JAPANESE JOURNAL OF
TROPICAL MEDICINE AND HYGIENE

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マレーシアのサバ州で採集された新種２種および未記録種３種のブユについて（英文）高岡 宏行 …………………………………………………………………………………………２
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家兎アカントアメーバ角膜炎の臨床像および組織所見（英文）山田 誠一，山田利津子，モアゼム ホセイン，月! 説子，藤田紘一郎 ………………２
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マレーシアのサラワク州で採集された２新種ブユの記載（英文）高岡 宏行 …………………………………………………………………………………………２
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日本熱帯医学会 MK 山道 草野 白石

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日 本 熱 帯 医 学 会

Original article
Takaoka, H.
Two New and Three Newly Recorded Species of Black Flies (Diptera: Simuliidae)
from Sabah, Malaysia ……………………………………………………………………………２
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Yamamoto, T., Shimada, M., Moji, K. and Mizota, T.
Research Activities on Emerging and Reemerging Diseases ……………………………………２
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Yamada, S., Yamada, R., Hossain Md. M., Tsukidate, S. and Fujita, K.
Clinical and Pathological Features of Experimental Acanthamoeba Keratitis in Rabbits ………２
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Takaoka, H.
Description of Two New Species of Black Flies (Diptera: Simuliidae) from Sarawak, Malaysia……………………………………………………………………………………………………２
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Ishak, H., Matsuse, I.T., Kamimura, K., Takegami, T. and Funada, H.
Comparative Oral Susceptibility of Dengue Vector Mosquitoes from Japan and Southeast Asia
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Otsuka,Y., Aoki, C., Saito, K., Hadi, U.K., Suzuki, H. and Takaoka, H.
Phylogenetic Analyses of a Blackfly Subgenus Simulium (Nevermannia) Based on
Mitochondrial 16S Ribosomal RNA Gene Sequences ……………………………………………２
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Vol. 29 No. 2

日熱医会誌

Hygiene

会報・記録
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ミトコンドリア１
６Ｓリボゾーム RNA 領域の塩基配列によるホソスネブユ亜属（Nevermannia）の系統学的研究（英文）大塚靖，青木 千春，斎藤 一三，Upik K. Hadi，鈴木博，
高岡 宏行 …………………………………………………………………………………………２
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Medicine

日本産と東南アジア産のヒトスジシマカとネッタイシマカのデング１型ウイルス感受性について（英文）
ハサヌディン・イスハック，松瀬イネス倶子，上村
清，竹上
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Journal of Tropical

新興・再興感染症を取り巻く研究の現状（英文）山本 太郎，嶋田 雅曉，門司 和彦，溝田JUNE ２０
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CONTENTS

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Japanese

原

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Jpn. J. Trop.
Med. Hyg.

JAPANESE SOCIETY OF TROPICAL MEDICINE

背は３mm
（決定）

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Jpn. J. Trop. Med. Hyg., Vol.２
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TWO NEW AND THREE NEWLY RECORDED SPECIESOF BLACK FLIES (DIPTERA: SIMULIIDAE)
FROM SABAH, MALAYSIAHIROYUKI TAKAOKA
Received December 12, 2000/Accepted February 9, 2001

Abstract: Two new black-fly species, Simulium (Gomphostilbia) guniki sp. nov. and S. (Nevermannnia) borneoense sp. nov., are described from adult flies emerged from pupae, collected from Sabah in Malaysia. S. guniki
is assigned in the ceylonicum species-group, and is characterized by the darkened legs in the adults of both sexes,
eight pupal gill filaments almost sessile, and the small postgenal cleft of the larva. S. borneoense, which is assigned to the feuerborni species-group, is most distinctive by having the four pupal gill filaments per side (in placeof usual six filaments). In addition, three species of Simulium (Gomphostilbia), i.e., S. dentistylum, S. parahiyangum, and S. sheilae, are newly recorded from Sabah.
Key words: Simuliidae, Black fly, Malaysia, Sabah, New species, Fauna

Edwards (1933) described five new species of black
flies (Diptera: Simuliidae) from Sabah (formerly British
North Borneo) in Malaysia, and then Smart and Clifford(1969) described additional six, one of which, Simulium sabahense, was found also in Sarawak, and another two, S. tualanense and S. kiuliense, were later synonymized with S.
aureohirtum Brunetti and S. nobile de Meijere, respectively(Crosskey, 1973). Takaoka (1983) illustrated female genitalia of five known species (S. aeneifacies Edwards, S. crassimanum Edwards, S. laterale Edwards, S. nigripilosum Edwards, and S. sabahense Smart and Clifford), and Takaoka(1996) described one more new species, S. beludense,
based on the pupal and emerged adult specimens, whichwere wrongly associated with female holotype of S. nigripilosum by Smart and Clifford (1969).
In 1998, I made faunistic surveys on Simuliidae in several localities of Sabah in Malaysia, and collected 12 species of Simulium s.l. including two new species and three
newly recorded ones. I herein describe these two new species, of which one is assigned to the subgenus Gomphostilbia and the remaining one, to Nevermannia.
The morphological features and terms used herein follow mostly those of Crosskey (1969), and partially those of
Takaoka (1983). All type specimens of new species will bein due course deposited at the Natural History Museum,London, U.K.

DESCRIPTION OF NEW SPECIES
Simulium (Gomphostilbia) guniki sp. nov.
Female. Body length ca. 2.1 mm. Head. Somewhat narrower than width of thorax. Frons (Fig. 1) narrow, darkbrown, subshiny in certain angle of light, thinly white pruinose, covered with ca. 10 dark simple long hairs interspersed with 5 or 6 whitish-yellow scale-like recumbent
hairs along each lateral margin; frontal ratio 1.9:1.0:3.5;
frons-head ratio 1.0:6.1. Fronto-ocular area (Fig. 2) welldeveloped, narrow. Clypeus dark brown, subshiny in certain angle of light, white pruinose, covered with 22-24 dark
simple long hairs interspersed with ca. 12 whitish-yellow
scale-like recumbent hairs on each side. Proboscis ca.
0.54× as long as clypeus. Antenna composed of 2+9 segments, brownish black except scape, basal 1/2 of pedicel
and base of 1st flagellar segment medium brown. Maxillary
palp composed of 5 segments, light to medium brown, proportional lengths of 3rd, 4th and 5th segments 1.0:1.2:2.6;
3rd segment somewhat swollen; sensory vesicle (Fig. 3) ellipsoidal, ca. 0.33× as long as 3rd segment, with medium
opening medially. Maxillary lacinia with 10 or 11 inner
teeth and 14 or 15 outer ones. Mandible with ca. 24 inner
teeth and 7 or 8 outer ones. Cibarium (Fig. 5) mediallyforming round sclerotized plate folded forward from posterior margin, and with well sclerotized, medial longitudinalridge near rounded tip. Thorax. Scutum brownish black,with 3 faint dark longitudinal vittae (1 medial and 2 subme-

Department of Infectious Disease Control, Oita Medical University, Hasama, Oita 879-5593, Japan２２２dial), shiny, whitish pruinose, densely covered withyellowish-white or golden-yellow scale-like recumbent
hairs intermixed with dark similar hairs; scutellum darkbrown, covered with yellowish short hairs as well as dark
long upright hairs along posterior margin. Postscutellum
dark brown, shiny, white-pruinose, bare. Pleural membranebare. Katepisternum dark brown, longer than deep, moderately covered with dark hairs. Legs. Light to dark brown
except base of mid and hind tibiae, basal 1/2 of hind basitarsus and basal 1/2 of 2nd tarsal segment whitish (Fig. 6);
all tibiae densely covered with whitish fine hairs (shiny inlight) on outer surface (fore tibia), or on posterior and outersurfaces of a little less than basal 1/2 (mid tibia), or on posterior and outer surfaces of basal 3/5 (hind tibia); fore tarsus
with moderate dorsal hair crest; fore basitarsus moderately
dilated, ca. 6.6× as long as its greatest width; hind basitarsus (Fig. 6) narrow, nearly parallel-sided, ca. 6.3× as longas wide, and ca. 0.7× and ca. 0.6× as wide as greatest
width of tibia and femur, respectively; calcipala ca. 1.2× aslong as wide, and ca. 0.6× as wide as distal portion of basitarsus. All femora, tibiae and parts of tarsus densely covered with dark (and also pale) scale-like hairs. Claws (Fig.
8) each with large basal tooth, ca. 0.4× as long as claw.
Wing. Costa with dark spinules as well as dark hairs; subcosta with dark hairs except near apex bare; basal portion ofradius fully haired; tuft hairs at base of radial vein darkbrown; basal cell absent; length ca. 2.0 mm. Abdomen. Basal
scale dark brown, with fringe of dark hairs with apical portion pale. Dorsal surface of abdomen brownish black except tergites 2, 3, 4 and 5 medium brown, moderately covered with short dark hairs; tergites of segments 2, 6, 7 and 8
wide and shiny, while those of segments 3, 4 and 5 narrow,nearly quadrate, subequal in size to one another, and all
dull; ventral surface brownish black except median large
portion of segment 2 pale; sternal plate on segment 7 (Fig.
9) weakly developed, triangular. Genitalia (Figs. 10-12).
Sternite 8 bare medially, with 18 or 19 long stout hairs and2 or 3 short hairs on each side. Anterior gonapophyses
nearly triangular, with round medioposterior corner, thin,membraneous, covered moderately with microsetae, interspersed with 4 or 5 short setae; inner margins sinuous, moderately sclerotized, close together. Genital fork of usual
inverted-Y form, with arms of moderate width; arm moderately folded medially. Paraproct moderately produced ventrally, with 17 long hairs on ventral and lateral surfaces, andwith 6-8 short hairs on inside surface. Cercus ca. 0.5× aslong as wide, rounded. Spermatheca ellipsoidal, ca. 1.5×
as long as wide, well sclerotized except tube, and withmany fissures on surface; internal setae appear to be absent;
both accessory tubes slender, subequal in diameter to major
one.

Male. Body length 2.1 mm. Head. Slightly wider thanthorax. Holoptic; upper eye consisting of 14 vertical columns and 16 or 17 horizontal rows of large facets. Facebrownish black, white pruinose. Clypeus brownish black,shiny, thinly white pruinose, moderately covered with dark
simple long hairs. Antenna composed of 2+9 segments,
dark brown except base of 1st flagellar segment somewhat
paler; 1st flagellar segment elongate, ca. 1.7× as long as
2nd flagellar segment. Maxillary palp with 5 segments,
light to medium brown, proportional lengths of 3rd, 4th and5th segments 1.0:1.1:2.9; sensory vesicle (Fig. 4) nearly
globular, small, ca. 0.2× as long as 3rd segment, and withsmall opening. Thorax. Scutum brownish black, shiny(in certain angles of light, white pruinose broadly on shoulders, along lateral borders, and on prescutellar area), anddensely covered with whitish-yellow (or golden-yellow inlight) scale-like recumbent hairs, intermixed with brown
ones; other features as in female. Legs. Light to darkbrown except extreme base of mid and hind tibiae and basal
1/3 of hind 2nd basal segment whitish yellow, and basal 1/3
of hind basitarsus somewhat paler; all tibiae densely covered with whitish fine hairs (shiny in light) on outer surface(fore tibia), or on posterior and outer surfaces near base(mid and hind tibiae); fore tarsus with moderate dorsal haircrest; fore basitarsus somewhat dilated, ca. 7.6× as long as
its greatest width; hind basitarsus (Fig. 7) enlarged, gradually widened toward midpoint, then parallel-sided, and narrowed toward apex, ca. 3.91× as long as wide, and ca.
0.85× and ca. 0.96× as wide as greatest width of tibia andfemur, respectively; calcipala small, nearly as long as wide,and ca. 0.27× as wide as greatest width of basitarsus. All
femora, tibiae and parts of tarsus moderately or densely
covered also with dark scale-like recumbent hairs. Wing.
Length ca. 1.9 mm; other features as in female except subcosta bare. Abdomen. Basal scale brownish black withfringe of dark hairs. Dorsal and ventral surfaces of abdomen almost brownish black to black except median portionof ventral surface of 2nd segment pale, and covered withdark hairs; segments 2, 5, 6 and 7 each with pair of shiny
dorsolateral patches, of which those on segment 2 broadly
connected in middle to each other, and those on other segments not connected to each other. Genitalia (Figs. 13-20).
Coxite in ventral view ca. 1.6× as long as wide. Style inventral view ca. 0.8× as long as coxite, gently bent inward,
with apical spine having median slit; style tapered frombase to midpoint, then nearly parallel-sided apically whenviewed ventrolaterally; style with round apex (though small
incision present in right style) when viewed posteriorly.
Ventral plate in ventral view transverse, nearly parallelsided (though posterior 1/2 slightly narrowed), with posterior margin somewhat concave medially, and densely cov-

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Figures 1-20 Morphological characters of S. guniki sp. nov. 1, female frons; 2, female fronto-ocular area; 3 and 4, 3rd segment ofmaxillary palp with sensory vesicle (3, female and 4, male); 5, female cibarium; 6 and 7, tibia, basitarsus and 2nd tarsal segment of hind leg (6, female and 7, male); 8, female claw of hind leg; 9, sternite 7 of female abdomen (ventralview); 10, sternite 8, anterior gonapophyses, genital fork and spermatheca of female genitalia in situ (ventral view);
11 and 12, paraproct and cercus in situ (11, ventral view and 12, lateral view); 13, coxites, styles, and ventral plate ofmale genitalia in situ (ventral view); 14-16, right style (14, ventrolateral view, 15, medial view and 16, end view); 17,ventral plate (end view); 18, ventral plate and median sclerite (lateral view); 19, left paramere and aedeagal membrane(end view); 20, left cercus (end view). Scale bars: 0.1 mm for figs. 6 and 7; 0.04 mm for figs. 1 and 2; 0.03 mm forfigs. 3-5 and 9-20; 0.01 mm for fig 8.

２２４ered with microsetae on ventral surface; basal arms of moderate length, somewhat diverged and then slightly converged; ventral plate in lateral view somewhat produced
ventrally; ventral plate in end view produced ventrally, withventrolateral margins nearly straight, with dorsal margin
much depressed, and densely covered with microsetae on
posterior surface. Parameres of moderate size, each with 3
distinct stout hooks (2 long and 1 medium) and several
smaller ones close together near apex; aedeagal membranemoderately setose. Median sclerite thin, plate-like, wide.
Ventral surface of 10th abdominal segment moderately sclerotized anteriorly forming dorsal plate, without short setae
near posterior margin on each side. Cerci rounded, each encircled with 18 or 19 setae.
Pupa. Body length 2.5 mm. Head. Integument light tomedium brown, moderately or densely covered with large
tubercles, each having minute tubercles on surface (Figs. 21
and 22); antennal sheath normal, with no spinous projections, and almost bare; face with 1 pair of simple long very
stout dark trichomes with coiled apex (Fig. 23), and frons
with 3 pairs of similar trichomes; 3 frontal trichomes oneach side arising close together, subequal in size to one another. Thorax. Integument light to medium brown, moderately covered with large tubercles similar to those on frons,
and with 9 pairs of trichomes (3 dorsally, 2 anterolaterally,
1 posterolaterally, and 3 ventrolaterally), all similar to
those on face and frons except 2 of 3 ventrolateral pairs medium and slender, and 1 of these 2 trichomes with uncoiled
apex. Gill (Fig. 24) composed of 8 slender filaments, allarising almost directly from short basal common stalk,which has somewhat swollen transparent portion ventrally
at base (though partially broken); apical portions of filaments lost except 2 filaments which are intact, and ca. 1.6mm long; all filaments light to medium brown, subequal in
thickness and also probably in length to one another, and allnearly the same size along basal 1/2, then gradually tapered
toward apex; outer surface of filaments lacking annularridges (though furrows present irregularly), and densely
covered with minute tubercles. Abdomen. Tergum 1 paleyellow, bare, with 1 simple slender medium-long seta oneach side; tergum 2 pale yellow anteriorly, bare, with 1 simple slender medium-long seta and 5 short setae on eachside; terga 3 and 4 mostly pale, each with 4 hooked spinesand 1 short seta on each side; tergum 5 lacking spinecombs; terga 6-8 each with distinct spine-combs in transverse row, together with comb-like groups of minute spines
on each side; tergum 9 with comb-like groups of minutespines, and 1 pair of distinct flattened terminal hooks,
which are nearly triangular when viewed posteriorly (Fig.
25). Sternum 4 with a few simple slender minute setae oneach side. Sternum 5 with 1 pair of bifid hooks submedially and a few short simple slender setae on each side;sterna 6 and 7 each with 1 pair of bifid inner and simple
outer hooks somewhat spaced from each other, and a few
simple short slender setae on each side. Each side of segment 9 with 3 grapnel-like hooklets (Fig. 26). Cocoon.
Wall pocket-shaped, neatly and thickly woven; anterior
margin somewhat thickly woven, and narrowly connectedventrally; posterior 1/2 with floor roughly woven; individual threads visible; 2.5 mm long × 0.9 mm wide.
Mature larva. Body length 4.2 mm. Body entirely greyish
black. Head capsule dark brown except eye-spot region oneach side. Cervical sclerite composed of 2 small rod-likepieces, not fused to occiput, widely separated medially fromeach other. Antenna consisting of 3 segments and apicalsensillum, longer than stem of labral fan; proportional
lengths of 1st, 2nd and 3rd segments 1.0:1.0:0.8. Labral fan
with ca. 40 main rays. Mandible (Fig. 27) with 2 usual
mandibular serrations; comb-teeth composed of 3 teeth,
gradually decreasing in size from 1st to 3rd; supernumerary
serrations absent. Hypostomium (Fig. 28) with a row of 9
apical teeth; median and corner teeth well developed; 3 intermediate teeth on each side subequal in size to one another; lateral serrations undeveloped; 4 hypostomal bristles
lying parallel to, or slightly divergent posteriorly from, lateral margin on each side. Postgenal cleft (Fig. 29) small,nearly quadrate in shape, and ca. 0.4× as long as postgenal
bridge. Thoracic cuticle very sparsely covered with simple
minute setae. Abdominal cuticle covered with simple setaevery sparsely on segments 1-5, sparsely on other posterior
segments except last segment densely covered with colorless simple (and a few bifid) setae on each side. Rectal papilla (retracted) appearing to be composed of 3 simple lobes.
Anal sclerite of usual X-form, with posterior arms nearly as
long as anterior ones; basal portion of arms widely sclerotized. Accessory sclerite absent. Ventral papillae large,
conical, placed ventrally, then, well discernible when thelarva is viewed laterally. Posterior circlet with ca. 80 rowsof up to 14 hooklets per row.
TYPE SPECIMENS. Holotype, female, reared from pupa,
collected at Timpohon, at alt. 1,970 m, upstream of thewater reserve just above the Carson Fall, in Kinabalu National Park, Sabah State, 13.!.1998. Paratype 1 male,reared from pupa, 1 mature larva, same data and date as
holotype.
ECOLOGICAL NOTES. Two pupae were collected from
slender tree sticks in water of a small clean stream flowing
down on the rocky streambed, shaded, in natural forest,

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Figures 21-29 Morphological characters of S. guniki sp. nov. 21-26, pupa, and 27-29, larva. 21 and 22, tubercles on thoracic integument (21, dorsal view and 22, lateral view); 23, facial trichome; 24, right gill filaments (lateral view); 25, terminal hooks (end view); 26, grapnel-like hooklet; 27, apical portion of mandible; 28, hypostomium (bristles omitted,
ventral view); 29, ventral surface of head capsule showing hypostomium and postgenal cleft. Scale bars: 0.1 mm for
fig. 24; 0.2 mm for fig. 29; 0.02 mm for figs. 23 and 28; 0.01 mm for figs. 21, 22, 25, 26 and 27.

with water temperature of 13°
C. This species was collected
together with S. aeneifacies, S. borneoense sp. nov. and S.
nigripilosum.

also very distinctive by having the small postgenal cleft (Fig.
29) and very darkened unicolored body.
Simulium (Nevermannia) borneoense sp. nov.

