entirely brown, tibiae with a long pale ring, tarsi

are almost uniformly brown, gonocoxite with a single
thumb‑1ike inner lobe with rounded margin, and inner
margin of gonostylus is strongly expanded (Pinder,
1978). Among the species of this subgenus recorded
from Europe, this species is somewhat related to C.
pallidipes Edwards, 1929, in body coloration being

bituberculatus, in body coloration and in the structure of

Hypopygium in Figs. 19 i, j. Anal point absent, ninth

brown, abdominal tergites I and IV entirely white andother tergites almost entirely brown, setae on abdominal
tergites 11 to VI are reduced and arranged roughly into

the median and the lateral groups, and inner lobe ofgonocoxite is single and foot‑shaped.

20. Cricotopus ( Cricotopus) mongolpequeus sp. nov.
(Figs. 19 a‑j)lobe of gonocoxite longer than wide and rounded, bear‑ing short setae. Gonostylus simple, inner margin strong‑ly expanded.

Remarks. This species is a typical member ofsubgenus Cricotopus van der Wulp, 1874, and is espe‑

almost entirely brown, but differs from it in that inner
10be of gonocoxite is longer, narrower and with numer‑
ous short marginal setae, inner margin of gonostylus is

Three males were collected at Bogd on August 13.

more broadly rounded, and AR is larger (1.36 in C.

1996 (# 11). Holotype : No. 309:O1. Paratypes: No. 309:

pallidulus, after Hirvenoja, 1973, p. 243; Pinder, 1978,
Fig. 100O . Among the species recorded from Japan, thepresent species is similar in body coloration and struc‑ture to C. togaspadix Sasa et Okazawa, 1992, but in thelatter inner lobe of gonocoxite is boot‑shaped, abdomi‑

02, 03.

Male. BL 3.96, 4.14, 4.08 mm, WL 1.64, 1.74, 1.78mm, WW/WL 0.32, 0.32, 0.33. Ground color of scutum
yellow, stripes dark brown, scutellum brown, postnotum

169

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Cricotopus mongolpequeus sp. nov. a: head; b: antepronotum; c: scutum and scutellum; d: wing; e: tip of front
tibia; f: tip of middle tibia; g: tip of hind tibia; h: abdominal tergites; i: hypopygium; j: inner lobe of gonocoxite

and gonostylus. Fig. 20 Cricotopus mongolquereus sp. nov. a: head; b: antepronotum; c: scutum and scutellum;d: wing; e: tip of front tibia; f: tip of middle tibia; g: tip of hind tibia; h: pale and dark areas on abdominal tergites;

i: distribution of setae on abdominal tergites; j: hypopygium.

170

nal tergites 111 to V with more numerous setae distribut‑

setae on inner margin. Gonostylus simple, with a

ed almost evenly, gonostylus is not expanded, and AR isrounded preapical tooth.

smaller (1.00‑1.12). It is also related in body coloration

Remarks. This species belongs to the group with
one inner lobe on gonocoxite, and it is most closely

to C. carbonarius Kieffer, 1923 recorded by Tokunaga(1936c) from Taiwan, but the latter has large pulvilli
and probably belongs to a different subgenus, Isocladiusrelated to C. festivellus (Kieffer, 1906) among the Eur‑

Kieffer, 1909.

opean species of this genus, since inner lobe of gonocox‑
ite is acutely angulate, fore tarsi uniformly dark, all

21. Cricotopus ( Cricotopus) mongolquereus sp. nov.

tibiae with conspicuous median pale ring, and setae on
abdominal tergites are highly reduced (Hirvenoja, 1973;(Figs. 20 a‑j)Pinder, 1978). However, the coloration of abdominal

Eleven males were collected at Bogdt (# lO) on

tergites of the latter is quite different from that of theAugust 13, 1996. Holotype: No. 309 : 13. Paratypes: No.

present species, and tergite IV has a pale band along

309 : 14, 15, 27, 30‑32; 311 : 49‑52.

posterior margin in C. festivellus, while in the present

Male. BL 3.12‑4.34 (3.51 in average of 6) mm, WL
1.50‑2.12 (1.72) mm, WW/WL 0.30‑0.33 (0.32). Ground

species, pale band is along the anterior margin of IV andcolor of scutum yellow, stripes brown, scutellum yellow,

VI, and the dark bands in the posterior part of these
tergites are produced anteriorly in the middle. The

postnoturn brown. Femora largely pale and with a distalbrown ring, tibiae largely pale and with a proximal anddistal brown ring, front tarsi brown, middle and hind

distribution of setae on abdominal tergites 111 and IV istarsi I and 11 yellow, the distal tarsomeres brown.
Distribution of pale and dark areas on abdominal ter‑lines in C. festivellus.

gites in Fig. 20 h; I with a caudal dark area, 11 and 111

22. CricQtopus ( Cricotopus) tremulus (Linnaeus,

largely dark and with pale bands along oral and caudal
margins, IV and V with an oral pale area and a caudal
dark area which is produced orally towards middle, VI
and VII Iargely dark and with a pale band along caudal
margin, VIII entirely dark.
Head in Fig. 20 a. Eyes pubescent, with a wedge‑

also different, in 5 Iongitudinal lines in the present
species, while it is distributed roughly on two transverse

1758)(Figs. 21 a‑i)

Two males were collected at the side of River
Orkhon (# 34, 37) on August 18, 1996, 1,775 m high fromsea level. No. 309 : 09, 10. BL 4.20, 4.24 mm, WL 1.57,

shaped dorsomedial extension, ER 0.83‑1.07 (0.99).

1.54 mm, WW/WL 0.31, 0.31. Ground color of scutum
dark brown, stripes, scutellum and postnotum black,femora largely dark brown, tibiae with a basal andAntenna with 13 flagellar segrnents, AR 1.37‑1.82 (1.53) ,,apical dark ring and a long pale area between them, fore

AHR 0.52‑0.56 (0.54). P/H 0.89‑1.05 (0.95). SO com‑posed of 0‑2 median and 4‑8 Iateral setae. CL 10‑18

tarsus I brown, entire length of 11 and basal half of 111(13.3). Antepronotum (Fig. 20 b) united in the middle,

white, other tergites largely dark. Head in Fig. 21 a.
Eyes pubescent, each with a dorsomedial extension, ER

with 3 or 4 Iateral setae. Distribution of setae on scutum

and scutellum in Fig. 20 c. DM 16‑28 (19.3), DL 15‑32(22.6) , both all minute and arising from very small pits.

PA 3‑6 (4.3), SC 6‑11 (8.8), both well developed. Wingbare, venation in Fig. 20 d. Squama with 10‑22 (13.3)
fringe hairs. Anal lobe expanding inwards. RR 0.48‑

pale, IV and V brown. Abdominal tergites I and II

0.74, 0.61. Antenna with 13 flagellar segments, AR 1.57,
1.55, AHR 0.60, 0.54. Palp long, P/H 1.14, 1.23. SO 15:
15, 12 : 12, CL 16, 20. Antepronotum (Fig. 21 b) united

in the middle, PN all 2. Distribution of setae on scutum

0.56 (0.52), VR 1.11‑1.17 (1.14), R/Cu 1.04‑1.08 (1.06).

and scutellum in Fig. 21 c. DM both 24, DL all 34, both
all minute. PA all 5, SC both 12.

Tips of tibiae in Figs. 20 e, f, g. fLR 0.52‑0.55 (0.54),

Wing bare, venation in Fig. 21 d. SQ 16 : 18, 20 : 20,

mLR 0.42‑0.47 (0.44), hLR 0.49‑0.54 (0.52), fTR 0.13‑
0.14, fBR 1.4‑2.0 (1.7), mBR 2.0‑2.7 (2.3), hBR 2.3‑3.0

tibiae in Fig. 21 e. f. g. fLR 0.58, 0.59, mLR 0.47, 0.49,(2.6). Pulvilli absent.

Distribution of setae on abdominal tergites in the

RR 0.53, 0.57, VR 1.15, 1.14, R/Cu 1.04, 1.05. Tips ofhLR 0.51, 0.52, fTR 0.12, fBR 2.3, mBR 2.5, hBR 2.7.
Pulvilli absent. Setae on abdominal tergites 11 to VII

holotype in Fig. 20 i, 48 on I, 11 on II, 18 on 111 and IV,

are distributed roughly into the lateral, the anterior, and20 on V, VI and VII, and 35 on VIII, and those on 11 to
VI are arranged into the median and the lateral groups.

the posterior groups, the numbers being 36 on I, 40 on II
and 111, 44 on IV and V, and 48 on VI to VIII (Fig. 21 h) .

Hypopygium in Fig. 20 j. Anal point absent, ninth

Hypopygium in Fig. 21 i. Anal point absent. Inner lobeof gonocoxite single and foot‑shaped. Gonostylus witha broad and rounded preapical tooth.

tergite with sorne 10 setae in the middle portion. Inner

10be of gonocoxite acutely angulate, and bears strong

171

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Cricotopus tremulus (Linnaeus, 1758) a: head; b: antepronotum; c: scuturn and scutellum; d: wing; e: tip offront tibia; f: tip of middle tibia; g: tip of hind tibia; h: abdominal tergites; i: hypopygium. Fig. 22 Cricotopus
mongolreseus sp. nov. a: head; b: antepronotum; c: scutum and scutellum; d: tip of front tibia; e: tip of middle
tibia; f: tip of hind tibia; g: front tarsus V; h: abdominal tergites; i: hypopygiurn.

172

Remarks. The above body coloration, measure‑ment data and structure of the present specimen arealmost in accordance with those of C. tremulus (Lin‑naeus) described by Hirvenoja (1973) and Pinder (1978)

0.53, fTR 0.15, fBR 2.7, mBR 2.5, hBR 2.7. Tips of tibiae

in Figs. 22 d, e, f. Tip of tarsomeres V with anempodium, a pair of claws and a brush‑like pulvilli (Fig.
22 g, front tarsus V) .

from Europe, and also by Tokunaga (1940) from Sak‑

Distribution of setae on abdominal tergites in Fig.

halin. This species is especially characteristic in thecoloration of abdominal tergites, I and 11 pale and othertergites dark, and of the fore tarsi, entire length of II

22 h, 36 on I, 22 on II,26 on 111 to VII, and 32 on VIII, andand proximal half of 111 are white and other portions
dark. The shape of inner lobe of gonocoxite being leg‑

those on 111 to VI are arranged into 2 to 4 median andthe rest in lateral groups. Hypopygium in Fig. 22 i.
Anal point absent, ninth tergite with 10 short setae in the

body coloration, measurement data and external struc‑ture of the above specimens are almost coincident withthose of C. triannulatus described by various authors

median portion. Inner lobe of gonocoxite single, about
as long as wide and rounded, with strong setae on inner
margin. Gonostylus strongly expanded near apex.
Remarks. This species is regarded as belonging to
the subgenus Isocladius, since legs with well developed
pulvilli. According to Pinder (1978), European speciesof this subgenus is divided into two groups according to
the shape of basal portion of inner margin of gonocox‑
ite, either rounded or produced into a hump, but in thepresent species it is straight. The setae on abdominalfrom Europe, and also by Tokunaga (1936c) and by

tergites 11 to V are arranged roughly into the lateral andSasa and Kikuchi (1995, p. 55) from a nurnber of local‑

the posterior rows, and does not fit to that of any

ities in Japan. Especially characteristic is the bodycoloration, femur is largely brown, tibiae largely pale
and with basal and apical dark rings, abdominal seg‑

European species. The peculiar coloration of abdominal
tergites, 11 to VI with a large oval dark mark, also
differs from the previously known speci s of this sub‑ment 11 with a broad pale band in oral half, IV and V are

genus.

form is another distinguishing character of this species.

23. Cricotopus ( Cricotopus) triannulatus (Macquart,
1826)

Twelve males were collected at Karakorum on
August 18, 1996 (# 26). No. 309 : 04‑07; 311 : 53‑60. The

largely pale, inner lobe of gonocoxite is composed of ananterior and a posterior arm, the former is sharply
angutate, and gonostylus is strongly expanded in the

25. Cricotopus (Isocladius) sylvestris (Fabricius,
1794)

A male was collected at Karakorum (# 27) on

middle.

August 18, 1996. No. 309 : 08. BL 4.66 mm, WL 2.26 mm,

24. Cricotopus (Isocladius) mongolreseus sp. nov.
(Figs. 22 a‑i)

A male was collected at Bogd (# 10) on August 13,
1996. Holotype: No. 309 : 11.

Male. BL 3.48, WL 1.56. WW/WL 0.34. Groundcolor of scutum yellow, stripes dark brown, scutellum
yellow, postnotum dark brown, proximal half of femora
yellow, distal half brown, tibiae largely yellow and withnarrow brown rings at both ends, front tarsi uniformly
brown, entire length of middle and hind tarsi I, 11 andproximal half of tarsus 111 yellow, the rest tarsal seg‑ments brown. Abdominal tergites 11 to VII each with anoval brown area in the middle, VIII Iargely brown (Fig.
22 h) .

WW/WL 0.30. Ground color of scutum pale, stripes
dark brown, median stripes reaching to only about
middle of scutum, scutellum brown, postnotum darkbrown, femota basally brown and distally dark brown,
tibiae largely yellow and with a basal and a distal brown
ring, front tarsomeres entirely brown, middle tarsomere

I Iargely yellow with a basal and an apical dark ring,hind tarsomere I Iargely yellow and with a basal brown
ring, the distal tarsomeres brown. Abdorninal tergite I
entirely pale, IV Iargely pale and with a small brownspot in the middle, VII Iargely pale and with a brownband along anterior margin, II, 111, V and VI Iargely
brown and with pale bands along anterior and posterior
margines, VIII and IX entirely brown.

Head in Fig. 22 a. Eyes pubescent, ER 0.91, AR 1.60,

Eyes pubescent, each with a wedge‑shaped dor‑

AHR 0.56. P/H 0.84. SO 0+3, 0+3, CL 12. Ante‑scutellum in Fig. 22 c. DM 24, DL 24 : 24, both all minute

somedial extension, ER 1.00. Antenna with 13 flagellar
segments, AR 1.74, AHR 0.54. P/H 1.03, SO 1+4 : 1+4,
CL 10, PN 5 : 5. DM 18, DL 18 : 20, both all minute. PA
5: 5, SC 10, all well developed. SQ 24 : 24, anal lobe

and arising from small pits. PA 4 : 4, SC 8. SQ 8 : 10.

expanding inwards. RR 0.53, VR 1.17, R/Cu 1.07. fLR

RR 0.57, VR 1.16, R/Cu 1.06. fLR 0.59 mLR 0.45, hLR

0.58, mLR 0.45, hLR 0.51, fTR 0.16, fBR 2.0, mBR. 0.21,

pronotum (Fig. 22 b) united in the middle, with only one

tiny lateral seta. Distribution of setae on scutum and173

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Paratrichocladius mongolseteus sp. nov. a: head; b: scutum and scutellum; c: wing; d: tip of front tibia; e:
tip of middle tibia; f: tip of hind tibia; g: abdominal tergites; h: hypopygium; i: inner lobe of gonocoxite, andgonostylus. Fig. 24 Paratrichocladius rufiventris (Meigen, 1830) a: head; b: antepronotum; c: abdorninal
tegites; d: hypopygium. Fig. 25 Orthocladius uraaubaatur sp. nov. a: antepronotum; b: hypopygium; c: inner
lobe of gonocoxite, dorsal view; d: inner lobe of gonocoxite, ventral view; e: gonostylus.

174

hBR 0.22. Pulvilli large, brush‑1ike. Setae on abdominal

ing to the genus Paratrichocladius Santos Abreu, 1918,

tergites 11 to VI are arranged roughly into the medianand the lateral groups. Anal point absent, inner lobe ofgonocoxite single, rounded and bearing strong setae,
inner margin of gonostylus expanded in the middle.

since eyes are pubescent, dorsolateral setae of scutum isextend beyond tip of R4+5, which is distal to tip of Cul,

Remarks. The above stated body coloration, mea‑

Cu2 is nearly straight, and anal point is absent. Species

surement data and the structures indicate that the pres‑ent specimen belongs to the pale form of C. sylvestris, aof this genus have fringe hairs on squama, but unfortu‑
nately both squamae are lost. This species is howeverquite unusual as a member of this genus in that maxill‑

cosmopolitan species recorded from many lakes in theworld.

well developed and arising from large pale pits, wing

venation is the Cricotopus type and costa does not

ary palp is very short, P/H only 0.51, and terminal comb
of hind tibia is composed of only 6 spines.

26. Paratrichocladius mongolseteus sp. nov.
(Figs. 23 a‑i)

A male was collected at Bogd (# 18) , about 1,500 m
high from sea level, on August 13, 1996. No. 309 : 45

Male. BL 4.42 mm, WL 2.24 mm, WW/WL 0.30.
Scutum, scutellum and postnotum entirely black, only

27. Paratrichocladius rufiventris (Meigen, 1830)(Figs. 24 a‑d)Nine males were collected at Karakorum (# 30) on
August 18, 1996. No. 309 : 34‑49; 311 : 72‑74. BL 2.78‑
3.32 (3.06 in average of 6) mm, WL 1.51‑1.85 (1.72 in

the bases of DL setae are pale. Leg segrnents andaverage of 9) mm, WW/WL 0.31‑0.33 (0.32). Groundabodominal tergites almost evenly dark brown. Head in
Fig. 23 a. Eyes pubescent, reniform and without dor‑somedial projection, ER 1.43. Antenna with 13 flagellar
segments, AR 2.45, AHR 0.66. Palp unusually short, Plcolor of scutum brown, stripes, scutellum and postnotum

H only 0.51. SO 3 : 3, all arising in the median portion.

CL only 4. Antepronotum lost. Distribution of setae on
scutum and scutellum in Fig. 23 b. DM 8, all minute. DL
10 : 10, all well developed and arising from large pale
pits. PA 10 : 10 (all very thin) . SC 8.

dark brown, Iegs and abdomen uniformly brown. Headin Fig. 24 a. Eyes pubescent, each with a conspicuous
dorsomedial extension, ER 0.50‑0.67 (0.60). Antenna
with 13 flagellar segments, AR 1.14‑1.42 (1.28), AHR
0.52‑0.56 (0.54). P/H 1.04‑1.26 (1.18), SO composed of
1 or 2 inner and 4 or 5 Iateral groups. CL 12‑16 (14.5).
Antepronotum (Fig. 24 b) united in the middle, with 4 or
5 (4.5) Iateral setae. DM 8‑10 (9.0), DL 10‑14 (12.6), all

Wing bare, very finely granular, venation in Fig. 23

well developed. PA 3 or 4 (3.3), SC 10‑14 (12.0), SQ

c. Squama lost. Anal lobe strongly produced inwards.

8‑12 (9.5), RR 0.48‑0.54 (0.52), VR 1.09‑1.13 (1.11) , 1.03‑

Costa does not extend beyond tip of R4+5. R2+3

1.05 (1.04), fLR 0.58‑0.62 (0.60), mLR 0.49‑0.51 (0.50),
hLR 0.57‑0.59 (0.58), fTR 0.13‑0.14, fBR 2.2‑2.6 (2.4),
mBR 2.6‑3.0 (2.8), hBR 2.6‑3.2 (2.9). Pulvilli small,brush‑like.

separated, RR 0.63. VR 1.09, R/Cu 1.13. Cu2 almost
straight. Tip of front tibia (Fig. 23 d) with a long andcurved spur, 88 pm long and 1.5 times as long as thediameter of front tibia at the tip. Tip of middle tibia(Fig. 23 e) with two short spurs, both 20 pm long. Tipof hind tibia (Fig. 24 f) with a long (38 pm) and a short(17 pm) spur, and with a terminal comb of only 6 spines,
and other 12 spines are distributed in the distal portion.

