量SSN O304−2146

Japanese

Joumal of Tropical

Medicine

Hygiene平成元年9月15日

第17巻第3号
容

内
原

and

著

マレーシア，キナバル山のウツボカズラより採集されたクシヒゲカとオオカの2新種（英文）…一……………・…………一一…・……塚本

増久

215−228

寄生虫病薬の7年間の治療成績の検討
一稀用薬の入手緩和を求めて一（英文）

・田辺

清勝，尾辻

義人，中林

敏夫，

大友 弘士，田中寛
マンソン孤虫に対する硫酸パロモマイシン，ビチオノール，メベンダゾール

229−236

およびフルベンダゾールの駆虫効果に関する実験的研究（英文）一牧純，柳沢十四男

237−241

一高岡 宏行
P伽吻04∫％吻加惚h切のsporogonyについて，蚊の飼育温度と純化ookineteの発育に対するmembrane feeding液の影響（英文）243−257

ミャンマー（ビルマ）のブユについて（英文）一矢野健一，Karl Maramorosch，Albina Kozlowska

259−267

研究ノート

Surveyof

P伽吻o伽吻翅吻α郷郷in

Makurdi，Nigeria−Annie John269−271

症例報告
膵臓およびリンパ節内に存在するB型肝炎ウイルス表層抗原（英文）

一千馬
会

正敬，山下

裕人，板倉

英吾

273−276報

平成元年度第1回幹事会記録

277−278

投稿規定

日熱医会誌

Japan．」．TrOP．

Med．Hyg．

日本熱帯医学会

Japan. J. Trop. Med. Hyg., Vol. 17, No. 3, 1989, pp. 215 228

215

TWO NEW MOSQUITO SPECIES FROM A PITCHER
PLANT OF MT. KINABALU, SABAH, MALAYSIA=CULEX RAJAH A N D TOXORHYNCHITES RAJAH(DIPTERA= CULICIDAE)
MASUHISA TSUKAMOTO
Received March 20 1989/Accepted April 21 1989

Abstract: Culex (Culiciomyia) rajah n. sp. and Toxorhynchites ( Toxorhynchites) rajahn. sp. were collected from the pitcher plant Nepenthes rajah at high elevations of Mt.
Kinabalu, northern Borneo. Cx. rajah n. sp. is characterized by: adults with a dark stripe
on upper parts of the pleura and a distinct pale basal band on terga II‑VII; pupa withsingle 4‑VIII setae and broad paddles; and larvae with short setae, 7‑C and 8‑P, a slendersiphon with usually 5 pairs of 4‑6 branched 1‑S tufts, and long gills. Tx. rajah n. sp. ischaracterized by: adults with a combination of long rm crossvein of wings, absence ofbrown fusiform scales on mesokatepisternum of thorax; and absence of well‑developed
caudal tufts on terga VI‑VIII; pupae with setae 11,12‑CT single, 1‑II single, 6‑1‑VII Iongand single, and broad shape of paddles; and larvae with 2‑branched setae 1‑III,IV, single
4‑VI, single 6‑P and 7‑M, single 11‑V, and a long siphon with 2‑branched 1‑S.

INTRODUCTION
During mosquito surveys in Malaysia in 1986, mosquitoe larvae were collected fromwater in pitchers of Nepenthes rajah Hooker f. on Mt. Kinabalu, Sabah, Malaysia. According
to Kurata (1976) , this species of pitcher plant is endemic to this mountain and it occurs at

high elevations (1,650‑2,650 m). Together with several known mosquito species, Iarvae of
two unknown species belonging to subgenus Culiciomyia of the genus Culex and to the genus
Toxorhynchites, respectively, were also collected.

The former was initially thought to be Cx. shebbearei because of a previous record by

Edwards (1931) and a citation by Barraud (1934), Knight and Stone (1977) and Beaver(1983) . According to the redescription of Cx. shebbearei by Sirivanakarn (1977), the larval

characters illustrated for this species are notably different in morphology from the newly

collected Culiciomyia larvae from Mt. Kinabalu.

Department of Medical Zoology, School of Medicine, University of Occupational and Environmen‑tal Health, Kitakyushu 807, Japan
This study is a part of the research project supported by a Grant‑in‑Aid for Overseas Scientific
Survey in 1986 (No. 61041070) from the Ministry of Education, Science and Culture of the Japanese
Government. The surveys were carried out with the permission of the Socio‑Economic Research
Unit, Prime Minister's Department of the Malaysian Government.

216

Another species of Culiciomyia recorded from the same mountain is Cx. javanensis, andlarvae of these recorded species are similar to each other. However, adults of Cx. javanensis
are distinctly different from those of Cx. shebbearei and from the present new materials, andthere is no fear of confusion in identification, as will be discussed later. Results of further

taxonomic examinations on the new materials led to the conclusion that this must be a new
species, although it has been misidentified as Cx. shebbearei for many years.
From the morphology of adults, pupae and larvae of Toxorhynchites. Steffan and Even‑huis (1985) classified 36 known Asian species into 7 species‑groups. The newly available

specimens of Toxorhynchites reared from the collected larvae, however, does not coincidewith any of the known species, indicating a possibility of another new species.

MATERIALS AND METHODS
Mosquito larvae were collected by the author together with Dr. Motoyoshi Mogi on 24
September 1986 on Mt. Kinabalu at elevations of about 1,600 m to 1,800 m. Larval collection
was carried out carefully in order not to destroy any pitcher plants, with the permission ofand regulated by the Department of Saba National Park, Kota Kinabalu, Saba, Malaysia,
some of whose staff members accompanied us.
Some of the larvae collected were used to study biochemical systematics by means ofelectrophoresis (Tsukamoto et al.. 1989), but to obtain pupae and adults for further
identification, some larvae were reared under laboratory conditions in the University of
Malaya, Kuala Lumpur, Peninsular Malaysia. Probably due to sudden changes of environ‑mental conditions during the transportation, such as shaking, temperature and/or air pres‑sure, some adults of Toxorhynchites failed to emerge from pupae. Both larval and pupal skinsof the holotype of Cx. rajah n. sp. were lost in an accident. Therefore, description of pupa andlarva was based on whole body paratypes preserved in a 70% ethanol solution.
The descriptions of the new species are based mainly on the format used by Bram (1967)
and by Sirivanakarn (1977) for Culex, and by Steffan and Evenhuis (1985) for Toxorhynchites.

With slight modifications, tables for pupal and larval chaetotaxy are also based on the form

used by Evenhuis and Steffan (1986) and Tanaka et al. (1979).
Abbreviation of generic and subgeneric names of mosquitoes followed the proposal by
Reinert (1985) .

DESCRIPTIONS
Culex (Culiciomyia) rajah, n. sp.
(Figures la, 2; Table 1)

The 4th instar larvae are characterized by a combination of I ) a short 7‑C hair, 2 ) avery short 8‑P hair with 2‑4 branches, 3 ) a slender siphon with usually 5 pairs of 4‑6
branched hair tufts, and 4 ) Iong gills. In the adult, thorax shows an indistinct dark stripe

at upper pleura and a dark spot in middle of sternopleuron. Abdominal terga II‑VII havebroad pale basal bands.
MALE. Wing: 3.6‑3.8 mm. Head: Proboscis entirely dark with scanty median ventral
tuft; palpus dark, about 1.2 times length of proboscis, segment 111 at the apical half with adistinct ventral row of about 5 Iong translucent scales. Thorax: Integument of pleuron tan

217

Cul ex ( Cul iciom yia)

Toxorh ynchttes ( Tox. ) raiah
n. sp.

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n. sp.

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a: Culex rajah, b: Toxorhynchites rajah.

with a typical dark upper stripe, and another isolated dark spot on middle of sternopleuron.
Wing with cell R2 (forked celD 1.7‑1.9 times length of vein r2+3 (stem) . Abdomen: Terga
II‑VII dark with distinct broad basal pale band covering an area about equal to dark portionof each tergum II‑V or more than half of each tergum VI‑VII. Male genitalia: Not
described.

FEMALE. Wing: 4.1‑4.3 mm. Generally similar to male. Head: Segment IV of palpus
longer than (or about) twice length of segment 111. Thorax: Wing with cell R2 2.35‑2.5 times

length of vein r2 + 3. Abdomen: Terga II‑VII with pale basal bands covering less than half
length of each tergum.

PUPA. Seta 4‑VIII single, 9‑VIII 3‑8 branched. Paddle, 0.9 mm length, broad anddeformed oval, index (length/width) about 1.2, apex slightly projected (Figure la) .

LARVA (Figure 2, Table l). Head: Width about 1.3 mm. Antennal tuft 1‑A 13‑15
branched, attached at 2/5 Iength of the shaft from base; seta 1‑C fine and simple; 4‑C single;

5‑C 3,4 branched; 6‑C 3 branched; 7‑C short (about 1/2 Iength of 5,6‑O , 4 branched; 8,lO‑C
single; 9‑C short, 2‑3 branched. Thorax: Seta O‑P minute, about 9 branched; 1‑P double; 2,3‑P
single; 4‑P double; 5,6‑P single; 7‑P 3‑4 branched; 8‑P very short (1/6 to 1/4 Iength of 7‑P)

forked with 2‑4 branches, scarsely single; 14‑P single. Seta 1‑M shorter than 3,4･M.
Abdomen: Setae 6‑1,II Iong, 3‑4 branched, 6‑III,IV,V,VI Iong, 2‑5 branched; 7‑1 Iong, single
or 2 branched; 7‑II,III,IV,V very short, 2‑6 branched; 1‑1,II,III,VI minute or short, 1‑IV,V Iong,single or 2 branched, 1‑VII single, about the same as length of segment VII, 1‑VIII 3,4
branched; 2,4‑VIII single; 3‑VIII 6‑8 branched; 5‑VIII double; comb consisting of 45‑60
narrow fringed scales. Saddle shorter; caudal margin with many strong spicules; l‑X single;
2,3‑X single slightly less than length of siphon or gills; 4‑X 4 pairs, weak and short, especiallya precratal pair shortest and usually single, other 3 pairs 2‑4 forked. Siphon slender, 1.7‑
1.8 mm length (n= 17) , not inflated in middle but gradually tapering to apical; siphon index

218

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Culex (Cuticiomyia) ralah n. sp.

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2

220

4.0‑5.5, 1‑S mostly 5 pairs (occationally 4 or 4.5 pairs) , individual tuft hair 4‑6 branched andas short as 0.4‑0.5 of the width of siphon at the point of attachment; pecten teeth 10‑12, each

tooth short and wide with a strong distal spine and 4‑5 strong lateral denticles. Upper analgill about the same as length of siphon, Iower gill slightly shorter than upper gill. Larval

chaetotaxy is given in more detail in Table 1.

TYPE DATA. Holotype male reared from a 4th instar larva collected on 24 September
1986 by M. Tsukamoto, from a pitcher of Nepenthes rajah in Mt. Kinabalu. Sabah, Malaysia.
Paratypes: 5 males, 6 females reared from larvae, and 15 Iarvae collected by M. Tsukamoto;lO pupae (4 females, 5 males and I skin with a half‑emerged male from it) and 30 Iarvae

collected by Dr. M. Mogi on the same date at the same place. Holotype and some ofparatypes will be deposited in the National Museum, Tokyo, Japan.

DISTRIBUTION. Known only from the type locality, Mt. Kinabalu, Sabah, Malaysia.
BIONOMICS. Larvae of Cx. rajah n. sp. occur in pitchers of Nepenthes rajah at highelevations in association with larvae of Culex (Lophoceraomyia) jenseni (De Meijere) ,Tripteroides (Rachionotomyia) sp. No. 2 of Mattingly (1981), Uranotaenia (Pseudoficalbia)
moultoni Edwards, and Toxorhynchites ( Toxorhynchites) rajah n. sp. ' Body surface of mostlarvae are covered by Vorticella‑like protozoa. Nothing is known of their adult biology,
habitat, or medical importance as a vector of diseases.

TAXONOMIC DISCUSSION. About 30 species of mosquitoes belonging to subgenus
Culiciomyira of the genus Culex are known in Asian countries (King, 1946; Sirivanakarn, 1973,

1977; Sirivanakarn and Kurihara, 1973; Knight and Stone, 1977; Toma et al.. 1984; Apiwath‑nasorn, 1987; Harrison, 1987). From North Borneo 6 species are so far known of thissubgenus: Cx. fragilis Ludlow, Cx. nigropunctatus Edwards, Cx. shebbearei Barraud, Cx.
spathlfurca (Edwards) , Cx. papuensis (Taylor) , and Cx. javanensis Bonne･Wepster.
The original record of Cx. shebbearei by Edwards (1931) is based on the collection by Mr.

H. M. Pendlebury in 1929 from a giant pitcher plant, Nepenthes rajah, on Mt. Kinabalu as
follows: "The adults reared from these larvae were Culex shebbearei. Barraud, a species which

had been found on one previous occasion only, when Barraud obtained larvae in the water ina hollow tree in the eastern Himalayas". At that time neither taxonomic discussion nor
basis for this identification was given by him. However, this record was cited by Barraud(1934), Knight and Stone (1977), Beaver (1983) and Apiwathnasorn (1986) without any
further evidence, although Sirivanakarn (1977) mentioned that the record of Cx. shebbearei

from Borneo was doubtful.
According to the redescription based on the type specimens and illustration of Cx.
shebbearei by Sirivanakarn (1977) , Iarvae of this species have 4 pairs of siphonal hair tufts(1‑S) , "first proximal pair double; second proximal pair triple; 2 distal pairs double, 1.5‑2.0

times as long as siphonal width at point of attachment". However, Iarvae of Cx. rajah n. sp.
possess 5 pairs (occasionally 4 pairs in either one or both sides) of the siphonal hair tufts, andeach hair is short (about 1/2 width of siphon at point of attachment) and has a different
number of branches (4‑6). Relatively long anal gills and broad shape of pecten teeth arealso different from those illustrated by Sirivanakarn (1977) for a larva of Cx. shebbearei.

From the morphology of larvae described above and from these situations about thepresent species, therefore, it seems more likely to conclude that the species is a new species
which has been misidentified as Cx. shebbearei for a long time, rather than to think that bothCx, shebbearei and Cx. rajah n. sp. are living in pitchers of Nepenthes rajah on Mt. Kinabalu.

221

Culex javanensis was also recorded from Mt. Kinabalu by Sirivanakarn (1977), but thisspecies can be readily distinguished from the new species in having fewer number (4 pairs)
and fewer branches (2‑3) of siphonal tufts in larvae, and by absence of basal pale bands on
terga in adults.

Some important larval characters among known east and southeast Asian species of
Culiciomyia are compared in Table 2, where Cx. azurini (with a single short seta 1‑A and an
incomplete saddle) and Cx. termi (with an unusually long siphon) are not included because

they are easily distinguishable from other members by their peculiar morphology (Toma etal., 1984; Thurman, 1955). Cx. (Thaiomyia) dispectus Bram and Cx. (Thaiomyia) hainanensis

Chen are also excluded, although Harrison (1987) proposed to include the subgenus
Thaiomyia into the subgenus Culiciomyia. Culex harrison Sirivanakarn, Cx. ceramensis
Sirivanakarn and Kurihara, and sometimes Cx. nailoni King and Hoogstraal also have 5 pairsof the siphonal tufts, but they differ from Cx. rajah n. sp. in having double branched 2,3‑P,
3,4‑branched 4‑P and 5‑VIII, respectively. In addition, Iarvae of both Cx. ceramensis and Cx.
nailoni have double 5,6‑C hairs, and the shape of the siphon in the former species is not
slender but more or less elliptical. Culex harrisoni resembles the present new species inhaving a minute hair 8‑P, Iong gills, branch numbers of several setae and tufts, but distinguish‑

able by length of anntenna (longer in Cx. rajah n. sp.) , branches of 7‑C (fewer in Cx. rajah) ,2,3‑P and 5‑VIII hairs.

Regardless of subgenera, only limited members of the genus Culex have minute or short
hair 8‑P: for example, Cx. (Cux.) alis. Cx. (Eum.) tenuipalpls. Cx. (Lop.) curtipalpis. Cx.

(Lop.) pholeter. Cx. (Lop.) umformis, etc. Especially in the subgenus Culiciomyia, only
harrisoni. rajah n. sp., dispectus and hainanensis fall into this category in Asia. Therefore, thelength of the hair 8‑P must be a good feature quite helpful in confirming a particular species.
In adults, Cx. rajah n. sp., Cx. harrisoni. Cx. shebbearei. Cx. viridiventer, and Cx. bailyi are

rather similar, all having dark marks or a distinct stripe on upper pleura of the thorax anddistinct pale basal bands on terga II‑VII of the abdomen. However, these basal bands are
narrow in Cx. shebbearei but broad in Cx. rajah n. sp. Table 3 compares taxonomicallyimportant characters in adults of several known species in Asia.

Toxorhynchites (Toxorhynchites) rajah, n. sp.
(Figures lb and 3; Tables 4,5)

Adults are characterized by a combination of I ) absence of well‑developed lateral tufts
on terga VI‑VIII, 2 ) absence of brown fusiform scales on mesokatepisternum, 3 ) absenceof pale greenish lateral scale portion of scutum, and 4 ) Iong rm crossvein of wings. Larva
is characterized by I ) a single and long 6‑P hair, 2 ) bristles 7‑P, 13‑M and 7‑T 2 branchedand 3 ) Iong siphon and saddle.

MALE. Wing: 5.5‑6.5 mm. He,ad: Proboscis dark without pale or lighter marking,maxillary palpus‑1 dark purple, with a small spot of silvery‑white scales at the apex ofsegment I, segments II‑V without dorsal lighter marking. Thorax: Scutum with metallic
greenish blue scales, without pale green lateral stripe zones, anterolateral portion of meso‑

katepisternum bare. Wings with long rm crossveins. Legs practically without white scales
on all tibiae and tarsi except faint lighter scales on baso‑ventral portions of midtarsi 1‑II.