DISTRIBUTION. Sabah.
ETYMOLOGY. The species guniki is named after Dr. Gunik
Gunsalam, Chief entomologist, Research and Education Division, Kinabalu Conservation Center, Sabah Parks (Kinabalu
Park), Sabah.
REMARKS. This new species is assigned in the ceylonicum
species-group, defined by Takaoka and Davies (1996), by
having the enlarged male hind basitarsus (Fig. 7). Within
this species-group, it is easily separated from the otherknown species by having the darkened legs in the adults ofboth sexes, narrow frons in the female (Fig. 1), enlarged tubercles on the frons and thorax (Figs. 21 and 22), gill filaments almost sessile (Fig. 24), and triangular terminal
hooks in the pupa (Fig. 25). The larva of this new species is

Female. Unknown.
Male. Body length 3.1 mm. Head. Slightly wider thanthorax. Holoptic; upper eye consisting of large facets in 15
vertical columns and 17 horizontal rows. Clypeus medium
brown, dull, thinly whitish grey pruinose, moderately covered with dark simple hairs. Antenna composed of 2+9 segments, dark brown except base of 1st flagellar segment paleyellow, and scape and pedicel light to medium brown; 1stflagellar segment much elongate, ca. 2.4× as long as 2ndflagellar segment. Maxillary palp brown, composed of 5
segments, proportional lengths of 3rd, 4th and 5th segments
1.0:1.0:1.6; 3rd segment (Fig. 30) of moderate size; sensory
vesicle small, ellipsoidal, ca. 0.22× as long as 3rd segment.
Thorax. Scutum medium to dark brown, subshiny, thinly
whitish-grey pruinose, densely covered with yellow recum-

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Figures 30-48 Morphological characters of S. borneoense sp. nov. 30-40, male, 41-44, pupa and 45-48, larva. 30, 3rd segment ofmaxillary palp with sensory vesicle; 31, basitarsus and 2nd tarsal segment of hind leg; 32, coxites, styles, and ventral
plate of genitalia in situ (ventral view); 33 and 34, right style (33, ventrolateral view, 34, end view); 35, ventral plate(end view); 36, ventral plate and median sclerite (lateral view); 37, left paramere (anterodorsal view); 38, mediansclerite (end view); 39, aedeagal membrane and dorsal plate (posterodorsal view); 40, cerci (end view); 41, facial
trichome; 42, frontal trichomes; 43, right gill filaments (only basal portion shown, lateral view); 44, left terminal
hook (lateral view); 45, whole body of larva showing faint brownish markings on thorax and abdomen (dorsal view);
46, apical portion of mandible; 47, hypostomium (bristles omitted, ventral view); 48, ventral surface of head capsuleshowing hypostomium and postgenal cleft. Scale bars: 1.0 mm for fig. 45; 0.1 mm for figs. 31, 43 and 48; 0.03 mm
for figs. 30, 32-40 and 47; 0.02 mm for figs. 41, 42 and 44; 0.01 mm for fig. 46.

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bent hairs, and with several black upright hairs on prescutellar area. Scutellum light brown, with many dark uprighthairs as well as yellow recumbent hairs. Postscutellum medium to dark brown, not shiny, and bare. Pleural membranebare. Katepisternum medium to dark brown, longer thandeep, and bare. Legs. Foreleg: coxa and trochanter dark
yellow to light brown; femur light brown with apical cap
medium to dark brown; tibia medium brown with apical cap
dark brown; tarsus dark brown; basitarsus slender, slightly
dilated, ca. 10.0× as long as its greatest width at apex.
Midleg: coxa medium brown; trochanter yellow or a little
darker; femur light brown with apical cap medium brown;
tibia medium brown with apical cap dark brown; tarsus darkbrown. Hind leg: coxa medium brown; trochanter yellow;
femur light brown with apical cap dark brown; tibia medium brown with base light brown and apical cap darkbrown; basitarsus medium brown, and other tarsal segments
dark brown except basal 1/3 of 2nd segment dark yellow orlight brown; basitarsus (Fig. 31) enlarged, ca. 3.87× aslong as its greatest width, ca. 1.07× and ca. 1.11× as wide
as hind tibia and femur, respectively. Calcipala well developed, ca. 0.9× as long as wide; pedisulcus well developed.
All femora moderately covered with yellow hairs on outer
and/or posterior surface of almost all of shaft, and fore tibia
moderately covered with yellow hairs on outer surface ofbasal 4/5 of shaft, and mid and hind tibiae also moderately
covered with yellow hairs on posterior and outer surface ofbasal 3/4 of shaft. Wing. Costa with dark spicules andhairs; subcosta with several hairs on basal 2/3; basal portionof radial vein fully haired; tuft hairs at base of radial veindark; basal cell absent; length 2.6 mm. Abdomen. Basal
scale dark brown, with fringe of dark long hairs. Dorsal
surface of abdominal segments dark brown, with dark simple hairs; ventral surface light to medium brown. Genitalia(Figs. 32-40). Coxite subquadrate, much longer than wide.
Style much shorter than coxite, broad, nearly parallel-sided
from base to near apex, then abruptly tapered apically andbent inwards, and with distinct apical spine directed inward
and forward. Ventral plate lamellate, much shorter thanwide, well sclerotized except along anterior margin thin,membraneous, with posterior margin sinuous (when viewed
ventrally), and moderately covered with fine short setae on
ventral and posterior surfaces; arms of moderate length,
stout, and curved outwardly and dorsally. Parameres ofnormal form, each with 8 distinct hooks. Median sclerite
simple, club-shaped, narrow medially. Dorsal plate well developed, broad, thin, with medial portion widened. Aedeagal membrane moderately covered with spinous microsetae.
Cerci small, rounded, each with 6 or 7 simple hairs.
Pupa. Body length 3.2 mm. Head. Integument yellow,

sparsely covered with round tubercles; face with 1 longsomewhat stout simple trichome with coiled apex (Fig. 41)
on each side, while frons with 2 short slender simple
trichomes (Fig. 42) on each side. Thorax. Integument yellow, sparsely covered with round tubercles, with 3 long
slender simple trichomes mediodorsally, 2 slender simplelong trichomes (1 long, and 1 medium-long) mediolaterally,
1 medium slender simple trichome posterolaterally, and 3
short slender simple trichomes ventrolaterally, on each side.
Gill (Fig. 43) with 4 long slender filaments arranged in inner and outer pairs, each pair sessile or with very short stalk,arising from medium-long basal common stalk; all filaments lying close together, directed forward, yellowish or
yellowish brown, subequal in length and thickness to one
another (length 3.8-4.1 mm), and much longer than pupal
body; cuticular surface with distinct annular ridges and furrows (though ridges becoming indistinct on apical 1/2), anddensely covered with minute tubercles. Abdomen. Terga 1
and 2 yellowish, almost bare; tergum 1 with 1 medium
slender seta on each side; tergum 2 with 1 medium-longslender seta and 5 short spinous setae on each side; terga 3
and 4 each with 4 hooks and a few spinous setae on eachside; terga 5-8 each with a transverse row of spine-combs
and comb-like groups of minute spines directed backwardon each side (though those on tergum 5 small in number);tergum 9 with 1 pair of distinct horn-shaped terminal hooks(Fig. 44), and comb-like groups of minute spines. Sternum
4 with a few slender setae on each side; sternum 5 with 1
pair of bifid hooks submedially and a few slender setae oneach side; sterna 6 and 7 each with 1 bifid hook submedially, 1 simple hook laterally, and a few slender setae oneach side. Each side of 9th segment with a single slenderseta. Cocoon. Simple, wall-pocket-shaped, compactly
woven without open spaces in web, very thin, with anterior
margin thickly woven, and extending ventrolaterally; anterodorsal projection absent; posterior 1/2 with floor neatly
woven; individual threads visible; 4.0-4.2 mm long × 3.03.4 mm wide.
Mature larva. Body length 6.0 mm. Body creamy white,with light brown dorsal markings on thorax and abdomen as
follows: thoracic segment 1 with light brown transverse
band, abdominal segments 3 and 4 each with light browntransverse band medially disconnected, abdominal segments
5-8 each with faint light brown transverse band (Fig. 45).
Cephalic apotome yellow with posterior margin somewhat
darkened; head spots distinct; 3 isolated spots below, and 2
larger spots behind, eye-spot region on lateral surface ofhead capsule also distinct; eye brow faint, not connected
posteriorly to upper larger lateral spot. Cervical sclerite
composed of 2 small rod-like pieces, not fused to occiput,

２
２
８

widely separated medially from each other. Antenna consisting of 3 segments and apical sensillum, longer than stemof labral fan; proportional lengths of 1st, 2nd and 3rd segments 1.0:0.9:1.1; segmentation between 1st and 2nd segments indistinct. Labral fan with ca. 33 main rays. Mandible (Fig. 46) with 2 usual mandibular serrations; comb-teeth
composed of 3 teeth, 1st tooth largest, 2nd and 3rd teeth
subequal in size to each other; supernumerary serrations absent. Hypostomium (Fig. 47) with a row of 9 apical teeth,
median and corner teeth well developed; median tooth of 3intermediate teeth on each side smallest; lateral serrations
weakly developed apically; 4 or 5 hypostomal bristles lying
parallel to, or slightly divergent posteriorly from, lateral
margin on each side. Postgenal cleft (Fig. 48) small, shallow, round or nearly quadrate, and ca. 0.6× as long as postgenal bridge. Gill histoblast with 4 filaments arising frommedium-long basal common stalk. Thoracic cuticle bare.
Abdominal cuticle bare except last segment moderately
covered with colorless short setae on each side of analsclerite. Rectal papilla of 3 lobes, each lobe with 16-20
finger-like secondary lobules. Anal sclerite of usual X-form,
with posterior arms subequal in length to anterior ones; basal portion of arms widely sclerotized. Accessory sclerite
absent. Ventral papillae large, conical, placed ventrally,
then, well discernible when the larva is viewed laterally.
Posterior circlet with ca. 72 rows of up to 14 hooklets per
row.
TYPE SPECIMENS. Holotype, male, reared from pupa, collected at Timpohon (same locality as S. guniki sp. nov.), at
alt. 1,970 m, upstream of the water reserve just above the
Carson Fall, in Kinabalu National Park, Sabah State, Malaysia, 13.!.1998. Paratype 2 males, reared from pupa, 1
pharate male, and 1 mature larva, same data and date as
holotype.
ECOLOGICAL NOTES. All the four pupae and 2 larvae collected were attached on glass leaves (except one pupa on
slender tree stick) in water of a small clean stream (samestream in which S. guniki sp. nov. was collected). This species was collected together with S. aeneifacies, S. guniki sp.
nov. and S. nigripilosum.
DISTRIBUTION. Sabah.
ETYMOLOGY. The species borneoense refers to the island
name, Borneo.
REMARKS. The feuerborni species-group within the subgenus Simulium (Nevermannia) is a small and relatively homogenous taxon, consisting of 13 named species in the Ori-

ental and Palaearctic Regions (Crosskey and Howard, 1996;
Crosskey, 1999; Takaoka and Saito, 2000).
S. borneoense sp. nov. is readily assigned to the feuerborni species-group by the characteristic male genitalia
with a simple lamellate ventral plate, short inwardly-twisted
styles, several parameral hooks, and a simple narrow median sclerite (Figs. 32-38).
It is worthwhile to note that the pupa of S. borneoense
is easily distinguished from those of the nine known species
within this species-group by the number of gill filaments
per side, i.e., four (Fig. 43) in place of six.
The other four known species of the feuerborni
species-group were described from adult males (and also females in one species) alone, and then, their pupal and larvalstages have remained unknown. Among these, S. fuscinervis was recorded from Sabah (Edwards, 1933), but it differsfrom the new species by having the reduced number oflarge eye facets, i.e., ca. 12 vertical columns and 12-14 horizontal rows (not 15 vertical columns and 17 horizontalrows), and paramere with 10 hooks (not eight); S. bryopodium described from Palawan Island, Philippines (Delfinado, 1971), also differs by having the subcosta bare (nothairy), ventral plate with basal arms straight (not diverged),and paramere with six hooks (not eight); S. senile, described from West Himalaya (Brunetti, 1911), is different
by having the bicolored hind basitarsus and the style with
no apical spine; S. rufithorax, described from a male andfour females collected from India (Brunetti, 1911), has a
reddish brown thorax according to the original description.

NOTES ON NEWLY RECORDED SPECIES
Simulium (Gomphostilbia) dentistylum Takaoka andDavies, 1995
Simulium (Gomphostilbia) dentistylum Takaoka andDavies, 1995: 51-55 (male, pupa and larva); Kuvangkadilok and Takaoka, 2000: 173.
SPECIMENS EXAMINED. 1 male, reared from pupa, collected from a stream (alt. ca. 490 m), crossing the road, near
Entrance of Hot spring Park, Poring, Sabah, 12.!.1998; 4mature and 1 immature larvae, collected from a small
stream (alt. ca. 550 m) flowing inside Hot spring Park, Poring, Sabah, 12.!.1998.
ECOLOGICAL NOTES. The pupa of this species was collected from a trailing grass in a moderately-flowing stream
with its width of 3-6 m, partially shaded. Five larvae of thisspecies were collected from fallen leaves in a slowlyflowing shaded stream with its width of ca. 20 cm. The
water temperature of the stream was 23-25°
C. This species２２
９

was collected together with S. beludense, S. laterale, S.
parahiyangum, S. sabahense and S. sheilae, or with S. sabahense and S. sheilae.
DISTRIBUTION. Peninsular Malaysia, Sabah (new record),
Thailand.
REMARKS. S. dentistylum was originally described from
Peninsular Malaysia (Takaoka and Davies, 1995), and later
it was also recorded from Thailand (Kuvangkadilok andTakaoka, 2000).
The male and its associated pupa, and larvae from Sabah agree well morphologically with the original descriptions except a difference of the leg coloration of the male.
All the tibiae of the present male specimen are much darker
than those of the holotype male in the original description,
and also have a subbasal dark band. It is considered that theholotype male specimen from Peninsular Malaysia might
have had a faded leg coloration when described since it had
been preserved in ethanol for nearly 20 years. In fact, additional male specimens of S. dentistylum recently collected
in Johor State, Peninsular Malaysia, have the darker tibiae
with a dark subbasal band, as in the present male specimen(unpublished data). The revised description of the male, as
well as the first description of the female of this species,
will be given in a separate paper.
Simulium (Gomphostilbia) parahiyangum Takaoka andSigit, 1992
Simulium (Gomphostilbia) parahiyangum Takaoka
and Sigit, 1992, 135-141 (female, male, pupa andlarva); Takaoka and Davies, 1995: 50-51; Takaoka
and Saito: 1996, 165; Takaoka et al ., 2000: 161.
SPECIMENS EXAMINED. 1 mature and 1 immature larvae,
collected from a stream (alt. ca. 490 m), crossing the road,near Entrance of Hot spring Park, Poring, Sabah, 12.
!.1998.
ECOLOGICAL NOTES. The larvae of this species were collected from trailing grasses in a moderately-flowing stream
with its width of 3-6 m, partially shaded. The water temperature of the stream was 25°
C. This species was collected
together with S. beludense, S. dentistylum, S. laterale, S.
sabahense and S. sheilae.
DISTRIBUTION. Java, Sumatra, Thailand, Peninsular Malaysia, Sabah (new record).
REMARKS. S. parahiyangum was originally described fromJava (Takaoka and Sigit, 1992), and it was also recorded

from Peninsular Malaysia (Takaoka and Davies, 1995),
Thailand (Takaoka and Saito, 1996), and Sumatra (Takaoka
et al., 2000). The larva of this species is easily distinguished from other Gomphostilbia species by having theprominent dorsal protuberances on abdominal segments 1-5,
several dark stout spines on the abdominal segments 6-8,
and the deep postgenal cleft reaching the posterior marginof the hypostomium (Takaoka and Sigit, 1992).
Simulium (Gomphostilbia) sheilae Takaoka and Davies,
1995
Simulium (Gomphostilbia) sheilae Takaoka andDavies, 1995: 60-65 (female, male, pupa and larva);
Kuvangkadilok and Takaoka, 2000: 172-173;
Takaoka et al., 2000: 161.
SPECIMENS EXAMINED. 6 females, 5 males, all reared
from pupae, collected from a small stream (alt. ca. 550 m)
flowing in a forest in the Hot spring Park at Poring, Sabah,
12.!.1998.
ECOLOGICAL NOTES. The pupae and larvae of this species
were collected from fallen leaves in a slowly-flowing
stream with its width of ca. 20 cm, shaded. The water temperature of the stream was 23°
C. This species was collected
together with S. sabahense.
DISTRIBUTION. Peninsular Malaysia, Sabah (new record),
Sumatra and Thailand.
REMARKS. S. sheilae was originally described from Peninsular Malaysia, and was assigned to the ceylonicum speciesgroup by Takaoka and Davies (1995). It was also recorded
from Thailand (Kuvangkadilok and Takaoka, 2000) and Sumatra (Takaoka et al., 2000).
This species is separated from other related species ofthe same species group by the enlarged oblong female sensory vesicle (ca. 0.7 times as long as 3rd maxillary palpal
segment) and the almost brown male hind basitarsus(though basal 1/3 or a little less somewhat paler). The
reared adult female and male specimens, as well as pupal
and larval ones examined in this study, are morphologicallyalmost the same as those originally described, with an exception that the scutum of the present male specimens is
whitish pruinose on each shoulder, along both lateral margins and on the prescutellar area (not entirely whitish pruinose, as in the type male specimen).