Tarsi I and 11 of middle and hind legs without preapicalspur. fLR 0.69, mLR 0.46, hLR 0.56, fTR 0.18, fBR 2.0,
mBR 2.0, hBR 2.0. Pulvilli absent.
Setae on abdominal tergites (Fig. 24 g) are arran‑ged roughly into the lateral and the median groups, 16 on
I, 36 on II, 40 on 111 to VII, and 36 on VIII. Hypopygium

in Fig. 23 h. Anal point and virga absent. Ninth tergite
bearing 8 short setae in the middle portion. Inner lobeof gonocoxite prominent, nearly rectangular but withrounded margin, with strong marginal setae. Gonos‑

Distribution of setae on abdominal tergites as inFig. 24 c, the numbers are 28 on I, 32 on 11 to VII, and 40

on VIII, and those on 11 to VII are arranged roughly into

7‑10 Iateral groups and the anterior and posterior rows.
Hypopygium in Fig. 24 d. Anal point absent. Inner lobeof gonocoxite acutely angulate. Gonostylus with alarge, rectangular preapical tooth.

Remarks. The above measurement data andstructure of the present specimens are almost coincident

with those of P. rufiventris (Meigen, 1830), which arecommonly recorded from Europe and Japan. Its mor‑
phology was described in details by Sasa (1981) incomparison with the related species, P. tamaater Sasa,
1981, and is especially characterised by that abdominal
tergites 11 to VI with 7 or more lateral setae (only 4 intylus (Fig. 23 i) simple, with srnall rectangular preapicalP. tamaater). The present specimens also differs fromtooth.

the above species, P. mongolseteus, in that palp is long,

Remarks. This specimen is considered as belong‑eyes with a long dorsomedial extension with smaller ER

175

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Eukiefferiella mongolteuus sp. nov. a: head; b: antepronotum; c: scutum; d: wing; e: tip of front tibia; f:
tip of middle tibia; g: tip of hind tibia; h: tip of middle tarsus I; i: tip of middle tarsus II; j: tip of hind tarsus
I; k: tip of hind tarsus II; m: abdominal tergites; n: hypopygium. Fig. 27 Eukiefferiella mongoluveus sp. nov.

a: head; b: antepronotum; c: scutum and scutellum; d: wing; e: tip of front tibia; f : tip of middle tibia; g: tipof hind tibia; h: abdominal tergites; i: hypopygium.

176

values, and hind tibia with 10 or more terminal comb
spines.

bripennis group of subgenus Orthocladius which are atpresent rather difficult to be classified into separate

species with adult males (Sasa and Kikuchi, 1995, p.

28. Orthocladius (Orthocladius) ulaanbaatus(Figs. 25 a‑e)

sp. nov.
A total of 33 males and 17 females were collected at
Ulaanbaatur on August 1, 1995. Holotype: male, No. 306 :05. Paratypes: males, No. 306 : 06‑23, 84‑88; 311 : O1‑09;

females: 306 : 24‑29, 90‑100.

Male. BL 3.26‑3.58 (3.39 in average of 8) mm, 1.66‑

2.16 (1.85 in 8) mm, WW/WL 0.31‑0.34 (0.32). Scutumlargely brown, with the exception of humeral areas
being yellow, scutellum brown, postnotum dark brown,legs almost uniformly brown; abdominal tergites 11 to V
brown for median and lateral areas and brownish yellow
for both sides of median dark area, VI to VIII Iargely170). The structures and measurement data of theabove specimens collected in Mongol are closely related
to those obtained with specimens of this group collectedin the summer season in Japan, but they are considered
as a different new species, because the body size, thevalues of AR, mLR and the numbers of most scutal setae
are srnaller, and the numbers of lateral setae of ante‑

pronotum are 9‑12 in the present specimens and muchlarger than 3 to 6 obtained with the Japanese specimens

examined by us (Sasa and Kikuchi, 1995, p. 170).

29. Eukiefferiella mongolteuus sp. nov.
(Figs. 26 a‑n)brown and with a yellow band along caudal margin.
Eyes bare, each with a dorsomedial projection, ER 0.97‑
1.32 (relatively high) . Antenna with 13 flagellar seg‑ments, AR 1.34‑1.48 (1.41, relatively smalD . Palp short,
P/H 0.92‑0.95 (0.94). SO 8‑16, most frequently 12, mean

12.3. CL 8‑14 (12.1). Antepronotum (Fig. 25 a) very
narrowly united in the middle, with 9‑12 (most frequent‑ly 10, mean 10.8, very many) Iateral setae. DM 12‑20(14.3), all minute. DL 6‑10 (most frequently 8, mean8.4), PA 4‑6 (4.5), SC 8‑12 (10.0).

Wing bare, brownish and very finely granular, anal
lobe strongly produced inwards. Costa does not extend
beyond tip of R4+5. RR 0.33‑0.42 (0.38), VR 1.06‑1.12(1.10), R/Cu 1.03‑1.08 (1.06). Cu2 almost straight,
slightly curved near apex. fLR 0.61‑0.64 (0.62), mLR
0.45‑0.47 (0.46, very small), hLR 0.54‑0.57 (0.56), fTR
0.15‑0.17 (0.16), fBR 1.9‑2.7 (2.4), mBR 2.6‑5.2 (3.4),
hBR 2.6‑5.4 (4.6). Tarsi I and 11 of middle leg, andA male was collected at Orkhon Gol on August 18,
1996 (# 45) at an altitude of 1,775 m. Holotype: No. 306:
02.

Male. WL 1.77 mm, WW/WL 0.34 (very wide).
Scutellum and postnotum are lost from the mounted
specirnen. Ground color of scutum yellow, stripes
brown, Iegs brownish yellow, abdominal tergites brown.
Head in Fig. 26 a. Eyes bare, reniform, inner margin

concave but without dorsomedial extension, ER 1.37.
Antennal flagellum 13 segmented, AR 0.86, AHR 0.45,
apical portion strongly expanded, without apical seta.

Palp short, P/H 0.88. SO 2+4 : 2+4, CL 4. Ante‑pronotum (Fig. 26 b) very narrowly united in the middle,
without setae. Distribution of setae on scutum in Fig. 26

c. DM 16, all minute. DL 6 : 6, all arising from large
pale pits. PA 3:3.

Wing bare, membrane brownish and slightly granu‑

with lateral setae, and distal half narrow and withlar, venation in Fig. 26 d. Squama with 20 ': 20 fringehairs, anal lobe obtuse. Costa extending slightly beyond
tip of R4+5, which is proximal to tip of Cul, R/Cu 0.93.
R2+3 separated, RR 0.34. VR 1.16. Cu2 nearly straight.
Tip of front tibia (Fig. 26 e) with a long spur, 56 pm
10ng and 1.6 times as long as the diameter of front tibia

sharply pointed apex. Virga 30 pm long and 20 pm

at the tip. Tip of middle tibia (Fig. 26 f) with two spurs,

wide, composed of some 20 strong spines. Inner lobes ofgonocoxite Fig. 25 c, dorsal, 25 d, ventraD are composedof almost completely overlapping two layers, the dorsal
one broader and rounded, the ventral one narrower andangulate. Gonostylus (also in Fig. 25 e) simple, without
preapical tooth, nearly straight and with rectangularly

one longer (30 pm) and the other shorter (17 pm). Tipof hind tibia (Fig. 26 g) with a long spur (54 pm), a
short spur (12 pm) , and a comb composed of 12 simple
spurs. Tips of middle and hind tarsomere I (Figs. 26 h,j) with two simple subterminal spurs, tips of middle andhind tarsomere 11 (Figs 26 i, k) with one simple subter‑
minal spur. fLR 0.66, mLR 0.52, hLR 0.58, fTR 0.17, fBR
2.3, mBR 3.2, hBR 4.4. Pulvilli absent.

tarsus I of hind leg each with 2 subterminal spurs, othertarsomeres, including 11 of hind leg, without subterminal
spurs. Pulvilli absent.

Hypopygium in Fig. 25 b. Anal point widest at base,truncate apex, or widest at about middle and tapering
towards apex (the shape of such differences are prob‑
ably due to the angle of observations) .

The numbers of setae on abdominal tergites are

Remarks. The above structure and measurement

relatively large, 64 on 11 to VI, 48 on VII, and 44 on VIII,

data indicate that these specimens belong to the gla ‑as in Fig. 26 m (left half) . Hypopygium in Fig. 26 n.

177

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Chaetocladius mongolveweus sp. nov. a: head; b: antepronotum; c: scutum and scutellum; d: wing; e: tip offront tibia; f: tip of middle tibia; g, h: tip of hind tibia; i: abdominal tergites I to IV, Ieft half; j: hypopygium.

Fig. 29 Metriocnemus oiraquintus Sasa, 1991 a: head; b: antepronoturn; c: scutum and scutellum; d: wing; e:
tip of front tibia, f: tip of middle tibia; g: tip of hind tibia; h: hypopygium.

178

Anal point absent, ninth tergite without long setae in the

middle portion. Inner lobe of gonocoxite large andDistribution of setae on abdominal tergites in Fig.
27 h, 36 on I, 40 on II, 111 and IV, 32 on V and VI, 28 onrectangular. Gonostylus simple, with a small preapical

VII and VIII, and those on 11 to VII are arranged into thetooth.

anterior row, the posterior row, and the latarel groups.
Hypopygium in Fig. 27 i. Anal point long, widest at baseRemarks. This species is considered as belonging
to the genus Eukiefferiella Thienemann 1926, in wider
sense, and seems to belong to the chuzeocta a group of
this genus, since eyes are bare, squama fringed, R2 + 3 isseparated from both R1 and R4+5, and anal point isabsent, and is very closely related to E. chuzeoctava
Sasa, 1984, in measurement data and structure (Sasa,
1984, p. 73). However, this specimen is considered as
belonging to a different new species, since body colora‑tion is much paler (almost entirely black in E.
chuzeoctava) , eyes with concave inner margin and ER is

smaller (inner margin convex, ER 1.41‑1.54 in thelatter) , palpi are shorter and tip of R4+5 is locatedmore proximally (P/H 1.02, R/Cu 0.99, in the holotypeof E. chuzeoctava). The present specimen has only one
terminal spur on tarsomere 11 of middle and hind legs,
which could be another differentiating character fromE. chuzeoctava, in which tarsomeres I and 11 of middle
and hind legs all have two subterminal spurs.

30. Eukiefferiella mongoluveus sp. nov.
(Figs. 27 a‑i)

A male was collected at Ulanbaatur on August l,
1995. Holotype: No. 306 : 03.

and apically pointed, with a pair of setae on base,otherwise ninth tergite without setae in the middle
portion. Virga is absent. Inner lobe of gonocoxite
single, Ionger than wide and rounded. Gonostylus sim‑ple, narrow and with a small preapical tooth.

Remarks. This species belongs to the tamaflava
group of genus Eukiefferiella, since R2+3 is in contact
with R4 + 5, tip of R4 + 5 is proximal to tip of Cul and Rl

Cu < 1.0, squama with fringe hairs, and anal point ispresent (Sasa and Kikuchi, 1995, p. 157). It is mostclosely related to E. tamaflava Sasa, 1981 among the
known species of this group, and the shape of eyes, anal
point and gonostylus as well as body coloration are quite
similar, but E. tamaflava differs from the present species

in that AR is 0.37 and smaller, the numbers of setae on
abdominal tergites are much fewer (only 13‑16 on ter‑gites 11 to VD , virga is present, ninth tergite has a pairof rounded lobes flanking anal point, and inner lobe ofgonocoxite is broader (Sasa, 1981). This species differsfrom the preceding one, E. mongolseteus, in that anal
point is present, inner lobe of gonocoxite is rounded,

antepronotum with a lateral seta, and the setae on
abdominal tergites are fewer.

Male. BL 2.65 mm, WL 1.52 mm, WW/WL 0.33.
Ground color of scutum yellow, stripes and postnotum
dark brown, scutellum brownish yellow, Iegs and abdo‑
men brownish yellow. Head in Fig. 27 a. Eyes bare,reniform, without dorsomedial extension, ER 1.25. Palp

31. Chaetocladius mongolveweus sp. nov.

with 13 flagellar segments, AR 0.72, AHR 0.43. SO 8 : 8.

Holotype: 306 : 42. Paratypes: 306:43‑51, 310:10‑16.

CL 7. Antepronotum (Fig. 27 b) slightly separated inMale. BL 4.72‑5.28 (5.05 in average of 8) mm, WL
2.88‑3.08 (2.97)mm, WW/WL 0.26‑0.28 (0.27). Groundcolor of scutum brownish yellow, stripes dark brown,

the middle, with I : 2 Iateral setae. Distribution of setaeon scutum and scutellum in Fig. 27 c. DM O, DL 13 : 14,
PA 3 : 3, SC 6, all well developed.
Wing bare, brownish, venation in Fig. 27 d. SQ with(Figs. 28 a‑j)

Seventeen males were collected on Mount Bogdrhan(# 6), on an elevation of 2,400 m, on August 5, 1995.

scutellum brownish yellow, postnotum dark brown, Iegs
and abdominal tergites brownish yellow. Head in Fig.28

16 fringe hairs. Anal lobe obtuse. Costa extending

a. Eyes bare, each with a wedge‑shaped dorsomedialslightly beyond tip of R4 + 5, which is much proximal to

tip of Cul, R/Cu 0.94. R4+5 in contact with R4+5. FCu

projection, ER 0.44‑0.90 (0.70). Antenna with 13 flagel‑lar segments, AR 1.92‑2.34 (2.08) , AHR 0.58‑0.65 (0.62).

much distal to R‑M, VR 1.47. Tip of front tibia (Fig. 27e) with a long spur, 43 pm long and 1.6 times as long as

Palp relatively short, P/H 0.86‑0.96 (0.92). SO 12‑22(16.7), CL 6‑12 (9.6). Antepronotum (Fig. 28 b) united

the diameter of front tibia at the tip. Tip of middle tibia

in the middle, with 8‑16 (11.9, very many) Iateral setae.

(Fig. 27 f) with two spurs, 20 and 24 pm long. Tip ofhind tibia (Fig. 27 g) with a long (53 pm) and a short(22 pm) spur, and a comb composed of 10 free spines.
All tarsal segments without terminal spur. fLR 0.65,
mLR 0.46, hLR 0.54, fTR 0.13. Tarsi with long beards,
fBR 3.9, mBR 3.2, hBR 6.8. Pulvilli vestigial.

Distribution of setae on scuturn and scutellum in Fig. 28

c. DM 22‑34 (27.0), DL 18‑26 (22.7), PA 11‑18 (12.8),
SC 14‑22 (18.3), all very many.

Wing membrane bare but conspicuously granular,
venation in Fig. 28 d. Squama with 12‑24 (17.6) fringehairs. R2+3 separated, ending closer to tip of R4+5

179

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Mongolcladius mongolwexeus sp. nov. a: head; b: antepronotum; c: scutum and scutellum; d: wing; e: tip offront tibia; f: tip of middle tibia; g: tip of hind tibia; h: abdominal tergites; i: hypopygium. Fig. 31
Mongolyusurika mongolxeyeus sp. nov. a: head; b: antepronotum; c: scutum and scutellum; d: wing; e: tipof front tibia; f: tip of middle tibia; g: tip of hind tibia; h: abdominal tergites, Ieft half; i: hypopygium; j: caudal
portion of ninth tergite; k: inner lobe of gonocoxite, and gonostylus.

180

than to tip of R1, RR 0.60‑0.66 (0.63). VR 1.06‑1.12(1.09), R/Cu 1.06‑1.10 (1.08). Costa extending onlymm, WW/WL 0.30‑0.33 (0.31). ER 1.35‑1.65 (1.50).

front tibia (Fig. 28 e) with a long spur, 115 pm long andAntennal flagellum 11 segmented in 1, 12 in 2, and 13 in
1, AR 0.60‑0.68 (mean 0.64), AHR 0.35‑0.51 (0.46). Palp
short, P/H 0.78‑0.88 (0.83). SO 3‑5 (4.0), CL 6‑10 (8.5).

1.7 times as long as the diameter of front tibia at the tip.

DM O in 3, 4 in 1, DL 9‑16 (12.8), PA 5 or 6 (5.8) , SC all

Tip of middle tibia (Fig. 28 f) with two spurs, 57 and 50

4.

slightly beyond tip of R4 + 5. Cu2 nearly straight. Tip ofpm long. Tip of hind tibia (Figs. 28 g, h) with a long

Wing bare, granular, SQ 1‑3 (2.0), RR 0.31‑0.36(113 pm) and a short (40 pm) spur, and a comb com‑

(0.34), VR 1.28‑1.33 (1.31), R/Cu 1.04‑1.09 (1.06). fLR

posed of 16 free spines 33‑68 pm long. Tips of middle

0.47‑0.52 (0.49), mLR 0.43‑0.46 (0.45), hLR 0.52‑0.58(0.55), fTR 0.13‑0.14, fBR 2.3‑2.7 (2.5), mBR 2.5‑3.1

and hind tarsi I and 11 each with two simple subterminal
spurs. Pulvilli absent. fLR 0.63‑0.69 (0.66), mLR 0.41‑(2.9), hBR 3.1‑4.3 (3.6).

Remarks. This species is identified as L.

0.45 (0.43), hLR 0.49‑0.53 (0.51), fTR 0.12‑0.13, fBR 2.8‑

4.5 (3.5), mBR 3.0‑3.7 (3.5), hBR 3.3‑4.1 (3.7).

minimus, since the above measurement data and the

Setae on abdominal tergites (Fig. 28 i, I to IV, right

structures are within the variation ranges of this specieshalf) are very many but short and weak, and almost
evenly distributed, 128 on I, 132 on II, 111, and IV, 140 onrecorded from Europe, and also described in detailes by
Sasa and Kikuchi (1986) with the Japanese specimens.

V and VI, 134 on VII, and 100 on VIII, in the holotype.
Hypopygium in Fig. 28 j. Anal point long, widest at base

33. Metriocnemus oiraquintus Sasa, 1991
(Figs. 29 a‑h)and tapering towards pointed apex, but very faint,
without microtricia and without lateral setae. NinthA male was collected at Mt. Bogdrhan on August 5,

tergite with some 16 short setae in the middle portion.

1995 (# 7) , at about 2,400 m high from sea level. No. 306:

Virga absent. Inner lobe of gonocoxite single, verybroad, obtuse and with rounded corner. Gonostylus

81.

simple, widest at about middle, and without preapical(very narrow) . Body almost uniformly dark brown.
Head in Fig. 29 a. Eyes bare, reniform and without

tooth.