Abdomen: Terga bluish purple, with yellowish lateral markings on I, and small baso‑laterallight marks on terga 111‑VII. Terga VI‑VIII with only poor black lateral setae not forming8目眉目︑H・H＃ε03の一酒ε三go且筑
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N 鴫 Nφり ゆ頃 の嶋めo頃Hoo ooマの頃寸的 寸国φり oのφ｝マ｝N N寸的NOり的N 一一N 寸の︐H咽の＜￠哨．ααの︵曇ミO遷心ミO︶廟Oの﹄㊤一〇邸﹄邸︑黛ぺ譜 ．燕︾ 一qンPQ属出9コ邸＞﹄

き幅ミ飛嘱燵り§醤黛o景愚ミ

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ミきミミミ
ミミoミ§醤繋舵寒軸ミ蕗爲唱︑§︑S
的矯簑鋤O嘱O＆

弩N鳶避碁
超︑寒
§ミ黛﹄ミ
k恥蝸ミ旬為㎞噂・§塙︑ミ竃ミミリ違ミ遠ミ

聡雲恥§ミ塁藝§鳶隠騒ミ飛
憩整超ミ冒
︑暴

著簑§魯軋
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〇一煽＞﹄oo一一q邸一﹄oαヨ哨﹄自邸〇一εogO図邸り︸o＝Qの咽﹄OαgOQ﹂﹇

ω五≦罵＞￠りog口o咽駕ε﹄o岩H一泊匿国一3︸O ﹄Oρ∈コ9 ﹄〇一口吋﹄的

222

「J
『寸・寸

oり
H
IIIIllNIIIIIII−IIIHININ寸9§寸や㊤の寸め早マ寸▽ωoo寸め§oりマ的oりoり
ぱ⊃uつ

uつ

の
的

uつu⇒u⇒ooσ｝

ゆゆ

Cつ

軋』⇒

r寸

頃
・ ・oo一寸寸 の寸頃頃鴫o・・oooトト
00H一一一
ママ寸Oト寸のゆLΩOのトO頃OO
1マllIIII嶋め頃ゆIIIII卜IIll1
rぜ寸
o
① 一

寒

Oり哨⇒σのOΣ

N①一ρ邸﹂ピ223

Table 3 Grouping of adult mosquitoes of subgenus ( Culiciomyia)

Without pale bandin Asia

Thorax (Upper pleura)

AbdomenTerga II‑VII

s pp.

Without dark markWith d.ark marks or a stripe

bahri, javanensis,

ceramensis. pa puensis, s pathtfurca

fragilis, nailoni.

scanloniWith pale spot markWith pale lateral markWith pale basal band

dispectus, fusctctn ctus

ramalingami
lam pangensis

ryukensis, kyotoensis, shebbearei,
bail ri:, viridiventer, harrisoni.

rajah n. sp., barrinus, pullus,

pallidothorax, thurmanorum
nigropunctatus

With pale joint band

With pale apical bandazurini, tricuspis,d elfinad oae

With dark basal band

termi

Adult unknown: spiculothorax,

No case in point.

either well‑defined lateral or caudal tufts. Male genitalia: Not described.

FEMALE. Head: Apex of maxillary pulpus silvery‑white scaled. Thorax: Generally
similar to males. Forelegs without distinguishable marks. Midlegs with light scaled markson basal 1/2 to 2/3 of T‑1, almost all T‑II yellowish white except dark scales at dorso‑apicalportion, T‑III, IV, and V entirely dark scaled. Hind T･II with light scaled marking atbaso‑ventral portion.

PUPA (Figure lb, Table 4). Setae lO‑CT 3 forked, 11,12‑CT single; 1･1 multibranched(about lO) , 2,4,5,9‑1 single, short, 3,6‑1 single, Iong; 1,3,5‑II single (length of 5‑II about 1.6 times

length of 1‑ID , 2‑II single, minute, 4‑II 3 branched, short; 1,3,5‑III single, Iong, 4‑III 3 single

short; 1,5‑IV single, Iong, 2,3‑IV single, short, 4‑IV 2 branched short; 1,5‑V single, Iong, 2,3‑V

short, single; 4‑V 3 branched short; 1,2,4‑VI single, short, 3‑VI 2 branched short, 5‑VI single,

long; 1,2,3,4‑VII single, short, 5‑VII single, Iong; 4,9‑VIII single, short. Paddle (Figure lb)

broad and subrhomboid‑ovate, Iength 2.4 mm, width 1.4 mm; with scattered dark spots or
subbasal mottling line; slightly emarginated at apex of midlib; with minute marginal spicules

near apex of lobe.
LARVA (Figure 3, Table 5) . Head: Integument brown without special dark dorsal mark;setae 9,lO‑C single. Thorax: Satae 1,2,3‑P single, 1‑P Ionger than 2‑P on the same tubercle, 3‑P
isolated, very short, 4‑P isolated, forked with 7 branches, 5‑P stout, single, 6‑P Iong, single, 7‑P

strong, 2 branched, 10‑P Iong, single; 1,2‑M isolated and very short, 3‑7･M single on the samesclerotized plate, 6‑M stout, 7‑M minute, 10‑M single, Iong, 13‑M stout 2 branched; 1‑T Iong,single, 2‑T short, single, 3,4‑T small, separated, 6‑T stout, single, 7‑T stout, 2 branched, 8‑T

minute, 4 branched, 9,13‑T stout, single, 10‑T slender, single. Abdomen: Setae 1‑1,II,V,VI,VII
single, 1‑III,IV 2 branched, 3,4‑1 strong, 2 branched; 2‑1 minute, single and isolated; 2‑II‑VI

short, isolated; 4‑1,II 2 branched; 4‑Ill‑VII single; 10‑1 short, single; 10‑II‑V, 2 branched;lO‑VI small, single; lO‑VII short; single; Il‑1‑IV 2 branched; 11‑V‑VII single; 12‑1 minute,branched; 12‑II‑VII minute or short, single; 13‑1‑VI Iong, single; 1‑VIII small, single and224

Table 4 Chaetotaxy of the pupae of Toxorhynchites (Toxorhynchites) rajah n. sp.

Abdomen

Seta Cephalo‑
No.

thorax

I

II

III

IV

V

VI

VII VIII

IL

1?

Im

Im
Im

Im
Im
2m

1

Im

O
l

IL

lO

1

2

Im
Im
Im
Im

Im

Im

IL
Im

IL

IL

1

Im
Im

3‑4

l

2

2‑3

1

1

IL
IL

IL
IL

IL
IL

IL
IL

Im
Im
Im

Im
Im
Im
Im
Im

Im
Im
Im
Im
Im

IL
IL
Im

3
4

6

l

IL

IL
IL

7

IL
IL

1

1

1

8

9

1

1

Im
Im

Im
Im

10

3

l

1

11

1

Im

Im

12

1

5

Im

Im

Im
1
1

Im
1‑2m

l

Im
Im

L: Iong, m: minute, ‑ : absent, ? : Iacked.
Specimen examined: a single pupal skin associated with a larval skin.

isolated; 2,3‑VIII small, single; 4,5‑VIII stout, single. Siphon long, 1.3 mm, siphon index about

2.5; 1‑S stout and 2 branched. Saddle narrow, about same as length of siphon, 2 times lengthof basal width; 1‑X single, stout; 2･X 6 branched, one of them longer than others (about 2times length of the shortest) ; 3‑X Iong (about 2.8 times length of siphon) , 3 branched; 4‑X 6
pairs on grid, their length ranging from I to 1.4 Iength of saddle. Gills very short.

TYPE DATA. Holotype male reared from a larva collected in Mt. Kinabalu, Sabah,
Malaysia, by M. Tsukamoto on 24 September, 1986. Paratype I male, 2 females, 2 pupal
skins, and I Iarval skin, collected by M. Tsukamoto and M. Mogi on the same date.
DISTRIBUTION. Known only from the type locality, Mt. Kinabalu, Sabah, Malaysia.
BIONOMICS. Larvae of this species were collected in water of Nepenthes rajah, inassociation with larvae of Cx. (Cui.) rajah n. sp., Cx. (Lop.) jenseni. and Ur. (J {c.) moultoni.

Adult do not suck blood but other biology is unknown.

TAXONOMIC DISCUSSION. In Asia at least 40 spp. of mosquitoes belonging to thegenus Toxorhynchites are counted. Steffan and Evenhuis (1985) assigned 36 known species (atthat time) into 7 species‑groups by the combination of various morphological characters.

Based on one female from Mt. Kinabalu, at about 1,300 m elevation, Tx. pendleburyi wasdescribed by Edwards (1930) . This species possesses well‑developed caudal tufts on terga VI,
VII and VIII, belonging to the splendens species‑group of Steffan and Evenhuis (1985) . Since

the present new species has poor caudal tufts, this cannot be Tx. pendleburyi. The acaudatus
group is characterized by I ) absence of well‑developed caudal tufts, 2 ) Iong rm crossveinof wings, 3 ) presence (in some species) of brown fusiform scales on anterolateral region of

mesokatepisternum in adults, 4 ) presence (in some species) of subbasal mottling on paddlesof pupae, and 5 ) pitcher plant dwelling immatures. The present new species, Tx. rajah, havelong rm crossvein of wings, without distinct lateral tuft on terga VI‑VIII; this tends toward

acaudatus group, but the new species does not have brown fusiform scales in anterolateral

225

TOxorhynchites ( Tox ) raiah n.

sp .

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9

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1:Figure 3 Larva of Toxorhynchites rajah'
A: antenna, C: head' P: Prothorax' M: mesothorax

T: metathorax, S: siPhon'

1 ::‑:‑::;1:=

: .

i

3

226
Table 5 Chaetotaxy of the 4th instar larva of Tonorh nchites ( Tonorhvnchites) rajah n. sp.

Seta Head Thorax
Abdomen
II 111 IV

No. C p M T V VI VII
O

Im

2

1

Im

Im
2L

4

2

4m

Im
2L
2L

Im

2

3

1

Im

4

1

7

5

5m

IS

6

1

7

Im

2S

8

1

?

Im
5m

4m

9

1

10

1

IS
IL

IS
IL

IS
IL

Im

Im

Im

11
12

l

13

2

IS

2S

IS
2S

IS

Im
Im
2L
3m
IL

?

1?

1

2

2

l

l

l

l

Im
2L
2L
Im
2L
2L
Im
Im
2L
2L
Im

Im
2L

Im
2L

Im

Im

Im

1‑2 L

1

1

Im
Im

IL

IL

1

1

Im

Im
2L
2L
Im
Im
2L
2L
Im

Im
2L
2L
Im
Im
2L
2L
Im

Im
IL
IL

IL
IL

5m
IL

1‑A

2m
Im

2m
Im

7m
Im

1‑ S

2

1

1

IL

IL

IL

IL

IL

IL

IL

1

Im

6m

14

15

1

1?

1?

?.

VIII

l
l

IL

IS
IS

2S

1‑X

IS

2‑ X
3‑ X

6L
3L

4‑ X

IL

(6 pairs)

1?

Im

L: Iarge, S: stout, m: minute, ‑: absent, ? : not detected.
Specimen examined: a single larval skin associated with the pupal skin.

region of thorax. More recently Evenhuis and Steffan (1986) have described Tx. angustiplatus

from the Malay Peninsula as a new species which also does not have the brown fusiform
scales. Pupae of the latter species, however, can be easily distinguished from those of thepresent new species because Tx. rajah has paddles with subrhomboid‑ovate shape and single
setae 11,12‑CT and 1‑II (like in Tx. nepenthis) instead of elongate paddles of Tx. angustiplatus

pupae and the branched setae in question. Pupa of Tx. rajah is also distinguished from thatof Tx. nepenthis by a branched seta lO‑CT and a single long seta 6‑VII. In addition, Iarvaeof the present new species are unique by showing elongate siphon and saddle, and bychaetotaxy completely different from that of Tx. nepenthis by long and stout setae 5,7,9‑P,
6,9,13‑M, 6,7,9,13‑T, 4,5‑VIII and l‑S. Number in branches of these setae also different. Larval

chaetotaxy of Tx. rajah is shown in Table 5 in more detail.

ACKNOWLEDGMENTS
I am grateful to Professor I. Miyagi, University of the Ryukyus, Okinawa, and Professor

Yong Hoi‑Sen, University of Malaya, Kuala Lumpur, for their support, encouragement andarrangement for the survey to Mt. Kinabalu. Permission and local guiding by Mr. Thomas
Yussop, Park Naturalist, and his staff, for collecting mosquito larvae from pitcher plants in

the Mt. Kinabalu National Park, are also deeply appreciated. Dr. M. Mogi, Saga Medical
College, Sagzi, offered many valuable suggestions and cooperation in giving paratype mate‑rials. Sincere appreciation is also due to Dr. M. Horio, University of Occupational and227

Environmental Health, Kitakyushu, for preparing the type specimens, and to Dr. T. Toma,
University of the Ryukyus, Okinawa, for her useful suggestion and taxonomic discussion. I
also thank Professor T. Kurihara, Teikyo University, Tokyo, for his kindly sending a reprintof the useful literature.

R

ERENCESl ) Apiwathnasorn, C. (l 86) : A List of Mosquito Species in Southeast Asia. Museum andReference Centre, SEAMEO‑TROPMED National Centre of Thailand, Mahidol University,
Bangkok, pp. 74
2 ) Barraud, P.J. (1934): The Fauna of British India, including Ceylon and Burma. Diptera, Vol.
V, Family Culicidae. Subgenus Culiciomyia Theobald, 1907, 376‑387, Taylor and Francis,

London3 ) Beaver, R.A. (1983): The communities living in Nepenthes pitcher plants: Fauna and foodwebs. In: Phytotelmata: Terrestrial Plants as Hosts of Aquatic Insect Communities, (Frank,
J.H. and Lounibos, L.P. ed.), 129‑159, Plexus Publ., Marlton, New Jersey
4 ) Belkin, J.N. (1962): The Mosquitoes of the South Pacific (Diptera, Culicidae) . Vol. I.
Subgenus Culiciomyia Theobald, 228‑234. University of California Press, Berkeley5 ) Bram, R.A. (1967): Contributions to the mosquito fauna of Southeast Asia. ‑ II. The genus
Culex in Thailand (Diptera: Culicidae) . Subgenus Culidomyia Theobald 1907, Contrib. Amer.
Entomol. Inst., 2(1) , I13‑1806 ) Chen, H.‑P. (1977) : A new species of Culex (Diptera: Culicidae), Acta Entomol. Sinica, 20,

95‑98 (in Chinese with English summary)7 ) Edwards, F.W. (1931): Mosquitoes breeding in plant pitchers, Natur. Hist. Mag., 3, 25‑28

8 ) Evenhuis, N.L. and Steffan, W.A. (1986): Classification of the subgenus Toxorhynchites(Diptera: Culicidae), II. Revision of the Toxorhynchites acaudatus group, J. Med. Entomol., 23(5) , 538‑574

9 ) Harrison, B.A. (1987): Culex subgenus Thaiomyla Bram, a synonym of Culex subgenus
Culiciomyia Theobald (Diptera: Culicidae) , Mosq. Syst., 19(1) , I11‑116

10) King W.V. (1946) : The New Guinea species of Culex (Culiciomyia) , with descriptions of two
new species, Proc. Biol. Soc. Washington, 59, 143‑154

lD Knight, K.L. and Stone, A. (1977): A Catalog of the Mosquitoes of the World (Diptera:
Culicidae) , 2nd Ed. Thomas Say Foundation, Vol. VI, Entomol. Soc. Amer., College Park, pp.
61112) Kurata, S. (1976): Nepenthes of Mount Kinabalu. Sabah National Parks Publications No. 2,
Sabah National Parks Trustees, Kota Kinabalu, pp. 8013) Mattingly, P.F. (198D: Medical Entomology Studies ‑ XIV. The subgenera Rachionotomyia.
Tricholeptomyia and Trip'teroides (Mabini group) of genus Tripteroides in the Oriental Region(Diptera: Culicidae) , Contrib. Amer. Entomol. Inst., 17(5), 1‑147

14) Miyagi, I., Toma, T., Tsukamoto, M., Mogi, M., Horio, M., Cabrera, B.D. and Rivera, D.G.
(1985) : A survey of the mosquito fauna in Palawan, Mindanao and North Luzon, Republic of
the Philippines, Mosq. Syst., 17(2), 133‑145

15) Reinert, J.F. (1975): Mosquito generic and subgeneric abbreviations (Diptera: Culicidae),
Mosq. Syst., 7(2) , 105‑llO16) Sirivanakarn, S. (1973): Three new species of Culex subgenus Culiciomyia Theobald fromSoutheast Asia and a redescription of the type of C. tricuspis Edwards from Alor, Lesser Sunda
Island, Indonesia (Diptera: Culicidae) , Proc. Entomol. Soc. Washington, 75(1) , I12‑124

17) Sirivanakarn, S. (1977): Redescription of four Oriental species of Culex (Culitiomyia) and the228
description of a

new species fromThailand（Diptera：Culicidae），Mosq．Syst，9（2），93−11118）Sirivanakam，S．and Kurihara，T．（1973）：A new species of Culex，subgenus Culiciomyia
Theobald from Ceram，Indonesia（Diptera：Culicidae），Proc．Entomol．Soc．Washington，75（2），220−22419）Steffan，W．A．and

Evenhuis，N．L．（1985）：Classi丘cation

of

the

subgenusTo蜘名h翅oh漉s（Diptera：Culicidae）．1．Australasian，eastem Palaearctic，and Oriental specie3groups，J・Med．

Entomol．，22（4），421−44620）Tanaka，K．，Mizusawa，K．and

Saugstad，E．S．（1979）：A revision of the adult and larval
mosquitoes of Japan（including the Ryukyu Archipelago and the Ogasawara Islands）andKorea（Diptera：Culicidae），Contrib．Amer．Entomol．lnst．，16，1−98721）Thu㎜an，D．C．（1955）：C燃（C％伽御吻）孟8甑an unusual new mosquito
Proc．Entomol．Soc．Washington，57，13−23

fromThailand，

new

crab

22）Toma，T．，Miyagi，1．and Cabrera，B．D．（1984）：C％伽（C擁吻吻卿）σ鍬吻4amosquito species from the Philippines，Mosq．Syst．，16（2），172−182

23）Tsukamoto，M．，Horio，M．，Rivera，D．G．，Sucharit，S．，Khamboonruang，C．and（1989）：Zymogram comparison of Southeast Asian
enzyme，Trop．Biomed．，6（in press）

mosquito

larvae．1．Lactate

hole

Yong，H．S．
dehydrogenase

and malic

マレーシア，キナバル山のウツボカズラより採集された
クシヒゲカとオオカの2新種
塚本増久

1986年度文部省科学研究費による海外調査でマレーシアの蚊相を調べた際，ボルネオ島キナバ
ル山の高所に自生する，食虫植物ウツボカズラの壺から数種の蚊幼虫を採集する機会があったので，その系統分類学的研究を行った。それらのうちのクシヒゲカの1種は1929年に採集され，C％伽s加ゐ66α勉として記録されていたものであったが，これを精査したところ東ヒマラヤ原産のこの学名の種とは全く異なり，新種であることが判明したので，C％嬬（C％」麺o吻釦）吻αhオ

ウサマクシヒゲカ（新称）と命名して詳しい記載を与えた。また，同種のウツボカズラから発生するオオカも未記載の新種であることが確認されたので，これも7bκoz勿解hJ趣 （710∬oz勿n−oh吻s）吻αhオウサマオオカ（新称）と命名し，成虫，蠣，幼虫などの形態について記載を行った。学名および和名は，これらの蚊が採集された巨大なウツボカズラ漉餌窺h召s吻αhの種小名（王様の意）に基づくものである。なお，同じ水域にはC銘伽U昭no吻n∫o（R6％4φoα」伽）吻oπ」も発生していた。

産業医科大学医動物学教室（Loρho68耀o吻吻）ノ6％s佛4

on4丁吻伽o漉5（Rαoh勿no伽n吻）sp．No．2などの蚊幼虫

Japan. J. Trop. M ed . Hyg ., Vol. 17, No. 3, 1989, pp. 229 236

229

MANAGEMENT AND UTILIZATION OF ORPHANDRUGS AGAlNST PARASITIC INFECTIONSIN RECENT 7 YEARS IN JAPANKIYOKATSU TANABE*, YOSHIHITO OTSUJ12, TOSHIO NAKABAYASHI'HIROSHI OHTOMO' AND HIROSHI TANAKA5,

Received March 22 1989/Accepted May 8 1989
Abstract: Since many important antiparasitic drugs are not registered in Japan, aresearch group, for the past 7 years, has managed the use of non‑registered orphan drugs.
Initially there were 16 orphan drugs under control of the group, these were as follows;

chloroquine, Fansidar, Fansimef, quinine (iv), primaquine, mebendazole, praziquantel,
thiabendazole, quinacrine, dehydroemetine (iv) , pentamidine, Pentostam, suramin, stibo‑phen, nifurtimox and pyrimethamine. Of these drugs, Fansidar, thiabendazole, mebend‑azole and praziquantel were recently registered. The number of cases treated with thesedrugs was nearly one thousand (920) . The number of malaria cases treated in this groupwas 201, corresponding to 22% of the total cases treated with orphan drugs. Mebendazole
was used in 210 cases, praziquantel in 163, thiabendazole in 120, quinacrine in 125,
pentamidine in 67, dehydroemetin in 28, Pentostam in 5 and suramin in l. Most drugs were
evaluated as effective in their antiparasitic actions but severe adverse reactions occurredat relatively high incidences.