ACKNOWLEDGEMENTS
I am grateful to Prof. Yong Hoi Sen, Malaysia Univer-２３０sity, Dr. F.S.P. Liew and Dr. Gunik Gunsalam, Sabah Parks,
for their kind help and arrangement of collections of black
flies in the fields. Thanks are also due to Dr. A. Shelley, theNatural History Museum, London, U.K., for his kind arrangement of the loan of the type specimen of S. fuscinervis.
This work was supported by the Grant-in-Aid of Japan Society for Promotion of Science (the year 1997) to HT.

REFERENCES
１）Brunetti, E. (1991): New Oriental Nematocera. Rec. IndianMus., 4, 259-316
２）Crosskey, R.W. (1969): A re-classification of the Simulii-dae (Diptera) of Africa and its islands. Bull. Br. Mus.
(Natur. Hist.) (Entomol.) Suppl. 14, 1-195
３）Crosskey, R.W. (1973): Family Simuliidae. p. 423-430. In:
Delfinado, M.D. & Hardy, D.E. eds., A catalog of the Diptera of the Oriental Region. Vol.!. Suborder Nematocera.
University Press of Hawaii, 618p.
４）Crosskey, R.W. (1999): First Update to the Taxonomic andGeographical Inventory of World Blackflies (Diptera: Simuliidae). 10 pp., The Natur. Hist. Mus., London.
５）Crosskey, R.W. and Howard, T.M. (1996): A New Taxonomic and Geographical Inventory of World Blackflies(Diptera: Simuliidae). 144p., The Natur. Hist. Mus., London.
６）Delfinado, M.D. (1971): Some Simuliidae and Curtonotidae from the Philippines and the Bismarck Islands (Insecta,
Diptera). Steenstrupia 1, 131-139
７）Edwards, F.W. (1933): Diptera Nematocera from Mount
Kinabalu. J. fed. Malay St. Mus.,17, 223-296

８）Kuvangkadilok, C. and Takaoka, H. (2000): Taxonomic

notes on Simuliidae (Diptera) from Thailand: Descriptionof a new species and new distributional records of nine
known species. Jpn. J. Trop. Med. Hyg., 28, 167-175
９）Smart, J. and Clifford, E.A. (1969): Simuliidae (Diptera) of
Sabah (British North Borneo). Zool. J. Linn. Soc., 48, 9-47
１０）Takaoka, H. (1983): The blackflies (Diptera: Simuliidae) of
the Philippines. #+199p., Japan Society for the Promotionof Science, Tokyo
１１）Takaoka, H. (1996): Description of a new species of Simulium (Simulium) from Sabah, Malaysia (Diptera: Simuliidae). Jpn. J. Trop. Med. Hyg., 24, 157-161
１２）Takaoka, H. and Davies, D.M. (1995): The Black Flies(Diptera: Simuliidae) of West Malaysia. "+175p., Kyushu
University Press, Fukuoka
１３）Takaoka, H. and Davies, D.M. (1996): The black flies(Diptera: Simuliidae) of Java, Indonesia. "+81p., Bishop
Mus. Bul. Entomol. 6, Bishop Museum Press, Hawaii.
１４）Takaoka, H. and Saito, K. (1996): A new species and newrecords of black flies (Diptera: Simuliidae) from Thailand.
Jpn. J. Trop. Med. Hyg., 24, 163-169
１５）Takaoka, H. and Saito, K. (2000): Description of a new
species of Simulium (Nevermannia) from Japan (Diptera:
Simuliidae). Jpn. J. Trop. Med. Hyg., 28, 19-24
１６）Takaoka, H. and Sigit, S.H. (1992): A new blackfly speciesof Simulium (Gomphostilbia) from Java, Indonesia (Diptera: Simuliidae). Jpn. J. Trop. Med. Hyg., 20, 135-142
１７）Takaoka, H., Yunus, M., Hadi, U.K., Sigit, S.H., and Miyagi, I. (2000): Preliminary report of faunistic surveys on
black flies (Diptera: Simuliidae) in Sumatra, Indonesia. Jpn.
J. Trop. Med. Hyg., 28, 157-166

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Jpn. J. Trop. Med. Hyg., Vol.２
９, No．
２, ２
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０１, pp.２
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４

RESEARCH ACTIVITIES ON EMERGING AND
REEMERGING DISEASESTARO YAMAMOTO1, MASAAKI SHIMADA2, KAZUHIKO MOJI3
AND TSUTOMU MIZOTA4
Received December 21, 2000/Accepted February 5, 2001

Abstract: This paper showed the trend in the number of paper published on emerging and reemerging diseases byusing the list of the Medline, which is one of the most valuable databases in the clinical and biological medicine.
The number of research papers published on the emerging and reemerging infections in that database decreased in
1997, in comparison with those between 1994 and 1996, in spite of the fact that the total number of the paper published has been increasing year by year. There is also a report showing that the number of the published papers ofnoticeable infectious diseases such as AIDS, Hepatitis B, Gonorrhea, Pertussis and Tuberculosis declined by 15%
or more in the USA from 1993 to 1995. It also became clear that USA is the highest in rank for the number of papers published and it was well ahead of the other countries. However, as for Vibrio cholerae O139, Human Tlymphtropic Virus type 1 and Plague, a lot of papers have been published by responding countries like India, Japanand Russia, respectively. This result might be showing that research activity of each country for each disease relates to the extent of their concern over each disease.
Key words: Emerging disease, Reemerging disease

INTRODUCTION
There was a paper titled “Infectious Diseases-A global
health threat” published by US government in 1995, whichshowed the importance of arising awareness of the infectious disease. According to the WHO and CDC in Atlanta,
United States, the term of “emerging and reemerging diseases” refers to diseases of infectious origin whose incidence in human has either increased within the past two
decades or threatens to increase in the near future (Instituteof Medicine, 1992). And CDC listed at least thirty diseases
as representatives of emerging and reemerging diseases.
National opinion leaders generally had considered thethreat of infectious diseases to be of only historical interestin 1960s and 1970s (Lederberg et al., 1992; Garrett, 1994;
Martine, 1996; Sande, 1996; Schwartz, 1997; Stephens etal., 1998). However, the emergence of HIV or Ebola virus
as well as the resurgence of Tuberculosis or Malaria haschanged the political circumstances that surround the infectious diseases.
Infectious diseases also remain the leading courses ofdeath not only in the developing countries but also in developed countries like USA (World Bank, 1993; McGinnis etal., 1993). Further, emerging and reemerging infections

have been attracting greater attention from not only the public health viewpoint but also national security viewpoint inrecent years. Under those circumstances, we had a strong
interest in whether research activities on emerging and reemerging infections have changed or not, and how.
We thought that the number of paper published is oneof the appropriate indicators to estimate their research activities on those infectious diseases.

METHODS
Medline on Internet was chosen for this survey because it has a broader coverage about biomedical studies.
Papers written in English were also selected. There are105,676 papers in total. The reason why papers written inother languages were excluded is that those might be inferior in quality to the international journals written in English and less reading worldwide. Medline is published by
National Library of Medicine (NLM) in the United Statesand is the main database for secondary reference materials
in the field of medicine. Papers were selected by the title
including the name of pathogens and were categorized bycountry, in which corresponding institutions were located,even if studies were conducted through international col-１ Department of Global Health and Socio-epidemiology, Kyoto University, School of Public Health, Yoshida-Konoe-cho, Sakyo-ku, Kyoto
606-8501, Japan. Tel. +81-75-753-4355, Fax. +81-75-753-4355 (Correspondent author)
２ Information and Reference Center of Tropical Medicine, Institute of Tropical Medicine, Nagasaki University
３ School of Allied Medical Science, Nagasaki University
４ Department of Social Medicine, Institute of Tropical Medicine, Nagasaki University

２
３
２

Table 1 The number of published paper of emerging diseases
Pathogen

80

81

82

83

84

85

HIV
Hepatitis C

9

11

11

9

18

10

86

87

88

89

90

91

92

93

94

95

96

97

Total

108

977

2,035

2,267

2,853

3,244

3,482

3,787

3,728

3,610

3,856

2,514

32,461

16

19

24

66

285

528

747

879

1,106

1,379

1,213

904

7,234

7

213

409

390

589

564

809

913

589

4,483
3,703

Helicobacter pylori
HTLV-1
Rotavirus

97

3

6

31

115

366

613

291

192

230

230

259

269

280

235

228

229

126

122

126

182

166

139

167

181

204

197

199

164

151

155

152

123

161

66

2,752

12

39

53

73

118

106

131

167

194

164

145

143

97

1,442

Borrelia burgdorferi
Campylobacter jejuni

44

60

99

11

130

72

114

88

91

61

77

63

72

80

77

95

61

40

1,335

Legionella pneumophila

47

70

66

85

66

64

74

72

77

76

57

65

75

49

67

63

50

23

1,146

Hepatitis E

26

31

27

19

23

23

29

18

18

30

32

69

72

78

102

107

92

77

873

4

3

8

17

22

26

30

23

42

59

63

95

90

102

158

109

851

2

1

6

12

20

15

26

17

22

22

42

48

65

80

92

470

2

1

3

11

12

10

8

21

14

48

83

65

53

31

362

1

5

5

3

13

36

29

34

53

41

72

46

338

7

11

15

16

17

21

21

18

25

18

33

23

241

3

1

1

3

6

2

7

8

5

45

30

16

164

13

40

51

38

16

158

Prion
enterohemorrhagic
Eschrichia coli O157

Hantavirus
Cryptosporidium parvum
Ehrlichia

6

1

4

1

Ebola virus

10

6

10

10

4
1

Vibrio cholerae O139
Bartonella

1

Encephalitozoon

103

Staphylococcus aureus
toxin shock

1

1

3

2

1

6

4

14

2
6

2

3

3

6

5

10

9

24

7

Cyclospora
HHV 6

3

6

4

7

46

52

31

156

7

4

15

11

13

17

16

132

3

4

5

9

2

7

2

99

6

13

20

26

18

84

1
1

1

3

3

2

1

1

1

3

6

Enterocytozoon bieneusi
HHV 8

2

1

1

2

2

5

10

11

5

9

3

7

5

77

3

3

3

8

5

10

8

9

49

12

36

2

2

2

Guanarito

3

2

5

1

1

1

Sabia
Total

250

309

364

356

538

724

1,223

1,790

2,820

3,171

4,173

5,121,

5,609

6,394

6,597

7,043

3

1

1

2

7,306

4,863

58,651

No.of papers on Medline 263,310 265,496 275,406 287,713 297,353 249,485 333,854 258,200 378,147 369,645 376,690 375,903 377,414 207,871 388,433 398,231 403,323 405,173 6,011,647

Table 2 The number of published paper of reemerging diseases
Reemerging disease

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

Total

Tuberculosis

808

772

968

825

813

578

901

1,055

1,030

895

1,120

1,070

1,023

1,186

1,172

1,451

1,399

773

17,839Malaria

221

271

325

368

376

367

387

494

555

688

568

596

624

606

617

577

580

396

8,616

Pertussis

55

86

89

122

141

183

216

240

341

312

309

342

280

261

255

235

238

188

3,893

Schistosomiasis

164

160

150

166

180

143

196

307

244

222

151

123

166

135

136

135

156

67

3,001

Leishmaniasis

85

83

107

122

134

117

184

195

163

146

188

174

186

239

226

226

224

94

2,893

Cholera

137

142

141

127

130

88

127

142

142

169

169

232

231

216

194

203

156

90

2,836

Rabies

121

143

171

104

122

93

150

140

197

122

135

125

145

145

144

129

141

73

2,400

Toxoplasmosis

122

111

114

127

136

78

118

113

126

143

158

135

171

138

138

134

121

68

2,251

Dengue

44

49

57

62

62

41

51

97

58

87

84

79

78

101

100

100

96

62

1,308

Plague

47

38

51

54

40

32

39

39

46

46

47

35

57

46

103

70

35

38

863

Echinococcus

22

32

35

34

41

22

47

52

55

44

60

58

56

61

52

52

42

43

808

Yellow fever

15

33

17

23

25

17

35

25

16

24

28

18

21

31

14

31

25

15

413

Diphteria

1

1

1,842

1,921

1,907

47,124

Total

1
2,225

2,134

2,200

1,759

2,451

2,899

2,973

2,898

3,017

2,987

3,038

3,165

3,151

3,343

3,214

3

No.of papers on Medline 263,310 265,496 275,406 287,713 297,353 249,485 333,854 258,200 378,147 369,645 376,690 375,903 377,414 207,871 388,433 398,231 403,323 405,173 6,011,647

laboration.
The total ranking list was made by giving scores to thecountries. In terms of the number of papers, the first, sec-

ond and third ranking countries were given five, three andone, respectively and all the scores for each country wereadded up at the end.

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RESULT AND DISCUSSION
Trend in the number of research papers: In terms of emerging diseases, 58,552 papers had been published in Medline
between 1980 and 1997, in total (Table 1). Human immunodeficiency virus (HIV), Hepatitis C virus (HCV) andHelicobacter pylori were the first, second and third in ranking for the number of papers, respectively. However, except
for enterohemorrhagic Escherichia coli O157 and Human
herpes virus 8 (HHV 8), the number of publication in 1997
is much fewer than the average number between 1994 and1996. In recent year, enterohemorrhagic E. coli O157 endemic occurred in some developed countries and HHV 8
was shown to be associated with Kaposi’s sarcoma.
Regarding the reemerging diseases, 47,124 papers
were published in total in Medline between 1980 and 1997
(Table 2). The papers related to Tuberculosis take the firstplace and papers on Malaria take the second in number. The
number of research papers listed as reemerging diseases decreased in 1997, compared with the average number of papers published between 1994 and 1996.
It is unknown whether the decreased number of the papers found may reflect reduced concern over emerging andreemerging diseases. The number of papers published reflects to some extents of concerning over those infectious
diseases in the last several years. Thus, this result might beshowing the decreasing of the concerning over emerging
and reemerging diseases despite the fact that there are a lotof papers which advocate the importance of the research on
emerging and reemerging diseases (Anthony, 1998;
MMWR, 1998).
High-ranking countries in the number of papers: Regarding
to the emerging diseases, USA was the highest in rank forthe number of papers published except for HCV, Human Tlymphotropic Virus type 1 (HTLV-1), Ebola and Vibrio
cholerae O139. Japan was the highest ranking for HCV
and HTLV-1, and Russia was the highest for Ebola, and India was the highest for V. cholerae O139. UK is the second
country for seven pathogens in terms of the number of paper published, and France and Japan were the second forfive pathogens, and USA was the second for four, and Germany was the second for three. Excluding USA, UK, Japan,
France and Germany, only Russia, India, Canada and Italy
were seen in top three countries.
In terms of reemerging diseases, USA were also thehighest in ranking for those except Leishmaniasis, Toxoplasmosis, Plague and Echinococcus. France was the highest in
ranking for Leishmaniasis and Toxoplasmosis, and Russia
was the highest for Plague, and New Zealand and Australia
was the highest for Echinococcus. USA are also the second
country for three pathogens in terms of the number of paper

Table 3 Total ranking of countries
１
２
３
４
５
６
７
８
９
１
０

Emerging
USA
Japan
UK
FranceGermany
RussiaIndia
CanadaItaly(score)
(117)
(28)
(24)
(22)
(15)
(5)
(5)
(3)
(1)

Reemerging
USA
FranceRussiaUK
NZ/Australia
India
Germany
Italy
Japan
Canada(score)
(54)
(18)
(12)
(7)
(5)
(5)
(5)
(3)
(3)
(1)

published, and France, UK and Russia were the second fortwo pathogens. Japan was never seen in top two countries.
As for the V. cholerae O139, HTLV-1 and Plague, a lotof papers have been published by responding countries like
India, Japan and Russia, respectively. This result might beshowing that research activity of each country for each disease relates to the extent of their concern over each disease.
The total ranking was shown in Table 3. Regarding
both emerging and reemerging diseases, USA were highest
ranking and it was well ahead of the other countries. Franceand UK published papers in the field of both emerging andreemerging diseases were ranking within top four countries.
On the other hand, Japan and Russia had tended to focus on
emerging diseases and reemerging diseases, respectively.
Although there is no clear reason why Japan and Russia had
those tendencies, the paper on “Cholera” and “Plague” contribute Russia to the high ranking in the field of reemerging
diseases.

ACKNOWLEDGMENT
We would like to thank Ms. Mayuko Kurashiki, Ms.
Mari Mori, Ms. Eiko Tara and Mr. Masataka Yamaki fortechnical assistance and also thank Dr. Masahiro Kihara andShigeru Katamine for invaluable discussion.