Remarks. This species is considered as belonging
to the genus Chaetocladius Kieffer, 1911, since wing isbare and granular, squama fringed, costa not extending
much beyond tip of R4+5, pulvilli absent, and anal point
is well developed but without lateral setae (Pinder, 1978;

Sasa and Kikuchi, 1995). This species is most closely
related among the European species of this genus to C.
melaleucus (Meigen) , but in the latter AR is about 1.5(smaller) and body is entirely black, anal point is muchsmaller, inner lobe of gonocoxite is narrower, rectan‑
gularly angulate and has a hook‑like process, and gonos‑

tylus is not expanded basally according to Edwards(1929) and Pinder (1978, Fig. 123D) . Five species havebeen recorded from Japan as members of this genus by
Sasa and Kikuchi (1995), among which the present one
is somewhat related to C. oyabevenustus Sasa, Kawai etUeno, 1988, in that inner lobe of gonocoxite is simple,
and anal point is long, and AR is about 2.2, but in thelatter pulvilli and virga are present, anal point is apical‑

Male. BL 3.06 mm, WL 1.63 mm, WW/WL 0.26

dorsomedial extension, ER 0.90. Antenna with 13 flagel‑lar segments, AR 1.06, AHR 0.51. Palp well developed,

10ng, P/H 1.17, and setae on segments I to 111 are
unusually long. SO 40 : 40 (very many) . CL 12. Ante‑pronotum (Fig. 29 b) united in the middle, with 18 : 18
basolateral setae (very many) . Distribution of setae on
scutum and scutellum in Fig. 29 c; DM 32, DL 54 : 56. PA

32: 34, SC 46, all very many and well developed.
Wing very narrow, anal lobe nearly flat, membranethickly clothed with macrotrichia on entire surface,
venation in Fig. 29 d. Both squamae are lost from themounted specimen. Costa slightly extending beyond tipof R4 + 5, which is slightly distal to tip of Cul, R/Cu

1.03. R2+3 separated, RR 0.27. VR 1.29. Cu2 nearlystraight. Tip of fore tibia (Fig. 29 e) with a long spur,

76 pm long and 1.5 times as long as the diameter of fore
tibia at the tip. Tip of middle tibia (Fig. 29 f) with two

spurs. Tip of hind tibia (Fig. 29 g) with a very long,stout and barbed spur, 120 pm, and a comb composed ofly rounded, and inner lobe of gonocoxite is higher and

12 free spines, but without the short spur present in mostnarrower.

32. Limnophyes minimus (Meigen, 1818)

other species of Orthocladiinae. Tips of middle and hindtarsi I and 11 each with two subterminal spurs. Pulvilli
absent. fLR 0.58; mLR 0.42, hLR 0.43 (both very srnalD .

Four males were collected at Bogdrhan on August
5, 1995, 2,400 m above sea level. No. 306 : 53‑56. BL

fTR 0.13, fBR 3.2, mBR 2.4, hBR 3.3.
Setae on abdominal tergites are relatively numer‑

1.92‑2.32 (2.13 in avarage of 4) mm, WL 0.98‑1.26 (1.09)

ous and distributed almost evenly, 72 on I, 90 on II, 72 on

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Pseudosmittia mongolzeaea sp. nov. a: head; b: antepronotum; c: scutum and scutellum; d: winf; e: tip offront tibia; f: tip pof middle tibia; g: tip of hind tibia; h: abdominal tergites; i: hypopygium; j: point; k: inner
10bes of gonocoxite; m: gonostylus. Fig. 33 Pseudosmittia mongolzebea sp. nov. a: head; b antepronotum; c:scutum and scutellum; d: wing; e: tip of front tibia; f: tip of middle tibia; g: tip of hind tibia; h: abdominal tergites;

i: hypopygium; j: anal point and inner lobes.

182

III and IV, 66 on V and VI, 80 on VII, and 64 on VIII.
Hypopygium in Fig. 29 h. Anal point narrow and long,on August 18, 1996, at 1,775 m high from sea level.

apically pointed. Inner lobe of gonocoxite low andMale. BL 2.08‑2.58 (2.38 in average of 8) mm, WL

broad. Gonostylus simple, without preapical tooth.
Remarks. This species is considered as belonging
to the genus Metriocnemus van der Wulp, 1876, since thebasic structures are same as those of the subfamily
Orthocladiinae, and wing with macrotrichia, eyes bare,Cu2 nearly striaght, costa extending beyond tip of R4 +5, and anal point is present (Sasa and Kikuchi, 1995) . It

is most closely related among the European species to
M. gracei Edwards, 1929, in that wing with macrotrichia
on entire surface, gonocoxite without inner lobe, andHolotype: 311 : 86. Paratypes: 311 : 87‑92; 309 : 42.

1.30‑1.52 (1.43) mm, WW/WL 0.34‑0.37 (0.35, verywide) . Ground color of scutum yellow, stripes brown,
scutellum yellow, postnotum brown, Iegs and abdominal
tergites brownish yellow. Head in Fig. 30 a. Eyes bare,reniform and without dorsomedial extension, ER 1.33‑
1.58 (1.43 in average of 8). Antenna with 13 flagellar
segments, AR 0.74‑0.85 (0.80), AHR 0.38‑0.46 (0.43).

Palp short, P/H 0.71‑0.89 (0.82). SO composed of 1

anal point in more robust, and gonocoxite with ainner and 4 or 5 Iateral setae. CL all 4. Antepronotum(Fig. 30 b) united in the middle, without lateral setae.
Distribution of setae on scutum and scutellum in Fig. 30
c. DM all O, DL all 6 : 6, excepting in a specimen withpreapical tooth (cf. Pinder, 1978, Fig. 131O. On the

7: 7. PA 2‑4 (2.9, most frequently 3), SC all 6, excepting

other hand, it is closely related to a Japanese species M.

in a specimen with 7.
Wing bare, brownish and finely granular, venationin Fig. 30 d. Squama with 5‑10 (7.5) fringe hairs. Costa

anal point is long, but in the latter AR is only about 0.5,

oiraquintus Sasa, 1991, described as based on a single

specimen collected at the side of the Oirase River,
Aomori, northern Honshu, in that narrow anal point ispresent, tarsi I and 11 of middle and hind legs with two
apical spurs, AR is about 1.0, hLR is 0.43 and very small,extending much beyond tip of R4 + 5, which is proximal
to tip of Cul, R/Cu 0.94‑0.97 (0.96). R2+3 ending about

midway between tips of R1 and R4 + 5, RR 0.45‑0.53

and since we cannot detect any essential difference from(0.49). VR 1.13‑1.17 (1.15). Cu2 nearly straight, anal

it, the present specimen is provisionally identified as
belonging to this species.

Mongolcladius gen. nov.

lobe obtuse. Tip of front tibia (Fig. 30 e) with a long
spur, 49 pm long and 1.6 times as long as the diameterof front tibia at the tip in the holotype. Tip of middle
tibia (Fig. 30 f) with two short spurs. Tip of hind tibia

A new genus belonging to subfamily Orthocladiinae.
Small species with wing length of about 1.5 mm. Eyes
bare, without dorsomedial extension. Antenna with 13
flagellar segments, AR about 0.8. Antepronotum united

spur, and a comb cornposed of 14 free spines. Tarsorner‑
es IV cylindrical and longer than V. Pulvilli absent.
Setae on abdominal tergites (Fig. 30 h) are relative‑(Fig. 30 g) with a long (58 pm) and a short (27 pm)in the middle, without lateral seta. DM absent, setae only small in the numbers, and are grouped roughly into

scutum, scutellum and abdominal tergites very small in
numbers. Wing bare, membrane granular, costa extend‑
ing beyond tip of R4+5, which is distinctly proximal to

the lateral and the median groups, 32 on I, 2 on II, 26 on

tip of Cul. Cu2 nearly straight. Squama with fringehairs. Anal point small, bare, narrow and sharply

III and IV, 20 on V and VI, 18 on VII, and 16 on VIII in

the holotype. Hypopygium in Fig. 30 i. Anal point
small, bare, narrow and sharply pointed apically. Ninthtergite without long setae in the middle portion, with 3
or 4 short setae on posterior margin flanking anal point.
Inner lobe of gonocoxite single, composed of a rectangu‑
lar base and an inner finger‑like process directed back‑pointed apically. Inner lobe of gonocoxite hook‑1ike.
This genus is most similar to Trissocladius Kieffer,
1908, in that wing is bare, granular and squama is frin‑ged, Cu2 is nearly straight and costa is extending beyond

wards. Gonostylus simple, widest at about middle,

tip of R4+5, pulvilli are absent, and anal point is small,without preapical tooth.

sharply pointed and without lateral setae and microtri‑

Remarks. This species can be differentiated fromchia, but differs from the latter in that hind tibial comb

the previously known species of this group by the char‑acters shown in the description of new genus.

is present (absent in Trissocladius) , and tip of R4 + 5 is

proxirnal to tip of Cul. At present, only one species
described here is included.

(Figs. 30 a‑i)

Mongolyusurika gen. nov.
A new genus of the subfamily Orthocladiinae.
Small midge with WL of about 1.5 mm, wing narrow,
WW/WL 0.27. Eyes bare, reniform. Antenna with 13

Eight males were collected at River Orkhon (# 43)

flagellar segrnents, AR 1.6‑2.0. Setae on scutum and

34. Mongolcladius mongolwexeus sp. nov.

183

scutellum very long. Squama fringed. Wing bare,

10be of gonocoxite small, rod‑1ike, and bearing 5‑7 short

granular. Cu2 nearly straight. Costa does not extend
beyond tip of R4+5. R/Cu > 1.0. Anal point absent.

but strong setae. Gonocoxite with rather long setae

Inner lobe of gonocoxite small, with strong setae.
The above characters indicate that it belongs to thesubfamily Orthocladiinae, and is most closely related
the genus Chaetocladius Kieffer, 1911 (Cranston et al.,with strongly expanded inner margin and nearly straightlateral margin, megaseta large and stout.
those of the related genera by the character indicated inaround base of inner lobe. Gonostylus (also in Fig. 31 k)

Remarks. This species can be differentiated from1989b) . However, it differs essentially from the latter inthe definition of this new genus. Especially noteworthy

that anal point is absent, setae on scutum are all welldeveloped and unusually long, and mid tibia has one long

are the presence of numerous long setae on scutum,
highly granular wing, presence of a long and a shortand one short terminal spurs. At present, only the

apical spur on middle tibia, absence of anal point, andpeculiar structure of inner lobe of gonocoxite.

following species is included.

35. Mongolyusurika mongolxeyeus sp. nov.

36. Pseudorthocladius mongolyezeus sp. nov.

(Figs. 31 a‑k)

(Figs. 34 a‑i)

Thirteen males were collected at Bogdrhan on

A male was collected at River Orkhon (# 41) on

August 5, 1995, 2,200 m high from sea level. Holotype:
No. 306 : 31. Paratypes: No. 306 : 32‑42, 311 : 17, 100
Male. BL 2.76‑3.04 (2.85 in average of 8) mm, WL

August 18, 1996. Holotype: No. 309: 43. Scutellum lost

1.44‑1.54 (1.49) mm, WW/WL 0.26‑0.28 (0.27, very

entirely yellow, only scutal stripes brownish yellow.

narrow) . Head in Fig. 31 a. Eyes bare, inner margin
concave but without dorsomedial extension, ER 0.85 ‑extension, ER 0.47. Antenna with 13 flagellar segments,

1.16 (0.95) . Palp slightly longer than width of head, P/H

AR 1.00. AHR 0.45. P/H 1.00. SO 10:10, CL 10.

1.02‑1.15 (1.06). SO 8‑12 (10.8), CL 8‑12 (9.8). Antepronotum (Fig. 31 b) united in the middle, with 6‑8

Antepronotum (Fig. 34 b) narrowly united in the middle,
with 5: 5 Iateral setae. Distribution of setae on scutum
and scutellum in Fig. 34 c. DM 16, all minute. DL 8: 8,
PA 5: 5. Wing bare, not granular, venation in Fig. 32 d.

(6.6) Iong lateral setae. Distribution of setae on scutum

and scutellum in Fig. 31 c. DM 8‑18, all unusually long.

from the mounted specimen.

Male. WL 1.76 mm, WW/WL 0.34. Body almost
Head in 34 a. Eyes bare, with narrow dorsomedial

DL 18‑28 (21.3), very many and all very long. PA 7‑10

Squama with 12 fringe hairs. R2+3 ending about(8.1), SC 10‑12 (10.8), on a transverse line.

midway between tips of R1 and R4+5, RR 0.47. Costa

Wing bare but conspicuously granular, venation in
Fig. 31 d. Squama with 5‑8 (6.2) fringe hairs, anal lobe

obtuse. Costa extending slightly beyond tip of R4 + 5,
which is slightly distal to tip of Cul, R/Cu 1.02‑1.06(1.04). R2+3 separated, RR 0.36‑0.42 (0.40). VR 1.21‑

extending mtich beyond tip of R4 + 5, which is slightly
distal to tip of Cul, R/Cu 1.04. FCu distal to R‑M, VR
1.15. Cu2 strongly curved. Tip of front tibia (Fig. 34 e)

with a long spur with many barbs. Tip of middle tibia(Fig. 34 f) with two barbed spurs. Tip of hind tibia (Fig.

1.30 (1.27). Cu2 nearly straight. Tip of front tibia (Fig.

34 g) with a long, and a short spur, and a comb com‑31 e) with a long and barbed spur, 72 pm and 1.8 times
as long as the diameter of front tibia at the tip in the

posed of 14 free spines. Tarsi without preapical spurs.

holotype. Tip of middle tibia (Fig. 31 f) with a long (482.9, hBR 3.6. Pulvilli absent (an unusual character as amember of this genus) .

pm) and a short (26 pm) spur, both barbed. Tip of hindtibia (Fig. 31 g) with a long (60 pm) and a short (30pm) berbed spur, and a comb composed of 16 free spines
in the holotype. fLR 0.56‑0.62 (0.59), mLR 0.43‑0.47(0.45), hLR 0.56‑0.61 (0.59), fTR 0.13‑0.14 (0.14). Tarsi

with long beards, fBR 2.6‑3.6 (3.2), mBR 3.6‑5.6 (4.6),
hBR 4.3‑4.9 (4.6). Pulvilli absent.
Setae on abdominal tergites (Fig. 31 h) are relative‑
ly numerous as midges of this size, and are distributed
almost evenly, 60 on I, 50 on 11 to V, 44 on VI and VII,and 34 on VIII. Hypopygium in Figs. 31 i, J. Anal point

absent. Ninth tergite with a broad and rounded lobe
bearing 12 marginal setae on posterior margin. Inner

fLR 0.70, mLR 0.51, hLR 0.60, fTR 0.19, fBR 2.6, mBR

Hypopygium in Fig. 34 h. Virga small, composed of
4 codes of 12 pm long. Inner process of gonocoxite (Fig.
34 i) quite peculiar in the structure, composed of thedorsal and the ventral lobes, the former narrow androunded, and with 6 short marginal setae but without
microtrichia, and the latter without setae and thickly
covered with microtrichia. Gonostylus simple, without
preapical tooth.

Remarks. This specimen is considered as belonging
to the genus Pseudorthocladius Goetghebuer, 1932, sinceeyes are bare, wing membrane bare and not granular,squama fringed, costa extending much beyond tip of184

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Pseudorthocladius mongolyezeus sp, nov. a: head; b: antepronotum; c: sutum; d: wing; e: tip of front tibia;
f: tip of middle tibia; g: tip of hind tibia; h: hypopygium; i: inner process of gonocoxite. Fig. 35 Pseudodiamesa
mongoleecea sp. nov. a: head; b: antepronotum; c: scutum and scutellum; d: wing; e: tip of front tibia; f: tipof middle tibia; g: tip of hind tibia; h: abdominal tergites I to IV, median and right lateral portions; i: hypopygium;

j: anal point; k: gonostylus.

185

R4 + 5, Cu2 is strongly curved, and anal point is stout,

rounded, covered by microtrichia and with numerous
lateral setae (Cranston et al.. 1989b) . A review of theHolarctic species of this genus was made by Saether andSublette (1983), and that of the Japanese species by
Sasa and Kikuchi (1995). The present species belongs
to the group with small virga, anal point well developed
and with lateral setae, and gonostylus not expanded andwithout preapical tooth, and AR is about 1.0, but differsfrom all the previously known species of this group in
the peculiar structure of the inner lobe of gonocoxite,
being composed of the double lobes, as stated above.

pm long and 12 pm wide at the base, situated on a cup
with the base on posterior 1/3 of ninth tergite, andentirely covered by microtrichia. Ninth tergite with 4

setae on both sides of anal point. Virga short but
prominent, composed of an U‑shaped cup and two codes
26 pm long. Gonocoxite with 3 inner lobes (Fig. 32 k,
dorsal view) , the basodorsal lobe is horn‑like, 22 pm

10ng and 4 pm in diameter near the base, and bears
numerous short and curved setae. The basoventral one
is nearly overlapped by the former, 22 pm long and 14

pm wide at the base, covered by microtrichia. The
distal lobe is peculiarly shaped, obtusely triangular anddarkly pigmented, 30 pm wide and 12 pm high, covered

A male was collected on Mount Bogdrhan, 2,400 m
high from sea level, on August 5, 1995. Holotype: No.

by numerous short spinelike microtrichia on dorsal sideand by longer and thinner microtrichia on ventral side.
The third lobe bears short triangular process on poste‑rior margin. Gonocoxite bears 4 peculiar setae arising306:52.

near the base of the distal lobe. Gonostylus (also in Fig.

37. Pseudosmittia mongolzeaea sp. nov.
(Figs. 32 a‑m)Male. BL 2.02 mm, WL 1.00 mm, WW/WL 0.34
(very wide) . Scutum largely brown and with a central
pale hole, scutellum yellow, postnotum dark brown, Iegs

almost evenly yellowish brown, abdominal tergites
largely brown and with conspicuous pale bands between
the tergites. Head in Fig. 32 a. Eyes bare, reniform andwithout dorsomedial extension, ER 1.50. Antenna with
13 flagellar segments, AR 1.11. Palp almost as long as32 m) simple, widest at base and tapering towards apex,
inner margin straight, without preapical tooth and withnumerous very long and fine setae on inner margin.
Remarks. This specimen is very closely related to
P. forcipata (Goetghebuer, 1921) recorded from Europe
in that gonocoxite with 3 inner lobes and the dorsal one
is not foot‑shaped but broad, and anal point is very
narrow, Iong, and covered by microtrichia, but is regard‑

the width of head, P/H 1.03. SO 0+3 : 0+3, CL 5.

ed as a diffenrent new species because the dorsal lobe is

Antepronotum (Fig. 32 b) tapering towards middle andwidely separated, without lateral seta. Scutum with a
median hole bearing two minute setae, DL 8 : 8, PA 4 :not quadrangular as in P. forcipata but is obtusely

5, SC 6, as in Fig. 32 c.

quadrangulate and with a horn‑Iike process on theagulate and highly chitinized, and with a small triangu‑
lar process on posterior edge, while in P. forcipata it isWing membrane bare, brownish, finely granular,anterior edge (cf. Pinder, 1978, Fig. 136D) . The colora‑

venation in Fig. 32 d. Squama bere, anal lobe nearly flat.

tion of abdominal tergites, which are largely brown andwith conspicuous pale bands along anterior and poste‑rior margins, is probably characteristic to this species.