INTRODUCTION
Tropical diseases are not endemic in Japan because of its geographical location in thetemperate zone. In addition, helminthic diseases, which prevailed in Japan for 2 decades afterWorld War II, have reduced incidence due to a change from use of night soil to chemicalfertilizer in agriculture, and due to improvements of public health and environmental hygiene.

Recent economical growth in Japan has created an urbanized life style for most people. On
the other hand, an increase of international relations has caused an irnportation of parasitic

diseases. In connection with the latter problem, effective antiparasitic drugs are necessary.

At the start of the present study, only a limited number of drugs effective against
parasitic infections were available on the market. Those were pyrantel pamoate, pyrvinium
1

2
3

4
5

Department of Infectious Diseases, Institute of Medical Science, The University of Tokyo,
Tokyo 108, Japan (Present address: Department of Medical Zoology, Faculty of Medicine,Kagoshima University, Kagoshima 890, Japan)
Second Department of Internal Medicine, Kagoshima University, Kagoshima 890, Japan
Department of Parasitology, Research Institute for Microbial Diseases, Osaka University,
Osaka 553, Japan
Department of Parasitology, Gifu University School of Medicine, Gifu 500, Japan
Department of Parasitology, Institute of Medical Science, The University of Tokyo, Tokyo
l08, Japan

230

pamoate, metronidazole, tinidazole, diethylcarbamazine, bithionol and quinine chloride (po,powder) , paromomycin and so on. Other drugs were not registered in Pharmacopoeia
Japonica, so‑called "orphan drugs". Although many antiparasitic drugs have been developed
in recent years and used widely in the world, pharmaceutical companies were not willing toregister the drugs in Japan, on account of the cost in application for approval, Iimited
business benefits and the occurrence of adverse reactions to these drugs. In order to collectclinical data on the orphan drugs, the Ministry of Health and Welfare in Japan (MOHW) set

up a research group for orphan drugs in 1980 and the members stored, managed and studied
16 different drugs. The clinical trials began in 1981 and the orphan drugs have been supplied

for use for 7 years. The present study deals with the results of the clinical trials and thepresent management of orphan drugs against parasitic diseases in Japan.

METHODS
Our research group managed non‑registered drugs and advertised the drug list. Eachmember of the group stored a set of drugs for distribution in accordance with the legal
responsibility of the member. An individual physician who wishes to use a particular drug,

contacts any member of the research group by telephone. The member sends out therequested drug along with prepared forms and guidelines for use of the drug. The doctor
sends back a form of ofricial request and an informed consent signed by the patient. The
doctor is obligated to make a report of the results of the trial. All drugs used in this study

were purchased from pharmaceutical companies in foreign countries.
The following drugs have been managed in stock: antimalarial drugs; (1) chloroquine
phosphate (Aralen, Resochin) , (2) sulfadoxine‑pyrimethamine (Fansidar), (3) mefloquinehydrochloride in Fansidar (Fansimef) , (4) quinine dihydrochloride (parenteral, Quinimax) ,(5) primaquine phosphate (Primaquine) ; and other antiparasitic drugs, (6) mebendazole(Vermox), (7) praziquantel (Biltricide) , (8) thiabendazole (MintezoD, (9) quinacrine hydro‑

chloride (Atabrine) , GO) dehydroemetine dihydrochloride (Dehydroemetine) , GD pentamidine

isothionate, pentamidine methanesulfonate (Lomidine), u2) sodium stibogluconate (Pentos‑tam) , a3) suramin (Germanin) , u stibophen (Fouadin or Neo‑Antimosan) , a5) nifurtimox(Lampit) and a6) pyrimethamine (Daraprim) .

RESULTSClinical data on the use of orphan drugs have been collected for 7 years from April, 1981

to March, 1988, and are shown in Table I for malaria cases, in Table 2 for cases treated withmebendazole, praziquantel and thiabendazole and in Table 3 for parasitic infections treatedwith other anthelmintics.

Antimalarial drugs
Clinical trials of antimalarial drugs were carried out in 189 cases, out of which 158 cases

were reported to this group as shown in Table 1. 106/158 (67%) cases were Plasmodium vivax
infections, 50 (32%) were P. falciparum, 2 (1%) were P. malariae, and in the other 26 cases
malaria species were not identified. The relationships of the species and antimalarial drugs

are shown in Table 1.

231

Table 1 The number of clinical trials in malaria cases by use of chloroquine,
Fansidar and quinine in 7 years

Species of plasmodium

Antimalarial drugs

Ch' Ch & Fa Qu' & othersd
Plasmodium falciparum

Plasmodium vivax
Plasmod ium malariae

16

7

9

18

61

36

9

106

(53) ‑

(32)

(7)

(92)
2

1

(1)

unable to identify

Total

50

(1)

31

11

1

43

(12)

(4)

(1)

(17)

109

55

19

(65)

(37)

(8)

18

201
(llO)

a : chloroquine phosphate.
b : Fansidar (pyrimethamine‑sulfadoxine).
c : quinine (parenteral, quinine dihydrochloride) .

d : other antima]arial drugs including chloroquine (6 cases),
Fansidar (6 cases) and both drugs (2 cases).

e : ( ) indicates the number of cases in which primaquine was used.

Primaquine was used in 92 cases (90%) of vivax malaria to prevent recurrence, and as
a result the recurrence occurred in 3 cases originating from Southeast Asia (Thailand).
Relapse was also seen in 2 patients with falciparum malaria. They had been treated withchloroquine or/and Fansidar, then quinine was given orally based on clinical evidence ofdrug‑resistant malaria. One fatal case of falciparum malaria occurred due to delayed
initiation of malaria therapy.

Adverse reactions were suspected in 3/42 cases (7%) treated with chloroquine (skineruption, photophobia and liver injury) but no adverse reactions were reported with Fansidar.

Mebendazole
Mebendazole was administered in 210 cases in which 126 (60%) were Trichuris trichiura(whipworm) infection (Table 2) . In most patients with trichuriasis, counts of eggs per gram

(EPG) were at low levels except for one individual with a high EPG over 5,000. In 53/55(96%) cases, EPG decreased after treatment. Adverse reactions were suspected in 5/75 (7%)
cases. Abdominal pain and skin eruption were the reported reactions.
This drug was also used for treatment of trichinosis during an epidemic in 1981 in which

about 60 persons were suspected of infecting the disease. The outbreak occurred in patronsof a restaurant in Yokkaichi in Mie Prefecture, where raw bear meat, possibly imported, wasserved to about 400 persons (Yamaguchi, 1983). There were two other epidemics in Japan;
15 out of 20 hunters in Aomori Prefecture in 1974 (Yamaguchi et al.. 1975) and 12 out of 94

persons who were served raw bear meat of local origin in Sapporo, Hokkaido in 1979 (Ozawa
et al.. 1981; Tebayashi et al.. 1981).

Gnathostomiasis caused by Gnathostoma spinigerum and G. hispidum from raw fishsometimes occurs in Japan. The source of a recent infection with G. hispidum was imported

232

Table 2 Parasitic diseases and effects of mebendazole, praziquantel and thiabendazole

Drugs andillness

No. ofCollected

Eff ect

cases

cards

(%)

C

R D

U

Yes No

73

1

22

5

Prognosis*

Side effectMebendazole

2

(89%)
(50%)
(75%)
(O%)
(100%)

210

l08 (51%)

92

87
17

trichuriasis

126

96

85

trichinellasis

55

4

2

gnathostomiasis

13

4

3

multi, echinococcus*

11

2

O

othersb

Total

2

5

3

1

3

1

70
4

3

l

2

1
1

1

(85%) 80

2

O 26

40

36 (90%) 38

2

11

8

7

2

(33%)
(o%)
(86%)

4

2

6 (7%)

81

11

25

2

6

Praziquantel
clonorchiasis

paragonimiasis

6

l

(73%)
(100%)l (lOO%)japonica29

93

haematobia
tape worm'

8

7

6

163

70 (45%)55 (79%) 56

4

2

106

75

l 16

Toxocara canis

7

gnathostomiasis

5

3

(78%)
2 (100%)(33%)(100%)
69 (85%)5

2

westermani
miyazakii
f asciolasis

1

2
l

1

l

schistosomiasis

othersd

Total

2

3

2

1

1

6

1

6

8

15 (27%) 40

8

Thiabendazole
strongyloidiasis

others*

Total

2

120

81 (68%)

65

53
2

57

5

20

55

1

2

1

2

1

l 18

25

(31%) 56

a : Echinococcus mutilocularisb : others include Angiostrongylus (2 cases), Toxocara canis (1 case), Iarval migrans (1 case) and
Capillaria philippinensis (1 case) .

c : Diphyllobothrium latum (fish tapeworm, 7 cases) , Taenia solum (pork tapeworm, 2 cases) andTaenia saginata (beef tapeworm, I case) .

d : others include Metagonium yohohawai (5 cases) and Sparganum mansoni (3 cases).
e : others include Capillaria philippi. (1 case) and Hymenolepis nana (1 case) .

* : C; cured; R; recurrent, D; died, U; unknown.

loaches from a neighboring Asian country (Ishii, 1983). Mebendazole was effective in
reducing complaints due to subcutaneous or visceral larval migration.
Mebendazole was examined for treatment of Echinococcus mutilocularis infection, alveo‑

lar hydatid disease (AHD) . Clinical cases were reported from endemic regions in theJapanese island of Hokkaido.
Praziquantel
Praziquantel was used in 163 cases, of which clonorchiasis(liver fiuke)

made up 87

233

Table 3

Parasitic diseases and drug effects (continued)

Drugs andNo. ofCollected

Eff ect

Prognosis'

illnessc a ses

cards

(%)

C R DUSide effectYes NoQuinacrine4226

25 (96%) 17

8

3

16

fish'

34

30

21

(70%)

ll

10

17

12

beefb

9

7

6

(86%)

5

2

4

2

giardiasis

tapewormtype unknown

40

18

4

(22%)

4

16

4

9

Total

125

81 (65%)

56

(69%)

37

36

28 (42%)39

67

41 (61%)20 (49%)529

7

12 (38%) 20

28

14 (50%)8 (57%)9l4

3 (25%)

93

22 (lOO%)11

1 (50%)

1

2

o

1

1

l (lOO%)1

Pentamidine
P, cariniic pneumonia 67

Dehydroemetine
hepatic amebiasis

PentostamKala‑Azar
leishimaniasis

Suramintry panosomiasis
a : Diphyllobothrium latum.

b : Taenia

inata.

c : pneumocystis carinii.

* : C; cured, R; recurrent, D; died, U; unknown.

(53%) (Table 2). In clonorchiasis, the cure rate was 90% at a dosage of 25 mglkg, given
three times a day for 2 days.

In paragonimiasis (lung fiuke) caused by Paragonium westernumi and P. miyazakii(rare) , praziquantel was shown to be effective with about a 70% cure rate.

The effect of praziquantel on Schistosoma japonicum infections could not be evaluatedproperly because new infections have ceased to occur in Japan since 1976 and all cases in thisstudy were at chronic stages. Four cases of S. haematobium (Japan Overseas Cooperation
Volunteers) originated from Egypt.
Adverse reactions to praziquantel were suspected in 15/54 cases (27% ) ; including
eruption (5 cases) , drowsiness (4), abdominal pain (4) and diarrhea (2).

Thiabendazole
Thiabendazole was used in 120 cases, mainly for strongyloidiasis (Table 2) . Infections

with S. stercolaris occur regionally in the Southwest Islands including Okinawa Island. The

cure rate was 87% but recurrence of the illness was often observed. Adverse reactions
reported with thiabendazole were abdominal discomfort (13 cases) , drowsiness (5) anderuption (2).

Other drugs
Quinacrine was used for the treatment of giardiasis (42 cases) and it provided a high rate

234

of cure. The drug was effective in recurrent cases after treatment with metronidazole. In one

case of giardiasis, toxic reactions including liver damage and eruption occurred after treat‑ment with quinacrine. Quinacrine was also used as an anthelminthic drug in 83 cases of tape'worm infection.
Cases of hepatic amebiasis and Pneumocystis carinii pneumonia are increasing in number

in Japan, resulting in increased uses of dehydroemetine and pentamidine, respectively.

A Japanese woman was infected with African trypanosoma, Tr)panosoma rhodesienseand fortunately cured by administration of suramin (Negishi et al.. 1984).

Newly registered drugs
The present study describes the success of this first step toward effective administrationof orphan drugs in this country. Among 16 orphan drugs listed previously, 4 drugs, thiabend‑azole, Fansidar, mebendazole and praziquantel were subsequently registered and are now
available on the market. Two other drugs, chloroquine and pentamidine, are under considera‑tion by the Central Pharmaceutical Affairs Council (CPAO in MOHW. The data collectedby this research group were widely utilized during evaluation for registration of these drugs.

DISCUSSION
Adverse reactions of the drugs studied by our group occurred often in this country, partly

on account of metabolic differences of Asian races including Japanese from other races. In
other instances, intoxication with chloroquine, chinoform and salidomide occurred by the

result of prolonged treatment or large dosages. The pharmaceutical bureau in MOHW andpharmaceutical companies were legally accused of fostering intoxication with these drugs.
The court trials concerning chinoform and salidomide were resolved by a compromise
between plaintiffs and the supplying companies, however, court trials regarding chloroquine
are still pending.

The reason why MOHW is also in litigation together with the supplying companies isbecause in Japan the approving organizations or review committees are charged with thesame responsibilities as the accused when untoward effects result from the approved prod‑
ucts. Because of the above legal liabilities and on‑going court trials, CPAC recently has been

careful in reviewing and approving new drugs. The same issues exist for decisions by ethicalreview committees in Japan for new medical trials on human subjects.
Due to careful attention to discussion and approval of drugs by CPAC, only data frompreclinical studies strictly following the standardized methods defined by CPAC and carriedout in Japan were the subject of consideration in the past. Nevertheless, data from preclinicalstudies conducted in foreign countries by methods, essentially equal to the Japanese stan‑dards, were recently considered valid by the Japanese CPAC.
Phase I and 2 clinical trials were conducted in normal and infected Japanese, respective‑
ly, by our research group. With the possibility of using preclinical studies from foreigncountries and the data from clinical trials conducted by our group, pharmaceutical companies
were encouraged and decided to apply for registration of a few orphan drugs and to offer
these drugs for commercial sale as an action of social contribution, even though commercial
merits would not be expected. Because of the above stated difficulties, 12 drugs still remain

as orphan drugs in Japan.

235

In the USA, the Centers for Disease Control (CDO Drug Service presently manages 9
drugs: diloxanide furoate (Furamide) , dehydroemetine, nifurtimox (Lampit) , sodium stibog‑

luconate (Pentostam), melarsoprol (Mel B), suramin, bithionol, ansamycin, and quinine
dihydrochloride (parenteraD as orphan drugs. Out of these 9 drugs, 5 are common to ourorphan drugs. Our supply system for these drugs is incidentally the same as that in the USA,although our organization is composed of researchers without assigned staffs.
There are few reports dealing with clinical trials using newly developed antiparasitic
drugs in Japan (Tanabe and Tanaka, 1986; Tanabe et al., 1987) . Data in the present study
may reflect a new trend in parasitic infections in Japan because it is difficult for individual

physicians to obtain antiparasitic orphan drugs from any other source by themselves.
In the present study, the total number of cases is not as large as that seen in tropicalcounties. However, in accordance with the increase in international travelers, parasitic
diseases have become an important problem even in developed countries. From this point ofview, the present study demonstrated an effective way for our country to provide orphan
drugs needed to treat parasitic diseases.

Many problems have remained unsolved regarding parasitic diseases in our country.
Echinococcus multilocularis infection occurs locally in Hokkaido and about 260 cases havebeen detected by clinical and pathological observations. According to recent reports basedon serological surveys, echinococcus infections have been increasing in number (Takahashi,1986). Amebiasis, giardiasis, strongyloidiasis and Pneumocystis carinii pneumonia are also
increasing in number in association with STD or as opportunistic infections. Treatment ofthese diseases remains a problem and should be solved by developing new types of drug.

ACKNOWLEDGMENTS
This work was supported by grant from the Scientific Research Fund of the Ministry of

Health and Welfare in Japan.