REFERENCES
１）Anthony, S.F. (1998): New and Reemerging Diseases: The

importance of biomedical research. Emerging Infectious
Diseases, 4 (3), 14-17２）Institute of Medicine (1992): Emerging Infections: microbial threats to health in the United States. Washington, DC:
National Academy Press.
３）Garrett, L. (1994): The coming plague. Newly emerging
diseases in a world out of balance. New York: Farrar,
Straus and Giroux, 30-52
４）Lederberg, J., Shope, R.E. and Oaks, S.C.Jr. (eds.) (1992):
Emerging infections: microbial threats to health in the２３４

United States. Washington DC: Institute of Medicine (National Academy Press).
５）McGinnis, M.J. and Foege, W.H. (1993): Actual causes ofdeath in the United States. JAMA, 270, 2207-2212
６）Martine, E.P. (1996): A global theme issue on emerging
and reemerging global microbial threats. JAMA, 276 (3),197７）MMWR (1998): Preventing emerging infectious diseases: astrategy for the 21st century overview of the updated CDC
plan. 47 (RR15), 1-14
８）Sande, M.A. (1996): Global microbial threats-The publichealth risk of emerging and reemerging infections. West J.
Med., 164 (1), 18-20

９） Schwartz, D.A. (1997): Emerging and reemerging infections-progress and challenges in the subspecialty of infectious
disease pathology. Arch. Pathol . Lab. Med., 121, 776784
１０）Stephens, D.S., Moxon, E.R., Adams, J. et al. (1998): Emerging and reemerging infectious diseases: A multidisciplinary
perspective. Am. J. Med. Sci., 315 (2), 64-75
１１）World Bank (1993): World development report, 1993. NY.
Oxford University Press.

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CLINICAL AND PATHOLOGICAL FEATURES
OF EXPERIMENTAL ACANTHAMOEBAKERATITIS IN RABBITSSEIICHI YAMADA, RITSUKO YAMADA, MD. MOAZZEM HOSSAIN,SETSUKO TSUKIDATE AND KOICHIRO FUJITA
Received November 10, 2000/Accepted February 13, 2001

Abstract: Experimental Acanthamoeba keratitis was induced in 24 female Dutch rabbits to examine the clinicaleffects of infecton in the eye and to study the usefulness of a new histopathological technique for evaluating rabbit
models of this infection. One eye of each animal in group A or B received an instillation of 1.3×104 (group A) or
1.3×105 (group B) amoeba cysts/eye; phosphate-buffered saline (PBS) solution was instilled into the contralateraleye as the control; animals in groups C and D received intrastromal injections of 5.0×103 (group C) or 1.5×104(group D) cysts/eye in one eye and injection of an equal volume of PBS in the contralateral eye. Animals were observed daily for 5 to 84 days. Two rabbits in group D were killed on day 5 and enucleated eyes were embedded inparaffin and stained with hematoxylin-eosin or iodine-potassium iodide. In groups A and B, clinical signs of corneal injury disappeared by 4 hours after inoculation and signs of infection disappeared by day 2. In contrast, alleyes that had been injected with Acanthamoeba (groups C and D) developed severe keratitis, including keratoneuritis and corneal ulcer, followed by neovascularization or corneal perforation. Histologic examination showed infiltrates of leukocytes, lymphocytes, eosinophils, plasmacytes and spindle-shaped cells. The most extensive cell infiltration, and also exocytosis, liquefaction degeneration and intrastromal trophozoites, were seen in the limbic conjunctiva and palpebral conjunctiva. In addition, there was evidence of migration of inflammatory cells to the ciliary
body and intravitreal space. This study showed that injection of Acanthamoeba into corneal stroma causes severe
infection of the cornea and other eye tissues and that iodine-potassium iodide staining of paraffin embedded specimens is useful to detect Acanthamoeba trophozoites and cysts.
Key words: Acanthamoeba keratitis, Rabbit, Corneal ulcer, Keratoneuritis, Pathological feature, Iodine-potassium
iodide staining

INTRODUCTION
The persistent and severe Acanthamoeba keratitis occurs occasionally in contact lens wearers and swimmers.
Clinical findings in such cases include granular superficial
corneal opacity, scleritis, discoid ulcer, pseudodendritis,
stromatitis with ring infiltration. Thinning of cornea leading to corneal perforation may also occur when there is delay in accurate diagnosis and effective treatment of this condition (Wright et al., 1985). However, accurate diagnosis
and effective treatment for amoebic keratitis are difficult because amoebae are encysted in infected tissues (Osato et al.,1991).
Rabbit models of Acanthamoeba keratitis have beenused to study more effective techniques for diagnosis andtreatment of this condition. The first rabbit model was reported by Font and colleagues, who demonstrated that stromal keratitis and the corneal ulceration can be induced by
the intrastromal injection of A. polyphaga after subconjunctival administration of corticosteroid for 4 days (Font et al.,1981). They reported that corneas were culture-positive atintervals, and encysted amoebas have been observed (Fontet al., 1981). Another rabbit model was reported by Lin etal. (1989), who investigated the effectiveness of steroid
treatment of Acanthamoeba keratitis. A rat model of Acanthamoeba keratitis was reported by Larkin and Easty (1990).
They induced Acanthamoeba keratitis in Wister rats by intrastromal inoculation of A. polyphaga cysts and reported
that on histopathologic examination, sections of corneas
showed liquefactive stromal necrosis and Acanthamoeba

Section of Environmental Parasitology, Department of International Health Development, Division of Public Health, Graduate School, Tokyo
Medical and Dental University
Corresponding author: Seiichi Yamada, M.D., Ph.D.,
Section of Environmental Parasitology, Department of International Health Development, Division of Public Health, Graduate School, Tokyo
Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8519
TEL: +81-3-5803-5193, FAX: +81-3-5684-2849, e-mail: yama.vip@tmd.ac.jp

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cysts in deep stroma (Larkin and Easty, 1990).
We undertook the present study to investigate the clinical and pathological features of several models of Acanthamoeba ocular infection, specifically the effects on tissues other than the cornea, such as the conjunctiva, ciliary
body, and vitreous; and secondarily, to determine whether
iodine-potassium iodide staining of paraffin-embedded sections might be useful in studying rabbit models of Acanthamoeba infection.

MATERIALS AND METHODS
Acanthamoeba isolated from a patient with Acanthamoeba keratitis were grown at 25°
C on 1% agar platewith an overlay of heat-killed Escherichia coli as a foodsource. On the basis of differences in the size and morphologic features of the cysts, the protozoan was identified
as Acanthamoeba polyphaga (group 2) (Fig. 1) (Font et al.,
1981; Visvesvara, 1991).
Acanthamoeba cysts were instilled or injected into theeyes of 24 female Dutch rabbits that weighed 2.6 (standard
deviation: 0.3) kg each. The rabbits were divided into fourgroups. Rabbits groups A (n=4) and B (n=4) received 9 linear latticed abrasions on the corneal epithelial layer, extending to the upper stromal layer. Then a suspension of cysts(1.3×104 amoebas/eye for group A or 1.3×105 amoebas/
eye for group B) was instilled into one eye. Contra-lateraleyes of rabbits in groups A and B were instilled with anequivalent volume of phosphate buffered saline (PBS) solution. Rabbits in groups C (n=8) and D (n=8) received an injection of 5.0×103 cysts/kg body weight (b.w.) (20 µl )(group C) or 1.5×104 cysts/kg b.w. (50 µl ) (group D); theinjection was made intrastromally at the 2 o’clock positionon the cornea, about 2.5 mm from the limbus. Contralateraleyes of rabbits in group C or D were injected with an equalvolume (20 µl or 50 µl, respectively) of PBS.
Slit-lamp examinations of the anterior segments of alleyes of all rabbits were performed hourly for the first 5 hr
and every day for up to 84 days after the inoculation.
Two rabbits in group D were killed and enucleated byintravenous injection of sodium pentobarbital on day 5, andall the other rabbits were sacrificed on day 84 after the inoculation. The enucleated eyes of rabbits were embedded
in paraffin and sectioned at 3-5 µm. Sections were thenstained with hematoxylin-eosin (H-E) or iodine-potassiumiodide and examined by light microscopy.

RESULTS
1. Clinical features of Acanthamoeba instillation! Group A and B
By 4 hrs after injury and instillation of Acanthamoeba
in eyes in groups A and B, all signs of corneal injury had
disappeared. On day 1 after the inoculation, hyperaemia
and a few dot haemorrhage were seen in the conjunctivas ofeyes that had received instillation of Acanthamoeba, butthese manifestations were disappeared on day 2. Daily examination of eyes in groups A and B for the remainder ofthe 12-week study showed on signs of corneal disorder andno recurrence of conjunctivitis. There was no apparent difference between group A and B eyes in the clinical response
to corneal injury and instillation of Acanthamoeba cysts.
" Group C and D
In inoculated eyes in groups C and D, corneal opacity
occurred at the site of inoculation within minutes after inoculation and disappeared within 5 hrs after the inoculation.
Simultaneously, hyperanemia and dot haemorrhages become evident in the palpebral conjunctiva. Cellular infiltration in an inoculated site of the cornea, keratoneuritis andcorneal ulcer were seen in the area of inoculation on day 2
after inoculation in eyes in group C and on day 1 after inoculation in eyes in group D (Fig. 2). In rabbits in group C,
the corneal lesions gradually increased in area until day 5
after the inoculation. On day 6, pannus appeared and corneal infiltration start to disappear. Small cystic lesions were
found in the cornea on day 8, and these enlarged until day
14. The corneal neovascularization was noted at the limbic
conjunctiva on day 14 and developed into a corneal ulcer.
After day, 28, the cystic lesions in the corneas began to decreased in size and replaced by nubecula. By day 49, only
small area of pannus and nubecula remained on the corneasof rabbits in group C (Fig. 3), and there were no signs of recurrent keratitis during the remainder of the 12-week period
after instillation of Acanthamoeba in the eyes of rabbits ingroup C.
In rabbits group D, the corneal lesion appeared granular and turbid on day 2 after inoculation and had enlarged to
involve half of the corneal surface. On day 4, a portion of
the corneas in 2 eyes (2 rabbits) in group D was noticeably
thinner (Fig. 4), and on day 7 perforation was noted at thesesite.
The significant clinical difference between rabbits ingroup C and those in group D was that corneal lesions ingroup C showed spontaneous resolution, while those ingroup D increased, resulting in cornel perforation.
2. Histological features of Acanthamoeba keratitis
In the 2 eyes of rabbits in group D that were killed on
day 5 after inoculation of Acanthamoeba, histological ex-２
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Figure 1 Iodine-potassium iodide stained Acanthamoeba cysts. Bar: 10 µm

Figure 2 Biomicroscopic appearance of a rabbit cornea in group D, 1 day after inoculation of Acanthamoeba. A corneal ulcer, stromatitis and keratoneurtis have developed

２３８

Figure 3 Biomicroscopic appearance of a rabbit cornea in group C at 7 week after inoculation of Acanthamoeba. The corneal ulcer has disappeared, and pannus and nubecula are seen.

Figure 4 Biomicroscopic appearance of a rabbit cornea in group D on day 4 after inoculation of Acanthamoeba. Thinning of the cornea is evident.

２３９Figure 5 Histologic appearance of a rabbit cornea 5 days
after inoculation with Acanthamoeba. The corneal stroma shows infiltration of cells, including
polymorphonuclear leukocytes (P), lymphocytes(L), eosinophils (E), plasmacytes (Q), spindleshaped cells (S) and trophozoites (T), surrounded by liquefaction products. Hematoxylin-eosin
staining. Bar: 10 µm.

Figure 6 Histologic appearance of a rabbit cornea 5 days
after inoculation with Acanthamoeba. Iodinepotassium iodide staining of the paraffin embedded section differentiates cysts from trophozoites(T). Bar: 20 µm

Figure 7 Histologic appearance of rabbit limbic conjunctiva 5 days after inoculation with
Acanthamoeba. Infiltrates of lymphocytes (L), polymorphonuclear leukocytes (P)and plasmacytes (Q) and several trophozoites (T) are visible. Lymphocytes andplasmacytes may adhere to the walls of dilated blood vessels (D). Bar: 50 µm

２４０Figure 8 Histologic appearance of rabbit palpebral
conjunctiva 5 days after inoculation of Acanthamoeba. Infiltration of inflammatory cells(P) into the palpebral conjunctiva, exocytosis(e) and migration of trophozoites (T), and dilated blood-vessels (D) are visible. Bar: 30µm

Figure 9 Histologic appearance of rabbit ciliary body 5 days after inoculation of Acanthamoeba. Polymorphonuclear leukocytic (P) infiltration and multinucleated giant cells (M) are visible in the ciliary
body and intravitreal space. Bar: 50 µm

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amination showed remarkable cell infiltration in the corneal
stroma, including polymorphonuclear leukocytes, lymphocytes, eosinophils, plasmacytes, spindle-shaped cells andtrophozoites, surrounded by an area of liquefactive degeneration (Fig. 5).
On iodine-potassium iodide stained paraffin embedded
sections of these eyes, it was possible to differentiate cysts
from trophozoites (Fig. 6). Changes in the limbic conjunctiva 5 days after inoculation of Acanthamoeba cysts were
infiltrations of lymphocytes, polymorphonuclear or eosinophilic leukocytes, plasmacytes and scattered trophozoites.
Dilatation of intravascular spaces and neovascularization
were also evident in the limbic conjunctiva of rabbits ingroup D 5 days after inoculation of Acanthamoeba cyste(Fig.7). In the palpebral conjunctiva, H-E staining showedin filtrations of inflammatory cells and trophozoites andexocytosis in the epithelial layer (Fig. 8). H-E staining
showed polymorphonuclear leukocyte infiltrations in theciliary body and the anterior vitreous, trophozoites in theciliary body, and multinucleated giant cells in the intravitreal space (Fig. 9).

DISCUSSION
Acanthamoeba has a life cycle of trophozoite and dormant cyst stages.
Acanthamoeba keratitis was first described clinicallymore than 30 years ago, however, starting in 1985 the number of cases of amoebic keratitis reported has grown eachyear (Warhurst and Mann, 1988; Wright and Buckley, 1988;
Wright et al., 1985).
The pathogenesis of Acanthamoeba keratitis has beenstudied using a variety of experimental models. Culbertson
et al. (1959) examined the results of intravenous injection
or intranasal inoculation of Acanthamoeba organisms intomice and monkeys, and others have developed experimental
models of amoebic keratitis in a variety of species, including rabbits, hamsters, swine and mice (Font et al., 1981;
Larkin and Easty, 1990; Klink et al., 1994; Alizaeh et al.,
1995; Ruddell and Easty, 1995; John et al., 1991). Larkinand Easty (1990) reported on the histological appearance ofthe cornea in eyes with Acanthamoeba keratitis. However,there is little information about the effects of Acanthamoeba infection on other eye tissues, including the conjunctiva, iris, intravitreal space and ciliary body.
In the present study, we created the first reported reproducible model of Acanthamoeba keratitis in rabbit eyesand documented changes in the cornea, limbic or palpebral
conjunctiva, ciliary body and anterior vitreous after intrastromal injection of a suspension of Acanthamoeba cysts.

Our results show that intra-corneal injection of Acanthamoeba cysts leads to the development of small cystic lesions in the cornea that can be observed clinically by slitlamp examination. In addition, paraffin-embedded sectionsof rabbit eyes obtained 5 days after inoculation showed thepresence of inflammatory cell infiltrates and Acanthamoeba
trophozoites in other parts of the eye, such as limbic conjunctivae, palpebral conjunctivae, ciliary body, as well as
the intrastromal spaces in the cornea.
Differentiating between the cyst and the trophozoite
stages of the Acanthamoeba life cycles helpful because almost all clinical specimen were found as the trophozoite
stages of the Acanthamoeba. In our study, corneal smears
and histologic sections stained with iodine-potassium iodide
were useful for differentiating cysts from trophozoites. As aresult, detailed studies of the Acanthamoeba life cycle inkeratitis using anti-amoebic monoclonal antibodies areplanned for the near future.
In patients, the course of Acanthamoeba keratitis isoften prolonged in spite of intense treatment. Corneal curettage is the most effective treatment, especially when
Acanthamoeba infection is confined to the cornea, because
most anti-amoebic drugs are not very effective against encysted Acanthamoeba. The results of our study suggest that
migration of Acanthamoeba trophozoites from the cornea toother parts of the eye, such as the palpebral or limbic conjunctiva, ciliary body and anterior vitreous, may help explain the difficulty of treating severe Acanthamoeba keratitis and the occurrence of complications such as endophthalmitis or panophthalmitis.