Costa does not extend beyond tip of R4 + 5, which ismuch proximal to tip of Cul, R/ Cu 0.84. R2+3 separat‑
ed, RR 0.52. FCu much distal to R‑M, VR 1.42 (verySuch species have not yet been recorded from Japan.

high) . Cu2 strongly curved. Tip of front tibia (Fig. 32e) with a long and barbed spur, 40 pm long and 1.7 times
as long as the diameter of front tibia at the tip. Tip of
middle tibia (Fig. 32 f) with two short barbed spurs of

38. Pseudosmittia mongolzebea sp. nov.

much different in length, 25 and 13 pm long. Tip of hindAugust 18, 1996. Holotype: No. 309: 44.

(Figs. 33 a‑j)

A male was collected at Karakorum (# 30) on

tibia (Fig. 32 g) with a long (39 pm) and a short (18Male. BL 2.32 mm, WL 1.27mm, WW/WL 0.30.

pm) barbed spur, and a comb composed of 14 free

Scutum, scutellum and postnotum almost entirely

simple spines. fLR 0.48 (unusually smalD, mLR 0.50,

brown, only the humeral area and a norrow zone on the
midline of scutum yellow. Legs and abdominal tergites
almost uniformly yellowish brown. Head in Fig. 33 a.
Eyes bare, reniform, ER 1.57. Antenna with 13 flagellar
segments, AR 0.98, AHR 0.55. Palp short, P/H 0.80. SO
6 : 6, CL 6. Antepronotums (Fig. 33 b) are united very
narrowly in the middle, without lateral seta. Distribu‑
tion of setae on scutum and scutellum in Fig. 33 c. DM

hLR 0.56, fTR 0.14, fBR 3.0, mBR 3.2, hBR 3.5.
The setae on abdominal tergites (Fig. 32 h) are verysmall in the numbers, O on I, 16 on 11 to VII, and 18 on
VIII, and those on 11 to VII are mostly arranged into the
4 groups, 3 in the lateral, 6 in the anterior, and 4 in theposterior portions. Hypopygium in Fig. 32 i. Anal point(also in Fig. 32 j) Iong, narrow and apically rounded, 38

186

7, all minute. DL 3:3 (very smalD, SC 4. Wing bare,
venation in Fig. 33 d. Squama bare. Costa does notextend beyond tip of R4 + 5, which is just above tip' of

40. Pseudodiamesa mongolzecea sp. nov.
(Figs. 35 a‑k)

Four males were collected at Bogd (# 5, # 22) , about

Cul, R/Cu 1.00. R2+3 separated but ending very close to1,500 m high from sea level, on August 13, 1996.

tip of R4+5, RR 0.90. FCu far distal to R‑M, VR 1.35.
Cu2 moderately curved. Tip of front tibia (Fig. 33 e)
with a long barbed spur, 46 pm and 1.6 tiems as long as

Holotype: No. 309 : 61. Paratypes: 309 : 62‑64.
Male. BL 5.84‑6.82 (6.40 in average of 4) mm, WL

3.62‑4.14 (4.00) mm, WW/WL 0.26‑0.28 (0.27). Body

the diameter of front tibia at the tip. Tip of middle tibia

alrnost entirely black, excepting tibiae which are largely(Fig. 33 f) with two short spurs. Tip of hind tibia (Fig.

yellow and with narrow dark basal and apical rings, andtarsi which are enirely yellow. Head in Fig. 35 a. Eyesb re, each with a long and narrow dorsomedial projec‑

33 g) with a long and a short spur, and a comb composedof 12 free spines. fLR 0.41, mLR 0.41,,hLR 0.49 (all very

smalD , fTR 0.13 fBR 3.3 mBR 3.8 hBR 5.3
Setae on abdorninal tergites (Fig. 33 h) are relative‑ly small in the numbers, 20 on I, 26 on 11 to VII, and 28

on VIII, and those on 11 to V are roughly arranged into

the anterior and the posterior rows and the lateralgroups. Hypopygium in Fig. 33 i. Anal point (also in
Fig. 33 j) very small, roughly ,semicircular, 19 pm highand 17 pm wide, highly chitinized, bearing short mar‑
ginal setae and microtrichia, and situated on about 1/3
level from posterior margin of ninth tergite. Virga very
long and narrow, 50 pm long. Ninth tergite with 8 shortsetae along posterior margin. Inner lobe of gonocoxite(also in Fig. 33 j) single, Ionger than wide and acutely
angulate. Gonostylus simple, widest at about basal 1/3,
with a small rectangular preapical tooth.
Remarks. This specimen is considered as belonging
to the genus Pseudosmittia Goetghebuer, 1932, since eyes

are bare and without dorsomedial extension, squamae
are bare, wing membrane bare and finely granular, costa

not extending beyond tip of R4+5, Cu2 is curved, andanal point is covered by microtrichia (Pinder, 1978;
Cranston et al.. 1989; Sasa and Kikuchi, 1995). This
species is however quite unusual as a member of thisgenus in that DM present, tip of R4+5 is not proximal
to tip of Cul but is situated just above it, anal point isvery small, virga is very long, and inner lobe of gonoco‑tion, ER 0.58‑0.77 (0.71). Antenna with 13 flagellar
segments, AR 3.20‑3.88 (3.50), AHR 0.70‑0.72 (0.71).
Palp long, P/H 1.07‑1.16 (1.11). SO composed of 10‑14
inner groups and 6‑8 Iateral groups. CL 20‑38 (27.5).
Antepronotum (Fig. 35 b) narrowly separated in the
middle, without dorsal setae and with 12‑16 (14.3)
basolateral setae. Distribution of setae on scutum andscutellum in Fig. 35 c. DM all O, DL 33‑44 (38.4), allarising from large pale pits. PA 24‑38 (31.5), SC 44‑94(54.0) .

Wing bare, bluish, very finely granular, venation in

Fig. 35 d. Anal lobe very strongly produced inwards,

squama with 104‑120 fringe hairs. Costa extending
beyond tip of R4+5, which is distal to tip of Cul, R/Cu
1.14‑1.16 (1.15). RR 0.63‑0.68 (0.66), FCu proximal to R‑

M and R‑Cu. VR 0.82‑0.84 (0.83). Cu2 nearly straight.
Tip of front tibia (Fig. 35 e) with a long and finely
barbed spur, 133 pm long and 1.4 times as long as thediameter of front tibia at the tip. Tip of middle tibia(Fig. 35 f) with two barbed spurs, 96 and 100 pm long.
Tip of hind tibia (Fig. 35 g) with a long (136 pm) anda short (75 pm) spur, and a comb composed of 14 freespines. Tarsomeres I and 11 of front leg, and I, 11 and 111

of middle and hind legs each with two simple subter‑
minal spurs. In addition, middle portions of mid tar‑xite is large, Ionger than wide and acutely angulate.

somere I with 8, 11 with I and hind tarsomere I with 12,
II with 4 simple spurs of similar structue in the holotype(Fig. 35 h, distal half of mid tarsomere D. Tarsomeres

39. Smittia aterrima (Meigen, 1818)
Fourty six males were collected at Mt. Bogdrhan,
2,400 m high from sea level on August 5, 1995. Male:IV are cylindrical and slightly longer than V, pulvilli
absent. fLR 0.67‑0.71 (0.69), mLR 0.46‑0.48 (0.47), hLR
0.54‑0.56 (0.55), fTR all 0.12. Tarsi with long beards,

No. 306 : 58‑66, 72‑75; 309 : 51‑55; 311; 18‑38, 93‑99.

fBR 4.9‑9.4 (7.0), 3.8‑4.2 (4.0), hBR 4.7‑7.6 (6.2).

Female: 306 : 77. This is a very common species
probably distributed all over the world, and have been

Abdominal tergites with large numbers of setae
which are arranged roughly into the median and therecorded widely from Europe, and also from a numberof localities in Japan by Tokunaga (1940) and by Sasa
and coworkers (Sasa and Kikuchi, 1995).

m, showing bases of the median and the right lateralgroups) . The total numbers on tergites I to V in par‑lateral groups, especially in the first 5 segments (Fig. 35

atype No. 309 : 62 are 156 in I, 130 on II, 108 on 111, 106

on IV, and 92 on V, with the numbers of the mediangroup of 20, 44, 32, 32, 28, respectively; the abdominal

187

setae are more evenly distributed on tergites VI to VIII.

these problems in the country of Mongol in Central Asia,

Hypopygium in Fig. 35 i. Anal point (also in Fig. 35 j,frorn which no information on the chironomids haveventral view) is needle‑1ike, very narrow, Iong, andtapering towards sharply pointed apex, arising frommiddle portion of ninth tergite. Ninth tergite bears
sorne 10 short setae on both sides of the base of anal
point. Inner lobe of gonocoxite (Fig. 35 j) Iarge,

been available. As the results, specimens of as many asrounded and with numerous short marginal setae.
Gonostylus (Fig. 35 k; Ieft, dorsal; right, ventral view)elongate oval, apical spur very tiny and thin, seen only

from the ventral view.
Remarks. This species is considered as belonging

to the genus Pseudodiamesa Goetghebuer, 1939 of the

42 species belonging to 23 genera were collected andidentified, and only 13 species among them were shown
to be in common with those recorded from Europe orJapan, and the rest 29 are described as new species inthis paper. These facts suggest that about 1/3 of thespecies of this family are widely distributed at least
throughout the Holarctic Region, but the rest 2/3 have
evolved to indigenous species in this rather isolatedareas in Central Asia. These evidences are obviously
very important contributions to the world taxonomy

similar in the structure to P. arctica (Maloch, 1919), an

and bilogy of this group of insects. Furthermore, as theresults of this study most of the important chironomid
species breeding in Mongolia have become feasible for
identification by biological and medical scientists, andthe authors hope that the role played by these chir‑onomid species in the environmental and medical fields

Arctic species illustrated by Oliver (1989, p. 151) , in thatwill be gradually clarified also in this country, and will

wing without macrotrichia and anal lobe is strongly
produced inwards, anal point is very narrow, Iong andsharply pointed, and apical spur (megaseta) of gonos‑

contribute to the improvements of the health of theMongolian people.

subfamily Diamesinae, since cross vein M‑Cu is distal toFCu, antepronotum is bare dorsally, and eyes are strong‑ly produced dorsomedially. Reviews of this subfamily
were made by Saether (1969) and Oliver (1989) . Amongthe previously known species of this genus, it is verytylus is very narrow and tiny, but differs from the latterin that anal point is not parallel‑sided but tapering
towards pointed apex, ninth tergite without a pair of

ACKNOWLEDGEMENTS
Thanks are due to Dr. Masao Kamiya, Professor ofof gonocoxite is much higher and with rounded margin,

Hokkaido University and the project leader of the
programm under the Grants in Aid of Overseas Scien‑and gonostylus is stouter and not tapering towards apex.

tific Researches No. 07041156 <'Diversity of Parasites

10bes on posterior margin flanking anal point, inner lobe

and its maintenance mechanism in Central Asia" and41. Tanypus punctipennis (Meigen, 1818)

also to Prof. D. Samiya, Institute of Biological Training

Sixteen males were collected at Bogd (# 8), about

and Research, Mongolian State University, and to

1,500 m high from sea level, on August 13, 1996. No. 309:

members of this research project of the two countries,

66‑81. This species has been recorded widely from
Europe, and has also been collected from Japan by
Tokunaga (1937) and by Sasa and coworkers (Sasa andfor providing us the facilities of collecting the materials

Kikuchi, 1995) .

used in the present study.

REFERENCES

42. Procladius crassinervis (Zetterstedt, 1838)
Two males were collected at Karakorum (# 25) on
August 18, 1996. No. 309: 83, 84. This species has beenrecorded widely from Europe, and also from Japan by
Tokunaga (1937) , and by Sasa and coworkers (Sasa andReferences to the taxonomy of Chironomidae werecompiled by Fittkau et al. (1976) , and supplemented by

Hoffrichter and Reiss (1981). Keys to genera were
propared by Wiedelholm (Ed. 1989). An useful key tospecies of British Chironomidae were published by

Pinder (1978), and a monograph of Japanese Chir‑
onomidae was compiled by Sasa and Kikuchi (1995),

Kikuchi, 1995) .

In view of the recent advances in the studies on the

including references to taxonomy and biology of chir‑onomids of Japan and the Oriental Region.
Abbreviation: NIES = National Institute for Envi‑

importances of the insects of the family Chironornidae inronmental Studies, Tsukuba, Ibaraki 305. TPEP=

the environmental and medical sciences, this study is
considered as to have contributed to the first step ofToyama Prefectural Environmental Pollution ResearchCenter, Kosugi, Toyama 939‑03.

DISCUSSION

1881 ) Cranston, P.S., Dillon, M.E., Pinder, L.C.V. and Reiss, F.

(1989a): The adult males of Chironominae of theHolarctic region‑Keys and diagnoses. Entom. Scand.
Suppl., 34, 353‑502
2 ) Cranston, P.S., Oliver, D.R. and Saether, O.A. (1989b) :The adult males of Orthocladiinae of the Holarctic
region‑Keys and diagnoses. Entom. Scand. Suppl., 34,
165‑3523 ) Edwards, F.W. (1929): British non‑biting midges(Diptera, Chironomidae) . Trans. Roy. Entom. Soc.
London, 77, 279‑429
4 ) Fittkau, E.J. (1962): Die Tanypodinae (Diptera, Chir‑
onomidae) ; die Tribus Anatopinini, Macropelopiini undPentaneurini. Abh. z. Larvensystematik der Insekten,
Nr. 6: 433pp. Akademie Verlag, Berlin5 ) Fittkau, E.J., Reiss, F. and Hoffrichter, O. (1976): A
bibliography of the Chironomidae. Gunnedria, 1‑177
6 ) Hirvenoja, M. (1973): Revision der Gattung Cricotopus

van der Wulp und ihrer Verdandten (Diptera, Chir‑

onomidae) . Ann. Zool. Fen., 10, 1‑363 "7 ) Hoffrichter, O. and Reiss, F. (1981): Supplement to abibliography of the Chironomidae. Gunneria, 37, 1‑688 ) Makarchenko, E.A. (1987): New or little known chir‑onomids of Podonominae and Diamesinae from the
USSR. Entom. Scand. Suppl., 29, 205‑209
9 ) Makarchenko, E.A. (1993): Chironomids of the sub‑family Diamesinae from Japan. I. Sasayusurika aenig‑
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10) Makarchenko, E.A. (1994): Chironomids of the sub‑family Diamesinae from Japan. II. Sympotthastia
Pagast, 1947. Bull. Natl. Sci. Mus. Tokyo A. 20(1),51‑5811) Oliver, D.R. (1989) : The adult males of Diamesinae ofthe Holarctic region‑Keys and diagnoses. Entom.
Scand., Suppl. 34, 129‑154

12) Pinder, L.C.V. (1978): A key to adult males of the
British Chironomidae. Freshwater Biol. Assoc. Sci.
Publ., No. 37, 169pp+189 figs.

13) Ree, H.1. (1981): Studies on Korean Chironomidae(Diptera) . 2. Description of a new genus and a new
species of Chironomidae. Korean J. Zool., 24, 217‑220

14) Ree, H.1. and Kim, M.S. (1981): Studies on chir‑onomidae (Diptera) in Karea. Taxonomical study on
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15) Ree, H.1. and Kim, M.S. (1988): Studies on Korean

18) Saether, O.A. (1980): Glossary of chironomid morphol‑ogy terminology. Ent. Scand. Suppl., 14, 5‑51
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haetocladius Wuelker, and Pseodorthocladius Goetgh‑ebuer. Entom. Scand. Suppl., 20, 1‑10020) Sasa, M. (1979): A morphoiogical study of adults andimmature stages of 20 Japanese species of the family
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the Tama River. Pt. 3. Res. Rep. NIES, No. 29, 1‑78
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23) Sasa, M. (1988): Studies on the chironomid midges
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as allergens of bronchial asthrna. Kansen, Ensho,
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68‑8826) Sasa, M. and Kamimura, K. (1987) : Chirtnomid midges
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37, 17‑39

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29) Strenzke, K. (1959): Revision der Gattung Chironomus
Meig. I. Die Imagines von 15 norddeutschen Arten und
Unterarten. Arch. Hydrobiol., 56, 1‑4230) Tokunaga, M. (1936a): Chironomidae frorn Japan. VI.
Diarnesinae. Philipp. J. Sci., 59, 525‑552

31) Tokunaga, M. (1936b) : Chironomidae from Japan. VII.
New species and a new variety of the genus Chironomus
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33) Tokunaga, M. (1937): Chironomidae from Japan. IX.
Tanypodinae and Diamesinae. Philipp. J. Sci., 62, 21‑65

34) Tokunaga, M. (1938) : Chironomidae from Japan. X.

Chironornidae (Diptera) 111. Description of two unrecor‑

New or little known midges. with descriptions of theded species from Korea and three new species. Korean

metamorphoses of several species. Philipp. J. Sci., 65,
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16) Saether, O.A. (1969): Some Nearctic Podonominae,
Diamesinae and Orthocladiinae. Bull. 170, Fishries Res.

Board Canada, 154 pp.

35) Tokunaga, M. (1940): Chironomidae from Japan. XII.
New or little known Ceratopogonidae Chironomidae.
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17) Saether, O.A. (1977): Taxonomic studies on chir‑onomidae: Nanocladius. Pseudochironomus, and the36) Townes, H.K. (1945): The Nearctic species of Ten‑

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Wiederholm, T. Ed. (1989): Chironomidae of the39) Yan Jing‑song and Ye Cang‑jiang (1977): Notes on thelarvae of some chironomid midge (Diptera, Ten‑
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198

J pn. J. Tro p.

191

Med. Hyg., Vol. 25, No. 4, 1997, pp. 191 195

TR YPANOSOMA RANGELI= SEM PROFILESDURING THE MIGRATION FROM THE
MID‑GUT TO THE SALIVARY GLANDS OF
A REDUVIID BUG RHODNIUS PROLIXUS
VIA DORSAL VESSELMARIA CARLOTA MONROYI AND MASATAKA KOGA'
Received August 25, 1997/Accepted October 3, 1997

Abstract: Metamorphosis of Tmpanosoma rangeli during the migration in the insect host, Rhodonius
prolixius, was first documented by scanning electron microscopy (SEM) . After infection, the parasites reachthe insect alimentally tract and then they penetrate into the mid‑gut and eventually emerge in thehemolymph of the abdominal cavity. From the abdominal cavity, both the parasites and the hemolymph arepropelled to the thorax by the dorsal vessel (aorta pump) , the end of which is terminated very near thesalivary glands. Thus, once these parasit s invade the abdominal cavity, they finally appear in the salivary
glands via the transport by the dorsal vessel. The p rasites gradually change thernselves in shape during the
migration. In the present study, we focused on the morphological characteristics of T. rangeli at individual
stage in the vector by SEM and revealed some new findings in morphology and confirmed a migration routeof this species. These results suggest that Guatemalan T. rangeli is mainly transmitted through the salivary
gland.

Key words: Trypanosoma rangeli, insect vector, circulatory system, hemolyrnph, parasite ultrastructure,

SEM

The prevalence frequency of Trypanosoma rangeli

this context, some trials were performed by using vari‑
ous biological methods to distinguish T. cruzi from T.
rangeli, as seen in the reports of Anthony et al. (1981),

Tejera, 1920 was from 6 to 10 times or more than of T.