REFEREN CES
1 ) Ishii, Y. (1983): Morphological eomparison of the gnathostomes obtained from imported fresh
water fish, Jpn. J. Parasitol., 32, supp., 4 (in Japanese)2 ) Negishi, M., Young, C., Masuda, G., Shimizu, N., Takeuchi, T., Kobayashi, S., Nishijima, S. andOkuuchi, Y. (1984): A case of African trypanosomiasis, J. Jpn. Assoc. Infec. Diseas., 58, 435‑

440 (in Japanese with English summary)3 ) Ozawa, E., Nakada, K.. Yokokawa, M. and Kobayashi, M. (1981): A case of trichinosis,
Kansen Ensho Men‑eki (Infection Inflammation Immunity), 11, 233‑240 (in Japanese)4 ) Takahashi, A., Yamaguchi. T.. Inaba, T. and Hayashi. H. (1986) : A review of multilocularechinococcosis cases reported from Honshu. Japan, during a period from 1926 to 1984, Jpn. J.
Parasitol., 35, 95‑107 (in Japanese with English summary)5 ) Tanabe, K. and Tanaka, T. (1987): A clinical evaluation of mebendazole on trichuriasis inJapan, J. Jpn. Assoc. Infec. Diseas., 61, 161‑166 (in Japanese with English summary)6 ) Tanabe, K., Ohtomo, H., Otsuji, Y., Nakabayashi, T. and Tanaka, H. (1987): A clinicalevaluation of chloroquine on malaria in Japan, J. Jpn. Assoc. Infec. Diseas., 61, 1064‑1069 (inJapanese with English summary)7 ) Tebayashi, A., Murakoshi, T., et al. (1981): An epidemic of trichinosis transmitted by frozen

236
bear

meat，JPn．Medical

J．，2971，46−49（in Japanese）

8）Yamaguchi，T．，Takeda，N．，8∫8乙（1975）：The first epidemic ofMedicalJ．，2668，16−21（in9）Yamaguchi，T．（1983）：Trichinosis in1427−1432（intrichinosis

in Japan，JPn．

Japanese）

Japan，Byori to

Rinsho（Pathology

andClinics），1，Japanese）

寄生虫病薬の7年間の治験成績の検討
一稀用薬の入手緩和を求めて一田辺

清勝1・尾辻 義人2・中林 敏夫3
大友 弘士4・田中
寛5

昭和55（1980）年に発足した厚生省「輸入熱帯病の治療薬に関する研究」班では，寄生虫病の治療に稀用される薬剤の入手緩和を計ることを検討してきた。わが国の薬事で承認されていない
寄生虫薬で，海外においては一部で既に使用されている16種の薬剤，すなわちファンシダール，
クロロキン，プリマキン，キニーネ（注射液），ファンシメフの抗マラリア薬とそれ以外のミンテ
ゾール，メベンダゾール，プラジカンテル，ペンダミジン（注射液），アテブリン，デヒドロエメ

チン（注射液），ペントスタム（注射液），スラミン，スチィボフェン，ランピット，ピリメタミンを選定して輸入し，治験用に供給して臨床試験を実施してきた。昭和56（1981）から62（1987）年までの7年間に，920症例（マラリア患者では2種以上の治験薬が投与されても1症例として集計した）に薬剤を配布してきた。症例の内訳は201例（22％）がマラリア患者で，マラリア以外の投薬症例数はメベンダゾールが210例，プラジカンテル163例，アテブリン125例，ミンテゾール120
例，ペンタミジン67例，デヒドロエメチン28例，ペントスタム5例，スラミン1例である。このうち治療カードの回収できた症例について，有効性と副作用を検討して概略をまとめた。なお，
今日ではファンシダール，ミンテゾール，メベンダゾール，プラジカンテル，ペンタニジンの承認が済んでいる。

東京大学医科学研究所感染症内科（現所属：鹿児島大学医学部医動物学教室）
鹿児島大学医学部第二内科
大阪大学微生物病研究所寄生虫病学部
岐阜大学医学部寄生虫学教室
東京大学医科学研究所寄生虫研究部

Japan. J. Trop. Med. Hyg., Vol. 17, No. 3, 1989, pp. 237‑241

237

STUDIES ON EFFECTS OF PAROMOMYClN
SULPHATE, BITHIONOL, MEBENDAZOLEAND FLUBENDAZOLE IN THE TREATMENTOF MICE INFECTED WITH PLEROCERCOIDS
OF DIPHYLL OBOTHRIUM ERINA CEI
JUN MAKI AND TOSHIO YANAGISAWA
Received March 22 1989/Accepted May 15 1989

Abstract: Mice infected with Diphyllobothrium erinacei plerocercoids were adminis‑
tered orally with paromomycin sulphate, bithionol, mebendazole and flubendazole at the
total doses of l0,000 mg/kg for 200 days or subcutaneously with the drugs at the total
doses of 2,500 mg/kg for 25 days. In spite of the high doses, no difference was observed
between the number and weight of larvae recovered from mice in experimental groups andthose in control groups.

Human cases infected with plerocercoids of Diphyllobothrium erinacei have been found
one after another in Japan (Mukai et al., 1977; Sasaki et al., 1980; Kusunoki et al., 1980;
Mukai et al., 1981). As reviewed in these papers, some Japanese eat inadequately cooked
fiesh of snakes harbouring the plerocercoids for healthy life or in the hope of finding health.

Human sparganosis thus resultant often involves such difflculties that it cannot be treatedsatisfactorily. In some cases surgical removal of the larvae in subcutaneous tissues success‑fully leads to rapid recovery. However, in other cases with visceral larvae, their discovery

and treatment are very difncult even if immunological diagnosis is made. This is why
establishment of suitable chemotherapy is required. According to Faust et al. (1970) ,injection of ethanol with procaine (free of epinephrine) into the lesion and intravenous
administration of norvarsenobenzol are effective for the treatment of ocular sparganosis in

Vietnam. However, no experimental studies except a preliminary study by Maki and
Yanagisawa (1983) have been reported regarding chemotherapy of sparganosis to the present
authors' knowledge. This communication describes the first trial of drugs in infected mice.

The following drugs were examined with the background of previous reports. Paromomycinsulphate was reported to be effective against various tapeworms including D. erinacei adults(Kagei and Hayashi, 1979; Orima et al., 1981; Suzuki et al., 1982). Bithionol is effective inelimination of D. erinacei adults (Oshima, 1976; Yoshimura, 1976). Both mebendazole andflubendazole have been found to be effective against hydatids of Echinococcus granulosus andE. multilocularis in animals (Schantz et al., 1982; Van den Bossche, 1982) . According to thereport of Heath et al. (1975) , mebendazole has lethal effect on cysticerci of Taenia pislformis

and tetrathyridia of Mesocestoides corti as well as hydatids of E. granulosus. Furthermore,
Department of Parasitology, Kitasato University School of Medicine, Sagamihara 228, Japan

238

fiubendazole was reported to be effective on cysticerci of Taenia solium in pigs (Tell6z‑Gir6n
et al., 1981) .

The anterior parts (about 4 mm long) of plerocercoids of D. erinacei isolated fromnaturally infected snakes, Elaphe quadrivirgata and washed with saline containing 1,000 unitsof penicillin and 500 pg of streptomycin per ml were inoculated subcutaneously into female
mice of ICR‑strain, 4 weeks old at the centre of the back using a hypodermic syringe and aneedle (10 head parts/mouse) .

The infected mice were divided at random into experimental and control groups of 6‑7

mice each. Paromomycin sulphate (potency: 715 pglmg; kindly supplied by Kyowa Hakko
Kogyo Ltd.) dissolved in 1% (v/v) Tween 80, and pure powdered bithionol (kindly supplied
by Tanabe Pharmaceutical Co., Ltd.) , mebendazole and fiubendazole (pure powdered drugsof Janssen Pharmaceutica, Belgium provided by the courtesy of Dr. K. Okano, Animal Health
Development, Fujisawa Pharmaceutical Co. Ltd.) suspended in 1% Tween 80 were given to
the mice of experimental groups orally via a stomach tube or subcutaneously with a tubercu‑
lin needle and syringe (1 ml, 26 G X 112). The control mice were orally or subcutaneously

given 1% Tween 80 alone. Medication was done as follows. In the first study, mice wereadministered with one of the 4 drugs orally at 100 mg/kg/day, every other day, 21‑219 days
post‑inoculation. In the second study, mice were administered with one of the 3 drugs,
paromomycin sulphate, bithionol and mebendazole at 100 mg/kg/day for 25 consecutive days
(98‑122 days post‑infection) , unless otherwise indicated, subcutaneously at the centre of the

back. Thus 10,000 and 2,500 mglkg of the drugs in total doses were given in the first andsecond study, respectively to all the mice of experimental groups except those which died
before the termination of medication. The skin of mice killed after the medication or thosewhich died during the medication period was removed to recover the larvae from various
body regions including the thoracic and abdominal cavities. This autopsy was carried out on
234‑235 or 125 days post‑infection in the first and second experiment, respectively with the

exception that the mice which died during the medication period were examined for the worm
recovery within 24 h after the death of mice. All the larvae recovered from each mouse were
found to be alive. They were immersed in 10% (v/v) formalin, washed in saline, dried at

lOO'C, 3 h and weighed to the nearest mg.
Oral administration of paromomycin sulphate, bithionol, mebendazole or flubendazole
even in the total dose of l0.000 mglkg had no effect on the plerocercoids in mice (Table 1) .

There was the possibility that no efficacy as shown in Table I was due to very low
concentrations of the drugs absorbed from the intestine of mice used. Therefore, subcutane‑

ous administration of paromomycin sulphate, bithionol and mebendazole was carried out.
However, no difference was seen between the number of worms recovered from medicatedand non‑medicated groups as shown in Table 2. Mean dry weight of worms recovered frommice given 2,500 mglkg in total dose was similar to that from control mice (Table 2) .
In view of the fact that fairly many mice did not survive the administration of high doses(Tables 1, 2), the total doses of 2,500 (subcutaneous administration) and 10,000 (oraladministration) mg/kg are higher than tolerable doses. Nevertheless no efiicacy was seen in
the 4 drugs against D. erinacei plerocercoids in mice.

Paromomycin sulphate and bithionol were reported to be effective against adult D.
erinacei, and the 2 benzimidazoles, mebendazole and fiubendazole to be effective against anumber of larval tapeworms (cyclophyllidea) in host tissues as mentioned above. The 4

239

Table 1 Plerocercoids recovered from mice orally given paromdmycin sul phate, bithionol,
flubendazole or mebendazole
Medication
mg/kg/day x days

total doses

Number of plerocercoids[Mean:!:S.D.

recovered per mouse(No. mice)]

Dry weight of plerocer‑

coids recovered permouse (mg)[Meand:S.D.
(No. mice) lParomomycin sulphate
N. M.

lOOx 64

6 , 400 '8100x 81

8 , 100'

9lOO x 100

10 , OOO

8, 9, 9 10 10lOOx 45

4 , 500'

lO, lOlOOX 59

5 , 900*

7

lOO x 100

10 , OOO

9 , 9 , 10, 10

N. M.
[9 . 2 :!: O . 8(5)]87, 88, 92. 107. 121[98 . 9 :!: 14 . 6(5)]Bithionol

N. M.
N. M.
[9.5:!:0.6(4)]91, 126, 132, 149[ 124 . 5 d: 24 . 4(4)]MebendazolelOOX 3300'

6lOOx 33

3 , 300'

lO

N. M.

lOOX 68

6,800'

7

N. M.

100X 70

7 , OOO'

7

100 X 100

10 , OOO

7, 9[8 . O :!: I . 4(2)]l04, 106[ 105 . O

l0,0008 , 9 , 9 , 10, 10, 10, lO[9 . 4 : : o . 8(7)]79, 85, 86, 104, 105,

[99 . O i 16 . 4(7)]7, 8, 8, 9, 9,l0,lO[8 . 7 i I . 1(7)]

N. M.

N. M.
I . 4(2)]

Flubendazole
100 X 100l07, 126

Controlo48, 77, 84, 88, 98,[85 . 9 : 19 . 4(7)]lOO, 105

Female mice of ICR strain 4 weeks old were subcutaneously given 10 head parts of plerocer‑
coids, orally administered with either of the drugs at 100 mg/kg/day, every other day, 21‑219

days post‑infection and autopsied on 234‑235 days post‑infection. *Mice died during the
medication period, being autopsied within 24 h after death. N. M.=not measured.
drugs were not effective against larval D. erinacei in mouse tissues. The reason for this fact
is an open question to be clarified.

Bithionol was administered to patients suffering from larval D. erinacei infections but noevidence indicating curative efficacy of the drug was obtained (Araki et al., 1976; Fujiwara
et al., 1978) . If the 4 drugs are not effective in human cases as in infected mice of the present

studies, the drugs including bithionol should not be administered. Further studies are needed
to find suitable drugs against sparganosis.

ACKNOWLEDGEMENTS
Dr. Nakamura, T., Department of Parasitology, Kitasato University School of Medicine
is gratefully acknowledged for the infection of mice with the plerocercoids. Thanks are also
given to Mrs. Honda, M. in the same laboratory for her excellent technical assistance. This
investigation is financially supported by a Scientific Research Grant from the Ministry of
Education, Science and Culture, Japan.

240

Table 2 Plerocercoids recovered from mice subcutaneously given paromomycin sul phate,
bithionol or mebendazole
Medication
mg/kg/day X days

Number of plerocer‑ [Mean:!:S.D. Dry weight of plerocer‑

coids (No. mice) ltotal doses

coids recovered permouse (mg)recovered per mouse[Mean S.D.
(No. mice) lParomomycin sulphate
N. M.

100X 222 , 200*

7

100X 25

2 , 500'

7, 7 ,

500X 1500X 3

500'

8,8

t

10, lO, lO, 10

E9 . OI . 5(6)]9 , 10[8 . 8 i I . O(4)][8.5:!:0.7(2)]63, 67, 91, 95, 105, 109

N. M.
N. M.

l , 500*

8, 9

5 , 500 *

7

N. M.

100X 13l , 300*

7

N. M.

lOOX 15l , 500'

7

N. M.

lOOx 222 , 200*

lO

N. M.

100X 24

2 , 400*

3

lOOX 25

2 , 5007, 9500X 11[88 . 3 :!: 19 . 2(6)Bithionol

N. M.
[63 . 5 :!: 12 . O(2)][8 . O d: I . 4(2)]55, 7232, 86, 91, 99, 126[86 . 876, 78, 81, 85, 92, 98,[87 . 6 :!: 10 . 3(7)]Mebendazole
N. M.

lOOX 23

2 , 300 '7

lOOX 25

2 , 500

3, 9, 9, lO, lO[8 . 2 d: 2 . 9(5)]lO, lO, 10, 10, 10, 10,[ 10 . O :!: O(7)]Controlo34 . 3(5)]

l03

10

Female mice of ICR strain 4 weeks old were subcutaneously given 10 head parts of plerocer‑
coids, subcutaneously administered with either of the drugs at 100 (or 500) mg/kg/day for 25
consecutive days (98‑122 days post‑infection) and autopsied 125 days post‑infection. *Micedied during the medication period, being autopsied within 24 h after death. N. M.=notmeasured.

REFERENCESl ) Araki, T., Nakazato, H., Imaoka, A., Fukunaga, A., Osawa, C., Matsuki, N. and Yasuhara, M.
(1976): Two cases of sparganosis mansoni: presumption of the parasitism under pericardium
and movement of the parasite during seven years, Jpn. J. Parasitol., 25, 343‑349
2 ) Faust, E.C., Russell, P.F. and Jung, R.C. (1970) : Sparganum and sparganosrs In "Cralg and
Fausts Clinical Parasitology", 8th ed. 515‑517, Lea & Febiger, Philadelphia3 ) Fujiwara, T., Yamaguchi, K., Ohtomo, H. and Araki, K. (1978): Cases of spargan6sis mansoni,
including one with the complication of pericarditis, Jpn. Med. J., 2847, 27‑32 (In Japanese)4 ) Heath, D.D., Christie, M.J. and Chevis, R.A.F. (1975): The lethal effect of mebendazole onsecondary Echinococcus granulosus, cysticerci of Taenia pislformis and tetrathyridia of
Mesocestoides corti, Parasitology, 70, 273‑285

5 ) Kagei, N. and Hayashi, S. (1979) : Aminosidine as an anti‑tapeworm drug, Shinryo to
Shinyaku, 16, 2807‑2817 (In Japanese)6 ) Kusunoki, M., Kimata, T., Hara, Y., Kinoshita, T., Araki, T. and Tanaka, S. (1980): Spar‑ganum mansoni found in interphalangeal joint of thumb, Jpn. J. Parasitol., 29 (Suppl.) , 52 (InJa panese)7 ) Maki, J. and Yanagisawa, T. (1983): A comparison of the effects of fltibendazole andthiabendazole on the larvae of Angiostrongvlus cantonensis. Trichinella spiralis. Diphylloboth‑rium erinacei and Hymenolepis nana in mice, Parasitology, 87, 525‑531

2418）Mukai，H．，Yamate，T．，Saito，R．and

Ito，Y．（1977）：Sparganosis

mansoni，Rinsho−Hifuka，31，551−555（InJapanese）
9）Mukai，H．，Saito，R．and

Ito，Y．（1981）：Sparganosismansoni，Hifubyo−Shinryo，3，219−222（InJapanese）
10）Orima，H．，Uno，Y．，Sasao，Y．，Tasaka，K．and Ichiki，H．（1981）：Paromomycin as anthelmintic
for」0ゆh翅oδo孟h吻御㎜％soηげ，JPn．J．Vet．Med．，34，1−511）Oshima，T．（1976）：The
outbreak in

Naganobiology，epidemiology

Prefecture，Shinshu

12）Sasaki，K．，Yokota，T．，Fujita，K．and
Parasitol．，29（SupPl．），52（ln13）Schantz，P．M．，Van

cosis

in

animals

den

and

Igaku

and

treatment of

Ikeda，T．（1980）：A

case

of

the

adult

Japanese）

orbitalsparganosis，Jpn．J．

Japanese）

Bossche，H．and

humans：Report

Eckert，J，（1982）：Chemotherapy

of

a

for

larvalechinococ・

workshop，Z．Parasitenknd．，67，5−2614）Suzuki，N．，Kumazawa，H．，｝Iosogi，H．and Nakagawa，0．（1982）：A
with

diphyllobothriasis：Recent

Zasshi，24，191−204（Incase of human infectionof助吻勉6渉勉爾ηαoθづ（Rudolphi，1819）Faust，Campbell

andKellogg，1929，Jpn．

」．Parasitol．，31，23−26

15）Tell6z−Gir6n，E．，Ramos，M．C．andoθ〃％Jos4召in16）VanMontante，M．（1981）：Effectden Bossche，H．（1982）：Present

world

and

omubendazole

onのsだ08κ欝

pigs，Am．J．Trop．Med．Hyg．，30，135−138

Ours（ed．Mettrick，D．F．and

state

of

mebendazoleandHubendazole，Parasites・一Their

Desser，S．S．），336−339，Elsevier

Biomedical

Press，

Amsterdam17）Yoshimura，H．，Kondo，K．，Moritani，S．andin

Hokuriku

District：Number

of

cases

Ohnishi，Y．（1976）：Studies

and

their

on

diphyllobothriasis

treatment，J．Therapy，58，953−956（InJapanese）

マンソン孤虫に対する硫酸パロモマイシン，

ビチオノール，メベンダゾールおよびフルベン
ダゾールの駆虫効果に関する実験的研究
牧純・柳沢十四男

マンソン孤虫を感染させたマウスに硫酸パロモマイシン，ビチオノール，メベンダゾールまたはフルベンダゾールを総量10，000mg／kg・200日間経口投与，或いは総量2，500mg／kg・25日間皮下投与した。しかし実験群と対照群の間に，回収した虫体の数，および重量について差が認められなかった。

北里大学医学部寄生虫学教室

Japan J Trop Med Hyg Vol. 17, No. 3, 1989, pp. 243‑257

243

NOTES ON BLACKFLIES (DIPTERA= SIMULIIDAE)

FROM MYANMAR(FORMERLY BURMA)
HIROYUKI TAKAOKA
Received April 24 1989/Accepted June 5 1989

Abstract: Adult pinned specimens of Burmese Simuliidae held in British Museum(Natural History) were examined. A total of eight taxa recognized are all assigned in thegenus Simulium Latreille s. l. and are further placed in the following subgenera:
Nevennannia Enderlein (3 species including 2 new species) , Gomphostilbia Enderlein (1new species) , Himalayum Lewis (1 species) and Simulium Latreille s. str. (3 species).
Descriptions and illustrations for 3 new species and I unnamed one are presented.