REFERENCES
１）Alizaeh, H., He, Y.G., AcCulley, J.P., Ma, D., Stewart, G.L.,

Via, M., Haehling, E. and Niederkorn, J.Y. (1995): Successful immunization against Acanthamoeba keratitis in a
pig model. Cornea, 14, 180-186
２）Culbertson, C.G., Smith, J.W., Chen, H.K. and Minner, J.R.
(1959): Experimental infection of mice and monkeys by
Acanthamoeba. Am. J. Pathol., 35, 185-197
３）Font, R.J., Tapert, M.J., Robinson, N.M., Osato, M.S. andJones, D.B. (1981): An animal model of Acanthamoeba
keratitis. Invest. Ophthalmol. Vis. Sci., 20 (Suppl), 8４）John, T., Lin, J., Sahm, D. and Rockey, J.H. (1991): Effectsof corticosteroid in experimental Acanthamoeba keratitis.
Rev. Infect. Dis., 13, 440-4425
５）Klink, F.V., Alizaeh, H., He, Y.G., Mellon, J.A., Silvany, R.
E., AcCulley, J.P. and Niederkorn, J.Y. (1993): The role ofcontact lenses, trauma, and Langerhans cells in a Chinese
hamster model of Acanthamoeba keratitis. Invest. Ophthalmol. Vis. Sci. 134, 1937-1944
６）Larkin, D.F.P. and Easty, D.L. (1990): Experimental Acanthamoeba keratitis: I. Preliminary findings. Br. J. Ophthal-

２４２mol., 74, 551-555

murine model of Acanthamoeba keratitis. Eye, 8, 142-144７）Lin, J., John, T., Sahm, D. and Rockey, J.H. (1989): Animal

１０）Visvesvara, G.S. (1991): Classification of Acanthamoeba.

model of Acanthamoeba keratitis: effect of corticosteroids.
Invest. Ophthamol. Vis. Sci., 30 (Suppl), 37
８）Osato, M.S., Robinson, N.M., Wilhelmus, K.R. and Jones,
D.B. (1991): In vitro evaluation of antimicrobial compounds for cysticidal activity against Acanthamoeba. Rev.
Infect. Dis., 13 (Suppl 5), 431-435
９）Ruddell, T.J. and Easty, D.L. (1995): Drug therapy in aRev. Infect. Dis., 13 (Suppl), 369-372, successfully
treated medically. Br. J. Ophthalmol. 1985, 69, 778-782.
１１）Warhurst, D.C. and Mann, P.G. (1988): Acanthamoeba
keratitis. Br. Med. J., 296, 568
１２）Wrigth, P. and Buckley, R.J. (1988): Acanthamoeba keratitis. Br. Med. J., 296, 568-569
１３）Wright, P., Warhurst, D. and Jones, B.R. (1985): Acanthamoeba keratitis. Br. J. Ophthalmol., 69, 778-782

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DESCRIPTION OF TWO NEW SPECIES OF BLACK FLIES(DIPTERA: SIMULIIDAE)
FROM SARAWAK, MALAYSIAHIROYUKI TAKAOKA
Received February 27, 2001/Accepted April 8, 2001

Abstract: Two new black-fly species, Simulium (Gomphostilbia) lehi sp. nov. and S . (G .) sarawakense sp. nov.,
are described from adult flies reared from pupae, collected in Sarawak State, Malaysia. In addition, three known
species of Simulium s.l., i.e., S . (G .) sheilae, S . (Nevermannia) aureohirtum, S . (Simulium) laterale, are newly recorded from Sarawak.
Key words: Simuliidae, Black fly, Malaysia, Sabah, New species, Fauna

Since Smart and Clifford (1969) recorded Simulium
sabahense from Sarawak, Malaysia, no other black-fly species has been added to the simuliid fauna of this state.
In 1998, I made faunistic surveys on Simuliidae in several localities of Sarawak in Malaysia, and collected six
species of Simulium s.l. consisting of S. sabahense, two
new species and three newly recorded ones. I herein describe these new species.
The morphological features and terms used herein follow mostly those of Crosskey (1969), and partially those of
Takaoka (1983). All type specimens of new species will bein due course deposited at the Natural History Museum,London, U.K.

DESCRIPTION OF NEW SPECIES
Simulium (Gomphostilbia) lehi sp. nov.
Female. Body length 1.8-2.0 mm. Head. As wide as thorax. Frons (Fig. 1) brownish black, semishiny, densely covered with yellowish white scale-like recumbent hairs, andwith several dark stout hairs in vertical row along each lateral margin; frontal ratio 1.9:1.0:2.4; frons-head ratio 1.0:
4.7. Fronto-ocular area (Fig. 2) well developed, narrow.
Clypeus brownish black, semishiny, densely covered withyellowish-white scale-like recumbent hairs interspersedwith 10-12 dark hairs on each side. Proboscis ca. 0.54× aslong as clypeus. Antenna composed of 2+9 segments, medium brown except scape, pedicel and base of 1st flagellar
segment pale yellow. Maxillary palp composed of 5 segments, light brown, proportional lengths of 3rd, 4th and 5th
segments 1.0:1.1:2.9; 3rd segment (Fig. 3) widened dis-tally; sensory vesicle of small (or moderate in the other female, Fig. 4) size, nearly ellipsoidal, 0.21× (or 0.26× inthe other female, Fig. 4) as long as 3rd segment, with medium (or large in the other female, Fig. 4) opening. Maxillary lacinia with 7 or 8 inner teeth and 9 or 10 outer teeth.
Mandible (Fig. 6) with 12-14 small inner teeth and lacking
outer ones. Cibarium (Fig. 7) smooth, without any processes or tubercles. Thorax. Scutum dark brown with anterolateral calli somewhat paler, subshiny, thinly white pruinose, with 3 faint longitudinal vittae (1 medial and 2 submedial), densely covered with yellowish-white scale-like recumbent hairs; scutellum medium brown, covered withshort, yellowish-white scale-like hairs as well as long upright dark hairs along posterior margin. Postscutellum darkbrown, bare. Pleural membrane bare. Katepisternum
brownish black, longer than deep, with fine short dark hairs.
Legs (Coloration appeared to be still incomplete). Foreleg:coxa pale yellowish white; trochanter and femur lightbrown; tibia light to medium brown; tarsus medium to darkbrown, without distinct dorsal hair crest; basitarsus slightly
dilated, ca. 6.7× as long as its greatest width. Midleg:coxa dark brown; trochanter dark yellow; femur lightbrown; tibia medium brown with basal 2/5 dark yellow; tarsus medium brown with basal 2/3 of basitarsus, and base of
2nd tarsal segment dark yellow or light brown. Hind leg:coxa light brown, trochanter pale yellow; femur dark yellow
or light brown with apical cap medium brown; tibia yellowish white on basal 3/4 and medium brown on distal 1/4;
tarsus medium brown except basal 2/3 of basitarsus, basal
1/2 of 2nd tarsal segment yellowish white; basitarsus (Fig.
8) narrow, nearly parallel-sided, ca. 6.0× as long as wide,and ca. 0.63× and ca. 0.48× as wide as greatest width of

Department of Infectious Disease Control, Oita Medical University, Hasama, Oita 879-5593, Japan２
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tibia and femur, respectively; calcipala (Fig. 8) ca. 1.2× aslong as wide, and ca. 0.8× as wide as distal portion of basitarsus. All femora, tibiae and parts of tarsus densely covered
with dark (and also pale), scale-like hairs on outer and posterior surfaces. Claws (Fig. 10) each with large basal tooth,
0.47× as long as claw. Wing. Length 1.6 mm. Costa withdark spinules as well as dark hairs except basal hair tuft
pale. Subcosta haired except near apex bare. Hair tuft onstem vein pale. Basal portion of radius fully haired. Abdomen. Basal scale light brown, with fringe of pale hairs. Abdomen dark brown except base of 2nd segment paler,sparsely or moderately covered with short dark hairs; tergites of segments 2, 6, 7 and 8 subshiny; sternal plate on
segment 7 undeveloped. Genitalia (Figs. 11-13). Sternite
8 bare medially, with 21-28 long hairs and a few short hairs
on each side. Anterior gonapophyses nearly triangular,
rounded posteromedially, thin, membraneous, covered
densely with microsetae (though posteromedial corner
somewhat widely bare), interspersed with 1 or 2 short setae;
inner margins sinuous, narrowly sclerotized, moderately
separated from each other. Genital fork of usual inverted-Y
form; arms slender, folded medially, with a small projection
directed forward. Paraproct somewhat produced ventrally,
with 12-16 long hairs on outer surface, and with 4 or 5 sensilla on inside surface. Cercus ca. 0.5× as long as wide,
rounded posteriorly. Spermatheca ellipsoidal, ca. 1.5× aslong as wide, well sclerotized except near tubal juncture
and tube unsclerotized, and with many fissures on surface;internal setae appear to be absent; both accessory tubes
slender, subequal in diameter to major one.
Male (dissected out of the pupa). Body length 2.0 mm.
Head. Much wider than thorax. Upper eye consisting of
10 vertical columns and 11 horizontal rows of large facets.
Face dark brown, white pruinose. Clypeus dark brown,
thinly white pruinose, moderately covered with yellow simple and scale-like hairs (except median portion widely bare),intermixed with several dark longer simple hairs on eachside. Antenna composed of 2+9 segments, dark brown.
Maxillary palp with 5 segments, light to medium brown;
3rd segment (Fig. 5) widened apically; sensory vesicle ellipsoidal, 0.24× as long as 3rd segment, with medium
opening apically. Thorax. Scutum light brown, shiny,densely covered with yellow scale-like recumbent hairs.
Scutellum pale, covered with yellow scale-like short hairs
and several dark hairs near posterior margin. Postscutellum
light brown, bare. Pleural membrane bare. Katepisternum
as in female. Legs. Colorings (though incomplete) nearly
as in female; fore basitarsus ca. 7.2× as long as greatest
width; hind basitarsus (still bent) (Fig. 9) slender, nearlyparallel-sided like that of female. Calcipala (Fig. 9) en-

larged, ca. 1.6× as long as wide. Pedisulcus well developed. Wing. As in female except subcosta bare; length notmeasurable. Abdomen. Basal scale light brown, withfringe of dark hairs. Dorsal surface of abdomen light brown,covered with dark hairs; segments 2, 5, 6 and 7 each with apair of shiny dorsolateral patches. Genitalia (Figs. 14-20).
Coxite in ventral view, subquadrate, ca. 1.5× as long as
wide. Style ca. 0.9× as long as coxite, gradually tapered
toward apex, curved inward, with apical spine (right stylewith additional spine near apex though somewhat smaller
than main one). Ventral plate transverse, densely covered
with microsetae on ventral surface (except along anterior
and lateral margins rather broadly bare) and on posterior
surface (except both lateral areas bare); basal arms directed
forward, then converged apically. Parameres of moderatesize, each with 3 large hooks (of which 1 is very wide) andca. 10 smaller ones. Aedeagal membrane moderately covered with microsetae, weakly sclerotized along dorsal baseforming dorsal plate. Median sclerite thin, plate-like, wide,with apex rounded. Ventral surface of 10th abdominal segment without short setae near posterior margin on each side.
Cerci small, rounded, each with 12 or 13 short hairs.
Pupa. Body length ca. 2.2 mm. Head. Integument yellowish, densely covered with round tubercles; antennal
sheath normal, with no spinous projections, and almost
bare; face with 1 pair of simple long trichomes with coiled
apex, and frons with 3 pairs of simple long trichomes withcoiled apex; 3 frontal trichomes on each side arising close
together, subequal in length to one another and slightly
longer than facial one. Thorax. Integument yellowish,
densely covered with round tubercles, with 3 pairs of simple long trichomes dorsally, 2 pairs of simple trichomes (1long and 1 medium) anterolaterally, 1 pair of simple
medium-long trichomes posterolaterally, and 3 pairs of simple trichomes (1 long and 2 medium) ventrolaterally; alllong trichomes with coiled apex. Gill (Fig. 21) composedof 8 slender filaments, arranged in groups of 3+3+2 filaments from dorsal to ventral, with somewhat swollen transparent organ ventrally at base; stalks of all 3 groups of filaments as well as basal common stalk short; all filaments
pale yellowish, subequal in length (ca. 2.0 mm) and thickness to one another; cuticular surface of filaments with distinct annular ridges and furrows gradually becoming indistinct apically, and densely covered with minute tubercles.
Abdomen. Terga 1 and 2 pale yellow, almost bare; tergum
1 with 1 simple slender medium-long seta on each side; tergum 2 with 1 medium-long simple slender seta and 5 shortsetae (of which 2 or 3 spinous), submedially on each side;
terga 3 and 4 pale, each with 4 hooked spines and 1 shortsomewhat spinous seta on each side; tergum 5 lacking

２
４
５

Figures 1-20 Morphological characters of S. lehi sp. nov. 1, female frons; 2, female fronto-ocular area; 3-5, 3rd segment of maxillary palp with sensory vesicle (3 and 4, female; 5, male); 6, apex of female mandible; 7, female cibarium; 8 and 9,
basitarsus and 2nd tarsal segment of hind leg (8, female; 9, male); 10, female claw of hind leg; 11, sternite 8, anterior
gonapophyses, genital fork, spermatheca, paraproct and cercus of female genitalia in situ (ventral view); 12, paraproctand cercus in situ (lateral view); 13, genital fork (lateral view); 14, coxite and style of male genitalia in situ (ventralview); 15, right style (ventrolateral view); 16-18, ventral plate (16, ventral view; 17, lateral view; 18, end view); 19,
left paramere and aedeagal membrane (end view); 20, right cercus (end view). Scale bars: 0.1 mm for figs. 8 and 9;
0.05 mm for figs. 1 and 2; 0.02 mm for figs. 3-5, 7, and 11-20; 0.01 mm for figs. 6 and 10.

２４６

Figures 21-25 Morphological characters of S. lehi sp. nov. 21 and 22, pupa, and 23-25, larva. 21, right gill filaments (apical 1/2
omitted, lateral view); 22, right terminal hook (end view); 23, apical portion of mandible; 24, hypostomium (bristles
omitted, ventral view); 25, ventral surface of head capsule showing hypostomium and postgenal cleft. Scale bars:
0.1 mm for fig. 21; 0.04 mm for fig. 25; 0.02 mm for fig. 24; 0.01 mm for figs. 22 and 23.

spine-combs; terga 6-9 each with distinct spine-combs intransverse row, together with comb-like groups of minutespines on each side; tergum 9 with 1 pair of distinct platelike terminal hooks, extending laterally, and with serrated
outer margin (Fig. 22). Sternum 4 with 1 simple hook(somewhat smaller than those on sternum 7) and a few simple minute setae on each side; sternum 5 with 1 pair of bifid
hooks submedially on each side; sterna 6 and 7 each with 1
pair of bifid inner and simple outer hooks somewhat spacedfrom each other, and a few simple short minute setae oneach side. Each side of segment 9 with 1 simple hooklet
and 1 minute seta, but lacking grapnel-like hooklets. Cocoon. Simple, wall-pocket-shaped, moderately woven
without open spaces in webs; some cocoons with a narrowanteroventral collar; posterior 1/2 with floor roughly
woven; individual threads distinct; 2.6-2.9 mm long × 0.80.9 mm wide.

numerary serrations absent. Hypostomium (Fig. 24) with arow of 9 apical teeth; median and corner teeth well developed; inner and median teeth of 3 intermediate teeth oneach side subequal in size to each other, and somewhat
shorter than outermost tooth; lateral serrations undeveloped;
6 hypostomal bristles lying parallel to lateral margin oneach side. Postgenal cleft (Fig. 25) medium, arrow-head
shaped, and ca. 2.9× as long as postgenal bridge. Thoracic
and abdominal cuticle almost bare except last segment
densely covered with colorless simple setae on each side.
Rectal papilla of 3 lobes, each lobe with 7 or 8 finger-likesecondary lobules. Anal sclerite of usual X-form, with posterior arms ca. 1.3× as long as anterior ones; basal portionof arms widely sclerotized. Accessory sclerite absent. Ventral papillae large, conical, placed ventrally, then, well discernible when the larva is viewed laterally. Posterior circlet
with ca. 100 rows of up to 17 hooklets per row.

Mature larva. Body length 4.3-5.1 mm. Body entirely
greyish green. Cephalic apotome yellow, moderately covered with minute setae, with distinct dark usual head spots.
Cervical sclerite composed of 2 small rod-like pieces, notfused to occiput, widely separated medially from each other.
Antenna consisting of 3 segments and apical sensillum,
longer than stem of labral fan; proportional lengths of 1st,
2nd and 3rd segments 1.0:1.0:0.5. Labral fan with ca. 38
main rays. Mandible (Fig. 23) with 2 usual mandibular serrations; comb-teeth composed of 3 teeth, 1st tooth largest ofall, 2nd and 3rd ones subequal in size to each other; super-

TYPE SPECIMENS. Holotype, female, reared from pupa,
collected at Pueh, Sematan, Sarawak, Malaysia, 18.III.1998.
Paratype 1 female, reared from pupa, 1 pharate male, 3 pupae, 8 mature larvae, same data and date as holotype; 1pharate male, collected in Hot spring Park, at poring park,at Poring, Sabah, 12.III.1998.
ECOLOGICAL NOTES. Six pupae were collected, three
from slender tree sticks, and the others from rocks, in waterof a small clean shaded stream (0.5-3.0 m wide) flowing
down on the rocky streambed in natural forest of Mt. Pueh.