Marinkelle et al. (1985) , Vallejo et al. (1993) and Mellocruzi in certain regions of Central America (Sousa,1972). This parasite was first detected in humans in

et al. (1995). Especially, the last one has reported
remakable defferences that T. rangeli in the homocoelof R. prolixus multiplied very rapidly, while T. cruzi
showed no division and disappeared eventually from thehomocoel of the vector.
Though several controversial data have been obser‑

INTRODUCTION

Guatemala (De Leon, 1949) and later found in some
reduviid vectors and wild reservoirs (Barrett andOliveira, 1977). It is not easy to differentiate the species

among the flagellar forms of trypanosomes in vectors

differtiating species have failed because of cross‑ved regarding to the transmission routes, namely, either
salivary toute or fecal one (D'Alessandro, 1963; Anez,

reactivity ( Anthony et al., 1979; Lopez et al.. 1981;
O'Daly et al., 1994). However it is necessary to distin‑

1982; Hoare, 1972) and whether T. rangeli persisted
intracellularly as the amastigote form or not in theguish between the pathogenic and non‑pathogenic

insect gut (Tobie, 1961; Anez, 1983; Hecker et al.. 1990) ,trypanosomes for epidemiological investigations. On

nontheless it was commonly accepted that this proto‑(Vallejo et al.. 1988). Several immunological trials for1 Universidad de San Carlos de Guatemala, Facultad de Ciencias Quimicas y Farmacia, Escuela de Biologia, Ciudad
Universitaria, Zona 12, Guatemala City, Guatemala, C.A.
2 Departrnent of Parasitology, Faculty of Medicine, Kyushu University, Fukuoka 812‑82, Japan
Authors correspondence should be sent to M. Koga, Kyushu University, Fukuoka 812‑82, JapanE‑mail address: masakoga @parasite. med. kyushu‑u. ac. jp (Masataka Koga) '192

zoan parasite was pathogenic to its vector insect

MATERIALS AND METHODS(Schaub, 1994; Molyneuz, 1983).

The present paper reports the metamorphism of T.
rangeli during the migration from the mid‑gut throughthe abdominal cavity and dorsal vessel to the salivary
glands of R. prolixus. The morphologicai alteration of
the parasite in the mid‑gut, hemolymph and salivary
glands were traced by a scanning electron microscopy(SEM) .

Insect and parasite
A Iaboratory colony of R. prolixus which was origi‑
nally obtained in Zacapa, Guatemala, C.A. in 1993 andmaintained thereafter was used. Parasite was of
Guatemaian T, rmgdi strain recentl, y isolated from thesalivary gland of R. prolixus.
Infection and Dissection
Young nursing mice of the White Swiss strain were
iufected withr. rangeli by injecting the contents of thesalivary gland of R. prolixus into the peritonium. Whenhigh Parasitemia appeared about 7 days after infection

Figure 1
Figure 2
Figure 3
Figure 4

Trypanosoma rangdi metacyclic trypomastigote in the salivary gland. Bar=1 pm.
Trypamastigote Of, T. 'angdi from the hemolymph. Bar = 2 ;imLong epimastigote of 1'. rmgeli from the herriolymph. Bar = 3 ,Im
Massive penetration of epimastigotes of 'r. r(Ingeli in the slender midgut. Bar = I ;Iml 93

sected, and the hemolymph, salivary glands and gut
contents were examined under a light microscopy. The
heavily infected bugs were processed ior SEM examina‑tion. Cancurrently Giemsa's stained smear specimens of

fixed over night in 3% glutaraldehyde in O.1 M sodium
cacodylate buffer, washed I hr in phosphate buffer andpostfixed in osmium tetroxide in 2 hours. They werethen dehydrated in a graded series of ethanol, dried at
critical point in a Hitachi HCP dryer, coated with gold
in a JEOL ionspatter coater, and examined under JEOL
U‑3 scanning electron microscope at 15 kv.
In addition, the hemolymph and the excrement werevarious tissues were prepared at pH 6.5 for 2 hrs.

placed on a round cover glass previously coated by poly‑

Material preparation for SEM

L‑Lysine solution and th, en dried. The glass was gently
placed and attached on the tap of a stub. The materials

in the mice, various stages of R. prolixus reared inlaboratory conditions were fed on the blood of infected
mice,
Periodically the infected triatomine bugs were dis‑Dissected tissues were washed in saline solutl'on and

Figure 5

Final ph, ase af penetrafion to the midgut of the host. Posterior portion of epimastigote of T. r(mgdi isevident. Bar=2 pm
Figure 6 Spheromastigote of T rmgdi in the vector's gut. Bar=2 kem
Figure 7 Epimastigote af T. rungeli in the dorsal vessel of R. protixus; the contractile elements and striated muscle
fibers were seen. Bar = 2 pm
Figure 8 Epimastigote of T. rangeli lying in the darsal vessel attached with some bacteria (arrowheads) which arecommonly found in the gut of the vector. Bar=1 ;Im194

were allowed to stay on the glass for I hr and were thenquently seemed to have penetrated into the salivary

fixed with glutaraldehyde. Post‑fixation by osmium
because these small parasites often disappeared fromglands by themselves. Fig. 8 shows ' the parasite sur‑rounded by some bacteria (arrowheads) that were prob‑
ably brought with the parasite from the intestine of thethe tissues of the insect by the conventional SEM speci‑

insect into the vessel.

tetraoxide was omitted before SEM examination,

men preparing procedure.

DISCUSSIONRESULTSThe flagella in all stages of T. rangeli were clearlyseen in insects. There were three shapes in trypomas‑
tigotes in the insect, one in the feces, another in thehemolymph and the last one in the salivary gland. Fig.
1 showed the metacyclic trypomastigote from the sali‑vary glands. The parasite from this gland is the smal‑

lest in all and somewhat stout. On the contrary, thetrypomastigotes in the abdominal cavity were bigger
and slender (Fig. 2), and very similar to those in thefeces. The trypomastigotes in the feces had't infectious
ability to mice in experimental infection.
Epimastigotes were dominant in the insect hosts.
Their bodies were also short, medium and very long insize. However the trypomastigotes distinguished fromthe epimastigotes by their shape and size. They often
segregated in a same host.
Fig. 3 showed one of the longest epimastigotes in
the mid‑gut.
Fig. 4 showed that the medium sized epimastigotes
which were penetrating into the gut epithelium. Fig. 5
showed the last step of the penetration of epimastigote

Some authors reported that T. rangeli trypomas‑
tigote from feces could infect mice (D'Alessandro, 1963;
Hoare, 1972), while the other noted that only the para‑sites from the salivary glands had infectivity (Tobie,
1961; Anez, 1982) . Present study showed that there weresome differences in shapes and sizes of the trypomas‑
tigotes and epirnastigotes in various regions of thevector insect, and this rapid morphological changes are
related to this parasite infectivity. In mice infection

with trypomastigotes from the salivary glands, wealways saw parasiternia, whereas we could not see itwith the parasites from the bug excrements. On theother hand, mice infected with the parasites from thehemolymph, no parasitemia occurred. These results
agreed with the findings of Tobie (1961) and Anez(1982). From these findings, we want to suggest thatthe salivary glands play an important role for the trans‑mission of T. rangeli to mice. It is reported that there
is a difference in the tissue affinity in bugs between T.

rangeli and T. cruzi; T. rangeli favors to live in theslender mid‑gut, whereas T. cruzi prefers the rectal sac

to the mid‑gut. As seen in this figure, the body penetra‑in the same host. Furthermore T. cruzi attached to therectal wall tightly (Zeledon et al.. 1984), while T.

tion elicited intense spiral movement which was acceler‑

rangeli crawled freely in the mid‑gut. In addition, theated with its flagellum. This flagellum movement

epimastigotes of T. rangeli in the hemolymph and thedorsal vessel were provided with very long and slender

seemed to play a very important role in the penetration.
The trigger of the invasion is unknown. Fig. 6 shows thebody, which is one of the characteristics of this species.

spheromastigote that is developing from the amastigote

For these characteristcs mentioned above, we(which means a round form parasite in the mig‑gut
epithelium or hemocyte described by Hecker et al..

facilitated the differentiation of each species by careful

1990) and is just shifting to epimastigote. This spher‑omastigote is also a very small in size.

Fig. 7 also showed the epimastigote on the contrac‑
tile fibers of the folded muscle tissues in the dorsal
vessel. Once the epimastigotes intrude into the host's
abdominal cavity filled with hemolymph, multiplication
occurs there and eventually a great number of parasites

separation of the vector's hemolyrnph, salivary glands,
rectal sac and the slender mid‑gut.

Spheromastigotes have been reported in both
species of T. cruzi and T. rangeli (De Lucena et al..
1973; Vallejo et al.. 1988; Marinkelle et al.. 1985) as ashifting stage from amastigote to epimastigote.
Present study is the first report for documenting

spheromastigote form of T. rangeli by SEM.

is able to be found in the cavity. The dorsal vessel of theIt has been described that in T. rangeli infection, thebugs absorbs the hemolymph into the aorta which acts
as the pumping organ. By this mean , the vessel soaked

symbionts (some bacteria) were small in number in thehernolymph of the bugs (Schaub, 1994). In the present

up the parasites together with the liquid and conveyed tothe thorax region. As the vessel is terminated v,ery nearto the salivary glands in the thorax, the parasites conse‑

study, however, we observed many bacteria in thehemolymph and even in the circulatory system of thevector by SEM. This is probably because of the tissue

195

damage of the insect gut due to massive invasion of thethe reduviid bug Rhodnius prolixus. Parasitol. Res., 76,

parasites.
In this study, trypomastigotes in the salivary glands10) Hoare. C. (1972): The trypanosomes of mammals.

as well as some stages of epimastigotes and spheromas‑
tigotes of T. rangeli in R. prolixus, the vector were first

dernonstrated by SEM. The authors here emphasizethat flagella of trypanosomes seem to play a major rolein the penetration to the gut wall.

ACKNOWLEDGEMENT
We are grateful to Prof. I. Tada, Department of
Parasitology, Faculty of Medicine, Kyushu University,
Japan for his critical reading the manuscripts. This
work was supported by JICA‑Guatemala Tropical Dis‑ease Project (GJET‑125).

311‑318
Blackwell Sci. Pub. Oxford, pp. 288‑314
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10, C. A. and Young, A. (1988): Longitudinal studies of

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Exp. Parasitol., 11, 1‑920) Vallejo, G., Marinkelle, C., Guhl, F. and De Sanches, N.
(1988) : Comportamiento de la infeccion y diferenciacion
morfologica entre Trypanosoma cruzi y T. rangeli en el
intestino del vector Rhodnius prolixus. Rev. Brazil Biol.,48, 577‑587
21) Vallejo, G., Chiari, E., Macedo, A. and Pena, S.D. (1993):

A simple laboratory method for distinguishing between

Trypanosoma cruzi and Tmpanosoma rangeli. Trans.
Roy. Soc. Trop. Med. Hyg., 87, 165‑166
22) Zeledon, R., Bolanos, R. and Rojas, M. (1984): Scanningelectron microscopy on the final phase of the life cycleof Trypanosoma cruzi in the insect vector. Acta Trop.,
41, 39‑43

197

Jpn. J. Tro p. Med. Hyg., Vol. 25, No. 4, 1997, pp. 197 207

NUCLEOTIDE AND DEDUCED AMlNO ACID SEQUENCES
OF THE WHOLE STRUCTURAL PROTElNS AND NS1NONSTRUCTURAL PROTEIN OF FOUR DENGUE 4 VIRUS
STRAINS, ISOLATED FROM PATIENTS IN BANGKOK,THAILAND, WITH DIFFERENT CLINICAL SEVERITIESCHARNCHUDHI CHANYASANHAl' 2
Received September 1, 1997/Accepted October 8, 1997

Abstract: A11 structural proteiri genes as well as one of the nonstructual protein NSI gene of four dengue

virus serotype 4 (DEN 4) strains recently isolated frorn patients admitted to the Children's Hospital in

Bangkok, Thailand in 1993, were sequenced and analyzed. Isolates from a dengue fever (DF) case, CT93‑
77, dengue hemorrhagic fever (DHF) grade I cases, CT93‑129 and CT93‑158 as well as DHF grade 11 case,
CT93‑74 were selected in this study. It was found that the DHF grade 11 isolate had the highest percentage

sequence divergence in the PrM and M regions from the published prototype strain (Caribbean, 814669). In

all four Thai isolates, the C region had the lowest divergence, while the PrM region had the highest
divergence both in nucleotide and amino acid sequence. In the whole analyzed regions, the percentage
divergence ranges from 8.02‑8.33% (nucleotide) and 3.97‑4.90% (amino acid) respectively. Three aminoacid replacements were found to be specific to the DF strain : at position 136, phenylalanine (F) instead ofleucine (L) ; at position 914, phenylalanine (F) instead of serine (S) and at position 1,042, glutamine (Q)instead of histidine (H). The mean hydrophobicity and isoelectric point value did not show significant
differences in the sequenced region for all analyzed isolates. When cornpare to other strains from different

geographical locations, four amino acid replacements were found unique to the Thai isolates. These wereat position 269, isoleucine (1) instead of valine (V) ; at position 374, methionine (M) instead of valine(V) ; at position 482, threonine (T) instead of lysine (K) and position 608, threonine (T) instead of alanine(A) . Phylogenetic analysis using the M/E junction showed that DEN 4 from different geographical areas
can be classsified into three enotypic groups, and the Thai isolates were divided into two genotypes.

Key words: dengue virus type 4, Thai strain, sequence, virulence, genotype, mutation, clinical severity

Dengue viruses belong to the monquito‑borne

The disease is caused by 4 dengue virus serotypes(DEN 1, DEN 2, DEN 3 and DEN 4) . Dengue viruses are
positive sense, single stranded RNA viruses possessing

flavivirus causing significant morbidity and mortality ofsequence of 5'‑C‑PrM/M‑E‑NS1‑NS2A‑NS2B‑NS3‑

children in tropical countries (Westaway et al., 1985a;

NS4A‑NS4B‑NS5‑3' Iike other flaviviruses (Westaway

Dengue virus infection may be

et al., 1985b; Castle et al., 1986; Rice et al., 1986, Coia etasymtomatic or may lead to illness in humans, varying
from a mild form of dengue fever (DF) up to the severeal., 1988) . Each of the dengue serotypes shares common
antigens with other flavivirus (Shope and Sather, 1979;

form of dengue hemorrhagic fever (DHF) (Nimman‑

Monath et al., 1986), which is supported by genetic

nitya, 1987), which can progress to a sudden and fatal

sequence data (Block et al., 1989; Chu et al., 1989; Trent

hypovelemic shock called dengue shock syndromeet al., 1989). Dengue virus presents a serious healthproblem in many tropical and sub‑tropical countries

INTRODUCTION

Wengler 1991).

(DSS) (Nimmannitya et al., 1969, Allen, 1992).
1

2

Department of Microbiology, Faculty of Public Health, Mahidol University, 420/1 Rajvithi Road, Bangkok 10400, Thailand
Department of Virology, Institute of Tropical Medicine Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan

198

including the Americas, Africa, Asia, Australia andPacific Islands (Gubler, 1996), especially causing great

medical and public health problems in Southeast Asia
(Halstead, 1980; Halstead, 1992).

Each serotype of dengue virus may have strains thatvary in virulence (Rosen, 1977, 1986). It is presently

unknown whether specific virus genotypes are respon‑sible for the more severe forms of the disease or forparticular widly spread epidemics. Seroepidemiologicalstudies suggest that severity of DHF/DSS may result
from immune enhancement in the secondary infection bypreexisting antibodies produced by preceding heter‑

Table 1 Clinical manifestation of patients from which four
selected DEN 4 isolates were isolated
Clinical manifestationof the patients'

DF

Dengue 4 isolates

DHF Grade I

CT93‑77
CT93‑129, CT93‑158

DHF Grade II

CT93‑74

Preparation of viral genomic RNA

The total genomic RNA was extracted from theinfected tissue culture fluid by a single step guanidiniurn

otypic infection (Halstead, 1981, 1988), or by maternal

thiocyanate method (Ausubel et al., 1991) with slightantibodies in the primary infection (Burke, 1988).

modifications. Briefly, 200 pl of infected tissue culture

There is another possibility that the severity of diseases

fluid was added to 500 pl GSM solution (5M

may be related to molecular differences of genomic

guanidinium‑thiocyanate, 25rnM sodium citrate, 0.1M 2‑
mercaptoethanol) and vortexed shortly, followed by astructure associated with the virulence of a particularstrain (Duangchanda et al., 1994; Thant et al., 1996).
The main objectives of this study is to determine thequick spin. One hundred microlitres of 2M sodium
acetate (pH 4.0) , 500 pl water saturated phenol and 100

nucleotide sequence of the whole structural (C, PrM/M,

pl chloroform : isoamyl alcohol (49:1) were added andE) and NSI gene of recent isolates of DEN 4 inmixed by vortexing. The mixture was then incubated

Thailand, and to correlate the disease severity to theobserved molecular differences.

on ice for 30 minutes and centrifuged at 15,000 rpm for
20 minutes at 4'C. The pellet was then washed with 500

pl 75% ethanol,

MATERIALS AND METHODS

ir dried and resuspended in RNase‑

free 10 pl of sterile distilled water and stored at ‑80'C
until use.

Virus strains

Four isolates of DEN 4 were selected out of 9
strains from the previous study (Chanyasanha et al.,1995). These isolates were obtained from patients serawith different clinical manifestations at Children's
Hospital, Bangkok, Thailand in 1993. Written consent
was obtained from the patient's parents or guardians
and the study protocol was approved by the ethicalreview research committee, established by the Ministryof Public Health, Thailand. Isolate (CT93‑77) wasfrom a dengue fever (DF) case, 2 isolates (CT93‑129

and CT93‑158) were from dengue hemorrhagic fever(DHF) grade I cases and another isolate (CT93‑74)
was from DHF grade 11 case (Table 1). Clinical sever‑ity grading and clinical diagnosis were classified accord‑

ing to the World Health Organization (WHO) criteria(Anonymous, 1986) . The viruses were isolated by inocu‑
lation of patients sera into Aedes albopictus clone C6/36
cell line (lgarashi, 1978) and serotypes were identified
by reverse transcriptase polyrnerase chain reaction (RT‑
PCR) using type specific dengue primer pairs (Morita etal., 1991; Tanaka, 1993 ) . They were then stored at‑80'C. The virus isolates were amplified againby grow‑ing in C6/36 cells, at 28'C for 7 days before use.