INTRODUCTION
The simuliid fauna of Myanmar (formarly Burma) has been poorly studied. No blackfly
species has been so far reported except Simulium (Himalayum) indicum Becher, 1985 which
was recorded from female specimens (Lewis, 1974). The early stages of this species haveremained unknown from Myanmar.
This paper reports additional seven blackfly species including three new species, basedon adult simuliid specimens loaned from British Museum (Natural History) , London.

MATERIAL AND METHODSAll the specimens examined were collected in 1934 by R. Malaise from Kambaiti, and in1938 by R. Kaulback from Nam Tamali Valley, in northern Myanmar.
Some pinned adult specimens were dissected under a stereoscopic microscope and theirhead and genitalia were observed in detail after being immersed in 10% KOH solution. They
were finally mounted on a glass slide, together with certain other parts of the body.
The laboratory procedures used in this work were almost the same as those described
previously (Takaoka, 1983) . ･ The measurement of the hind basitarsus and tarsal claw tooth
follows that of Davies and Gy6rkds (1987) .

The morphological features and terms used follow those of Crosskey (1969). The
classification of Crosskey (1981) is followed except one of the subgenera, i.e., Montisimulium,

which is not adopted in this study. One new species apparently belonging to this subgenus isincluded in the subgenus Nevernumnia Enderlein.

Division of Medical Zoology, Medical College of Oita, Hazama, Oita 879‑56, Japan

244

SPECIES AccouNTs
1 . Simulium (Nevermannia) burmense sp. nov.

Female. Wing length 2.8 mm. Head. Frons (Fig. 1) and clypeus black, heavily whitepruinose, and covered with numerous hairs. Frontal ratio 1.6:1.0:2.1. Frons‑head ratio 1.0:4.8.

Antenna composed of 2 + 9 segments, dark brown except scape and pedicel orange. Maxillary
palp with 5 segments, proportion of apical 3rd, 4th and 5th segments 1.3:1.0:5.9; sensory vesicle(Fig. 2) elongated, ca. 3/5 x length of 3rd segrnent. Maxilla with 15 strong teeth on each side.

Mandible with ca. 40 small inner teeth and 13 outer teeth. Cibarium (Fig. 3) without any
tubercles or teeth medially. Thorex. Scutum brownish black, entirely whitish grey pruinose
when viewed in certain angle of direct light, or with 2 pairs of white pruinose spots eachsubmedially and laterally (on prescutellar regions) near anterior margin when viewed inanother angle (Fig. 4); scutum densely covered with recumbent golden pubescence, andwithout any longitudinal lines. Scutellum brown with numerous golden pubescence andseveral upstanding dark hairs. Postscutellum blackish brown, white pruinose and bare.
Pleural membrane and katepisternum bare. Legs. All coxae and trochanters yellow except
mid and hind coxae dark. All femora yellow except apical small portion dark brown. A11
tibiae dark brown with median large portion yellow to light brown; all femora and tibiae
densely covered with golden hairs. All tarsi brown to dark brown. Fore basitarsus (Fig. 5)
slender, cylindrical, ca. 7.5x as long as its greatest width. Hind basitarsus nearly parallel

sided (Fig. 6) . Calcipala and pedisulcus moderately developed. Claw with basal tooth, ca.
1/2 x length of claw (Fig. 7). Wing. Costa with parallel rows of short spinules as well as
hairs. Subcosta with hairs. Basal section of radius fully haired. Hairs at base of stem veindark. Abdomen. Basal scale pale yellow, with golden fringe. Dorsal surface of abdomen
dark brown, with minute hairs, and somewhat shiny on segrnents 7‑9. Genitalia. Sternite 7
large and well demarcated. Sternite 8 bare medially and with ca. 12 Iong hairs on each side

(Fig. 8) . Anterior gonapophyses simple, nearly triangular, and membraneous with a few
microsetae; inner margin slightly curved and narrowly sclerotized; posterior border thin andtransparent. Genital fork with wide arms; stem and anterolateral margin of arms heavily
sclerotized; arms expanded posteromedially (Fig. 9) . Spermatheca almost globular in shape,strongly sclerotized and with reticulate pattern (Fig. 10) . Paraproct short, somewhat
produced ventrally and moderately haired (Figs. 11 and 12) . Cercus semicircular in side view,ca. 3/5 x as long as its width at base (Fig. 11).

Male, pupa and larva. Unknown.
T)pe specimens. Holotype female, slide‑mounted except thorax, Ieft fore and mid legs andright wing left on pin. MYANMAR: Kambaiti, 7,000 ft., 30. IV. 1934, R. Malaise. Paratype
female, pinned except terminal tip of abdomen on slide, same data as holotype.

Remarks. This new species is assigned to the vernum group of the subgenus Nevernumnia
defined by Crosskey and Davies (1972) , and shows similarities to S. gracilis and S. puri, both

described from India by Datta (1973) . Simulium burmense sp. nov. differs from the former
species by the shape of the posterior portion of cibarium, and from the latter by the shape ofthe genital fork.

245

i.'!
f'://;;:.,'i]=?!: i,

'

t;

' '*:

4

1

2
) 1･1 ̲i/l * ,r!':'S :;i ii̲̲¥.̲̲ ¥' ¥ ' "I>( 7,r[; :.r; )".

･̲

i (

( .‑,., ' 6, .,', ;‑ I . 5‑':*̲il ‑ . ̲*,r'+FL L:JL:JL:¥t¥ ‑ l: I O""
11

1 ̲ : !' ="'{=',"='=" 1';

Figs. 1‑12 Simulium (Nevermannia) burmense sp. nov. female. 1, frons; 2, 3rd segmentof maxillary palp showing sensory vesicle; 3, cibarium; 4, scutum showing two pairsof whitish spots; 5, fore leg (coxa and trochanter omitted) ; 6, hind leg (coxa andtrochanter omitted) ; 7, claw; 8, 8th stemite and anterior gonapophyses; 9, genital fork;

10, spermatheca; 11, paraproct and cercus in lateral view; 12, paraproct in ventral
view.

This species is also related to S. aberrans Delfinado, 1969 from Philippines (Takaoka,1983) and S. taulingense Takaoka, 1979 from Taiwan (Takaoka, 1979). However, S.
aberrans differs by the female antenna and legs entirely dark brown, and S. taulingense shows
the different shape of the cercus (triangular in side view) .

2 . Simulium (Nevermannia) kambaitense sp. nov.
Male. Wing length 2.8 mm. Head. slightly wider than thorax. Upper eye consisting of 19
horizontal rows and 19 vertical columns of somewhat enlarged facets. Clypeus brownish
black, whitish grey pruinose, and with dark hairs. Antenna composed of 2 + 9 segments, darkbrown except base of Ist fiagellar segnlent pale; Ist fiagellar segment elongate, ca. 2 x as longas 2nd flagellar segment. Maxillary palp with 5 segments, with 3rd, 4th and 5th segments inproportional length of 1.0:1.3:2.3; sensory vesicle small, ca. 1/4 x as long as 3rd segment (Fig.

24613) . Thorax. Scutum brownish black, whitish grey pruinose, densely covered with goldenrecumbent pubescence. Scutellum brown with numerous golden hairs and several dark hairs.

Postscutellum brownish black, whitish grey pruinose and bare. Pleural membrane andkatepisternum bare. Legs. All coxae and trochanters dark yellow except hind coxa brown.
All femora dark yellow to pale brown except apical portion dark brown. All tibiae darkbrown with median large portion somewhat pale. All tarsi dark brown except hind basitarsus
and basal l/2 of 2nd tarsal segment pale brown. Fore basitarsus slender, cylindrical, ca. 10 X

as long as its greatest width. Hind basitarsus enlarged, spindle‑shaped, its greatest widthalmost the same as the greatest width of hind femur and tibia (Fig. 18). Calcipala andpedisulcus well developed. Wing. Costa with 2 parallel rows of short spines as well as darkhairs. Subcosta bare. Basal portion of radius fully haired. Basal tuft hairs dark̲ brown.
Abdomen. Basal scale brown with golden hair fringe. Dorsal surface of abdomen brownish
black with golden and dark short hairs. Genitalia. Coxite enlarged, much longer than wide
(Fig. 20) . Style shorter than coxite, twisted dorso‑inwardly, and with a single apical spine
directed dorso‑inwards (Figs. 25‑28) ; posterodorsal surface thin, membran ous and smooth
except a few minute setae (Fig. 2,5). Ventral plate lamellate, well sclerotized, much wider
than long, and covered ventrally and posteriorly with minute setae in the middle (Fig. 21);ventral surface somewhat raised ventrally in the center (Fig. 23), and posterior borderdeeply concave (Fig. 21). Paramere with 4 stout spines (Fig. 24). Median sclerite simple,
rod‑like.

Female. Wing length 2.8 mm. Head. Frons (Fig. 15) and clypeus brownish black, thickly
whitish grey pruinose, and densely covered with golden pubescence, intermixed with sparse
dark hairs. Frotal ratio 1.7:1.0:2.3. Frons head ratio 1.0:4.8. Antenna composed of 2+9
segments, and dark brown except scape and pedicel greyish yellow. Maxillary palp com‑posed of 5 segments, with 3rd, 4th and 5th segrnents in proportional length of 1.0:1.0:1.5; 3rd

segrnent somewhat enlarged, with elongated sensory vesicle (Fig. 14) , slightly over 1/2 x as
long as 3rd segment. Maxilla with 10 or 11 inner teeth and 14 or 15 outer ones. Mandible withca. 28 inner teeth and without any outer teeth. Cibarium without any denticles. Thorax.
Scutum brownish black, thickly whitish grey pruinose, and densely covered with goldenrecumbent pubescence; no longitudinal lines discernible. Scutellum brown with golden

pubescence and several erect dark hairs. Postscutellum brownish black, whitish grey
pruinose, and bare. Pleural membrane and katepisternum bare. Legs. All coxae, trochantersand femora light yellow except mid and hind coxae, and apical tip of all femora brown. Alltibiae and tarsi brown to dark brown except middle large portion of tibiae dark yellow tolight brown. All femora and tibiae densely covered with golden hairs. Fore basitarsus (Fig.
16) slender, cylindrical, ca. 7.4 x as long as its greatest width. Hind basitarsus (Fig. 17)
parallel‑sided. Calcipala and pedisulcus well developed (Fig. 17). Claw with medium basal
tooth, ca. 0.37 x length of claw (Fig. 19) . Wing. Costa with 2 parallel rows of short spines
as well as hairs. Subcosta fully haired. Basal portion of radius fully haired. Basal hair tuft

dark brown. Abdomen. Basal scale dark yellow with golden hair fringe. Dorsal surface ofabdomen dark brown, with terga somewhat grey pruinose, and with light and dark shorthairs; terga 7‑9 shiny when viewd in light. Genitalia. Sternite of 7th abdominal segment
widely developed. Sternite 8 (Fig. 29) bare medially but furnished with ca. 36 stout hairs oneach side. Anterior gonapophyses (Fig. 29) thin, membraneous, produced posteromedially,

247

多

リ

ロ
ラ
，塩ll・1・ド，．

o

o

o

14

13

ーく−

愈
19

16

15

ノ

ー／

ll

1脚1

・：鼎

で1：一，

ノZ

17

21

22

r

31

譲露欝一、 ＼、

㌔融、

硬￥

／フ＼
23

︑ず

20

29

24

鐸

26

Figs．13−32S加％伽吻（ハ勧召競on％宛）肋吻δ癬2％s召sp．nov，male
14，3rdsegments

16，foreleg of female；17，hind leg

omittedinventralplateinmaxillarypalp（13forFigs．16−18）；19，claw

inview；24，paramere
style

of

of

male，14forfemale；18，hindlegof female；20，coxiteandwith4hooks；25，styleview；26，style

gonapophyses；30，genital

in

end

in

insidemale（coxa

style

in

view；28，style

side

of

ventral view；22，ventral plate

ventolateralandfemale．13and

female）；15，frons
infork；31，spennatheca；32，paraproctinventralview；21，plate

outsidestemiteandfemale；trochanter

viewl23，ventral

view；29，8th

of

and

and

cercusinendview；27，
anteriorinlateral248

appearing tongue‑like, narrowly sclerotized on inner border, bare and transparent nearrounded posteromedial border; a few short setae near anterior border which is not welldemarcated. Genital fork (Fig. 30. with well sclerotized stem and wide arms; arm with stout,

long projection directed forwards, as well as rounded projection directed posteromedially.
Spermatheca (Fig. 31) ovoid in shape, strongly sclerotized and with reticulate pattern.
Paraproct (Fig. 32) short, not produced under cercus, and moderately setose. Cercus (Fig.
32) short, ca. 2/5x as long as its width, and moderately setose.

T)pe specimens. Holotype male, slide‑mounted, MYANMAR: Kambaiti, 7,000 ft., 30. IV.
1934, R. Malaise. Allotype female, slide‑mounted, same data as holotype. Paratypes 3
females, slide‑mounted, same data as holotype; I male, pinned (genitalia on slide) , I female,pinned, 5.V.1934, same locality as holotype.

Remarhs. Simulium hambaitense sp. nov. seems to belong to the montium group of thesubgenus Nevermannia (Rubtsov, 1959‑64) (i.e., subgenus Montisimulium in Crosskey, 1981)
by the shape of the genitalia of both sexes.
The male of this species is similar to that of S. ghoomense Datta, 1975 from India (Datta,
1975) but differs from the latter by the shape of the style. This species is also similar to theother Indian related species, S. nemorivagum Datta, 1973 (Datta, 1973), from which this isreadily separated by the ventral plate with a depressed posterior border.
The morphological characters of the female (herein described as a probable female of S.

hambaitense) resemble those of S. nemorivagum (Datta, 1974) and S. chowi Takaoka, 1979
from Taiwan (Takaoka, 1979). There are slight differences in the shape of the genital fork
between these known species and the present new species. The female genitalia (e.g., theshapes of anterior gonapophyses and genital fork) of S. ghoomense illustrated by Datta(1975) is different from those of this new species.

3 . Simulium (Nevermannia) sp. A.
Female. Wing length 3.1 mm. Head. Frons and clypeus brownish black, thickly whitish grey
pruinose, and densely covered with golden pubescence, intermixed with sparse dark hairs.
Frontal ratio 2.2:1.0:3.4 (this ratio was variable, being 2.0:1.0:2.7 and 1.7:1.0:2.7 in 2 otherspecimens examined) . Frons head ratio 1.0:5.1 (also variable, i.e., 1.0:4.5 and 1.0:5.7 in 2 otherspecimens) . Antenna composed of 2 + 9 segments, and dark brown except scape and pedicel
yellow. Maxillary palp composed of 5 segments, with 3rd, 4th and 5th segments in propor‑
tional length of 1.3:1.0:1.9; 3rd segment somewhat enlarged, with elongated sensory vesicle, ca.

2 x as long as its width, and slightly over 1/2 x length of 3rd segrnent. Maxilla with 9 inner

teeth and 13 outer ones. Mandible with ca. 20 inner teeth and without any outer teeth.
Cibarium without any denticles. Thorau. Scutum reddish brown, whitish grey pruinose, with
3 Iongitudinal dark lines, and densely covered with golden recumbent pubescence. Scutellum
light brown, whitish grey pruinose and with golden pubescence and several erect dark hairs.

Postscutellum brown, whitish grey pruinose, and bare. Pleural membrane and katepisternum
bare. Legs. All coxae, trochanters and femora light to dark yellow except mid and hindcoxae, and apical tip of all femora light brown. All tibiae brown except middle large portion

dark yellow to light brown. All tarsi dark brown except most of hind basitarsus and basal
l/2 of hind 2nd tarsal segment light brown. All femora and tibiae densely covered with249

golden hairs. Fore basitarsus slender, cylindrical, ca. 8.5 x as long as its greatest width. Hind

basitarsus (Fig. 33) parallel‑sided. Calcipala and pedisulcus well developed (Fig. 33). Claw
with large basal tooth, ca. 0.4 x length of claw (Fig. 34). Wing. Costa with 2 parallel rowsof short spines as well as hairs. Subcosta fully haired. Basal portion of radius fully haired.

Basal hair tuft dark brown. Abdomen. Basal scale light brown with a fringe of golden hairs.

Dorsal surface of 2nd abdominal segment pale and somewhat pruinose in light; rest of
abdominal segments brown with golden and dark short hairs; terga 7‑9 semishiny. Genitalia.
Sternite of 7th abdominal segment widely developed. Sternite 8 (Fig. 35) bare medially but

furnished with ca. 16 stout hairs on each side. Anterior gonapophyses (Fig. 35) thin,membraneous, triangular in shape, with a few short setae; inner border narrowly sclerotized,

and posteromedian corner rounded, transparent and bare. Genital fork (Fig. 36) with wellsclerotized stem and wide arms; arm with stout, Iong projection directed forwards, as well as
rounded projection directed posteromedially. Spermatheca (Fig. 37) ovoid in shape, strongly
sclerotized and with reticulate pattern. Paraproct (Fig. 38) short, not produced under cercus,
and moderately setose. Cercus (Fig. 38) short, ca. 1/2 x as long as its width, and moderately
setose.

Specimens examined. I female, pinned, MYANMAR: Kambaiti, 7,000 ft., 30. IV. 1934, R.
Malaise, 3 females, slide‑mounted, same data as first specimen except collection date of 2
females, i.e., 2.V.1934 and 5.V.1934.

Remarks. This species belongs to the feuerborni group of the subgenus Nevermannia, as
defined by Datta (1973) , to which several species are assigued. The female of this species isvery similar to those of S. praelargum Datta, 1973 from India (Datta, 1973) , and S. chitoense

Takaoka, 1979 from Taiwan (Takaoka, 1979) in many features including the slender fore
basitarsus (width‑length ratio, 1:8.5) , but is distinguished from these two species by the

number of macrosetae on the eighth sternite (ca. 16 versus over 20 on each side) . The
characters of the fore basitarsus and genitalia of this species agree with those of S.
perulucidulum Takaoka, 1983 from Philippines (Takaoka, 1983) , but there are differences in

the number of teeth of the maxilla between the two species.
Despite these differences, it would be better to leave this species unnamed because it is
impossible to compare with other related species, e.g., S. senile Brunetti, 1911 from India(Brunetti, 191D. S. fuscinervis Edwards, 1933 from Borneo (Edwards, 1933), S. bryopodium

Delfinado, 1971 from Palawan (Delfinado, 1971) , and S. feuerborni Edwards, 1934 from Javaand Bali (Edwards, 1934) because the female of these species was still unknown. The pupa
and larva of an unnamed species of the same species group have been reported from Thailand
as S. (Eusimulium) sp. B by Takaoka and Suzuki (1984). Future studies may solve whether
the present specimens represent a new species or are the female of one of the related species

mentioned above.
There is a possibility that the female specimens examined consist of more than one
species because a frontal ratio of these specimens varied remarkably as noted above,
although the character of the genitalia was almost the same.