２４
７

One pupa of this species was collected from a slender stickin a slowly-flowing stream with its width of ca. 20 cm,
shaded, at Hot spring Park in Poring. The water temperature was 23-25°
C. This species was collected together with
S. laterale, S. sabahense, and S. sarawakense sp. nov. orwith S. sabahense and S. sheilae.
DISTRIBUTION. Sabah and Sarawak.
ETYMOLOGY. The species lehi is given after Dr. Charles
Leh, Curator, Zoology Department, Sarawak Museum,
Kuching, Sarawak State.
REMARKS. This new species is assigned to the batoense
species-group within the subgenus Gomphostilbia, definedby Takaoka and Davies (1996), by having 2+9 antennal segments, slender male hind basitarsus (Fig. 9), and 8filamented pupal gill (Fig. 21).
S. lehi is very similar to S. miblosi described from females and pupae collected from Mindanao Island, Philippines (Takaoka, 1983), by the mandible with serration only
on inner margin (Fig. 6), simple cibarium (Fig. 7) and enlarged calcipala (Fig. 8) in the female, and the absence ofthe grapnel-like hooklets on the last abdominal segment in
the pupa. However, this species differs from the latter in thefemale by the reduced number of small teeth on inner margin of the mandible (12-14 versus ca. 24), of stout hairs on
the eighth sternite (21-28 versus ca. 50 on each side), and in
the pupa by the usual number of trichomes on the frons (3versus 2 on each side).
The male of S. lehi is characterized by the very wideand high ventral plate (Figs. 16-18), and the paramere with
one of the three major hooks very wide (Fig. 19), together
with the reduced number of enlarged facets. The male of S.
rayohense, described from Sabah (Smart and Clifford,1969), has the similar ventral plate and the reduced numberof enlarged facets. However, this known species differsfrom S. lehi by the usual short calcipala. The pupal gill
filaments of S. rayohense are arranged in different manner,having the longer stalk of the ventral paired filaments; andthe larval postgenal cleft of this known species is much
deeper than that of S. lehi, according to the illustrations
given by Smart and Clifford (1969).
Simulium (Gomphostilbia) sarawakense sp. nov.
Female. Body length 2.0 mm. Head. Somewhat narrower
than width of thorax. Frons (Fig. 26) narrow, dark brown,dull, covered moderately with yellowish-white scale-like recumbent hairs, interspersed with several dark simple long
hairs along each lateral margin; frontal ratio 1.8:1.0:3.6;

frons-head ratio 1.0:5.8. Fronto-ocular area (Fig. 27) welldeveloped, narrow. Clypeus dark brown, covered moderately with yellowish-white scale-like recumbent hairs, interspersed with several dark simple long hairs on each side.
Proboscis ca. 0.67× as long as clypeus. Antenna composed of 2+8 segments (this female appears to be an abnormal indivisual, showing the incomplete segmentation between the 1st and 2nd flagellar segments, since both of the
1st flagellar segments are unusually longer, and that of theright antenna has an transverse incision, as shown in Figs.
28 and 29; normal females of this species must have 2+9antennal segments, since the pharate male examined shows
usual 2+9 antennal segments on both sides), whitish yellow
on basal 1/2 and gradually darkened toward apex, though its
border not well defined. Maxillary palp composed of 5 segments, light to medium brown, proportional length of 3rd,
4th and 5th segments 1.0:1.2:3.3; 3rd segment somewhat
swollen apically; sensory vesicle (Fig. 30) ellipsoidal, ca.
0.26× as long as 3rd segment, with medium opening medially. Maxillary lacinia with 9 or 10 inner teeth and 12 or 13
outer ones. Mandible with ca. 20 inner teeth and ca. 10
outer ones. Cibarium (Fig. 31) medially forming roundsclerotized plate folded forward from posterior margin, andwith well sclerotized, medial longitudinal ridge with forked
apex. Thorax. Scutum medium brown, with 3 faint dark
longitudinal vittae (1 medial and 2 submedial), shiny,densely covered with yellowish-white scale-like recumbent
hairs interspersed with a few dark upright long hairs onprescutellar area; scutellum medium brown, covered withyellowish short hairs as well as dark upright long hairs
along posterior margin. Postscutellum medium brown, bare.
Pleural membrane bare. Katepisternum medium brown,
longer than deep, moderately covered with dark hairs. Legs.
Foreleg: coxa whitish yellow; trochanter whitish yellow except outer surface slightly darker; femur light brown; tibia
light brown except base whitish yellow; tarsus dark brown,with moderate dorsal hair crest; basitarsus slightly dilated,
ca. 6.5× as long as its greatest width. Midleg: coxa medium brown with posterior surface dark brown; trochanter
whitish yellow; femur light brown except base yellow; tibia
whitish yellow on basal 2/5 with slightly dark subbasal
band, and medium brown on apical 3/5; tibia densely covered with whitish fine hairs on ourter and posterior surfacesof basal 2/5; tarsus medium brown with basal 1/2 of basitarsus whitish yellow. Hind leg: coxa light brown, trochanter
whitish yellow; femur light brown with base pale whitish
yellow and apical cap medium brown; tibia (Fig. 32) whitish yellow basally, slightly darkened toward apical 1/3, withdark subbasal band, and medium to dark brown on apical
1/3; tibia densely covered with whitish fine hairs on outerand posterior surfaces of basal 2/3; tarsus brownish black２
４
８

Figures 26-37 Morphological characters of S. sarawakense sp. nov. 26-35, female; 36 and 37, pupa. 26, frons; 27, fronto-oculararea; 28 and 29, antennae (dorsal view; 28, right; 29, left); 30, 3rd segment of maxillary palp with sensory vesicle;
31, cibarium; 32, tibia, basitarsus and 2nd tarsal segment of hind leg; 33, claw of hind leg; 34, sternite 8, anterior
gonapophyses, genital fork, spermatheca, paraproct and cercus of genitalia in situ (ventral view); 35, paraproct andcercus in situ (lateral view); 36, basal portion of right gill filaments (lateral view); 37, terminal hook (end view).
Scale bars: 0.1 mm for figs. 32 and 36; 0.05 mm for figs. 26-29; 0.02 mm for figs. 30, 31, 34 and 35; 0.01 mm forfigs. 33 and 37.

２
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９

except basal 3/4 of basitarsus, basal 1/2 of 2nd tarsal segment yellowish white; basitarsus narrow, nearly parallelsided, ca. 6.7× as long as wide, and ca. 0.67× and ca.
0.55× as wide as greatest width of tibia and femur, respectively; calcipala ca. 1.35× as long as wide, and ca. 0.72×
as wide as apical portion of basitarsus. All femora, tibia
and parts of tarsus also moderately covered with dark scalelike hairs. Claws (Fig. 33) each with large basal tooth,
0.51× as long as claw. Wing. Length 1.7 mm. Costa withdark spinules as well as dark hairs. Subcosta with darkhairs except near apex bare. Hair tuft on stem vein yellowish. Basal portion of radius fully haired. Basal cell absent. Abdomen. Basal scale dark yellow, with fringe ofpale fine hairs. Dorsal surface of abdomen dark brown except that of segment 2 yellow, moderately covered withshort dark hairs; tergites of segments 2, 6, 7 and 8 shiny;sternal plate on segment 7 undeveloped. Genitalia (Figs.
34 and 35). Sternite 8 bare medially, with 22 or 23 longstout hairs and 4 or 5 short hairs on each side. Anterior
gonapophyses nearly triangular, with round medioposterior
corners, thin, membraneous, covered moderately with microsetae, interspersed with no short setae; inner margins
nearly straight or slightly sinuous, moderately sclerotized,
and somewhat separated form each other. Genital fork ofusual inverted-Y form, with arms of moderate width; arm
moderately folded medially, with small projection directed
anterodorsally. Paraproct moderately produced ventrally,
with 22 long hairs on ventral and lateral surfaces, and with
3 sensilla on inside surface. Cercus ca. 0.5× as long as
wide, rounded. Spermatheca ellipsoidal, ca. 1.33× as longas wide, well sclerotized except tube and small area of tubal
base, and with many fissures on surface; internal setae appear to be absent; both accessory tubes slender, slightly
larger in diameter than major one.
Male. Unknown.
Pupa. Body length 2.2 mm. Head. Integument yellow,
moderately covered with round tubercles; antennal sheathnormal, with no spinous projections, and almost bare; facewith 1 pair of simple long trichomes with uncoiled apex,
and frons with 3 pairs of similar trichomes; 3 frontal
trichomes on each side arising close together, subequal insize to one another. Thorax. Integument yellow, moderately covered with round tubercles, and with 3 pairs of simple long trichomes with coiled or uncoiled apex dorsally, 2pairs of simple trichomes (1 long and 1 medium) anterolaterally, 1 pair of medium-long simple trichomes posterolaterally, and 3 pairs of simple trichomes (2 medium and 1
short) ventrolaterally. Gill (Fig. 36) composed of 8 slenderthread-like filaments, arranged in groups of 3+3+2 fila-

ments from dorsal to ventral; upper and middle triplets sharing short stalk; each triplet consisting of 2 paired filamentswith very short stalk and 1 individual filament; lower pairwith stalk of medium length; basal common stalk short,with somewhat swollen transparent organ ventrally; all filaments pale yellowish, subequal in length (1.2-1.6 mm) andthickness to one another except outer filament (ca. 3.0 mmlong) of lower pair ca. 2.0× length and thickness of theother filaments (when basal portion of each filament iscompared); cuticular surface of apical 3/4 of stalk of lower
pair and basal 1/2 of outer filament of lower pair with distinct annular ridges and furrows but those of other filaments
and stalks smooth; cuticular surface of all filaments densely
covered with minute tubercles. Abdomen. Tergum 1
mostly pale, bare, with 1 simple slender medium-long setaon each side; tergum 2 mostly pale, bare, and with 1 simple
slender medium-long seta and 5 short setae on each side;
terga 3 and 4 mostly pale, each with 4 hooked spines and 1
short seta on each side; tergum 5 lacking spine-combs; terga
6-9 each with distinct spine-combs in transverse row(though those on tergum 9 much smaller in size), together
with comb-like groups of minute spines on each side; tergum 9 with 1 pair of distinct plate-like terminal hooks, extending laterally, and with serrated outer margin (Fig. 37).
Sternum 4 with 1 simple or bifid hook and a few simple
slender minute setae on each side. Sternum 5 with 1 pair ofbifid or trifid hooks submedially and a few simple slender
minute setae on each side; sterna 6 and 7 each with 1 pair ofbifid or trifid inner and simple outer hooks somewhat
spaced from each other, and a few simple slender minute setae on each side. Each side of segment 9 with 3 grapnellike hooklets. Cocoon. Simple, wall-pocket-shaped, neatly
and moderately woven, somewhat extending ventrolaterally; anterior margin not thickly woven; posterior 1/2 withfloor roughly woven; individual threads visible; 2.5-2.8 mm
long × 1.5-1.9 mm wide.
Mature larva. Unknown.
TYPE SPECIMENS. Holotype, female, reared from pupa,
collected at Pueh, Sematan, Sarawak, Malaysia, 18.III.1998.
Paratype 1 pupa, same data and date as holotype.
ECOLOGICAL NOTES. Two pupae were collected, one froma dead leaf, and the other from a rock, in water of a small
clean shaded stream (0.5-3.0 m wide) flowing down on the
rocky streambed in natural forest of Mt. Pueh. The watertemperature was 25°
C. This species was collected together
with S. laterale, S. lehi and S. sabahense.
DISTRIBUTION. Sarawak.

２
５０

ETYMOLOGY. The species sarawakense is named after thename of state where this species was collected.
REMARKS. This new species is assigned in the batoense
species-group by the slender male hind basitarsus (dissected
out of the pharate male) as well as the usual 2+9 segmentsof the adult antennae. The pupa of S. sarawakense is characterized by the gill composed of one long and seven shortfilaments which are about half the length and thickness of
the longer filament (Fig. 36). The similar arrangement of
the pupal gill filaments, coupled with plate-like terminal
hooks with a serrated outer margin, has been seen in S.
epistum Delfinado and S. luzonicum Takaoka, both from
Philippines (Takaoka, 1983). However, stalks of upper andmiddle triplets of these two species share a very short common stalk. This new species is distinguished in the female
from these known species by the narrower frons and smaller
sensory vesicle. The frons-head ratio is 1:5.8 in this new
species, while that is 1:52 in S. epistum and 1:4.3 in S.
luzonicum; and the relative length of sensory vesicle to thethird segment is 0.26 in this new species but it is 0.4 in theother two species. The pupal gills of S. rayohense Smart
and Clifford described from Sabah, seem to be similar to
those of S. sarawakense, but seven shorter filaments of thisknown species are much longer (i.e., 2.0-2.3 mm), according to the illustration given by Smart and Clifford (1969).
The pupa of S. pegalanense Smart and Clifford, describedfrom Sabah, also differs from that of this new species by theterminal hooks not serrated. In the pupal gills of S.
pegalanense, it is not the outer filament of the ventral pairbut the counter inner filament that is somewhat thicker andlonger than its counter filament and six other filaments ofupper and middle triplets. This was confirmed with slidemounted type specimen.

NOTES ON OTHER SPECIES NEWLY RECORDED FROM SARAWAK
Simulium (Gomphostilbia) sheilae Takaoka and Davies,
1995
Simulium (Gomphostilbia) sheilae Takaoka and Davies,
1995: 60-65 (female, male, pupa and larva); Kuvangkadilok and Takaoka, 2000: 172-173; Takaoka
et al., 2000: 161.
SPECIMENS EXAMINED. 1 female, 1 male, both reared
from pupae, and 2 larvae, collected at Saya Bakti, 10 km
from Kuching, 17.III.1998; 6 females, 5 males, all reared
from pupae, and 10 larvae, collected from Jau R., at Santobung, 30 km from Kuching, 17.III.1998; 2 males, reared
from pupae, 2 larvae, collected at Damai Look-out Point, 17.
III.1998; 1 male, reared from pupa, and 3 larvae, collected

at Lundu, in Gunung Gading National Park, 18.III.1998.
ECOLOGICAL NOTES. The pupae and larvae of this species
were collected from fallen leaves and trailing grasses inslowly-flowing lowland streams 0.1-4.0 m wide, exposed tothe sun or partially shaded. The water temperature of thestream was 26 or 27°
C. This species was collected together
with S. sabahense.
DISTRIBUTION. Peninsular Malaysia, Sabah, Sarawak (newrecord), Sumatra and Thailand.
REMARKS. S. sheilae was originally described from Peninsular Malaysia, and was assigned to the ceylonicum speciesgroup by Takaoka and Davies (1995). It was also recorded
from Thailand (Kuvangkadilok and Takaoka, 2000), Sumatra (Takaoka et al., 2000) and Sabah (Takaoka, 2001).
The reared adult female and male specimens, as wellas pupal and larval ones examined in this study, are morphologically almost the same as those originally described,
with an exception that the scutum of the present male specimens is whitish pruinose on each shoulder, along both lateral margins and on the prescutellar area (not entirely whitish pruinose, as in the type male specimen). The same difference in the male scutal pattern has been noted for specimens of this species collected from Sabah (Takaoka, 2001).
Simulium (Nevermannia) aureohirtum Brunetti, 1911
Simulium aureohirtum Brunetti, 1911: 283-288 (male);
Edwards, 1934: 134-137 (female, pupa and larva).
Simulium (Nevermannia) aureohirtum: Ogata, 1956:
61-62; Ogata, 1966: 129; Crosskey, 1988: 459;
Takaoka and Roberts, 1988: 194-195; Crosskey and
Lowry, 1990: 204; Takaoka and Davies, 1995: 8788; Takaoka and Davies, 1996: 5-6. Crosskey andHoward, 1997: 47.
Simulium aureohirtum: Orii, Uemoto and Onishi, 1969:
1-13 (larva).
Simulium (Eusimulium) aureohirtum: Puri, 1933: 1-7(female, male, pupa and larva); Ogata and Sasa, 1954:
325; Ogata, Sasa and Suzuki, 1956: 73; Crosskey,
1973: 423; Takaoka, 1976: 170-171; Takaoka, 1979:
382-384 (female, male, pupa and larva); Datta, 1983:
225; Takaoka and Suzuki, 1984: 11-12.
Simulium (Eusimulium) tuaranense Smart and Clifford, 1969: 40-43. Syn. by Crosskey, 1973.
Simulium (Eusimulium) philippinense Delfinado,
1962: 47-62. Syn. by Takaoka, 1983.
SPECIMENS EXAMINED. 1 female reared from pupa, and 1
larva, collected from Bunga R., Kuching, Sarawak, 16.
III.1998.

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１

ECOLOGICAL NOTES. The one pupa and one larva of thisspecies were collected from grass leaves trailing in thewater of a stream with width of 2-5 m, running in a cacao
plantation, exposed to the sun. The water temperature was
26°
C. Many pupae and larvae of S. sabahense were collected together with this species.
DISTRIBUTION. Indonesia, Japan, Malaysia (Peninsular
Malaysia, Sabah, and Sarawak (new record)), Nepal, Pakistan, Philippines, Sri Lanka, Taiwan and Thailand.
REMARKS. This species belongs to the ruficorne speciesgroup of the subgenus Nevermannia (Crosskey, 1969) andis known to be widely distributed in the Oriental Region
and parts of the Palaearctic Region (Crosskey and Howard,1997). The female, its associated pupa and larva collectedfrom Sarawak agree in morphological characters with theredescription of this species given by Takaoka (1979).
Simulium (Simulium) laterale Edwards, 1933
Simulium laterale Edwards, 1933: 256 (female).
Simulium (Simulium) laterale: Smart and Clifford, 1969:22-26 (female, male, pupa and larva); Takaoka, 1983:
164 (only female genitalia illustrated).
SPECIMENS EXAMINED. 1 male reared from pupa, 2 pupae
and 5 mature larvae, collected at Pueh, Sematan, Sarawak,
18.III.1998.
ECOLOGICAL NOTES. Pupae and larvae were attached onstones and rocks in water of a small clean shaded stream
0.5-3.0 m wide, flowing down on the rocky streambed in anatural forest of Mt. Pueh. The water temperature was 25°
C.
This species was collected together with S. lehi, S. sabahense and S. sarawakense.
DISTRIBUTION. Sabah and Sarawak (new record).
REMARKS. This species was originally described from female specimens collected at Lumu Lumu, Sabah, by Edwards (1933). The male, pupa and larva were describedlater by Smart and Clifford (1969). Takaoka (1983) examined the female genitalia of one of the syntype specimens of
S. laterale preserved at The Natural History Museum, London, and assigned this to the melanopus species-group of
the subgenus Simulium s. str. The male, pupal and larval
specimens collected from Sarawak agree with the descriptions of this species given by Smart and Clifford (1969), except the difference of leg colorings: fore coxa white (notlight brown), fore tibia dark brown with a wide whitish area

on outer surface (not entirely dark brown); mid tibia darkbrown with a basal tip white (not entirely dark brown), andbasitarsus almost white with an apical tip dark brown (notlighter brown); hind tibia dark brown with a basal small
area white (not entirely dark brown), and basitarsus whiteon the basal 1/2, dark brown on the apical 1/2 and on a basal tip (not dark brown with the basal 1/3 light brown).

ACKNOWLEDGEMENTS
I am grateful to Prof. Yong Hoi Sen, Malaya University, and Dr. Charles Leh, Sarawak Museum, for their kind
help and arrangement of collections of black flies in thefields. Thanks are also due to Dr. A. Shelley and Dr. J.
Chainey, the Natural History Museum, London, U.K., fortheir kind arrangement of the loan of the type specimens of
S. rayohense and S. pegalanense. This work was supportedby the Grant-in-Aid of Japan Society for Promotion of Science (the fiscal year 1997) and in part by Grant-in-Aid for
Scientific Research C of Ministry of Education, Science,Sports and Culture, Japan (no. 11670246).