Primer designed for ampllfying target gene
The primers for amplifying the target regions were

designed with one pair for each of the C, PrM and M
gene region covering 839 bases. The E protein gene,which is 1,482 bases long, was divided into 3 regions ofapproximately 600 bases each. The NS1 gene which is
1,230 bases long was divided into 2 regions of approxi‑

mately 700 bases each. Both sense and complementary
primer pairs were constructed for each region according
to published sequences (Zhao et al., 1986; Mackow et al.,1987). Oligonucleotide prirners were synthesized using

an Applied Biosystem DNA synthesizer (Model 392,
California, USA) . A schematic diagram of nucleotide
sequencing strategy and DEN 4 primers for C, PrM/M,
E and NSI are shown in Figure 1. Sequences covering
the whole structural and nonstructural NS1 protein
were determined from cDNA clones of six overlapping
gene regions (Table 2). The above mentioned primers
were used to amplify the gene regions for each of thefour selected strains of DEN 4 virus.
Amplz 5ling the target gene region ly RT‑FCR and purzation

For complementary DNA (CDNA) synthesis, 10 plof RNA samples were incubated at 65'C for 5 minutes

199

Table2

Nucleotide

sequences

of

Code

DEN4primers

used

in

this

study
Position

Sequence（5仁31）

54−72

GGA−AGC−TTG−CTT−AAC−ACA−GT
ACA−TCT−TGT−TGC−CGT−AGT−TA

42−61

AAC−AGG−AAT−CCA−GCG−AAC−TG
TGT−TAG−TTC−TCC−ATA−GTC−CG

878−897

D4−1484C
D4−1376S
D4−1978CAGA−CAC−CCA−TGC−AGT−AGG−AA
ACT−TTT−TCC−TTG−TTT−FHF−TH

1376−1395

1978−1959

D4−1831S
Di−2517C

CAT−ACA−CGA−TGT−GTT−CAG−GATGT−ACT−GTT−CTG−TCC−AAG−TG

2517−2498

D4−2370S
D4−3031CTGC−ATA−GCT−GTT−GGA−GGA

2370−2387

CTC−TCT−CAA−TAA−CCC−AT

3031−3012

D4−2258S
D4−3031C

AAG−TGT−GTA−TAC−AAC−CAT−GT
CTC−TCT−ATC−CAA−TAA−CCC−AT

3031−3012

D4−2906S
D4−3674C

GTT−CAC−GAC−CAA−CAT−ATG−G
AHH−AGA−CAT−AGT−GTC−CCC−C

D4−959C†
D4−42S

D4−1160C
D4−878S

regionC，PrM，M

AAC−ACA−GOO−CTA−ACA−GTT−T
CCT−ACT−CCT−ACG−CAT−CGC−AT

D4−54S＊

Gene

959−940

C，PrM，M

1160−1141

E

1484−1465

EE

1831−1850

NS1
NS12258−2277

NS12906−2924
3674−3656＊S：Sense，†C：Complementary

and

mi琴ed

Moloney

withmurinetranscriptase

αo％吻90％4s69％6ηo初9げ4召ngz ひ40P呪4
The purified cDNA was ligated into pCRTM II

strandvector10μlofRT−mixture

leukemiavims

reverse（M−MLV−RT）1μ」（200U／μ1），5X

firstcontaining
buffer

（Gibco BRI）4μ1，0．1M dithiothreito1（DTT）4μ1，
deoxynucleosi（ie57−triphosphates（dNTPs）0．5μ1（10−mM／μ1），RNase

plementary

inhibitor

primer

O．5μ1（10μmole／μ1）and

O．5，μ1（50pmol／μ1）．The

com−

mixtureusing

TACloning

was incubate（1at37。C for30minutes．Five microlitersof cDNA product were used for PCR reaction．A PCR

enzymedigestion

targetprimers．Aftermixtureclones

were

broth

mediumdNTPs

of45μlof10X

TTH

buffer5μ1，

O．5μ1（10mM／μ1）71h6槻κs孟h8朋oゆh歪1％s（Tth）

polymerase
sense

O．25μ1（4U／μ1），distilled

O．5μlμ1）waswascomposed

and

added

carriedcomplementary

to

out

the

cDNA

for35cycles

primerwater37．5μ1，
O．5μ1（50pmo1／

product．The
using

a

PCR

reactionthermocycler（MiniKit（lvitrogen，USA）andwasused to transform JM−1090r Xレ1blue Esoh副6h毎oolJ（Hanahan，1983）． Plasmids from the transformant
colonies were isolatedby the boiling method（Sambrook
6渉α1．，1989）and inもertion was comfirmed by EooRl
and

PCR

grown

amplification

using

a

confirmation，target

in5ml

of

pair

of

recombinant

Lubria−Bertani（LB）

supplemente（1with

Ampicillin

ovemight，and the plasmid DNA containing inserted gene waspurified with QIAprep Spin Miniprep Kit（QIAGEN，
Germany）and used for sequencing．
Each cDNA target region was sequenced in bothcycler TM，MJ research，USA）with94。C denaturation
for l minute，53。C amealing for l minute and72。C

directions，using

primerandprimer，5｝一CCC−

primerAGT−CAC−GAH−GTT−GT−3｝asacomplementary

extension

for

l

The

PCR

products

isolatewaspurified

minute．

of
by

each

regionagarose

gel

from

each

DEN4

electrophoresis

andGAC−CAT−G−3

primer

by

as

the

primer，5a

sense

dideoxygerαα1．，1997），using

chain
the

一GGA−AAC−AGC−TAT−

terminationDNA

method（San・

sequencing

readysubsequently extracted using the Gene Clean II Kit（BioRad，GL 1131−05，Bio101，La Jolla，Califomia，USA）．Purified samples were electrophoresed in3％tion

kit（Applied

Biosystems，USA）．Cycle

was

done

GeneAmp（PerkinElmer，USA）andagarose

（10

at

DNA

was

gel
kept

to

confirm

at−20。C

the
until

DNA

band，and

used．

the

purifiedsecannealing

cent

in
95。C

and4minlabelled

CENTRISEP
USA）and

a
for

PCR

for

products
by373A

out

sec

I

at25cycles
at

50。C

extension）．The

was

COLUMNS（Princetonsequenced

system2400machine

carrieddenaturation；5at60。C

PCR

was

DNA

reac−

sequence

purifiedforfluoreS−using

Separations，Inc；
sequencer

machine200

50o I OOO

1 500

5' C PrM M

CDNA Eclones []3OOO 3500 4OOO bp
NS1

E] I :[

42 ‑ 1 160

2000 25oO

]

[I

l

]

2906 ‑ 3674

2258 ‑ 3031

Figure I Schematic diagram showing the sequencing strategies of the four Thai DEN 4 isolates. The cDNA
clone in each region of C, PrM/M, E, and NS1 are shown in open boxes with nucleotide numbers.

(Applied Biosystems, USA) . To avoid sequence varia‑
bility, 3 different clones from each amplified fragments

were isolated and sequenced.
Nucleotide and protein sequence data analysis

Homology comparison and analysis of nucleotide

the M and NSI regions, with the divergence range of
8.44‑9.33% and 8.03‑8.52%, respectively. The C regionis the most conserved as shown in Table 3.

Comparison of amino acid sequences among the four
DEN 4 isolates and the prototype strain

The deduced amino acid sequences of four DEN 4

and deduced amino acid sequences were carried out for
all 4 strains and compared with published sequences of
DEN 4 Caribbean strain 814669 isolated in Dominican
Republic 1981 (Zhao et al., 1986; Mackow et al., 1987),

position I to 113, followed by PrM, M, E and NS1

with a DNASIS‑MAC Version 2.4, NEW CD3 Software

regions at positions ll4 to 204, 205 to 279, 280 to 773, andSystem (Hitachi Software Enginerring Co., Ltd., Tokyo,
1994). The nucleotide sequence of the PrM/E junction

from GEN BANK, NCBI. Phylogenetic analysis was

774 to 1,184 respectively. In all four isolates, thededuced amino acid sequences exhibited high homology
to the prototype strain. However, amino acid replace‑ments at certain position were also observed. When the

carried out with the aid of the PHYLIP package soft‑percentage difference in each region of the four isolates

ware (Felsenstein, 1993). The statistical reliability ofwere compared with the prototype strain, highest amino

the phylogenetic tree were assessed using the bootstrap
method, with 100 replications (Felsenstein, 1985). The

acid variation was found in the PrM (6.59%) region,
followed by the NS1 (6.32%), E (4.45%), M (4.40%) andC (1.17%) regions.

and E region from other DEN 4 strains was obtained

sequence from a representative of dengue serotype 3
virus was used as an outgroup to confirm reliability ofthe phylogenetic analysis (data not shown) .

isolates, compared to the prototype strain (Caribbean
814669) are shown in Figure 3. The C region begins at

Comparison of amino acids among four Thai DEN 4
isolates in relation to the clinical severity of the disease

RESULTS

Specific amino acid replacement among four Thai
isolates in relation to the clinical severity of the disease

Comparison of nucleotide sequences between recent iso ‑are shown in Table 4. The DHF grade 11 strain had

lates of four DEN 4 Thai strains and the prototype
The nucleotide sequences of four new DEN 4 Thai

specific amino acid replacements at positions 119, 145,
207, 470, 520 and 598. The DF strain was shown strain‑
specific amino acid replacements at position 136 having

isolates and the published strain (Caribbean 814669) arephenylalanine (F) instead of leucine (L) , at position 914

shown in Figure 2. The C region begins at position 1
until 339, followed by PrM, M, E and NSI regions at

having phenylalanine (F) instead of serine (S) and at

position 340 to 612, 613 to 837, 838 to 2,319 and 2,320 to(H) .

strain3,552 respectively. All four newly sequenced Thai iso‑lates revealed nucleotide sequence homologous to theprototype strain, with the base replacements occurring
tnostly on the third codon resulting in silent mutations.

The nucleotide sequences in the PrM region of the four
Thai isolates had the highest divergence from the proto‑

type strain ; ranging from 9.84 to 11.36%, followed byposition 1,042 having glutamine (Q) instead of histidine

Comparison of amino acid residues at M/E junction
and E region of DEN 4 Thai isolates and other isolates
from dlfferent location
The amino acid sequence of the four Thai isolates
and the other 19 isolates from different geographicallocations were compared. The amino acids at position

201

l‑･c

12Q

814669CT93‑77
CT93‑129
CT93‑158
CTg3‑74

ATG JLACca JLcGW AGGTGGTTAGACCACCTTTCAATATGCTGAAAcccGncAGAAACCGCGTATC･ACCCCTCAAGGGTTGeTGAAGAGATTCTCAACCGGACTTTTTTCTGGG2

8la669CT93‑77
CT93‑129
CTg3‑lS8
CT93‑74

GGACCCTTACGGATGGTGCTAGCATTGATCACGTTTTTGCGAGTCCTTTCCATCCCAceuCAGCAGGGATTCTGAAGAGATGGeGAC J TTGAAaLunTAAGccCATG JLAGATACTG‑‑‑‑T‑‑‑‑*‑‑A‑‑‑‑‑‑‑‑‑ T‑‑‑‑‑‑T‑‑‑‑‑ ‑ ‑T‑G‑‑ ‑‑ ‑ ‑‑ T‑‑‑‑‑A‑ ‑ ‑ * ‑ ‑A‑ ‑ ‑ ‑ c‑ ‑ ‑ ‑‑‑‑‑A‑‑‑‑‑‑‑ ‑ ‑A‑‑ ‑ ‑ ‑ ‑ ‑ ‑A‑‑‑‑A‑‑‑‑‑‑‑‑‑ ‑ ‑T‑G‑
‑
‑
‑
‑
‑
‑T‑
‑
‑
‑
‑
‑
‑A‑
‑
‑
c‑
‑
‑
‑
‑ ‑ ‑A‑ ‑ ‑ ‑ ‑ ‑ ‑A
‑A‑ ‑‑‑‑‑T‑‑‑‑‑ ‑ ‑T‑G‑‑ ‑ ‑‑‑ ‑ ‑T‑‑ ‑ ‑ ‑ ‑ ‑ ‑A‑ ‑ ‑ ‑ c‑ ‑ ‑ ‑‑‑‑‑A‑‑‑‑‑‑‑‑‑ ‑ ‑A‑ ‑ ‑ ‑ ‑ ‑ ‑A

811669CT93‑77
CT93‑129
CT93‑158
CT93‑74

ATTGGATTCAGGMGGAGATAGGCCGcATGCTGMCATCCTGMCGGGAGA,WAeGTCAAeGATILACATTGCTGTGCTTGATTCCCACCGTAa:TGGCGTTTTCCTTGTca JLC JLAGAGAT‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑‑‑T‑‑A‑‑G‑‑‑‑‑‑‑‑‑‑‑A‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑T‑‑‑‑‑‑‑‑‑‑‑‑cA‑‑‑‑‑‑‑‑‑G‑c‑‑‑‑‑‑‑‑‑‑‑‑‑*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑‑‑T‑‑A‑‑G‑‑‑‑‑‑‑‑‑‑‑A‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑T‑‑‑‑‑‑‑‑‑‑‑‑cA‑‑‑‑‑‑‑‑‑G‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑‑‑T‑‑A‑‑G‑‑‑‑‑‑‑‑c‑‑A‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑e‑‑T‑‑ ‑‑‑‑*‑‑‑‑‑‑cA‑‑‑‑‑‑‑‑‑G. ‑...‑‑‑‑‑‑‑‑*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑‑‑ ‑‑ ‑‑ ‑‑‑‑‑‑‑‑‑‑‑ ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑ ‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑‑‑‑‑‑‑‑G‑A‑‑‑‑

8laG69CT93‑77
CT93‑129
CT93‑158
CT‑74

GGCG JuLCCCCTCATGATAGTGGCAA JuCATGAAAGGGGGAGACCTCTCTTGTTTAAGACAACAGAGGGGATCAACAAATGCACTCTCATTGCCATGGACTTeGTGAuTGTGTGAGGAC.‑T‑. ‑*‑‑‑‑‑‑...‑ ‑‑‑‑G‑‑‑‑ ‑c‑‑‑‑‑‑‑ ‑‑‑‑‑‑T‑T‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑. .A‑ ‑A‑ ‑ ‑ ‑.T..G‑‑.‑‑‑‑ .T‑.‑‑‑‑‑‑‑‑‑ ‑c‑‑‑‑‑‑ ‑G‑‑‑‑ ‑c‑‑‑‑‑‑.‑T‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑G‑ ‑ ‑ ‑‑c*‑‑‑‑‑‑‑‑‑‑‑ ‑T‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑. .A‑ *A‑ ‑ ‑ ‑‑T‑.G‑‑*‑‑ *‑‑1 ‑‑‑‑‑‑‑‑‑‑ ‑c‑‑‑‑‑ ‑.G‑‑‑‑ ‑c‑‑‑‑‑‑‑*T ‑‑‑‑‑‑..*.. ‑‑‑‑‑‑‑‑G.‑.‑ ‑c‑‑*‑‑‑‑‑‑‑‑*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑A‑ ‑A‑ ‑ * ‑ ‑T‑‑T‑‑.‑‑‑‑ ‑T.‑‑‑‑‑‑‑‑‑‑ ‑c‑‑‑‑‑‑ ‑G‑‑‑‑ ‑c‑‑‑‑‑‑‑‑T ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑G *‑‑‑‑c ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑ ‑‑‑ ‑ ‑‑ ‑‑**‑‑‑‑l ‑‑‑‑‑‑‑‑‑‑‑ c‑‑‑‑‑‑‑ G‑‑‑‑‑c‑‑‑‑‑‑

814669AaTGTCACGTATAAATGCCCCCTACTGGTCAATACCGAACCTCAa,GACATTGATTGCTGCTGCAACCTCACOTCTACATG BTCATGTATGGGAeATGeACCCACAGceGAGAACGGAGA‑‑c‑‑T‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑‑‑･‑‑‑‑‑‑‑‑‑‑‑‑‑‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑*‑**‑T*‑‑‑‑‑‑‑‑G‑(･‑‑‑‑T‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑A‑‑T‑‑‑‑‑‑‑‑A‑‑G‑*c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑T‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑*c‑‑‑‑‑‑*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑‑‑‑‑‑‑‑, c‑‑‑‑‑T‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑A‑‑T‑‑‑‑‑‑‑‑A‑‑G‑‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑T‑‑T‑‑‑‑‑‑‑‑‑‑‑‑‑G‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑‑‑‑T‑‑‑G‑C‑‑‑‑‑T‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑A‑‑T‑‑‑‑‑‑‑‑A‑‑A‑‑ ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑ ‑‑ ‑‑‑‑ ‑‑‑‑‑.‑. ..‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑.‑‑‑‑‑ ‑‑..‑‑‑‑‑ ‑ ‑‑‑‑. .‑...‑.‑**.‑.‑‑..‑.‑ ‑. ‑‑‑‑‑‑‑‑A‑‑G*‑‑c‑‑‑‑‑‑‑<;‑‑T‑‑‑‑‑‑c‑‑‑‑‑‑G‑‑‑c‑‑‑‑ ‑‑‑(:‑‑T‑‑‑‑‑‑‑c‑‑‑‑ ‑‑‑ ;‑‑T‑‑‑ ‑‑‑c‑‑‑‑‑‑ ;‑‑‑‑‑‑ ‑‑‑c‑‑‑
240‑‑‑T‑G‑‑‑ ‑‑‑‑A‑ ‑‑ ‑‑‑‑A‑‑‑ c‑‑‑ ‑‑‑‑A‑‑‑ ‑‑‑‑All,Prl(3GO

480

A A CT T

600

CT9 3 ‑ 7 7

CT93‑129
CTg3‑158
CT93‑74

c T T T c G T GT T A T
ll,bt

72o

814669CTg3‑77
CT93‑12g
CTg3‑158
CT93‑74CGAGAGAAGCGCTCAGTAGCTTTAACACCACATTCAGGAATGGGATTGGA JLACAAGAGCTGAGACATGGATGTCATOGGAAGGGGCTTGGAAGCATGCTCAGAGACTAGAGAGCTGGATA‑ ‑A‑‑‑‑‑‑ ‑ ‑ ‑ ‑c‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑G‑‑ ‑ ‑ ‑ *A‑ ‑‑.A.‑‑‑‑c‑‑c‑ ‑ ‑ ‑ ‑‑G‑‑.
‑ ‑‑T‑‑ ‑‑A‑ . . ‑‑ ‑ ‑ ‑ ‑ ‑c‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑G‑ ‑ ‑ ‑ ‑I r ‑ ‑ ‑ ‑ ‑‑ ‑‑A‑‑c‑ ‑c‑ ‑ ‑ .‑ G..‑
‑T‑ ‑ ‑

814669CT93‑77
CT93‑129
CT93‑lS8

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G‑ ‑ ‑‑‑T‑‑‑‑ ‑ ‑‑‑‑G‑‑‑‑‑‑G‑ ‑ ‑ ‑‑‑‑c‑‑c‑‑‑‑ ‑‑c‑‑‑‑ ‑c‑ ‑ ‑ ‑‑‑‑A‑‑‑‑‑ ‑ ‑A‑ * ‑ ‑ ‑‑‑cA*T‑‑‑‑* ‑ ‑CA‑g ‑ ‑ ‑ ‑‑‑‑T‑*‑‑ * ‑ ‑T‑‑‑G‑‑‑ ‑‑‑G‑‑‑*‑ ‑‑c‑‑‑ ‑‑‑‑A‑‑‑ ‑‑‑cA‑T*‑‑‑ ‑‑‑‑T‑‑‑‑ ‑ ‑c‑ ‑ c‑ ‑ ‑960
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CT93‑7a

GATTTTGAACTGACTAAGACAACAGCGAAG';MGTGGCTCTGTTA JL!GA'LCCTATTGCATTGuGCCTCAATATCAMCATMCTACGGCAACAAeATGTCCAACGCMGGAGAGCCTTAT‑c‑T‑‑C‑
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cT93‑129cT93‑158
cr93‑7a

TCGGGGAAGATAACAGGCAATTTGGTCCGMTTGAGAACCTTGAATACACAGTGGTTeTMCAGTCCACAATeeAGACACCCATGCAGTAGGwTGACACATCCnLTcnTGGAGTTACA

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CT93‑129
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CT93‑158
CT93‑74

Tc‑ ‑ ‑ ‑ ‑ ‑ ‑T‑ ‑c‑ ‑ ‑ ‑ ‑ ‑ ‑ ‑c‑ ‑ ‑ ‑ ‑ ‑ ‑c‑‑‑Tc‑‑‑‑‑‑*T‑‑c‑‑‑‑ ‑‑‑‑c‑‑‑‑‑‑c‑‑‑‑ ‑‑‑‑c‑‑‑c*‑‑Tc‑‑‑ ‑‑‑‑T‑‑c‑‑‑‑
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1320

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Figure 2 Comparison of nucleotide sequences of the structural (C, PrM/M, E) and nonstructural NSI protein
genes among four Thai DEN 4 isolates and the prototype strain (814669) . Nucleotide base positions

are numbered beginning from C gene.