4 . Simulium (Gomphostilbia) namense sp. nov
Male. Wing length 2.5 mm. Head. slightly wider than thorax. Upper eye consisting of 15

250r )1r‑t‑1 L‑̲

/ .

:: " .1'*' t' r ¥‑ "tr 1 ‑'I '" /"'‑i ' 11:
!'‑'*
/t'
'l

/'

'f 1 " ('‑)/:‑"'"J'i; ,)i ‑'*1‑/‑'/ >̲Jlr:
:1'1
' l‑r
/ /‑:

,37

!.

5

7,f7'./ .̲r ¥ ¥ ,rrT

i;1
Figs. 33‑38 Simulium (Nevern annia) sp. A female. 33, hind leg (coxa and trochanter
omitted) ; 34, claw; 35, 8th sternite and anterior gonapophyses; 36, genital fork; 37,
spermatheca; 38, paraproct and cercus in lateral view.

horizontal rows and 14 vertical columns of enlarged facets. Clypeus brownish black, whitish

grey pruinose, and with dark hairs. Antenna composed of 2 + 9 segments, dark brown except
base of Ist flagellar segment pale; Ist flagellar segment elongate, ca. 2 x as long as 2nd

fiagellar segment. Maxillary palp with 5 segments, with 3rd, 4th and 5th segments inproportional length of 1.0:1.2:2.7; sensory vesicle small, ca. 1/5.5x as long as 3rd segment(Fig. 39). Thorax. Scutum dark brown, whitish grey pruinose, with 3 faint dark longitudinallines, and densely covered with golden recumbent pubescence. Scutellum brown, whitish grey
pruinose, with numerous golden hairs and several dark hairs. Postscutellum brownish black,whitish grey pruinose and bare. Pleural membrane bare. Katepisternum haired. Legs. All
coxae dark brown except fore coxa whitish yellow. All trochanters whitish yellow. All
femora brown except apical portion dark brown. Fore tibia dark brown with median large
portion somewhat whitish yellow. Mid and hind tibiae whitish on basal 2/5 and dark brown
on apical 3/5. All tarsi dark brown except basal 1/4 of mid basitarsus somewhat pale andbasal 2/5 of hind basitarsus whitish. Fore basitarsus slender, cylindrical, ca. 9 x as long as

its greatest width. Hind basitarsus inflated distally, wedge shaped, ca. 3.3 x as long as its
greatest width; greatest width almost the same as the greatest width of hind tibia but slightly

wider than hind femur (Fig. 42). Calcipala and pedisulcus well developed. Wing. Costa with
2 parallel rows of short spines as well as dark hairs. Subcosta bare or with a few hairs. Basal

portion of radius fully haired. Basal tuft hairs golden yellow. Abdomen. Basal scale darkbrown with greyish yellow hair fringe. Dorsal surface of abdomen dark brown with darkhairs; terga 2,5,6 and 7 dorsolaterally grey pruinose and shiny. Genitalia. Coxite enlarged,
ca 1.8x as long as its width (Fig. 47). Style shorter than coxite, gently curved inwards andwith a single spine (Fig. 47). Ventral plate lamellate, well sclerotized, much wider than long,and covered ventrally, posteriorly and dorsally with minute setae (Fig. 48); ventral surface

251

somewhat raised ventrally in the center (Fig. 49) . Paramere with 4 stout spines, th o ugh
innermost spine short (Fig 51) . Median sclerite simple, wide plate‑like.
Female. Wing length 2.0 mm. Head. Frons and clypeus brownish black, thickly whitish grey
pruinose, and densely covered with whitish yellow pubescence, intermixed with sparse darkhairs. Frontal ratio 1.9:1.0:2.6. Frons head ratio 1.0:4.9. Antenna composed of 2 + 9 segrnents,

and dark brown except scape, pedicel and basal 1/2 of Ist fiagellar segrnent greyish yellow.

Maxillary palp composed of 5 segments, with 3rd, 4th and 5th segments in proportional lengthof 1.0:1.1:2.4; 3rd segment normal, with small, ovoid sensory vesicle (Fig. 40) , ca. 1/4 x as longas 3rd segrnent. Maxilla with 11 or 12 inner teeth and 14 outer ones. Mandible (Fig. 41) withca. 26 inner teeth and with 3 or 4 outer teeth. Cibarium without any denticles. Thorax.
Scutum brownish black, thickly whitish grey pruinose, with faint 3 Iongitudinal lines, anddensely covered with golden recumbent pubescence. Scutellum brown with golden pubescen‑ce and several erect dark hairs. Postscutellum brownish black, whitish grey pruinose, andbare. Pleural membrane bare. Katepisternum haired. Legs. All coxae and trochanterswhitish yellow except mid and hind coxae dark brown. Fore femur yellow on base, becoming
dark toward apex. Mid femur brown except basal 1/4 yellow. Hind femur light brown withapical tip dark brown. Fore tibia whitish with apical 2/7 dark brown. Mid tibia whitish on
basal 1/3, becoming dark towards apex, and dark brown on apical 1/3. Hind tibia whitish
with apical 1/4 dark brown; its border not well defined. All femora and tibiae densely covered

with whitish hairs. Fore tarsi dark brown. Mid and hind tarsi dark brown except basal 2/5of mid basitarsus and basal 2/3 of hind basitarsus and basal 1/2 of hind 2nd segment whitish.
Fore basitarsus (Fig. 43) slender, cylindrical, ca. 6.7x as long as its greatest width. Hind
basitarsus (Fig. 45) parallel‑sided. Calcipala and pedisulcus well developed (Fig. 45). Claw
with large basal tooth, ca. 1/2 x length of claw (Fig. 46) . Wing. Costa with 2 parallel rowsof short spines as well as hairs. Subcosta fully haired. Basal portion of radius fully haired.

Basal hair tuft golden yellow. Abdomen. Basal scale greyish yellow with golden hair fringe.

Dorsal surface of abdomen dark brown, with dark hairs; when viewed in light, tergum 2
somewhat grey pruinose and terga 7‑9 shiny. Genitalia. Sternite of 7th abdominal segment
undeveloped. Sternite 8 (Fig. 52) bare medially but furnished with ca. 20 stout hairs on eachside. Anterior gonapophyses (Fig. 52) thin, membraneous, triangular in shape, narrowly
sclerotized on inner border, bare and transparent near rounded posteromedial border; a few
short setae near anterior border. Genital fork (Fig. 53) with well sclerotized stem andlacking any projections on its arms. Spermatheca (Fig. 54) ellipsoidal in shape, strongly
sclerotized but without definite reticulate pattern. Paraproct (Figs. 55 and 56) short, notproduced under cercus, and moderately setose. Cercus (Fig. 56) short, ca. 2/5 x as long as
its width, and moderately setose.

T)pe specimens. Holotype male, slide‑mounted, MYANMAR: Kambaiti, 7,000 ft., 30. IV.
1934, R. Malaise. Allotype female, slide‑mounted, 2.V. 1934, same locality as holotype.
Paratypes I male, slide‑mounted, same data as holotype, I female, pinned and I female, slide‑mounted, both same data as allotype; I female, slide‑mounted and 2 females, pinned, Nam
Tamali, 3,000 ft., 8. VIH. 1938, R. Kaulback (BM 1938‑741).

Remarks. The male of S. namense sp. nov. is characterised by the inflated wedge‑shaped hind252

basitarsus (Fig. 42). This character is shared by several Gomphostilbia species, most ofwhich have been studied in detail by Davies and Gydrkds (1987) while they described S. ela
from Sri Lanka.
This new species seems to be similar to S. metatarsale Brunetti, 1911 described from asingle male specimen from Kurseong, India (Brunetti, 1911) , from which this is distinguish‑
able in the coloration of the fore femur ( metatarsale in parenthesis) : brown with apex darkbrown (dull yellowish) .

The male of S. namense shows similarities to that of S.metatarsale var. described by
Edwards (1934) from Java in the coloration of the scutal hairs, hairs at the base of stem veinand legs, but differs from the latter by the size of hind basitarsus: width‑length ratio l:3.3 (1:2.7 in metatarsale var. according to Davies and Gydrk6s, 1987) . The male of this new species

is much more similar to the specimens of "S. metatarsale" reported from Taiwan (Takaoka,1979) . There are slight differences in the following characters (parentheses in metatarsale ) :number of vertical and horizontal rows of large facets 14 and 15 (10 or ll and 13) ; color ofantenna dark brown except base of the first fiagellar segment pale (scape, pedicel and baseof the first flagellar segrnent pale, and the rest dark brown) . The female of S. namense also

has a close similarity to that of the latter species but is differentiated by. the very small

sensory vesicle, which is ovoid and about 0.25x as long as third segment of the maxillarypalp, whereas sensory vesicle is oblong and about 0.36 x length of third segment in Taiwanesemetatarsale .

Simulium namense is similar to S. nepalense from Nepal (Lewis, 1964) and S. tenuistylum
from India (Datta, 1973) , from which it differs also by the small sensory vesicle in the female

and by the shape of the ventral plate in the male.
Three Japanese species, i.e., S. ogatai Rubtsov, 1962 (Rubtsov, 1959‑1964), S. toharense

Takaoka, 1973 (Takaoka, 1973) and S. okinawense Takaoka, 1976 (Takaoka, 1976) , sharemany characters with the present new species. The male of S. namense is separated fromthese three species by the dark antenna and also from S. ogatai by the smaller number ofvertical and horizontal rows of large facets (17 rows in ogatai) . There are also slightdifferences in the size of the female sensory vesicle between this new species and three otherspecies (relative length of sensory vesicle against third segment of maxillary palp 0.25 innamense whereas 0.33 or more in others) .

Simulium sp. C reported from Thailand (Takaoka and Suzuki, 1984) has the similar
wedge‑shaped hind basitarsus. The number of vertical and horizontal rows of large facetsof this species is different from S. namense (i.e., 11 and 13 versus 14 and 15) .

Simulium ela from Sri Lanka (Davies and Gy rkds, 1987) is easily distinguished from the
new species by the brassy scutal hairs and dark hairs at the base of stem vein in both sexes,
and by the large sensory vesicle, basal 1/3 of the hind tibia pale and the small number of stout
hairs on the sternite 8 in the female.

This new species is distinguished from S. rosemaryae Takaoka et Roberts, 1988 from
Sulawesi (Takaoka and Roberts, 1988) by the small sensory vesicle and the number of teeth
on outer margin of the mandible in the female, and by the number of vertical columns of large
facets in the male.

5 . Simulium (Himalayum) indicum Becher, 1 885
Simulium indicum Becher, 1885: 199‑200.

253

へ・△
39

鵬

毒

41
緩灘・

争

男

46

44
42

43

45
1蟻

し＼

ψ︾

一

・

48

＼、
47

癖・

︾・

50

鵠・

56孝

53
Figs．39−56S吻％伽解（（｝o窺ρhos∫鋤毎）nα耀ns召sp．nov．male
male

and

female3rd

mandible；42，hind（coxaeand48，ventral

segment

leg

of

of

maxillary

male；43−45，fore，mid andtrochanters omitted）；46，female
plate

in

view；51，paramere

ventralhindclaw；47，coxite

view；49，ventral

with4hooks；52，8th

andfemale，39and40，

palp，respectively；41，apical tip

plate

stemite

in

end

and

legs

of

and

style

in

view；50，ventral

anterioroffemalefemale，respectively
ventral
plate

view；
in

side

gonapophyses；53，

genitalforkl54，spematheca；55，paraproctinventralview；56，paraproctandcercus
in side

view．

254

Simulium (Himalayum) indicum: Lewis, 1973: 462‑463; Lewis, 1974: 25‑33.

Material emmined. I female, pinned except head mounted on glass slide, MYANMAR:
Kambaiti, 7,000 ft., R. Malaise, 30. IV. 1934.

Distribution. Pakistan, India, Nepal, Bhutan, Myanmar, Bangladesh, China, Thailand.

Remarks. This species has been reported to be widely distributed in the Himalayas from
Assam to Kashimir (Brunetti, 1911; Lewis, 1974; Datta, 1983) . It should be remembered thatconsidering its wide range of distribution, S. indicum may be a species complex consisting of
more than one species, as already suggested by Takaoka and Suzuki (1984) . The pupal andlarval specimens are needed for final identification although the female specimen examined

agrees with the description of S. indicum given by Lewis (1974). The female specimen
examined is also similar to S. nigrogilvum Summers from Thailand (Takaoka and Suzuki,
1984) but the shape of the cibarial projection seems to be slightly different from each other.

S . Simulium (Simulium) chamlongi Takaoka et Suzuki, 1 984
Simulium (Simulium) chamlongi Takaoka et Suzuki, 1984: 27‑30.

Material eramined. I pinned female, MYANMAR: Kambaiti, 7,000 ft., 30. IV. 1934 R
Malaise.

Distribution. Thailand, Myanmar (new record) .

Remarks. The female specimen was provisionally identified as above because it almost
agrees with the' description of S. chamlongi described from Thailand (Takaoka and Suzuki,
1984). Further material is required for final identification. This is a first record of thevariegatum species group from Myanmar.
7 . Simulium (Simulium) novolineatum Puri, 1 933
Simulium (Simulium) novolineatum Puri, 1933: 817 (replacement name for S. (S.) Iineatum
Puri, 1932b: 1125‑30)

Material euamined. 2 females, pinned except genitalia of I female mounted on glass slide,
MYANMAR: Kambaiti, 7,000 ft., 30. IV. 1934, R. Malaise; I female, pinned, same data as 2other specimens except 5.V. Myanmar 1934.
Distribution. India, Myanmar (new record) .

Remarks. The genitalia of one female specimen examined conformed to those of S.
novolineatum Puri, 1933 and S. barraudi Puri, 1932, both described from India (Puri, 1932b) .

The identification of these female specimens as S. novolineatum was based on their colorationof the legs, i.e., basal 1/2 of the mid basitarsus yellow (not basal 3/4 as in barraudi).

Puri (1932b) indicated that the coloration of mid and hind femora of the female wasvariable by localities. The present Burmese specimens, which have all the femora (even fore

255

femur) almost entirely dark, are close to the specimens of S. novolineatum from Mercara but
are different from those from Marianbarie. Bengal Terai (type locality of this species) , whichshow the femora yellowish except apical tip dark (Puri, 1932b) .

This represents a first record of this species from Myanmar.
8 . Simulium (Simulium) rufibasis Brunetti, 1 91 1

Simulium rufibasis Brunetti, 1911: 282‑88; Rubtsov, 1959‑1964: 554.
Simulium (Simulium) rufibasis: Puri, 1932a: 899‑903; Ogata et. al., 1956: 94‑95; Crosskey,
1973: 428; Datta, 1974a: 19‑20; Takaoka, 1977: 213‑216; Takaoka, 1979: 395; Takaoka andSuzuki, 1984: 41‑42.

Material examined. 23 pinned females, 3 pinned males, and I male, slide‑mounted, MYAN‑
MAR: Kambaiti, 7,000 ft., R. Malaise, 30. IV. 1934; I pinned female, same data as others
except 2.V.1934; 2 pinned males, same data as others except 5.V. 1934.

Distribution. Pakistan, India, Thailand, Myanmar (new record) , Taiwan, Japan.
Remarhs. The female specimens almost agree with the description of S. rufibasis Brunetti,
1911 from India (Puri, 1932a) . The female of this species has been reported to be identicalto that of S. ramosum Puri, 1932 also from India, although these two species are distinguishedin the male and pupal stage (Puri, 1932a). It is therefore possible that some or all of thesefemale specimens are S. ramosum.
Three male specimens examined agree with the description of S. rufibasis given by Puri
(1932a) in which the coloration of the male hind basitarsus is pale on basal 1/3 (not on basal

l/2 as S. ramosum).
This species is for the first time recorded from Myanmar.

ACKNOWLEDGEMENTS
I am grateful to Dr. R. W. Crosskey, Department of Entomology, British Museum(Natural History) , London, U. K., for giving an opportunity to study the Burmese simuliid

specimens held in BMNH.
Thanks are also due to Dr. D. M. Davies, Professor Emeritus, and Mrs. Helen Gy6rk6s,
McMaster University, Ontario, Canada, for their kind review of the manuscript.

REFERENCES
1 ) Becher, E. (1885): A new species of Simulium from Assam, J. Asiatic Soc. Bengal, 53, 199‑2002 ) Brunetti, E. (1911): New Oriental Nematocera, Rec. Indian Mus., 4, 283‑288
3 ) Crosskey, R.W. (1969): A re‑classification of the Simuliidae (Diptera) of Africa and its islands,

Bull. Br. Mus. Nat. Hist. (Entomol.) Suppl., 14, p. 195

4 ) Crosskey, R.W. (1981): Simuliid Taxonomy‑The contemporary scene, p. 3‑18, In: Laird, M.
(ed.) Blackfiies: The future for biological methods in integrated control, Academic Press, p.