REFERENCES
１）Crosskey, R.W. (1969): A re-classification of the Simulii-dae (Diptera) of Africa and its islands. Bull. Br. Mus.
(Natur. Hist.) (Entomol.) Suppl. 14, 1-195
２）Crosskey, R.W. and Howard, T.M. (1997): A New Taxonomic and Geographical Inventory of World Blackflies(Diptera: Simuliidae). 144 p., The Natur. Hist. Mus., London.
３）Edwards, F.W. (1933): Diptera Nematocera from Mount
Kinabalu. J. Fed. Malay St. Mus., 17, 223-296
４）Kuvangkadilok, C. and Takaoka, H. (2000): Taxonomic
notes on Simuliidae (Diptera) from Thailand: Descriptionof a new species and new distributional records of nine
known species. Jpn. J. Trop. Med. Hyg., 28, 167-175
５）Smart, J. and Clifford, E.A. (1969): Simuliidae (Diptera) of
Sabah (British North Borneo). Zool. J. Linn. Soc., 48, 947
６）Takaoka, H. (1979): The Black Flies of Taiwan (Diptera:
Simuliidae). Pacific Insects, 20, 365-403
７）Takaoka, H. (1983): The blackflies (Diptera: Simuliidae) of
the Philippines. xi+199p., Japan Society for the Promotionof Science, Tokyo
８）Takaoka, H. (2001): Two new and three newly recorded
species of black flies (Diptera: Simuliidae) from Sabah,
Malaysia. Jpn. J. Trop. Med. Hyg., 29, 2, 221-230
９）Takaoka, H. and Davies, D.M. (1995): The Black Flies(Diptera: Simuliidae) of West Malaysia. viii+175p., Kyushu University Press, Fukuoka
１０）Takaoka, H. and Davies, D.M. (1996): The Black Flies(Diptera: Simuliidae) of Java, Indonesia, viii+81p., Bishop

２
５２Mus. Bul. Entomol. 6, Bishop Museum Press, Hawaii.
１１）Takaoka, H., Yunus, M., Hadi, U.K., Sigit, S.H., and Miya-gi, I. (2000): Preliminary report of faunistic surveys on
black flies (Diptera: Simuliidae) in Sumatra, Indonesia. Jpn.
J. Trop. Med. Hyg., 28, 157-166

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Jpn. J. Trop. Med. Hyg., Vol.２
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COMPARATIVE ORAL SUSCEPTIBILITY OF DENGUE VECTORMOSQUITOES FROM JAPAN AND SOUTHEAST ASIA TO TWO
DENGUE-1 VIRUS STRAINSHASANUDDIN ISHAK1, INES TOMOCO MATSUSE1, KIYOSHI KAMIMURA1,＊,
TSUTOMU TAKEGAMI2 AND HISASHI FUNADA1
Received January 12, 2001/Accepted April 11, 2001

Abstract: Five geographic strains of Aedes albopictus from Japan and Southeast Asia and three strains of Aedes
aegypti from Indonesia and Pakistan were compared for their susceptibility to oral infection with the human virus,
dengue-1 Mochizuki strain (isolated in 1943, Japan) and A88 strain (isolated in 1988, Indonesia). Female mosquitoes, aged 3-4 days, were infected with a virus-erythrocyte-sucrose suspension. After 14 days of incubation at 2530°
C, viral infection in mesenteron (midgut) or in head-thorax (salivary gland) of each individual mosquito was
identified by the reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of dengue-1 virus envelope gene. Although the results of susceptibility varied in some extent in different strains of mosquitoes and viralstrains, the oral susceptibility to both dengue-1 virus strains was not significantly different among dengue vector
mosquitoes from Japan and Southeast Asia.
Key words: Susceptibility, Vector mosquitoes, Geographic strains, Dengue-1 virus, Oral infection

INTRODUCTION
Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) have become a global
public health problem in recent years (WHO, 2000). In Japan there are 42 confirmed cases of DF 1988 (Yamada et al.,1999), most of them infected when visiting Southeast Asian
countries.
Aedes aegypti, which is found in most parts of thetropics and subtropics, has been considered to be main vector of DF/DHF. Another important vector, Aedes albopictus
is able to thrive in both cold and hot climates. The latter
species is commonly found in Japan and has been implicated as a vector of DF epidemics in Japan during World
War ! (Sabin, 1952; Hotta, 1998).
There are some studies on mosquito susceptibility tooral infection with dengue viruses using Asian strains of Ae.
aegypti and Ae. albopictus (Gubler and Rosen, 1976;
Gubler et al., 1979; Rosen et al., 1985; Tardieux et al.,1992). However Ae. albopictus from Tokyo, Japan is the
only one strain that has been tested for its vector competence to dengue virus (Boromisa et al., 1987). In this report,
the similarity in vector competence of this species from Japan with that from North America support the possibility

that this mosquito was introduced in North America withthe shipment of used tires from Northren Asia (Hawley etal., 1987). The development of transportation systems in
the world has stimulated the global spread of dengue via
travelers and trade.
The immmunofluorescence technique was used for detection of virus in most of previous investigation on vector
competence. Recently, several studies described the sensitivity and rapid generation of results of polymerase chain
reaction (PCR) or reverse trancriptase-PCR (RT-PCR) assay
for detecting and typing dengue viral RNA extracted frommosquitoes (Tardieux and Poupel, 1990; Tardieux et al.,
1992; Chungue et al., 1993; Chow, et al., 1998). The PCR
method that can detect the dengue virus in infected mosquitoes from extremely small amounts of sample was appliedin this study.
The dengue-1 viruses (DEN-1) used in this study are
Mochizuki and A88 strains. Mochizuki strain was isolated
from the blood of a DF patient during an epidemic of dengue fever in Nagasaki, Japan in 1943 (Kimura and Hotta,
1994) and was found to have lost its pathogencity for human (Hotta, 1952). The A88 strain was isolated from theblood of a DHF patient in an outbreak of DHF in Jakarta,
Indonesia in 1988 (Fujita et al., 1997).

１ Department of Biodefence Medicine, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan
２ Division of Tropical Medicine, Medical Research Institute, Kanazawa Medical University, Ishikawa 920-0293, Japan
Correspondent author: DR. KIYOSHI KAMIMURA, Department of Biodefence Medicine, Faculty of Medicine Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan
Tel.076-434-7246 FAX.076-434-5018

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Ae. albopictus strains from different areas of Japan
were tested for their susceptibility to infection with both
DEN-1 Mochizuki and A88 strains and then compared withthe susceptibility of Ae. albopictus and Ae. aegypti fromepidemic areas of Southeast Asia. Mochizuki and A88
strains were also compared for their abilities to infect orally
and replicate in mosquito vectors.

MATERIAL AND METHODS
Mosquito strains:
The origin of Ae. albopictus (5 strains) and Ae. aegypti(3 strains) and the number of generations used for oral infection are shown in Table 1. All mosquito strains werecolonized in our insectary room from field collected eggs indifferent areas of Japan and Southeast Asia. The mosquitoes were reared in plastic pans (33.5 cm×25 cm×11.5 cmhigh) containing 3 liters of aged tap water with a density of
100-200 larvae per container. The larvae were fed a mixture of yeast extract with a liver and vegetables powder andmaintained at 25°
C, 70% relative humidity and a 14:10 hrlight: dark photoperiod. Adult colony mosquitoes were provided with 10% sucrose solution.
Preparation of virus as source of virus solution:
DEN-1 Mochizuki and A88 strains used in this study
were prepared by growth in Vero cell cultures. A confluent
25 cm2 tissue culture of Vero cell monolayer was infected ata multiplicity of infection of 0.01 PFU/cell with addition of
5 ml of Eagle’s essential medium (E-MEM) containing 5%
heat-inactivated fetal calf serum (FCS). After 7 days of incubation at 37°
C, cells were scraped into the medium, centrifuged at 2,000 rpm for 5 min, and then re-suspended in
500-600 µl of the infected supernatant culture medium assource of viral suspension.

Oral infection of mosquito strains:
The 3-4 days old female mosquitoes were placed in asmall cylindrical cardboard container and deprived of sucrose solution 26-36 hr prior to the infection meal, and thenallowed to feel on a hanging drop virus solution. Virus solution was prepared by mixing equal parts of DEN-1 virus
suspension, washed human red blood cells and 10% (final
concentration) sucrose. Adenosine triphosphate (ATP) was
added at a final concentration of 5×10−3 M as a phagostimulant (Rutledge et al., 1964). The drop of virus solution was
warmed at 40°
C for 5-10 min and placed on a fine meshcovering a small cylindrical cardboard container containing
mosquitoes. In each experiment, a small aliquot of virus
solution was held at room temperature during mosquito
feeding (1 to 2 hr) and subsequently titrated by plaque assay
in Vero cell monolayers. Mosquito strains to be compared
were fed the same mixture at the same time. Mosquitoesthat took at least three-fourth of full blood meal were selected and held for 14 days at 30°
C or 25°
C and a 76% relative humidity in a desiccator. The infected mosquitoes were
killed by freezing and the virus was detected in mesenteron(midgut) and thorax-head (salivary gland) by RT-PCR amplification of envelope gene. The experiments using viruses
and infected mosquitoes were conducted in P3 level laboratory of Animal Research Center, Toyama Medical and Pharmaceutical University.
Sample preparation for virus assay:
Mesenteron and thorax-head were removed from individual mosquito and triturated by a pellet mixer in 150 µl of
phosphate-buffered saline with 30% FCS. Triturated samples were centrifuged at 10,000 rpm at 4°
C for 5 min. Twothird of the supernatant fluid was diluted, filtered through0.45 µm syringe filter unit and titrated by plaque assay on
Vero cell monolayers and the remaining fluid was treatedfor RNA extraction. The virus titer was calculated as

Table 1 Origin of Aedes albopictus and Aedes aegypti tested for oral infection with dengue
virus type 1 (DEN-1) Mochizuki and A88 strains
Year ofcolonization

Number ofgenerations

Toyama, Japan
Hyogo, Japan
Okayama, Japan
Bangkok, Thailand
South Sulawesi, Indonesia

1992
1998
1996
1986
1996

F88
F20
F38
F137
F30

West Timor, Indonesia
East Java, Indonesia
Karachi, Pakistan

1999
1986
1990

F10
F103
F81

Species/strains

Geographic origin

Ae. albopictus
ToyamaAwajishima
KojimaThailand
MakassarAe. aegypti
Timor
SurabayaPakistan２５５plaque forming units (PFU) per mosquito based on the estimation of blood meal size of Ae. albopictus (2 µl ) as previously reported (Konishi and Yamanishi, 1984).
RT-PCR amplification for viral detection in infected mosquitoes:
Viral RNA was extracted from triturated sample using
Isogen (Nippon Gene). The pellet was dried up and suspended in 10 µl of sterile water. RNA reverse transcription
was carried out at 42°
C for 60 min in a final volume of 20µl (5 µl of RNA template, 25 pmol of primer, 10 units of
RNase inhibitor, 10 mM of dNTP and 2.5 units of reverse
transcriptase). The reaction mixture also was held at 95°
Cfor 5 min and then quickly chilled in ice. For PCR, 20 µl of
cDNA was added with 25 pmol of sense primer, 25 pmol ofcomplementary primer, 10 mM of dNTP and 1.25 units of
Taq polymerase in a final volume of 50 µl . The reaction
mixture was overlaid with mineral oil and amplification was
performed in a Perkin Elmer thermal cycler. Each of the 30
cycles performed comprised a 1 min denaturation at 94°
C, 1
min annealing at 60°
C and 2 min elongation at 72°
C.
The cDNA primer, which was also the PCR complementary primer: D1-NS1N/2439, a 20-mer (5’-ACTGGAAAGGCAGAGAACTC-3’) and the PCR sense primer: D1ES/1552, a 20-mer (5’-GCTCGTCCACAAACAATGG-3’)
were synthesized based on the known nucleotide sequencesof dengue-1 Nauru strain (Puri et al., 1998). The PCR was
carried out to amplify a 900 bp target sequence of the envelope gene. PCR product samples were electrophoresed on
1.5% agarose gel in Tris-Borate-EDTA (TBE) buffer andthe bands were visualized by ethidium bromide staining. To
ensure that cross contamination did not occur between samples, a negative control was made without RNA for each
PCR experiment.

RESULTS
Oral susceptibility of mosquito strains to DEN-1:
For the experiment of the oral susceptibility to DEN-1
Mochizuki and A88 strains, 3 different viral concentrations
were used to infect mosquito strains. The proportions ofmosquitoes infected with Mochizuki and A88 strains at
30°
C incubation were estimated based on the PCR results(Tables 2 and 3). Ae. albopictus Kojima strain was the only
one strain orally susceptible to DEN-1 Mochizuki infection
after ingestion of low viral dose (1.56×10 PFU/mosquito)(Table 2). Most of Ae. albopictus strain ware infected afteringestion of higher viral doses (1.98×102 and 7.9×102
PFU/mosquito). However, all of mosquito strains showed
low susceptibility to DEN-1 Mochizuki strain even with ahigher viral dose (7.9×102 PFU/mosquito). Ae. aegypti
strains could be infected with DEN-1 Mochizuki strain
within 7 days incubation at 30°
C (data not shown), but theinfection rate decreased after 14 days incubation, therefore
the infections were not observed.
In contrast to Mochizuki strain, most of mosquito
strains were susceptible to DEN-1 A88 infection even withlow viral dose (8.55 PFU/mosquito) (Table 3). The susceptibility to A88 infection incresed in Ae. albopictus Toyama
strain as same as that of Ae. aegypti Timor and Pakistan
strains after ingestion of high viral dose (2.16×103 PFU/
mosquito). Ae. albopictus Kojima, Thailand, and Makassar strains showed same or similar proportions of infection
with 3 different infectious doses. In general, mosquito
strains incubated at 30°
C were more susceptible to DEN-1
A88 than to Mochizuki strain as shown in Table 4.
The above data were statistically analyzed by the multiple comparison Bonferroni test (SPSS 10.0J for Windowsbase system) and no significant differences were foundTable 2 Proportion of Ae. albopictus and Ae. aegypti strains orally infected with different
doses of DEN-1 Mochizuki strain after 14 days of incubation at 30°
C
Species/strains
Ae. albopictus
ToyamaAwajishima
KojimaThailand
MakassarAe. aegypti
Timor
SurabayaPakistan
NT=not tested.

Proportion of mosquitoes infected (n)Virus ingested (PFU/mosquito)
1.56×10
1.98×102
7.9×102
0.00!
0.00!
0.33!
0.00!
0.00!

0.75"
0.25"
0.25"
0.00"
NT

0.33!
0.33!
0.00!
0.67!
0.00!

0.00!
0.00!
0.00!

0.25"
0.00"
0.00"

0.00!
NT
NT

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Table 3 Proportion of Ae. albopictus and Ae. aegypti strains orally infected with different
doses of DEN-1 A88 strain after 14 days of incubation at 30°
C
Proportion of mosquitoes infected (n)Virus ingested (PFU/mosquito)
1.48×102
2.16×1010

Species/strains
8.55
Ae. albopictus
ToyamaAwajishima
KojimaThailand
MakassarAe. aegypti
Timor
SurabayaPakistan

0.33"
0.33"
0.00"

0.25#

0.67"

0.75#
0.25#

0.00"
0.00"

0.33"
0.67"

0.50!
NT

0.33"
0.67"

0.00!

0.25#
0.50#
0.00#

0.67"

1.00!
0.33"

NT
0.67"

NT=not tested.

Table 4 Comparative susceptibility of Ae. albopictus and Ae. aegypti strains orally infected with DEN-1 Mochizuki andA88 strain at 30°
C
Species/strains
Ae. albopictus
ToyamaKojimaMakassarAe. aegypti
Timor
SurabayaPakistanNo. (%) of mosquitoes infected with DEN-1
Mochizuki*
A88**
0/30 (0.0)
1/11 (9.1)
1/29 (3.4)

6/30 (20.0)
2/15 (13.3)
3/29 (10.3)

0/18 (0.0)
1/9 (11.1)
0/12 (0.0)

1/18 (5.6)
2/13 (15.4)
1/16 (6.3)* Virus ingested was 4.0×10 PFU/mosquito ** 6.0×10 PFU/mosquito

among oral susceptibility of mosquitoes to Mochizuki or
A88 strains.
Comparison of mesenteronal and head-thorax (salivary
gland) infections of mosquito strains:
The comparison of mesenteronal and disseminated(head-thorax) infections of mosquito strains with DEN-1
Mochizuki and A88 strains after 14 days of extrinsic incubation at 25°
C are shown in Figs. 1 and 2, respectively. In
Fig. 1, Ae. albopictus Kojima strain showed the highest
proportion of infection with Mochizuki strain in the mesenteron (14/17) and near half of them (7/17) showed disseminated infection. Toyama strain also showed high proportionof disseminated infection in contrast to Awajishima strain,
in which disseminated infection was not detected. The
same proportion of mesenteronal and disseminated infection was observed in Ae. albopictus Thailand strain and Ae.
aegypti Timor strain. Infection did not occur in Ae. aegypti
Surabaya and Pakistan strains.

Fig. 2 shows that all Ae. albopictus strains were infected orally with A88 strain except Awajishima strain.
However, not all of them developed disseminated infection.
Surabaya strain, among Ae. aegypti species, had higher
mesenteronal infection with A88 strain than other strains,
but the infected mosquitoes did not develop disseminatedinfection. Timor strain had the same proportion of mesenteronal and disseminated infection, while Surabaya strain
did not show disseminated infection although it showed
relatively high proportion of mesenteronal infection.
From the above results Ae. albopictus strains tended tobe more susceptible to oral infection with either DEN-1
Mochuizuki or A88 strain than Ae. aegypti strains.
Mesenteronal escape barrier of mosquito strains:
Based on the data of disseminated infection (Figs. 1
and 2), the mesenteronal escape barrier was estimated asshown in Fig. 3. The existence of mesenteronal escape barrier means that virus multiplies only in mesenteron and２５７

Figure 1 Proportions of mesenteronal (midgut) and disseminated (head-thorax) infections of Ae. albopictus
and Ae. aegypti strains orally infected with DEN-1 Mochizuki strain after 14 days incubation at
25°
C. The amount of virus ingested was 1.3×102 PFU/mosquito.