202

814669CT93‑77
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180a
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A‑ ‑G ‑‑‑‑‑‑‑‑‑‑‑G‑‑‑A ‑T‑‑‑‑‑‑‑‑A‑‑‑‑‑‑‑‑T‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑G‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑C‑‑‑‑‑‑‑‑‑‑‑T‑‑‑‑‑‑‑‑T‑‑‑‑‑‑‑‑‑‑‑‑

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CT93‑129
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1920

2040

G‑‑ ‑‑ ‑ ‑‑‑‑G‑‑‑G‑‑‑‑T‑‑‑‑‑‑‑G‑‑‑G‑‑‑‑T‑‑‑‑‑‑c‑‑‑
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2520

8la669cT93‑77
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8146G9TGGAAGCAMTMCCAACGAGCTAAACTATeTTCTCTGGGAAGGAGGACATGACCTaLCTGTAGTGGCTGGGGATGTGAAGGGGGTGTTGACCMAGGCMG,LGAGCACTCACACCCCCA

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CT93‑74
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CT93‑74‑ ‑ ‑A‑* ‑ ‑‑ ‑ ‑A‑‑ ‑ ‑2760
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CT93‑77
CT93‑129
CT93‑158
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814669CT93‑77
CT93‑129
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CT93‑7aATTA JLAGATCAewGCTGTGCATGCTGACATeGGTTATTGGATAGAGAGCTC:A, cCAGACTTGGCAGATACAGAILGCATCTCTTATTGAAGTGAn cATeTCTGTGGCCCAAG‑‑c‑‑‑‑ ‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑T..‑‑ ‑c‑‑*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑c‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑c‑‑‑‑ ‑c‑‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑c‑‑‑‑ ‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑T‑‑*‑ ‑c*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑c‑‑‑‑‑‑‑‑‑‑‑ ‑‑‑G‑‑‑‑ ‑c‑‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑c‑‑‑‑ ‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑T‑‑‑‑ ‑c‑‑‑‑‑‑‑*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑‑c‑‑‑‑‑‑‑‑‑‑‑‑‑ ‑G‑‑‑‑ ‑c‑‑c‑‑‑‑‑‑‑‑*‑*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑c ‑‑‑‑‑c ‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑T‑‑‑‑‑c ‑‑‑‑*‑‑*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑c ‑‑‑‑‑‑‑‑‑‑‑‑‑‑G ‑‑‑‑‑c‑‑c‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑

8146G9CT93‑77
CT93‑129
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‑‑‑T‑‑‑‑‑‑c‑‑T‑‑‑‑‑G‑‑‑‑‑‑‑G‑‑‑T**‑‑‑‑C‑‑T‑‑‑‑‑G‑‑‑‑‑‑A‑‑‑T‑‑‑‑‑‑‑‑T‑‑‑‑‑G‑‑‑‑･･‑‑G‑‑‑‑‑‑‑‑‑T‑‑‑ ‑‑‑c‑‑T‑‑‑‑‑G‑‑‑ ‑‑‑‑G‑A‑‑‑c‑‑ ‑‑‑ G‑‑‑‑ ‑ ‑ ‑A‑ ‑‑ ‑‑ ‑A‑ ‑ ‑ *‑‑‑C‑‑ ‑‑‑ ‑‑‑‑ ‑ ‑ ‑A‑ ‑‑ ‑‑ ‑A‑ ‑ ‑ ‑‑‑‑c‑‑ ‑‑‑.G‑‑‑ ‑‑‑‑A‑‑ ‑ ‑ ‑ ‑A‑ ‑ ‑‑‑‑C‑‑ ‑‑‑ ‑‑‑‑ ‑ ‑ ‑A‑ ‑‑ ‑‑CA‑ ‑ ‑ ‑3240
TGCCACTTAGGCA'uTTAGAGATAGACTTTGGAGAATGCCCCGGAACAACGGTCACAATTCAGGAGGATTGTGACCATAGAGGCCCATCTTTGAGGACCACCACTGCATCTGGAI ACTA‑‑‑‑‑A ‑‑G ‑‑‑‑‑‑‑‑G ‑‑‑*‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑‑
AT ‑‑‑
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3360

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C‑ ‑ ‑C* ‑ ‑ ‑ ‑ ‑C‑ ‑ ‑ ‑ ‑T‑ ‑A‑

C‑*‑C‑‑‑ ‑‑‑C‑‑‑‑‑1 ‑A‑

C‑ ‑ ‑C‑ ‑ ‑ ‑ ‑ ‑C‑ ‑ ‑ ‑ ‑T‑ ‑A‑

C‑‑‑C‑‑‑ ‑‑‑C‑‑‑‑‑T‑*A‑

Figure 2 continued.

‑‑‑‑T
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Figure 3 Comparison of deduced amino acid sequences of the structural (C, PrM/M, E) and nonstructural

NSI protein among four Thai DEN 4 isolates and the prototype strain (814669). Amino acidpositions are numbered beginning from C gene.

Table 3 Nucleotide and amino acid sequence divergence and percentage of divergence among the four new Thai
DEN 4 isolates compared with the prototype strain (814669)
Sequence divergence from 814669 strain

Gene region

CT93‑77nt * (%)

CPrM

aa t (%)

CT93‑129
nt(%)
aa(%)

CT93‑158
nt(%)
aa(%)

CT93‑74
nt(%)
aa(%)

22 (6 . 49)

2 (1 . 77)

22 (6 . 49)

2 (1 . 77)

20 (5 . 90)

1 (O . 88)

22 (6 . 49)

2 (1 . 77)

28 (10 . 26)

4 (4 . 40)

27 (9 . 89)

3 (3 . 30)

28 (10 . 26)

4 (4 . 40)

31 (11 . 36)

6 (6 . 59)

20 (8 . 89)

2 (2 . 67)

19 (8 . 44)

2 (2 . 67)

20 (8 . 89)

3 (4 . OO)

21 (9 . 33)

3 (4 . OO)

E
NS1

1 13 (7 . 62)

20 (4 . 05)

118 (7 . 96)

25 (4 . 05)

119 (8 . 03)

19 (3 . 85)

117 (7 . 89)

22 (4 . 45)

105 (8 . 52)

26 (6 . 32)

99 (8 . 03)

21 (5 . 11)

102 (8 . 27)

25 (4 . 87)

105 (8 . 52)

25 (6 . 03)

Whole

288 (8 . 11)

54(4 56) 285(8 02) 48(4 05) 289(8 14) 42(3 97) 296(8.33)

58 (4 . 90)

M

* nt

nucleotide, t aa

amino acid

269, 374, 482 and 608 were unique to the Thai isolates.
These was at position 269 having isoleucine (D insteadof valine (V), at position 374 having methionine (M)instead of valine (V) , at position 482 having threonine(T) instead of lysine (K) and at position 608 having
threonine (T) instead of alanine (A) as shown in Table
5.

204

Table 4 Position of salient amino acid changes from C to NSI regions of DEN 4 isolates withdifferent clinical severities

Amino acid replacements in
Isolates

CT93‑77
CT93‑129
CT93‑158
CT93‑74

Clmrcal seventy 119

DF
DHF Grade I
DHF Grade I
DHF Grade II

R
R
R

K

M

PrM
136

F
L
L
L

NS1

E

145 207 470 520 598
T
A I
K
N
K
T
A
N
K
N
A
T
V D
A
H
I
I

914

1042

F

Q

S
S
S

H
H
H

One letter symbol amino acids with abbreviations : A=alanine, D=aspartic acid, F=phenilalanine, H =histidine, I = isoleucine, K=1ysine, L=1eucine, N = asparagine, Q=glutamine, S =serine, T =threonine,
V = valine

Genot pe determination of Thai strains from M/E junc‑tion and E region
For the genotypic classification of the 7 Thaistrains, 1,530 nucleotides (map position 690‑2,219)
from the M and E regions, were used to construct aphylogenetic tree (Figure 4). The tree showed that the

DISCUSSION
The nucleotide and amino acids sequences of theentire analyzed genomic regions in four Thai strains inthis study differed from prototype strain, ranging equal
to or lower than 8.33% and 4.90%, respectively (Table

strains analyzed in this study could be divided into three3). The rates of variation among each strain weremajor groups. Group I consisted of I isolate (TC 2443)
from Thailand in 1963, group 2 included isolates fromCentral America, Brazil, Tahiti and Indonesia and group
3 included the remaining 6 isolates from Thailand, 2
isolates from the Philippines and I isolate frorn Sri

sirnilar to the other dengue serotype such as Thai DEN

Lanka.

2 sequences, which were isolated from Maha Sarakham
in 1986‑1987 (Duangchanda et al., 1994), and Nakorn
Phanom in 1993 (Thant et al., 1996), compared withDEN 2 published strains. However, dengue viruses also
seem to have lower nucleotide and amino acid change
than the other RNA viruses (Buonagurio et al., 1986;

Table 5 Position of salient amino acid changes in M/E junction and E region of DEN 4 isolates
compared with other strains isolated from different geographical areas
Strains

Geografical source

Year isolated

814669Dominican Republic

1981

1411, 6494

El Salvador

1983, 1994

1492

Mexico
New Caledonia

1984

5489
1650

Puerto Rico

1986

1385

Brazil1982

Tahiti

1985

S‑4475030153, 1036, 1132
H‑241, 16589‑64, 12123

Sri Lanca
Indonesia

1978

TC2443ThailandThailandThailandThailandThailandThailandThailand

D78‑01
D84‑024

CT93‑77
CT93‑129
CT93‑158
CT93‑74

One letter symbol amino acids with abbreviations

T =threonine, V=valine

E
374

482

608

V

V

K

A

1984

S‑44754, 114‑094‑85

PhillipinesM

269

1973, 1976, 1977
1956, 1964, 1984
1963

1978

I

1984

I

1993

I

1993

I

1993

I

1993

I

M
M
M
M

T
T
T
T

T
T
T
T

A= alanine, I = isoleucine, K=1ysine, M = metheonine,

205

Strain Geographlcal Yearsignificant differences (data not shown) .

SourceisolatedThalland'63

301 53

Indonesia

'73

1 1

Tahitl'85

1 1 32Indonesia

.77

1 65Q

Puerto Rico

.86

81 4669

Dominlcan Rep.

.81

1 492

Mexico'84

1 385

Brazil'825489

New Caledonia

'84

1411

El Salvador'83

S4475O

Sri Lanka.78

1 6589'64

Phili p pines' 64

1 21 23

Phili p plnes'84

CT93･74

Thailand.93

CT93･1 58

Thailand.93

CT93‑1 29

Thailand'93

evidence that dengue viruses change genetically andCT93‑77

Thailand' 93

some strains have greater epidemic potential (Gubler et

D84‑024

Thalland.84

D78‑O1Thailand'78

al., 1978; Lanciotti et al., 1994). According to theantigenic and genetic variation of arboviruses whichhave been described in many studies (Haishi, 1990; Ali

TC 2443094‑85O.･lFigure 4 Phylogenetic relationships among 19 DEN 4
strains isolated from different geographicalareas. Phylogenetic tree was constructed bycomparison of all nucleotide sequences (mapposition 690 to 2,219). Genetic distance wascalculated by DNA dist program. Distancematrix was constructed andphylogenetic tree

was deduced by UPGMA method in the
PHYLIP package program.

The phylogenetic tree showed that the dengue 4
isolates from different geographical ares segregatedinto three separate branches, suggesting the presence of
three genotypes (Figure 4). Interestingly, two isolates

obtained from Indonesia were found to be closely
related to the other American strains and the isolate
from Tahiti.

There were two main hypotheses concerning thepathogenesis of DHF/DSS. One was that immunopath‑ological mechanisms of hypersensitivity reaction orenhancement of virus replication resulting from secon‑
dary infection by heterotype of dengue viruses (Hal‑stead, 1970). On the other hand, several people suggest‑ed that virulence of infecting viruses might be related tothe severity of the disease (Rosen, 1977). There is also

and lgarashi, 1997; Thant, et al., 1995), the mechanism
which associated with the antigenic strain variation isstill not completely understood. It has been proposedthat particular amino acid replacements or combina‑
tions may lead to antigenic variation and its path‑ogenicity among strains. The evidence obtained in thisstudy shows that three positions of one DF strain andthree DHF Thai strains had strain‑specific amino acidreplacements at 136 of PrM region (F ‑ L), 914 (F ‑S) and 1,042 (Q ‑ H) of NS1 regions. These three

Rico‑Hesse et al., 1987; Nichol et al., 1989; Rico‑Hesse,
1990; Tanaka et al., 1991; Zheng, 1994; Ali et al., 1995).

positions may be critical for severity and are points ofMost of the amino acid replacements in the f ourinterest to be analyzed further by comparing nucleotide

Thai strains compared with the prototype strain did notalter the nature of the proteins. However there weresome amino acid replacements, that could significantly
change the nature of the proteins in comparison to theprototype strain : from hydrophobic to hydrophilic : atand amino acid sequences from various DEN 4 isolateswith different disease severity in the same particular

position 81 (1' T) , at 442 (M ‑> T) , at 608 (A ‑> T) , at901 (A ‑> T), at 1,085 (V ‑> O , and at 1,163 (1 ‑ T). On

the other hand, amino acid replacements in the opposite
direction from hydrophilic to hydrophobic were found at
position 186 (T ‑ A) , at 325 (T ‑ D , at 878 (T ‑ A) , attime and place.

ACKNOWLEDGEMENTS
The author would like to express my deepest thanks
to both of my advisors, Professor Dr. Akira lgarashi,
Department of Virology, Institute of Tropical Medicine,
Nagasaki University, Japan and Professor Dr. Savanut

1,019 (S ‑ A) and at 1,077 (S H･ F) (Figure 3). The

Tharavanich, Departynent of Microbiology and Im‑

amino acid at positions 269, 374, 482 and 608 in M and E

munology, Faculty of Tropical Medicine, Mahidol Uni‑

regions were found to be unique for the Thai strains

versity, Thailand for their valuable advice and encour‑(Table 5). These amino acid replacements may haveagement. Thanks should be given to Dr. Suchitra

led to variations in the antigenicity.

Ni nmanitya, Dr. Siriphen Kalayanarooj, Dr. David W

The mean hydrophobicity and isoelectric point val‑ue in each region had been analyzed. Both of this value

Vaughn, Dr. Khunying Anada Nisalak, Dr. Kouichi

in each region and whole study region, did not show

Morita, Dr. Futoshi Hasebe, Dr. Kyaw Zin Thant, Dr.
Wei‑Yun Zheng and Dr. Ronald Matias for their gener‑

206

ous support and advice.
This work is in partial fulfillment of the require‑ments for the PhD degree of Charnchudhi Chanyasanha,
in the Graduate School in Medical Sciences, Nagasaki

University, Japan. The author was supported by the

Ronpaku Program from The Japan Society for the
Promotion of Sciences (JSPS).
Financial support: This study was financially supportedby the budget of the Department of Virology, InstituteF., Nimmannitya, S., Tharavanij, S. and lgarashi, A.
(1995): Dengue virus isolation and viral genome detec‑tion by reverse transcriptase‑polymerase chain reaction

from serum specimens of dengue patients. Southeast
Asian J. Trop. Med. Public Health, 23, 495‑502
11) Chou, P.Y. and Fasman, G.D. (1978): Prediction of thesecondary structure of proteins from their amino acidsequence. Adv. Enzymol., 47, 45‑158

12) Chu, M.C., O'Rourke, E.J. and Trent. D.W. (1989):
Genetic relatedness among structural protein genes ofof Tropical Medicine, Nagasaki University and bydengue I virus strains. J. Gen. Virol., 70, 1701‑1702

Grant‑in‑Aid for Scientific Research No. 07459098 from
the Ministry of Education, Science, Sports and Cultureof Japan, with principal investigator of Professor Akira

13) Coia, G., Parker, M.D,., Speight, G., Byrne, M.E. andWestaway. E.G. (1988): Nucleotide and complete amino

lgarashi.

acid sequences of Kunjin virus : definite gene order andcharacteristic of the virus specified proteins. J. Gen.
Virol., 69, 1‑214) Duangchanda, S., Tanaka, M., Morita, K., Rojanasu‑

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J pn. J. Trop. Med. Hyg., Vol. 25, No. 4, 1997, pp. 209 213

Research Note

IN VITRO EFFECTS OF DECOCTIONFROM FIVE SPECIES OF CHINESE
PLANTS AGAINST TRICHOMONAS VAGINALISZHANG YONGSHENGl, JUN MAK12, DU JUNl AND YOHICHI IT02
Received May 16, 1997/Accepted October 2, 1997

Abstract: In vitro effects of extracts from the 5 species of Chinese herbal medicine against Trichomonas
vaginalis, one of the most important protozoal diseases in China, were investigated. It was demonstrated

that the extracts from Artemisia annua. Phellodendron amurense and Pulsatilla chinensis were highly
effective against the protozoa at I to 5 hr after the start of the exposure to the drugs. The extract from
Cnidium monnieri was much less effective and that from Sophora flavescens showed no activity even after
the exposure for 5 hr. After this screening for excluding non‑effective drugs, the parasites were exposed toall of the drugs except the extract from S. flavescens, the negative one in the above test, for 5 days at the

drug concentrations lower than those above. The extracts from A. annua and P. chinensis were highly
effective, immobilizing the parasites completely 2 or 3 days after the start of the exposure and afterwards.

Contrarily, those from C. monnieri and P. amurense were not so effective. The results obtained in thepresent studies were in favour of the prospect that further studies including those on side effects, would be
worthy of being carried out for the establishment of the chemotherapy of T. v ginalis infection with thecrude drugs from medicinal plants in China.
Key words: Trichomonas v ginalis. Chinese medicinal plants, therapy

INTRODUCTION

hopefully come in, based on scientific exploitation of
traditional medicinal plants like a global strategy for

There are a large number of obstinate parasitic

the control of obstinate parasitic diseases (Maki et al.,diseases still nowadays in Asian and other countries on

the earth. Though several excellent drugs have be n

1996). This is, at least in part, in accordance with thefamous fact that cloroquine‑resistant malaria is treatedsynthesized for their treatment and control, there still

nowadays with quingha0 u, originally a traditional

remain matters to be considered. Excellent synthesized

Chinese drug from the plant, Artemisia annua.
In this line of thought, the present authors have been

drugs are not readily available in local villages becauseof their high cost and inconvieniences in transportation.

testing efficacy of extract from medicinal plants on

This fact might be rather a minor one. Howev.er, there

various kinds of parasites (Maki et al., 1997) . Effects ofis an important problem. Some strains (isolates) of

decoction from a number of Chinese plants against

parasitic protozoas and helminths have been found to beinsensitive to excellent synthesized drugs (Boreharn,
1995; Geerts et al., 1997). For instances, according to

Trichomonas vaginalis have been one of the topics whichinterest them.