3995 ) Crosskey, R.W. and Davies, L. (1972): The identities of Simulium lineatum (Meigen) , S. Iatipes

256(Meigen) and S. vernum (Macquart) . Entomologists Gazette, 23, 249‑258

6 ) Datta, M. (1973): New species of black flies (Diptera: Simuliidae) of the subgenera
Eusimulium Roubaud and Gomphostilbia Enderlein from Darjeeling area, India, Orient. Insects,
7, 363‑402
7 ) Datta, M. (1974): New species of black flies (Diptera: Simuliidae) from the Darjeeling area,
India. Orient. Insects, 8, 457‑468

8 ) Datta. M. (1975): A new black fly species (Simuliidae‑Diptera) from the Darjeeling area,
India, Proc. Indian Acad. Sci., 81, 67‑74

9 ) Datta. M. (1983): A review of the Simuliidae (Diptera) from the Oriental Region. Orient.
Insects, 17, 215‑267lO) Davies, D.M. and Gy6rk s, H. (1987): The Simuliidae (Diptera) of Sri Lanka. Descriptions ofspecies in the subgenera Eusimulium and Gomphostilbia of the genus Simulium, Can. J. Zool.,
65, 1483‑1502

11) Delfinado, M.D. (1971): Some Simuliidae and Curtonotidae from the Philippines and theBismarck Islands (Insecta, Diptera) , Steenstrupia, l, 131‑139

12) Edwards, F.W. (1933): Diptera Nematocera from Mount Kinabalu, J. Fed. Malay States Mus.,17, 223‑296

13) Edwards, F.W. (1934): Deutsche Limnologische Sunda‑Expedition. The Simuliidae (Diptera)of Java and Sumatra, Arch. F. Hydrobiol. (Suppl.), 13, 92‑138
14) Lewis, D.J. (1964): Diptera from Nepal‑Simuliidae, Bull. Brit. Mus. Nat. Hist., 15, 291‑294
15) Lewis, D.J. (1973): The Simuliidae (Diptera) of Pakistan, Bull. Ent. Res., 62, 453‑470
16) Lewis, D.J. (1974): Man‑biting Simuliidae (Diptera) of northern India, Israel J. Ent., 9, 23‑53

17) Ogata. K., Sasa, M. and Suzuki, T. (1956): The Japanese black fiies and their control, DDT

Kyokai, Tokyo, pp. 162
18) Puri. I.M. (1932a): Studies on Indian Simuliidae Part 11 . Descriptions of males, females andpupae of Simulium rufibasis Brunetti, its variety fasciatum nov. var. and of three new species

from the Himalayas, Indian J. Med. Res., 19, 899‑913
19) Puri, I.M. (1932b): Studies on Indian Simuliidae Part HI. Descriptions of males, females andpupae of Simulium griseifrons Brunetti (1911) and of four new species with striped thorax,
Indian J. Med. Res., 19, 1125‑1143
20) Puri, I.M. (1933): Studies on Indian Simuliidae Part VII. Descriptions of larva, pupa and femaleof Simulium nodosum sp. nov. with an appendix dealing with S. novolineatum nov. nom. ( =S. Iineatum Puri) , Indian J. Med. Res., 20, 813‑817

21) Rubtsov. I.A. (1959‑1964): Simuliidae (Melusinidae) , In: Lindner (ed.), Fliegen Palaearkt.
Reg., 14, 1‑689

22) Takaoka, H. (1973) : Descriptions of 2 new species of blackflies. Simulium (Gomphostilbia)
tokarense and S. (Eusimulium) morisonoi (Diptera: Simuliidae) , from the Tokara Islands,
Japan, Jpn. J. Sanit. Zool., 23, 201‑207

23) Takaoka. H. (1976): Studies on black flies of the Nansei Islands, Japan (Simuliidae; Diptera).

II . On six species of the subgenera, Gomphostilbia Enderlein, Morops Enderlein, Odagmia
Enderlein and Gnus Rubzov with the description of Simulium (Gomphostilbia) okinawense sp.
nov., Jpn. J. Sanit. Zool., 27, 385‑398

24) Takaoka, H. (1977): Studies on black flies of the Nansei Islands, Japan (Simuliidae; Diptera)
III. On six species of the subgenus Simulium Latreille, Jpn. J. Sanit. Zool., 28, 193‑217
25) Takaoka, H. (1979) : The black flies of Taiwan (Diptera: Simuliidae) , Pac. Insects, 20, 365‑403

26) Takaoka, H. (1983): The black flies (Diptera: Simuliidae) of the Philippines, Japanese Society

for the Promotion of Science, Tokyo, p. 210

27) Takaoka, H. and Roberts, D.M. (1988): Notes on blackflies (Diptera: Simuliidae) from
Sulawesi, Indonesia, Japan. J. Trop. Med. Hyg., 16, 191‑219

25728）Takaoka，H．and

Suzuki，H．（1984）：The

blackflies（Diptera：Simuliidae）fromThailand，Jpn．

J．Sanit．Zool．，35，7−45

ミャンマー（ビルマ）のブユについて高岡宏行

大英自然史博物館に保管されていた，ミャンマー産ブユの成虫標本を，分類学的に検討した。
ミャンマーからは，これまで1種しか報告がなかったが，今回の研究の結果，さらに7種の分布が明らかになった。本論文では，このうち新種と思われる3種の記載を行った。

大分医科大学医動物学教室

Japan. J. Trop. Med. Hyg., Vol. 17, No. 3, ib89, pp. 259‑267

259

EFFECTS OF ENVIRONMENTAL TEMPERATURE
AND MEMBRANE FEEDING SOLUTIONS OFPURIFIED OOKINETES ON THE SPOROGONlC

DEVELOPMENT OF PLASMODIUM BERGHEIKENYICHI YANO*, KARL MARAMOROSCH AND ALBlNA KOZLOWSKA
Received April 27 1989/Accepted August 18 1989

Abstract: When Anopheles stephensi infected with Plasmodium berghei by bite were
kept at 21'C in darkness, degenerative changes occurred in 33.8% of oocysts, as contrasted

to 1.5% of such changes in mosquitoes kept at 21'C with 12‑hr light period. Temperature
shift of infected mosquitoes from 21'C for 24 hr to 25'C and 28"C increased the degeneration

rate of oocysts to 87.8% and 85.8% , respectively, and inhibited oocysts maturation. P.
berghei ookinetes were cultured from gametocytes of infected hamster blood and purified
by discontinuous Percoll gradient, using the modified procedure described by Munderloh
and Kurtti (1987) . Suspensions of purified ookinetes (1 x 10'/ml) fed through membrane
feeders permitted stable infection of A. stephensi. Mosquitoes fed purified ookinetes inphosphate buffered saline (PBS) and then maintained only with sugar, were able toproduce infective sporozoites. Fetal bovine serum, mammalian cell medium F‑12, andhamster red blood cell, added to PBS, improved infectivity of ookinetes to mosquitoes anddecreased degenerative changes during oocyst development. The system of feeding
purified ookinetes in simple chemical solution, PBS, would help to gain more information

about the transformation of ookinetes to oocysts.

INTRODUCTION
Most of the life cycle of the rodent malaria parasite, Plasmodium berghei can be studied
in in vitro culture systems: exoerythrocytic cycle in liver (Hollindale et al., 1983), eryth‑rocytic cycle in blood (Janse et al., 1984), gametocytogenesis (Mons et al., 1985) andookinete formation (Weiss and Vanderberg, 1977; Janse et al.. 1985a, b; Sinden et al.. 1985) .

The transformation of ookinetes to oocysts and the development of oocysts have not yet
been studied in an in vitro system. Progress in cultivation of ookinetes and their development

to mature oocysts has been hampered by the lack of information on the biophysiologicalconditions of sporogony in anopheline mosquitoes. We report the effect of environmental
temperature and darkness on sporogonic development of infected Anopheles stephensi and thenourishing effect of the feeding solution of purified P. berghei ookinetes as determined byexamination of the number and degenerative changes of oocysts in mosquito midguts.

Department of Entomology and Economic Zoology, Cook College, Rutgers University, New
Brunswick, New Jersey 08903* Present address; Department of Protozoology, Research Institute for Microbial Diseases, Osaka
University, Suita, Osaka 565, Japan

260

MATERIALS AND METHODSParasites and hosts: *The Plasmodium berghei (strain ANKA) ‑Anopheles stephensi‑golden hamster(Mesocriecetus auratus) complex was maintained as described by Vanderberg et al. (1968),

and used for experiments.
Usually hamsters, 17 to 24 days old, were inoculated with P. berghei. Older hamsters thanthese were injected with phenylhydrazine HCI ( 100 mg/kg) four days before infection (Janseet al.. 1985b) .

For infection, 40‑50 female mosquitoes 3‑7 days old, were collected from a stock colony

into small mosquito cages, using disposable 350 ml plastic caps. Infected mosquitoes weremaintained at 21'C with a daily cycle of 12 hr of light and darkness on 5% sucrose provided
on dental cotton roll.

Media:The ookinete culture medium consisted of Ham's F‑12 medium (GIBCO) without
NaHC03, supplemented with 10% heat‑inactivated fetal bovine serum (FBS: GIBCO) , 25 mM

BIClNE. The pH of the medium was adjusted to 8.0 with IN NaOH. The culture medium
F‑12, supplemented with 10% FBS, 25 mM HEPES and 0.2% NaHCO*, and adjusted to pH
7.3 with IN NaOH (washing medium) , was used for washing ookinetes, for dissecting
mosquito midguts and for diluting blood and 90% Percoll.
Culture and harvest of ookinetes:
Ookinetes were cultured and harvested according to the modified procedure described byMunderloh and Kurtti (1987) . One ml of the parasitized blood containing approximately 15
units of heparine/ml was inoculated into petri dishes (100 mm diameter) (Falcon) containing
lO ml of ookinete culture medium and incubated at 21'C for 20‑22 hr to develop ookinetes.
Purification of ookinetes:

Isotonic 90% Percoll was prepared by adding I volume of 10 X concentrated medium 199(GIBCO) to 9 volumes Percoll (Sigma). This was further diluted to 45% and 36% withwashing medium. The above cultures were collected into centrifuge tubes in an ice bath andcentrifuged at 150 X g for 8 min at 4'C. Both supernatant and upper greyish layer on thesediment were used to collect ookinetes. The supernatants were centrifuged at 175 x g for 10

min and the sediment were resuspended in 5 ml of 36% Percoll. The upper greyish layer ofthe sediment were collected into centrifuge tubes containing 10 ml of washing medium andalso centrifuged at 175 x g for 10 min. Then again, the upper greyish layers were carefullycollected and resuspended in 36% Percoll. Both 36% Percoll suspensions were separately
overlayed on 5 ml of 45% Percoll and then 3 ml of washing medium was overlayed on eachsuspension. The preparations were spun at 150Xg for 5 min and then for another 12 min at
700 X g. The interfaces between the 45% and 36% Percoll were removed with a Pasteur pipet,

and the number of ookinetes and contaminated red blood cells (RBO were counted in ahemocytometer. Samples containing RBC fewer than 30 per ookinete were washed once andresuspended with described membrane feeding solutions, then used for feeding.

261

Effect of environmental temperature and darkness on sporogonic development:
Mosquitoes in 2 small cages were allowed to feed on an infected hamster for 10 min at
21'C and then mosquitoes in other 2 cages were fed on the same hamster for 20 min at 21'C.
After acquisition of the infected blood, mosquitoes in one cage was kept at 21'C with
12‑hr light period as a standard control. Some cages were kept at 21'C for 24 hr and then kept
at 25'C or 28'C with 12‑hr light period, respectively. Other cages were kept at 28'C with 12‑hrlight period or at 21'C in darkness throughout the experiment.
Infected mosquitoes were dissected on day 11 post infection (PD (experiment 1) and day

12 PI (experiment 2 and 3) . The dissected midguts were examined with an inverted phase‑contrast microscope at 360 X maguification for the presence of oocysts.
Sporozoite infections of hamsters were carried out on day 15 PI in experiment I and 2,
and day 17 PI in experiment 3. In the experiment I and 2, anesthetized hamster pups wereplaced on the top of the mosquito cages. The infected mosquitoes were allowed to feedthrough the nylon net of the cage on the underside of the hamster for 20 min. In theexperiment 3, each mosquito was cut in half at the junction of the thorax and abdomen. The
head‑thoraxes of mosquitoes containing mature oocysts in the abdomen were kept in an
Eppendolf containing the washing medium, 0.2 to 0.5 ml in an ice bath according to thenumber of mosquitoes dissected, and crashed with a round head of a spatula, after all
mosquitoes were examined for the presence of oocysts. The suspension was centrifuged at
150 X g for 2 min to remove the large mosquito debris. When the number of sporozoites in the
supernatant could be counted in a hemocytometer, I x 103 sporozoites were used for infectionof a hamster by intracardial inoculation carried out under sodium pentobarbital anesthesia.
Table I Effects of environmental temperature and darkness on sporogonic development of

Phomodium berghei
Environmental

No, ofcondition']experiment

21'C

%

Inf ected(No. examined)

No, of oocysts/
inf ected

mosquitoSD

Degeneration

Infectivity ofrate ( % )sporozoites(No. of mosquitoesof oocysts(average)used )

l100 .o (15)30 . 2 :!: 33 . 9

On.c.b]

2

44 .o (18)

16 . 4:!:42.3

3.l+ (18)

3

lOO .O(8)

48 . 1 :!: 25 . 2

1.3

+ (3)

(1.5)

21'C in1

91 . 7(12)

52 . 9d: 26 . 6

24 . 4+ (17)

darkness3

lOO . O(9)43.0 27.6

43 . 2(33 . 8)+ (7)Temperature1

100.0 (5)19 . 6d:ll. 3

87 . 8n.c.

0 22. 7shift from
21'C to 25'C
100 . O

(7)

81.8

‑ (8)

2

66 . 6(24)

19. O i 32 . 5

86 . 2‑ (32)

3

90 . 6(lD

19 . 8d: 15 . 9

89 . 4(85 . 8)

‑ (2)

2

o

(23)

3

o

(15)

Temperature
shift from
21'C to 28'C

28'C

22 .

a) Mosquitoes were kept with 12‑hr light period except mosquitoes at 21'C in darkness.
b) Sporozoite infection was not checked.

262

If the number could not be counted, all the supernatant was used for infection of a hamster.

To examine infectivity of sporozoites, the final examination of parasitemia was carried out

on day 14 PI.
Nourishing effect of feeding solutions of purified ookinetes on the sporogonic development:

A. stephensi was infected by membrane feeding of purified ookinetes (1x 106/ml) to
study the essential nourishment of blood on sporogony of P. berghei. Phosphate bufferedsaline (PBS: 8.0 g of NaCl, 0.2 g of KC1, 1.15 g of Na2HP04 and 0.2 g of KH2P04 per litter)

was used as non nourishment. Five per cent glucose and washing medium were used forfeeding ookinetes as moderate nourishment. A experiment of mosquitoes infected withookinetes in PBS and in 5% glucose were maintained on 5% glucose in stead of 5% sucrose.
Heparinized fresh hamster blood was diluted two times with washing medium and used forfeeding ookinetes as blood meal (complete nourishment) , because a small volume of whole
blood easily evaporates during feeding and because it is too sticky for mosquitoes to engorge.

RBC of heparinized hamster blood (1 volume) were washed once with cold PBS (100volumes) and resuspended with PBS (1 volume) as RBC suspension.
To study the nourishing effect of blood component on sporogonic development of purified

ookinetes, PBS was supplemented with each 10% of hamster RBC suspension, FBS as serum
component and hamster whole blood. Each feeding solution was supplemented with 10 mM
of NaHC03, a phagostimulant for anopheline mosquitoes (Galun et al.. 1985; Yano et al., in
preparation) .

Feeding of ookinetes to mosquitoes:
Purified ookinetes were resuspended in 0.5 to 1.5 ml of each feeding solution to obtain theconcentration I x 106 ookinetes per ml.
Water‑jacketed membrane feeders (2.6 cm in an inside diameter) (Rutledge et al.. 1964)

fitted with a Baudruche membrane (Long and Long, Belleville, New Jersey) were used forTable 2 Nourishing effect of feeding solution of purified Plasmodium berghei ookinetes on the
sporogonic development
Feeding
solution

Dissecting

time(days)% Inf ected(No. examined)

No. of oocysts/
inf ected

mosquitoSD

Degeneration
rate (%)

Infectivity oflOO . On,c,a)

sporozoites

5% Glucose

12‑18

12 . 1(33)

1.3+ 0.5

PBS

8‑10

40 . 8(71)

3.2+ 3.7

4.3

12‑18

44 . l(183)

4.3+ 5.6

19 . O

415b)

8‑lO

68 . 4(19)

18 . 9+ 20 . 8

O

n,c.

12‑18

87 . O(46)

29 . 518 . 62/2

8‑10

45 . 5(11)

5.4 5.9

O

n,c.

12‑18

76 . 3(76)

6.9+ 6.8

11.0

6/6

8‑10

92 . 3(26)

42.5+41.1

4.8

n.c.

12‑18

87 . 4(143)

31 . 6:!: 34 . 7

7.9

5/5

PBS+10% FBS
Washing medium

Blood meala) Sporozoite infection was not checked.
b) No. of infection/No. of trials.

29 . 5n.c.

263

Table 3 Nourishing effect of blood component in the feeding solution of purified Plasmodium
berghei ookinetes on the sporogonic development

Feeding
solution% Infected(No. examined)

No. of oocysts/
infected mosquitod: SD')

Degeneration
rate (%)

PBS+10% FBS

lO0.0 (31)36 . 329 . 817 . 6

PBS+10% RBC

95.7 (23)25 . 835 . 6

11.6

PBS+10% Whole

100.0 (29)34 . 2 : 27 . 4

22 . O

94.7 (18)38 . 9 l 33 . O

12 . 9suspension

blood
Blood meala ) The presence of oocysts was examined on day 14 PI.

feeding. Feeders were pre‑warmed at 37'C in a 21'C‑incubator. Mosquitoes in small cagewere fed ookinetes in solution for 15 min. From day 8‑10 PI, and 12‑18 PI, the dissected
midguts were examined for the presence of oocysts. Sporozoite infection of hamsters was
carried out by intracardial inodulation of day 14‑18 PI as described before.

Degenerative rate of oocysts:

The degenerative changes of oocysts proceeded from a few granules observed in the
protoplasm of an oocyst, to coarse granules that filled the whole protoplasm of an oocyst.
Finally, the protoplasm with coarse granules shrunk away from the oocysts wall and concen‑
trated in the oocyst as an amorphous mass. Oocysts which under phase‑contrast microscopic

examination contained distinct co rse granules were counted as degenerative oocysts.
Degeneration rate of oocysts was caliculated from total degenerative oocysts per all oocystsof an experimental group.

RESULTS
Effects of environmental temperature and darkness on the development of malaria oocysts:

Table I summarizes the effects of environmental temperature and darkness on thedevelopment of malaria oocysts. The rate of infection and numbers of oocysts per mosquito
did not differ significantly among mosquitoes kept at 21'C with 12‑hr light period (21'C
mosquitoes) , at 21'C in darkness, and of temperature shift from 21'C for 24 hr to 25'C (21'C

to 25'C mosquitoes) or to 28'C (21'C to 28'C mosquitoes) . Almost all oocysts of 21'C
mosquitoes matured without degenerative changes but the oocysts of 21'C mosquitoes indarkness, 21'C to 25'C mosquitoes and 21'C to 28'C mosquitoes degenerated significantly
during their maturation; the average degeneration rates of oocysts were 33.8% , 87.8% and85.8% , respectively, in contrast to the 1.5% of 21'C mosquitoes. Infective sporozoites wererecognized both in mosquitoes kept at 21'C with 12‑hr light period and those kept in darkness.

Mosquitoes that were shifted from 21'C for 24 hr to 25'C or 28'C could not develop matureoocysts with sporozoites. In none of the surviving mosquitoes kept at 28'C with 12‑hr lightperiod could any development of oocysts be recognized in their midguts.
Nourishing effect of feeding solutions of purified ookinetes on the sporogonic development:

264

Mosquitoes fed purified ookinetes in PBS developed infective sporozoites but the in‑fective rate and the number of oocysts per mosquito were lower than those fed ookinetes in
PBS supplemented with 10% FBS, ookinetes in washing medium or in blood meal (Table 2).
Addition of 10% FBS to PBS improved the infection rate, 40.8% in day 8‑10 PI and 44.1% in
day 12‑18 PI to 68.4% and 87.0% , respectively, and the number of oocysts per mosquito, 3.2
3.7 oocysts in day 8‑10 PI and 4.3:!:5.6 oocysts in day 12‑18 PI to 18.9:!:20.8 and 29.5i29.5,
respectively.