Figure 2 Proportions of mesenteronal (midgut) and disseminated (head-thorax) infections of Ae. albopictus
and Ae. aegypti strains orally infected with DEN-1 A88 strain after 14 days incubation at 25°
C.
The amount of virus ingested was 1.1×103 PFU/mosquito.

does not invade tissues regardless of the length of extrinsic
incubation (Kramer et al., 1981). Ae. albopictus from Japan especially Kojima strain showed the highest mesenteronal escape barrier to infection with Mochizuki strain (Figs.
1 and 3). Ae. aegypit Surabaya strain showed high mesenteronal escape barrier to infection with A88 strain (Figs. 2
and 3). Ae. albopictus Toyama strain showed the samemesenteronal escape barrier to infection with both Mochizuki and A88 strains.

２
５８

Figure 3 Proportions of mesenteronal escape barrier＊ of Ae. albopictus and Ae. aegypti strains orally
infected with DEN-1 Mochizuki and A88 strains.
＊ Barrier: proportion of (mesenteron-head thorax) infection

Viral titers in mesenteron of mosquito strains:
The viral titer of DEN-1 A88 strain (2.16×103 PFU/
mosquito) in mesenteron of Ae. albopictus were measured
after 7, 14 and 21 days in one mosquito of each Toyama and
Makassar strains. The titers in Makassar strain decreasedslightly after 7 days (3.41×102 PFU/mosquito), 14 days
(2.1×102 PFU/mosquito) and 21 days (4.8×102 PFU/mosquito) of extrinsic incubation at 30°
C. The titers in Toyama
strain were 2.36×102 PFU/mosquito after 7 days and remained constant after 14 days (1.41×102 PFU/mosquito)
and 21 days (1.30×102 PFU/mosquito).

DISCUSSION
A RT-PCR method was applied with some modifications of the method of Sambrook et al. (1989) for rapid andsensitive detection of viral RNA in infected mosquitoes,
while the viral titer was detected with the method of plaqueassay. Since this method can detect rapidly a few doses ofinfected materials, it is expected to be a useful tool for early
warning monitoring system for dengue outbreaks in the future. The sensitivity and specificity of PCR method for viral detection allowed a comparison of susceptibility of mosquitoes to dengue viruses in the present study. For this purpose, low viral doses (about 1/1,000 of the viral concentrations reported so far) were used to infect the mosquitoes
orally. In the preliminary study, various infectious meals
and conditions were tested for oral infection. The blood
meal prepared with the mixture of infected cells and theirculture media was more effective to infect the mosquitoes

than the meal prepared with the infected cell culture supernatant. This result led us to add cells in infective meal.
The susceptibility is also dependent to the serotype ofdengue virus. The oral threshold of infection for DEN-1
has been reported to be higher than that for other dengue serotypes (Gubler and Rosen, 1976; Gubler et al., 1979). The
high concentration of virus in the blood meal is neccessary
to overcome this threshold, but it varies for different virus
strains in the same mosquito species and for the same virus
in different species or different geographic strains of thesame species as reviewed by Hardy et al. (1983).
Colonization of mosquito strains in the laboratory may
have some influences over susceptibility. This possibility
was shown by the proportions of orally infected mosquitoesthat were higher in Timor strain (F8) than Surabaya (F107)
or Pakistan strains (F81) of Ae. aegypti. The influence ofcolonization was not investigated in the present study but
concerning this, Lorenz et al. (1984) correlate the differences of susceptibility with genetic variation at the malate
dehydrogenase locus.
There was a tendency for a high susceptibility at highviral concentrations, i.e., it seemed to possess a dosedependent mesenteronal (midgut) barrier as discussed by
Gubler and Rosen (1976), Gubler et al. (1979) and Kramer
et al. (1981). The dissemination barriers and infections virus titer in mosquito determine vector competence for dengue virus (Bosio et al., 1998). In this experiment not all infected mosquitoes developed disseminated infection, whichindicates the existence of a midgut escape barrier. According to Kramer et al. (1981), there are two dissemination

２
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barriers (meseteronal escape barrier and salivary gland infection barrier) in Culex tarsalis infected with Westernequine encephalomyelitis virus. For dengue infection the
midgut basal lamina thickness in mosquito strains influences the viral dissemination (Thomas et al., 1993). On theother hand, Rosen et al. (1985) reported that dengue virus
usually replicate to about the same extent in orally infectedmosquitoes as well as in the parenterally infected specimensof the same species.
Mosquitoes were more susceptible to DEN-1 Mochizuki strain than to A88 strain after incubation at 25°
C, butinversely at the elevated temperature, 30°
C. The effect of
extrinsic incubation temperature on the viral susceptibilityof mosquitoes has been reported by Kramer et al. (1983).
The different abilities of DEN-1 Mochizuki (human attenuated virus) and A88 strains to infect mosquitoes orally may
be related to their genetic differences (Ishak et al., 2001). In
an experiment using dengue-2 vaccine virus Miller et al.
(1982) showed that it was markedly less efficient in its ability to infect mosquitoes orally.
Boromisa et al. (1987) reported that the susceptibilityof Ae. albopictus from Japan (Tokyo strain) to dengue 1 virus was not significantly different to that from Malaysia(Southeast Asia), but its transmission rate was lower than
Malaysia strains. Similarly, from the present results it was
concluded that Japanese strains of Ae. albopictus are susceptible to DEN-1 in almost the same extent as Ae. albopictus and Ae. aegypti from Southeast Asian countries.

ACKNOWLEDGMENTS
The first author was supported by the fellowship of Dr.
Takafumi Hayashi, Director of Yuai Hospital Association.
We are indebted to Prof. Dr. Chairuddin Rasjad of the Faculty of Medicine, Hasanuddin University for his invaluable
support, and to the Dean of the Faculty of Public Health,Hasanuddin University for the approval of this study. We
also thank Dr. Motoyoshi Mogi, Division of Parasitology,
Saga Medical School and Dr. Yuki Eshita, Department of
Infections Disease Control, Oita Medical University forthier kind help in this study.

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PHYLOGENETIC ANALYSES OF A BLACKFLY SUBGENUSSIMULIUM (NEVERMANNIA) BASED ON MITOCHONDRIAL
16S RIBOSOMAL RNA GENE SEQUENCES
YASUSHI OTSUKA1, CHIHARU AOKI1, KATSUMI SAITO2, UPIK K. HADI3,
HIROSHI SUZUKI4 AND HIROYUKI TAKAOKA1,*
Received April 2, 2001/Accepted May 20, 2001

Abstract: Nucleotide sequences of a subregion of the mitochondrial 16S ribosomal RNA gene of 10 species of a
blackfly subgenus Simulium (Nevermannia), which include four species of feuerborni species-group, two speciesof ruficorne species-group, three species of vernum species-group and an ungrouped species (S. konoi), were determined. Phylogenetic analyses of the sequences of the Nevermannia species and other species of related subgenera of Simulium s.l. showed that the feuerborni and vernum species-groups were closely related, but the ruficorne
species-group and S. konoi were not. Variations between the ruficorne species-group and other Nevermannia species were larger than those between Nevermannia species (excluding the ruficorne species-group) and other subgenera species. These molecular data suggest that revision of the definition of the subgenus Nevermannia isneeded.
Key words: Black fly, Simulium, Nevermannia, Phylogeny, Mitochondrial rRNA

INTRODUCTION
A blackfly subgenus Simulium (Nevermannia) is distributed worldwide. In Asia, there are 3 species-groups (i.e.
feuerborni, ruficorne and vernum species-group), and some
ungrouped species (Crosskey and Howard, 1997). To investigate the relationship within subgenus Nevermannia species and between subgenus Nevermannia and other subgenera, we analyzed sequence variations in a subregion of themitochondrial 16S ribosomal RNA (rRNA) gene of 10 species of subgenus Nevermannia and related species.

MATERIALS AND METHODS
Materials used in this study and their origin are listed
in Table 1. Total DNA was extracted from single larva using the crude STE boiling method (O’Neill et al ., 1992).
Polymerase chain reactions (PCR) were performed in a
50 µl reaction mixture using 2 µl of the DNA solution.
The primers (primer A, 5’-CGCCTGTTTATCAAAAACAT-3’; primer B, 5’-CTCCGGTTTGAACTCAGATC-3’)
were used to amplify the mitochondrial 16S rRNA region as
described by Xiong and Kocher (1991). The reaction mix-

ture contained 10 mM Tris (pH 8.3), 1.5 mM MgCl2, 50
mM KCl, 250 µM dNTPs, 2.5 units of Tag DNA polymerase and 50 pM of each of the primers. The thermal cycling conditions were 35 cycles of denaturation at 95°
C for
1 min, annealing at 50°
C for 2 min, extension at 72°
C for 2min and a final extension at 72°
C for 10 min. PCR products
were purified by using the QIAquick PCR purification kit(Qiagen), and cloned into pGEM-T Easy vector (Promega).
At least 4 independent clones from each blackfly sample
were sequenced to identify polymerase error using the fmol
DNA sequencing system (Promega). Sequences were deposited in DDBJ/EMBL/GenBank databases under accession numbers AB056728-AB056747.
The sequences were aligned by using the program
CLUSTAL W ver. 1.7 (Thompson et al ., 1994). Sites containing alignment gaps were removed in the following
analyses. The number of nucleotide substitution per site
was estimated between each pair of the sequences, using
Jukes-Cantor methods (Jukes and Cantor, 1969). Construction and bootstrap probability estimation of the neighborjoining tree (Satiou and Nei, 1987) were performed by
PHYLIP 3.57c (Felsenstein, 1995).

１ Department of Infectious Disease Control, Oita Medical University, Hasama, Oita 879-5593, Japan (*: Correspondent author)
２ Department of Parasitology, School of Medicine, Yokohama City University, Kanazawa-ku, Yokohama 236-0004, Japan
３ Department of Parasitology and Pathology, Faculty of Veterinary Medicine, Bogor Agricultural University, Taman Kencana 3, Bogor 16151,
Indonesia
４ Institute of Tropical Medicine, Nagasaki University, Sakamoto, Nagasaki 852-8523, Japan２
６
２

Table 1 Materials used in this study
Genus

Subgenus

Species-group

Species

Locality

GenBank Acc.

Simulium

Nevermannia

feuerborni

S. feuerborni Edwards

Java, Indonesia
Peninsular Malaysia
Kanagawa, Japan
Nagano, Japan
Yakushima, Japan
Kanagawa, Japan
Hakone, Japan
Indonesia
Peninsular Malaysia
Irian Jaya, Indonesia
Ogasawara, Japan
Kanagawa, Japan
Oita, Japan
Oita, Japan
Yakushima, Japan
Tochigi, Japan
Palau
Peninsular Malaysia
Irian Jaya, Indonesia
Oita, Japan
Oita, Japan
Oita, Japan

AB056728
AB056729
AB056730
AB056731
AB056732
AB056733
AB056734
AB056735
AB056736
AB056737
AB056738
AB056739＊
AB056740†
AB056741
AB056742
AB056743
AB056744
AB056745
AB056746
AB056747

S. mie Ogata & Sasa

ruficorne

vernumS. saitoi Takaoka
S. sasai Rubtsov
S. aureohirtum Brunetti
S. ornatipes Skuse
S. bonninense Shiraki
S. subcostatum Takahasi
S. uchidai Takahasi

ungrouped
Gomphostilbia
MoropsSimulium s.str.
Prosimulium

S. konoi Takahasi
S. palauense Stone
S. whartoni Takaoka & Davies
S. farciminis Smart & Clifford
S. aokii Takahasi
S. quinquestriatum Shiraki
P. kiotoense Shiraki＊ Sequence of S. subcostatum from Oita, Japan was identical to that from Kanagawa, Japan† Sequence of S. uchidai from Yakushima, Japan was identical to that from Oita, JapanRESULTSWe determined the mitochondrial 16S rRNA region of
10 Nevermannia species including three species-groups(feuerborni, ruficorne, vernum) and an ungrouped species(S. konoi Takahasi), five species of other subgenera andProsimulium kiotoense Shiraki, and aligned (Fig. 1). All of
the Nevermannia species had 516 bases in this region. As
for the five species (S. feuerborni Edwards, S. mie Ogata
and Sasa, S. aureohirtum Brunetti, S. subcostatum Takahasi,
S. uchidai Takahasi), we determined the sequences of twoor three samples from different localities. S. subcostatumand S. uchidai did not have any intraspecific variations, but
S. feuerborni, S. mie and S. aureohirtum had. These intraspecific variations were not larger than the interspecificvariations.
To study relationships between Nevermannia species
and between Nevermannia and other subgenera, a neighborjoining tree was constructed based on the estimated d values (the number of the nucleotide substitutions per site) between each pair of the samples (Fig. 2). P. kiotoense wasused as an outgroup. The three species-groups of Nevermannia were separated into different clusters with highbootstrap probabilities. The feuerborni and vernum species-groups were clustered, but the ruficorne species-group wasplaced in a distinct cluster. One of the objectives of thisstudy was to determine the relationship of the ungrouped
species, S. konoi, to the known species-groups. But S.
konoi was not related to any species-groups of Nevermannia in the tree.
Table 2 summarizes the average d values among
species-groups of Nevermannia and other subgenera. The
average d values between the ruficorne species-group andthe other species-groups of Nevermannia were higher thanthose between the species-groups (without the ruficorne
species-group of Nevermannia) and other subgenera, andwere approximately the same level as those between the
ruficorne species-group and other subgenera.

DISCUSSION
Our phylogenetic analyses of subgenus Nevermannia
based on the mitochondrial 16S rRNA gene sequences
showed that the feuerborni and vernum species-groups
were closely related, but the ruficorne species-group andthe ungrouped species, S. konoi, were not. The ruficorne
species-group was largely divided from other Nevermannia
species and other subgenus species. The ruficorne species-

２
６３

Figure 1-1 DNA alignment of mitochondrial 16S rRNA region for the 16 species. A period indicates the site identical to S. feuerborni (Indonesia); a dash indicates a gap site.

２
６
４

Figure 1-2

２
６５

Figure 2 The neighbor-joining tree of the mitochondrial 16S rRNA region for the 16 species. Numbers on the nodes indicate probabilities based on 1,000 bootstrap replicates. The probability of more than 50% is shown. Branch lengths are proportional
to genetic distance (scale bar).
Table 2 The average number of nucleotide substitutions per site among the species-groups of
Nevermannia and other subgenera
２
１

feuerborni s.g.

２

ruficorne s.g.
vernum s.g.

３

６

S. konoi
Gomphostilbia
Morops７

Simulium s.str.

４
５

0.076

３

４

５

６

７

0.029

0.045

0.043

0.059

0.050

0.070

0.072
0.040

0.080
0.044

0.075
0.045

0.071
0.043

0.042

0.054
0.054

0.041
0.045
0.056

group and the ungrouped species, S. konoi, have morphological characters which depart from the other species of
Nevermannia. One of such characters in the ruficorne
species-group is the male genitalia with ventral plate with adistinct median keel (Crosskey, 1969). On the other hand, S.
konoi has a female adult cibarium with a distinctive armature consisting of several oblique rows of denticles on eachside (Bentinck, 1955); its larval antennae have a few unpigmented annulations on the second segment (unpublisheddata). These molecular and morphological data taken into
consideration, revision of the definition of the subgenus

Nevermannia may be needed.

ACKNOWLEDGMENTS
This research was supported by the Grant-in-Aid for
Scientific Research (C) of the Ministry of Education, Science, Culture & Sports, Japan (No. 11670246 to HT & YO).

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マレーシアのサバ州で採集された新種２種および未記録種３種のブユについて（英文）高岡 宏行 …………………………………………………………………………………………２
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家兎アカントアメーバ角膜炎の臨床像および組織所見（英文）山田 誠一，山田利津子，モアゼム ホセイン，月! 説子，藤田紘一郎 ………………２
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日本熱帯医学会 MK 山道 草野 白石

June２
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日 本 熱 帯 医 学 会

Original article
Takaoka, H.
Two New and Three Newly Recorded Species of Black Flies (Diptera: Simuliidae)
from Sabah, Malaysia ……………………………………………………………………………２
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Yamamoto, T., Shimada, M., Moji, K. and Mizota, T.
Research Activities on Emerging and Reemerging Diseases ……………………………………２
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Yamada, S., Yamada, R., Hossain Md. M., Tsukidate, S. and Fujita, K.
Clinical and Pathological Features of Experimental Acanthamoeba Keratitis in Rabbits ………２
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Takaoka, H.
Description of Two New Species of Black Flies (Diptera: Simuliidae) from Sarawak, Malaysia……………………………………………………………………………………………………２
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Ishak, H., Matsuse, I.T., Kamimura, K., Takegami, T. and Funada, H.
Comparative Oral Susceptibility of Dengue Vector Mosquitoes from Japan and Southeast Asia
to Two Dengue-1 Virus Strains ……………………………………………………………………２
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Otsuka,Y., Aoki, C., Saito, K., Hadi, U.K., Suzuki, H. and Takaoka, H.
Phylogenetic Analyses of a Blackfly Subgenus Simulium (Nevermannia) Based on
Mitochondrial 16S Ribosomal RNA Gene Sequences ……………………………………………２
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Vol. 29 No. 2

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Hygiene

会報・記録
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and

ミトコンドリア１
６Ｓリボゾーム RNA 領域の塩基配列によるホソスネブユ亜属（Nevermannia）の系統学的研究（英文）大塚靖，青木 千春，斎藤 一三，Upik K. Hadi，鈴木博，
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Medicine

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Journal of Tropical

新興・再興感染症を取り巻く研究の現状（英文）山本 太郎，嶋田 雅曉，門司 和彦，溝田JUNE ２０
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