Boreham (1995), some lines of Giardia resistant toMuch attention has been paid to a large nurnber ofChinese medicinal herbs because of their possible utility

quinacrine and albendazole have been isolated.

for the control of various kinds of parasites (Shen, 1992;

The situation with these facts is thought to bewhere the possible development of new drugs would

Zhao, 1983; Zhao et al., 1991). Traditional medicinal

1

2

plants, A. annua. Cnidium monnieri. Phellodendron

Department of Parasitology, Norman Bethune University of Medical Sciences, Changchun 130021, P.R. China
Department of Parasitology, Kitasato University School of Medicine. Sagamihara 228, Japan

210

Table I Efficacy in scores of the extracts from the 5 speciesof Chinese plants against Trichomonas vc ginalis

duration of exposure

Extract diluted(4 to 64 times) I hr 2 hr 3 hr 4 hr 5 hr

Table 2 Efficacy of the extract from the 4 species of theplants in the reduction of the number of T. v ginalis

Extract diluted Reduction in %(64 to 512 times) 'Duration in days after the start of exposure

1 day 2 day 3 days 4 days 5 days

A. annua
4

4

4

4

4

8

4

4

4

4

4

16
32

4

4

4

4

4

4

4

4

4

4

64

3

4

3

4

4

1

1

1

2

1

1

1

1

C. monnieri

A. annua

64 95 93 100 100 100

128 78 83 72 86 86
256 37 18 56 82 75

512 52 31 5 69 65
64 86 84 79 92 88

C. monnieri
8

1

1

16

O

O

32
64

O

O

O

1

O

O

O

O

O

O

4

4

4

4

4

8

4

4

4

4

4

16
32

2

4

4

4

4

o

3

3

4

4

64

o

2

2

4

P. amurense

P. chinensis
4

4

4

4

4

4

8

4

4

4

4

4

16

4

4

4

4

4

32
64

4

4

4

4

4

3

4

4

4

4

o

O

S. flavescens

128 74 72 76 90 85
256 25 41 56 63 59

512 7 37 60 64 57
64 89 99 83 76 50

P. amurense

128 81 84 70 79 48
256 38 39 58 83 51
P. chinensis 512 44 35 69 75 51
64 86 100 100 100 100

128 69 98 99 99 84
256 74 65 57 41 23

512 8 39 54 39 33

The mean number of parasites per 10 /1 1 of the medium inthe control group with standard deviation was originally record‑ed. However, statistical analysis was not carried out because
the purpose of this study was to find such a decoction at a
certain concentration that will prohibit the movement of the

4

o

8

o

16
32
64

o

o

O

o

o

o

o

O

o

o

o

o

O

action. One of the components of P. amurense (bark),ooberberine has an activity against bacterial infection. P.

1oo

Number of T. vaginalis moving per field of the view (10X
10) was recorded with the result of more than 50 parasites incontrol groups. The efficacy of the extracts tested was expres‑sed in scores in this table. The efficacy score 4, 3, 2, I or O wasgiven to the groups in which the number of moving parasites wasO, 1‑5, 6‑lO, 11‑50 or more than 50, respectively.

parasites completely (see underlined parts) .

chinensis (roots) containing saponin is useful for thetreatrnent of Entamoeba histolytica infection andm tlaria. And S. flavescens (roots) is said to have ananthelmintic action.
It is intriguing to find out a possible efficacy of theextract from the plants including C. monnieri against T.

amurense. Pulsatilla chinensis, and Sophora flavescens

were chosen for the present study. These medicinal

vaginalis. The present communication describes thecomparative effects of the plant extracts against T.

plants are readily available in China, mostly seen in thevaginalis cultured in our laboratory.

fields of North‑East area of China.
The clinical utility of the 5 plants for therapy of
infectious diseases is described in a number of publica‑
tions including Japanese books. For example, according

Decoction tested were as follows. The commer‑

to Mitsuhashi (1988) and Namba (1993 a, b) , theseplants are useful for the treatment of parasitic andcially available medicinal plants in China, A. annua. C.
monnieri. P, amurense, P. chinensis, and S. flavescens

bacterial infections as follows. A. annua (whole herb)
is a plant of established utility for the treatment ofmalaria as mentioned abo, ve. Extract from C. monnieri(fruits) is described as a drug having anti‑T. v ginalis

was added to 100 ml of distilled water. They were each

MATERIALS AND METHODS

were washed and dried. Five grams of the materials
boiled for 30 minutes. The extract was filtered andpreserved as the first part of the drug extraction. Fifty

211

ml of distilled water was added to the residue and thiswas boiled again. The extract was filtered to obtain thesecond part of drug extraction. The first and second
portion were mixed. The mixed extract was boiled tobe concentrated to 10 ml. As a result, 10 ml extraction
was prepared using 5 g of the materials. This decoction
was diluted at two folds repeatedly up to nine times withsterilized distilled water. For the test in which theparasites were exposed to the extract for 5 hr, it had
been diluted at 4, 8, 16, 32 and 64 folds and for the
exposure for 5 days at 64, 128, 256 and 512 folds.

T. v ginalis were donated from School of Medicine,days. Ten pl media taken was subjected to the observa‑
tion and count of the moving and immobilized parasites
everyday for 5 days. The criterior for the distinction

between moving and immobilized parasites was basedon the observation above described. The test was
repeated three times.

RESULTS
Preliminary study to exclude non‑effective drugs
The results of the preliminary tests based on theobservation for 5 hr (Table 1) showed that the extract

Tokai University, Kanagawa Prefecture, Japan. Theyof A. annua. P. amurense and P. chinensis was highly

were maintained in Asami media containing about 1,500

effective in immobilizing the parasites. These 3 kinds ofparasites/ml before proliferation at 36.5 0.5'C

extraction showed high efficacy against the parasites as
early as I hr after the start of the exposure. Especiallythroughtout the present studies.
A preliminary study was first carried out in order to

exclude non‑effective drugs among the extracts fromthe 5 species of the plants. The decoction diluted withsterilized distilled water at a desired concentration (orthe water in the controls) and the culture medium
containing the parasites were mixed at the ratio of 1:5
throughout the present studies. In the first study, 10 plthe extract of A. annua and P. chinensis were found to
be extremely effective irrespective of the drug concen‑
trations tested at I hr after the start of the expdsure.

Compared with the efficacy of the extracts from A.
annua and P. chinensis, that from P. amurense ramained
less effective at lower concentrations of the extract
until 3 hr after the start of the exposure. At 4 and 5 hrof the suspension was taken onto glass slides for theirobservation at 1, 2, 3, 4 and 5 hr after the mixture had
been put into each pore of a plastic plate. Three fieldsof the light‑microscope view (10X 10) were checked tocount the number of the moving (including "moribund")after the start, there was scarcely any differences

or immobilized parasites. They were observed some‑times at l0><40 whenever their close examination wasnecessary. The test was repeated three times for the

extract from C. monnieri seemed to have somewhat

confirmation of the reproducibility. A special attention
was paid as follows to distinguish the moving parasites
from the immobilized ones during the preliminary study
for 5 hr and the subsequent one for 5 days. The normal
parasites like those in control groups were seen rotating

under the present experimental conditions.
Thus, the present preliminary study showed that theextracts from all the plants examined except S. flaves ‑cens had, more or less, an effect immobilizing or killingT. vaginalis in vitro following the exposure for 5 hr oractively or going back and forth with the movement ofless. As the decoction from S. flavescens showed no

the anterior flagella. The cytoplasm of the normal

efficacy, this was omitted in the subsequent study.

parasites was clear with transparent granules distribut‑
ed in the inside. The moribund parasites were attachedamong these 3 test materials.
The extracts of C. monnieri and S. flavescens were
found to be much less effective if they had any efficacy(Table 1). At 5 hr after the start of the exposure, theeffectiveness at 4‑ or 8‑fold dilution. However, noefficacy was detected in the extract from S. flavescens

Examination for finding such drugs that immobilize thewith more and more materials with the passage of time.
The movement of the anterior flagella and undulating

parasites completely at certain concentrations

membrane became slower and ceased eventually. ‑The

monnieri. P. amurense and P. chinensis at comparative‑
ly low concentrations were examined for 5 days (Table

cytoplasm of the immobilized parasites contained larger

The effects of the extracts from A. annua. C.

and obscure granules of poor transparency. Their

2) .

distribution in the cytoplasm was not clear.
The subsequent study was carried out in an attempt
to select drugs that immobilize the parasites completely
at a certain concentration. In this study, the test tubes

The extracts from A. anmua and P. chinensis were
found to be highly effective, immobilizing 100% of theparasites at 64‑fold dilution, 2 (P. chinensis) or 3 (A.

containing T. vaginalis and the drugs except the decoc‑tion from S. flavescens in the medium was stood for 5

annua) days after the start of the exposure and after‑wards. No complete immobilization was seen under thepresence of these extracts at 128‑fold dilution or less

212

concentrations.

a drug effective against the disease caused by T.

The extract from C. monnieri was shown to be lesseffective than those from A. annua and P. chinensis.
The efficacy of. P. amurense decoction against T.
vaginalis was nearly the same as that of C. monnieri
during 3 days from the start of culture.

vaginalis (Mitsuhashi, 1988; Namba, 1993a), the effec‑
tiveness was not high as far as the present study wasconcerned.
The extract frorn P. amurense was fairly effectivein the test of the exposure for 5 hr while it was not so
effective in that for 5 days. What was resposible for thisDISCUSSION
Infection with T. vaginalis was found whereversearch was made (Faust et al., 1970). In Japan, 5‑10%

female and 1‑2% male adults were infected with theparasite though the percentage was decreasing (Yo‑shida, 1992) . This sexually transmitted disease, severely

is a matter for speculation now. One of the possibilities

is that effective components in question are unstable.

Thus, the extracts described as a drug effective
against protozoal and bacterial diseases were found to
reduce the number of T. vaginalis in vitro. However,
the extract from S. flavescens, thought to be useful ineliminating parasitic helminths (Mitsuhashi, 1988;

harmful to fernale health, is still nowadays a problem ofNamba, 1993a) was not effective against the parasitic

public‑health importance in China. Though the infec‑
tion rate varies from region to region, one third offemales are presumably suffering from the infection

protozoa, T. vaginalis in the present study.
Based on the data so far obtained, the extracts frorn

with the parasite in some areas of China (Zhang, unpub‑
lished) . Thus, unfortunately too many people have been
infected with this parasite all over the world.

The curative efficacy of metronidazole (flagyD

A. annua and P. chinensis are thought to be promisingbecause they could suppress the movement of the para‑sites completely under certain conditions. However, thepossible side effects of the extracts under the conditions
still remain to be studied before clinical application.

against T. vaginalis is well‑known. However, this drug
is accompanied with such difficulties that it is rather
expensive for many patients as a local standard and is
not readily available because of transportation problems

cial for the basic study leading to the development of
new drugs from the wealth of Chinese medicinal plants
which have been believed to be useful from the ancient

as mentioned in INTRODUCTION.

times.

In conclusion, the present research wotild be benefi‑

Another way to treat the patients is the use of
extracts from medicinal plants. However, this also hasa problem to be resolved. The curative efficacy of crude
drugs from plants has been obscure and the incomplete
treatment of the disease seems to be responsible foranother transmission, producing chronic carriers whoare very dangerous from the viewpoint of epidemiology.
The aim of the present investigation on the 5 speciesof Chinese herbal medicine against T. vaginalis was to

ACKNOWLEDGEMENTS
Ms. Fusako Osada, Department of Parasitology,
Kitasato University School of Medicine, is gratefully
acknowledged for her excellent technical assistance.
Thanks are also given to the faculties in the department
for their cooperation and friendship during the stay of
the first author (Y. Zhang) of this paper in Japan.

select them scientifically for the development of the newantitrichomonal drugs available even in local areas in
China. It is hoped that the topical application of suchdrugs to patients would eventually control the furthertransmission of the parasitic disease. Needless to say,such plant‑derived drugs would have the possibility of1 ) Boreham, P.F. (1995): Dreamtime, Devastation andplaying an alternative role in the treatment of T.
vaginalis patients when this parasite is found to be2 ) Faust, E.C., Russel, P.F. and Jung, R.C. (1970): Tri‑
chomonas v ginalis, Craig and Faust's Clinical Par‑
asitology. 70, 8th ed. Lea & Febiger, Philadelphia

resistant to metronidazole in developed countries andareas.

In both the present preliminary and the subsequent
investigation, the extracts of A. annua and P. chinensis
were found to be highly effective against T. vaginalis,
immobilizing 100% of the parasites under certain condi‑
tions. Though extract from C. monnieri is described as

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the World in Colour, Hokuryukan, Tokyo7 ) Namba, T. (1993a) : The Encyclopedia of Traditional

12) Zhao, W., Wang, T. and Zhang, R. (1991): Chinese

Parasit. Int., 46 (suppl.), 65

Sino‑Japanese Medicines with Colour Pictures (D,Hoikusha, Osaka

tary Publishing House. Beijing, 56
Journal of Parasitology and Parasitic Diseases 9, 20

Jpn. J. Trop. Med. Hyg Vol. 25, No, 4, 1997, pp. 215‑216

215

Case Re port

ASYMPTOMATIC SCHISTOSOMIASISHAEMATOBIA= AN IMPORTED CASE
KENJI OHNISHI
Received August 21, 1997/Accepted October 13, 1997

Abstract: Stool and urine examinations revealed eggs of Schistosoma haematobium in a 31‑year‑old
asymptomatic Japanese female returned from Africa. She was treated with praziquantel, and good therapeu‑
tic result was obtained. Hawever, as S. haematobium is not indigeneous to Japan, most Japanese doctors andmedical technologists are unfamiliar with this parasitic disease, they need to be reminded of its existence
when they encounter persons who have visited or resided in endemic areas, and of the necessity of urine andstool examinations of such persons.
Key words: Schistosoma hoewatobium, schistosomiasis, praziquantel

INTRODUCTION
Schistosomiasis haematobia, a common helminth
disease caused by the infection with Schistosoma
haematobium in certain countries in Africa and western
Asia, is a major public health problem in endemic areas(Mahmond, 1988) . Although many Japanese now travel
to or stay in tropical or subtropical areas where S.
haematobium is endemic, only a very small number ofJapanese patients have been reported in Japan in the
1990's (Yamaguchi et al.; 1993; Kobari et al.. 1996;
Ohnishi, 1997). Recently, we treated an asymptomaticJapanese female patient infected with S. haematobium
who had recently returned from Africa. The purpose ofthis report is to emphasize the importance of par‑had no symptoms, she was referred to my outpatient
clinic on May 2, 1997, because a health examination on
her return to Japan had revealed Eletamoeba cysts in herstool. She was initially treated with oral administrationof metronidazole. Eggs of S. haematobium (Fig. l) wererevealed by centrifugation sedimentation stool examina‑tion on May 8, and urine examination on May 15, 1997,

but neither macroscopic nor microscopic haematuria
was found. The urine mentioned above was obtained atabout ll:OO a.m. Her biood count and serum biochemi‑
cal tests were normal, except for her blood eosinophil

count, which was 750/mm3 (WBC 9,600/mm3; 7.8%

asitological investigation for people who have visited or
resided in tropical developing countries, even if no symp‑

toms of infection are evident̲

CASE DESCRIPTION
A 31‑year‑old female Japanese stayed in Niger
from January 1995 to April 1997 as a member of Japan
Overseas Cooperation Volunteers. Subsequently, shetraveled in Co^te d'lvoire, Ghana, Togo, Cameroon and
Thailand, and she returned to Japan on May 2, 1997.
During this period, she came into contact with waterfrom both a river and a pond in Africa. Although she

Figure l Schistosoma haematobium egg with a distinet
terminal spine in stool ( x 380) .

Department of Infectious Diseases, Tokyo Metropolitan Bokutoh General Hospital, 4‑23 15 Kohtohbashi, Sumida‑City, Tokyo 130,
Ja pan216

eosinophils) on May 2, 1997. She was diagnosed ashaving S. haematobium infestation, and was treated withoral administration of 30 mglkg and 20 mglkg doses ofpraziquantel on the mornings of and evenings of May 22
and 23, 1997, respectively. Neither side effects nor

abnormal laboratory changes due to this drug were
found. Urine, which were obtained between 10:30 and 11:
30 a.m. and stool examination on May 29 and August 11,
1997 revealed no eggs of S. haematobium, and her blood

eosinophil count was 260lmm3 (WBC 5,200/mm3; 5.0 %eosinophils) on August 11, 1997.

DISCUSSION
The intermediate hosts of Schistosoma spp., several
types of snails, discharge cercariae, the larval stage of

Schistosoma spp., in fresh water. On contact with theskin of humans, cercariae of S. haematobium penetrate
into the skin, enter the venous circulation, and grow in

the intrahepatic portal vessels. Subsequently, theymigrate into the rectal veins and vesical and pelvic
plexuses, and the female worms produc eggs in thevenules (Beaver et al.. 1984). Eggs were found in boththe stool and urine in my patient, indicating that thefemale worms may have produced eggs in the vessels ofboth the lower part of the colon and urogenital systems.
Although many Japanese have traveled to or stayed in

S. haematobium ‑endemic areas and come into contact
with river or pond water, few cases of schistosomiasis

haematobia have been reported in Japanese patients.

This disease may have been overlooked in many
Japanese patients because cases infected with only small

number of worms are commonly asymptomatic andJapanese doctors and medical technologists are unfamil‑

iar with this disease. The number of Japanese contract‑
ing schistosomiasis haernatobia will increase as more

people travel to S. haematobium ‑endemic areas.
Japanese doctors and medical technologists should beaware of this disease when they encounter people whohave returned from such areas, and of the necessity of
the urine and stool examinations of these people.

ACKNOWLEDGEMEN T
I am grateful to Dr. H. Tanaka, Medical adviser,
Japan Overseas Cooperation Volunteers in Japan Inter‑national Cooperation Agency, for referring this patientto the Department of Infectious Diseases, Tokyo Metro‑
politan Bokutoh General Hospital.

REFERENCES
1 ) Mahmound. A.A.F. (1988): Schistosorniasis. In: J.B.
Wyngaarden and L.H. Smith Jr. (Ed.), Cecil Textbookof Medicine, 18th ed. pp. 1895‑1901, W.B. Saunders Co.,
Philadelphia

2 ) Yamaguchi, T., Masuda, G., Negishi, M.. Ajisawa, A.
and Tsuji, M. (1993): Two cases of schistosomiasis
haematobium. J. Jpn. Assoc. Infect. Dis., 67, 394 (inJapanese)3 ) Kobari, T., Kato, N., Endoh, K., Yoshigoe, H., Yamaza‑ki, H., Oishi. A. and Otomo, H. (1996) : A case of urinary

schistosomiasis. Clin. Parasitol., 7, 120 ‑ 122 (inJapanese)4 ) Ohnishi, K. (1997): Schistosomiasis haernatobium: acase imported by a Japanese patient. J. Jpn. Assoc.
Infect. Dis., 71, 672‑674

5 ) Beaver. P.A., Jung, R.C. and Cupp, E.W. (1984): Schis‑tosomes or blood flukes. In: Clinical Parasitology, 9thed. 415‑448, Lea & Febiger, Philadelphia

OECEMBER1997

VOL．25NO．4

CONTENTS
Original

ArticleSasa，M．and Suzuki，H．
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