Changes of PBS+10% FBS to washing medium (F‑12+10% FBS+25 mM HEPES+
0.2% NaHC03 ) decreased the degenerative rate, from 18.6% to 11.0% but the infected rate
was not significantly different. Mosquitoes fed ookinetes in blood meal had the highest
infection rates, 92.3% on day 8‑10 PI and 87.4% on day 12‑18 PI.
Young oocysts on day 8‑10 PI of mosquitoes fed ookinetes in PBS with lO% FBS andwashing medium did not degenerate but some later oocysts of mosquitoes fed ookinetes in all
five kinds of solutions contained degenerative granules; the degenerative rates were 19.0% in

PBS, 18.6% in PBS+10% FBS, Il.O% in washing medium and 7.9% in blood meal.
The infection rate of mosquitoes fed ookinetes in 5% glucose was very low, 12.1% of 33

mosquitoes in two experiments. The number of oocysts per mosquito was 1.3i0.5 and alloocysts were degenerated.
The addition of 10% RBC suspension to PBS decreased the degenerative rate of oocysts
as did the blood meal (Table 3) .

The number of oocysts in mosquitoes fed ookinetes in PBS+10% RBC suspension was
the lowest, 25.8d:35.6 in contrast to 36.3d:29.8 in PBS+10% FBS, 34.2d:27.4 in PBS+10%
whole blood and 38.9 33.0 in blood meal, respectively.

DISCUSSION
The temperature range which permits P. berghei ookinete formation in the natural vector
A. dureni, is 18'C to 21'C (Yoeli, 1965). In the experimental vector, A. stephensi, oocysts did

not develop at 28"C (Table 1). When ookinetes were allowed to form at 21"C for 24 hr after
feeding parasitized blood, and then the environmental temperature was shifted to 25'C or

28'C, A. stephensi could develop young oocysts at 25'C and 28'C, but no mature oocysts
formed at these temperatures.
Mosquitoes exposed to high temperature fail to develop sporozoites of P. berghei(Vanderberg and Yoeli, 1966) . Thus the oocysts of 21'C to 25'C mosquitoes and 21'C to 28"C

mosquitoes were highly degenerated during their later developmental stage. Oocysts without

apparent degenerative changes were smaller and their development seemed to be retarded.
Some had cloudy protoplasms with fine granules, indicating the start of degenerative changes.

The damaging action of high temperature would begin before sporozoite budding, for there
were no oocysts containing sporozoites.
In darkness, mosquitoes remained motionless in the cages. The degenerative changes ofoocysts of 21'C mosquitoes in darkness were high. We assume that poor nourishment ofmosquitoes, which did not feed on the 5% sugar solution in darkness was responsible for thisfinding. The transformation rate from ookinetes to oocysts increased in mosquitoes indarkness, compared to mosquitoes with 12‑hr light period. We assume that the motionlessof mosquitoes was responsible for this finding.

265

Mosquitoes fed ookinetes in PBS could develop mature oocysts and infective sporozoites
when they were provided solely 5% sugar or 5% glucose on cotton roll like avian malaria
ookinetes (Rosenberg and Koontz, 1984) (Table 2). Blood components were not essential on

the development from ookinete to sporozoite.
Mosquitoes fed ookinetes in 5% glucose were seldom infected and they developed onlya small number of degenerative oocysts per mosquito. The reason for this seems to be theweakly acidic glucose solution and its unstable pH.
The serum component. FBS, supplemented with PBS improved the viability of ookinetes
in membrane feeders, and increased infectivity and the number of oocysts per infectedmosquitoes almost the same as blood meal. The ookinetes kept in PBS in an ice bath for 30
min lost significantly their infectivity to mosquitoes compared to ookinetes kept in washingmedium that proved completely infective for mosquitoes (100% infective rate). The RBC
with PBS improved the viability of ookinetes to a lesser degree than FBS and whole blood(Table 3) but supplemented the nourishment and decreased the degeneration rate of develop‑
ing oocysts.

The oocyst maturation in mosquitoes fed ookinetes in blood meal preceded by more than

one day the maturation in mosquitoes fed ookinetes in PBS, PBS+10% FBS and washingmedium. The maturation of oocysts could explain the higher degeneration rate of oocysts inday 8‑10 PI of mosquitoes fed ookinetes in blood meal, because the degenerative changes

became dominant in late stages of oocysts.
There was a large difference between the degeneration rate of oocysts in mosquitoes fed

directly by bite on an infected hamster (1.5%) and those fed in vitro‑formed ookinetes inblood meal (7.9% in Table 2 and 12.9% in Table 3) . The gametocytes acquired by mosquitoes
by bite should transform to ookinetes within 20 hr not to be trapped within the peritrophic

membrane (Orihel, 1975). Thus ookinetes penetrating the peritrophic membrane to the
midgut basement membrane would be biologically stronger than those transformed later andtrapped within the peritrophic membrane, and could grow to mature oocysts and sporozoites
without degeneration.
The ookinetes acquired by mosquitoes through membrane feeders could easily reach the
midgut basement membrane before the peritrophic membrane hardened and had no selectionof biological strongness. Some ookinetes could not retain the capacity for maturation due tounknown factors.
The experimental transformation of ookinetes to oocysts in vitro remained unsolved.
The system of feeding purified ookinetes in simple chemical solution, PBS, would help to gain

more information about it.

ACKNOWLEDGEMENTS
The authors wish to thank to Ms. Rebecca Shamy for technical assistance. This work
was supported by Public Health Service research grant Al 18345 from the National Instituteof Allergy and Infectious Diseases.

REFERENCES
1 ) Galun, R., Koontz, L.C. and Swadz, R.W. (1985): Engorgement response of anopheline266
mosquitoes to blood fractions and artificial solutions, Physipl. Entomol., lO, 145‑149
2 ) Hollindale, M.R., Leland, P. and Schwartz, A.L. (1983): In vitro cultivation of the exoeryth‑rocytic stage of Plasmodium berghei in a hepatoma cell line, Am. J. Trop. Med. Hyg., 32,
682‑6843 ) Janse, C.J., Mons, B., Croon, J.J.A.B. and Van der Kaay, H.J. (1984): Long‑term in vitro
culture of Plasmodiuin berghei and preliminary observations on gametocytogenesis, Int. J.
Parasitol., 14, 317‑320

4 ) Janse, C.J., Mons, B., Rouwenhorst, R. J., Van der Klooster, P.F.J., Overdulve, J.P. and Van derKaay, H.J. (1985a): In vitro formation of ookinetes and functional maturity of Plasmodium
berghei gametocytes, Parasitol., 91, 19‑29

5 ) Janse, C.J., Mons, B., Rouwenhorst, R.J., Van der Klooster, P.F.J., Van der Kaay, H.J. and

Overdulve, J.P. (1985b): Development of Plasmodium berghei ookinetes in the midgut ofAnopheles atroparvus mosquitoes and in vitro. Parasitol., 91, 219‑225
6 ) Mons, B., Janse, C.J., Boorsma, E.G. and Van der Kaay, H.J. (1985) : Synchronized erythrocytic

shizogony and gametocytogenesis in Plasmodium berghei in vivo and in vitro. Parasitol., 91,

423‑430
7 ) Munderloh, U.G. and Kurtti, T.J. (1987): The infectivity and purification of cultured Plas‑
modium berghei ookinetes, J. Parasitol., 73, 919‑923
8 ) Orihel, T.C. (1975): In "Invertebrate Immunity" (K. Maramorosch and R.E. .Shope, eds.) pp.

65‑73, Academic Press, New York9 ) Rosenberg, R. and Koontz, L.C. (1984): Plasmodium gallinaceum: Erythrocyte factor essential
for zygote infection of Aedes aeg)pti. Exp. Parasitol., 57, 158‑164

10) Rutledge, L.C., Ward, R.A. and Gould, D.J. (1964): Studies on the feeding response of
mosquitoes to nutritive solutions in a new membrane feeder, Mosquito News, 24, 407‑419ll) Sinden, R.E., Hartley, R.H. and Wingor, L. (1985): The development of Plasmodium ookinetesin vitro: an ultrastructural study including a description of meiotic division, Parasitol., 91,

227‑244
12) Vanderberg, J.P. and Yoeli, M. (1966) : Effect of temperature on sporogonic development of
Plasmodium berghei, J. Parasitol., 52, 559‑564
13) Vanderberg, J.P., Nussenzweig, R.S. and Most, H. (1968) : Further studies on the Plasmodium

berghei‑Anopheles stephensi‑rodent system of mammalian malaria, J. Parasitol., 54,
1009‑l016
14) Weiss, M.M. and Vanderberg, J.P. (1977): Studies on Plasmodium ookinetes: II. In vitro
formation of Plasmodium berghei ookinetes, J. Parasitol., 63, 932‑934
15) Yoeli, M. (1965): Studies on Plasmodium berghei in nature and under experimental conditions,
Trans. R. Soc. trop. Med. Hyg., 59, 255‑276

267

P伽卿04伽吻68響h8づのsporogonyについて，蚊の飼育温度と純化ookineteの発育に対するmembrane feeding液の影響

矢野健一〜K．Maramorosch・A．Kozlowska

P伽勉04伽吻加響h8づ（ANKA株）一シリアンハムスターr4ηoρh8伽s妙h8㎜ の系を使用した。P加㎎宛θ」感染ハムスターを吸血したA．s妙h8ηsJでのookinete形成には，蚊の飼育温度を21℃に保つζとが必須条件であった。吸血蚊を21。Cの暗闇に保つとoocyst形成は増えたが，
oocystの発育中に33．8％が変性を起こした。吸血後，・4．s孟砂h伽sfを通常の飼育温度28。Cで飼育するとoocystの形成は全く見られなかったが，吸血後24時問21。Cに保った後28。Cで飼育したもの

では，oocystの発育は見られたが，sporozoiteへの発育は阻害された。

P加㎎hθ6感染ハムスター血を，10％FBS，25mM

Bicine加HamsF−12培地（pH8．0）で約10％に希釈し，21。Cで22−24時間培養し，ookineteを得た。Percollgradient（0〜36％〜45％）

で純化したookinete（lx106／mJ）を，5％グルコース，PBS，PBS−10％FBS，F−12−10％
FBS，ハムスター全血に再浮遊し，membrane feeding法で蚊に感染させた。血液成分を使用しなくても，PBSをfeeding液としてookineteを蚊に感染させた後，5％薦糖あるいは5％グルコースで飼育して，感染力のあるsporozoiteを得た。その場合の低い感染率（44．1％），oocyst数（4．3個）は，PBSに10％FBSを添加することにより感染率（87．0％），oocyst数（29．5個）とを，全血をfeeding液に使用した場合とほぼ同じ程度に回復させ得た。赤血球を全血の10％，PBSに添加した場合は，FBS添加より感染率，oocyst数は劣ったが，oocystの変性率は低下した。FBS

添加による感染率，oocyst数の回復効果は，oocyst発育過程での栄養供給よりも，membranefeeding中のookineteのviabilityの低下を防ぐ為と思われる。赤血球には，oocyst発育過程での栄養供給効果があると思われ，oocystの変性率を低下させた。

Department of Entomology and Economic Zoology，Cook＊現所属： 大阪大学微生物病研究所原虫学部門

College，Rutgers

University

Japan. J. Trop. Med. Hyg., Vol. 17, No. 3, 1989, pp. 269 271

Res*ar*h269'te

SURVEY OF PLASMODIUM FALCIPARUM
IN MAKURDI, NIGERIAANNIE JOHN
Received January 9 1989/Accepted June 16 1989

Studies on different aspects of Plasmodium falciparum have been reported by several
workers (Dietz et al., 1974; Aderounmu et al., 1981; Ibeziake et al., 1980; Krafsur et al., 1978;

Okeahialam et al., 1972). Current conservative estimate puts the annual mortality frommalaria among Nigerians as 100,000 according to the press note of Enwonwu (1983) . Thoughmuch work on P. falciparum has been conducted in different parts of Nigeria, no attempt has
so far been made to report the prevalence of malaria from the studied area. Therefore thepresent report is focussed on the results of a parasitological blood examination of individuals

from local schools and local clinics in North Bank area of Makurdi, Benue, Nigeria during
a period of three months (June‑August 1984) .
North Bank area is on the banks of River Benue and has a tributary River Guma on one
side. It has a tropical savannah climate and there are two marked seasons, the wet season(April to October 40 60" ramfalD and dry season (October to April O lO" ramfalD. The age(0‑30 years) and sex of the individuals (1,001) including the pregnant women were recordedat the time of blood sample collection. The blood smears were stained with Giemsa's for
detecting the parasites. The parasite in positive slides and age groups were expressed
according to the code followed by WHO (Black, 1968) .
Results are summarised in Table 1, P. falciparum was the only prevalent species in thearea and the infection rate was 1.2% in the studied population. Infections with P. malariae.
P. ovale and P. vivax were not detected. So there were no mixed infections. Parasite rate ofP. falciparum was 6.9% in the whole population being a measure of malaria prevalent in thearea which was at its maximum.
Children between 0‑5 months old were least affected, which may be due to parental
protection. It has been reported that congenital infections are rare (McGregor, 1978) . A
definite indication of recent transmission of malaria in the area was known by the significant

infant parasite rate which was 5.6% . It has been recently shown that P. falciparum invades

cells which contain Haemoglobin F (HbF) to a greater degree than cells which contain
Haemoglobin A (HbA) (Wilson et al., 1977) . The infection rate in the second year of life wasless than that in the first year of life in the present survey, similar to the findings of Dar es

Salaam (Okeahialam et al., 1972) , since the suppressive action of drugs gives a suppressive

cure of P. falciparum (Bruce‑Chwatt, 1982) . Maximum effect was recorded in the age groups
2‑4 years (7.7% ) . The present findings revealed that the parasite rate in children (1‑14 yrs)

was high '(22.45%) while in adults (15‑30 yrs) was low (13.1%), since immunity to malaria
Department of Zoology, University of Kerala, Trivandrum‑695 581, India

270

increases with age. In a survey conducted among the inhabitants of Ethiopia it was estimatedthat the parasite rates were two folds greater among children than adults (Krafsur et al.,1978). Okeahialam et al. (1972) reported at Dar es Salaam, that more than two third of thechildren admitted were less than 2 years of age, and there were more boys than girls. But inthe present findings, more than two third of children examined belonged to the age group 2‑14

years and there were more girls than boys. It has been noted that females are much more
frequently infected, while more males were infected than females as reported by Dutta et al.
(1975) .

The high parasite rate in women (15‑30 years) when compared to the other separate agegroups, indicates that placental malaria is a frequent event in pregnancy (McGregor, 1978) .
There appeared to be a decrease in parasite densities with age and the parasite density forthe studied population was 3.6.

This preliminary parasite survey, however reports the maximum prevalence of malaria
in the studied area being the wet season, and the prevalent parasite species in P. falciparumin Nigeria while infections with P. malariae are low and with P. ovale and P. vivax are scarce.

ACKNOWLEDGEMENT
I extend my thanks to Dr. J.A. Ajayi, Department of Zoology, University of Jos, Nigeria
and the Epidemiological Unit, Makurdi, Nigeria for their technical assistance during thecourse of this work. Indeed, I am indebted to Dr. P.V. George, Department of Zoology, Marlvanios College, Trivandrum, India for critically going through the manuscript.

Table 1 Plasmodium falciparum survey‑Consolidated report

Age Groups

No. of persons

Blood examinations

examined
Males Females

ositive slides
Males Fema I es

Parasiterate

Inf ection

Meanrate forP. falciparum

parasitedensity

0‑11 months( inf ants)

23

31

2

1

5 . 6%

o . 3%

6 . 48

38

20

1

1

3.4%

0.2%

4 . 98

41

36

3

3

7 . 7%

0.6%

3 . 24

177

220

ll

17

7 . 05%2.8%

2 . 76

113

ll9

2

8

4 . 3%

o . 9%

1 . 63

12

171

24

13 . 1%

2 . 3%

2 . 536 . 9%

l.

2%

3. 60

(3 . 5)(1. D(1. 8)

12‑23 months(children)

2‑4 years(pre school
children)

22 . 45%

5‑9 years( juveniles)

10‑14 years( adolescents)

15‑30 years(adults)

Total

404

597

19

Mean
SD

54

Locality: North bank area of Makurdi; Benue State; Nigeria.

Date:

June‑August, 1984. Season: wet

271

REFEREN CES1 ) Aderounmu. A.F.. Salako, L.A. and Walker, O. (198D: Chloroquine sensitivity of P. falciparumin lbadan, Nigeria. Correlation of in vitro with in vivo sensitivity, Trans. Roy. Soc. trop. Med.

Hyg, 75, 637‑669

2 ) Black, R.H. (1968): Manual of Epidemiology and Epidemiological Services in Malaria

Programmes, WHO Geneva, 27‑44
3 ) Bruce‑Chwatt, L.J. (1982): Chemoprophylaxis of Malaria in Africa, the spent magic bullet, Br.
Med. J., 285, 674‑676

4 ) Dietz. K., Molineaux, L. and Thomas, A. (1974): Malaria model tested in the African
Savannah, Bull. WHO, 50, 347‑357
5 ) Dutta, S.M. and Saha, B.C. (1975): Observations on the recent incidence of Malaria, J. Indian.

Med. Assoc., 65, 177‑178
6 ) Ibeziako, P.A. and Williams, A.1. (1980) : The effect of malaria chemoprophylaxis on im‑
munoglobulin levels of pregnant Nigerian women and the new born. Brit. J. Obstet. gynae., 87,
976‑9827 ) Krafsur, E.S. and Armstrong, J.C. (1978): Integrated view of entomological and parasiticalobservations on falciparum malaria, Trans. Roy. Soc. trop. Med. Hyg., 72, 48 ) McGregor, I. (1978): Tropical aspects of the epidemiology of malaria, Isra. J. Med. Sci., 14,

523‑5369 ) Okeahialam, T.C., Kilama, W.E. and Ramji. B.D. (1972): Clinical significance of malaria
parasitaemia in children. East. African Med. J., 49, 962‑969

lO) Wilson, R.J.M.. Pasvol, G. and Weatherall, D.J. (1977): Invasion and growth of Plasmodium
falciparum in different types of human erythrocytes, Bull. WHO, 55, 179

Japan J Trop. Med. Hyg., Vol. 17, No. 3, 1989, pp. 273‑276273

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LOCALIZATION OF HEPATITIS B SURFACEANTIGEN IN THE PANCREAS AND

LYMPH NODESMASACHIKA SENBA , HIROTO YAMASHITA2 AND HIDEYO ITAKURA*
Received June 17 1987/Accepted March I 1989

Abstract: An autopsy case of syphilitic aortic aneurysm with an infection of hepatitis

B virus in a 65‑year‑old woman is reported. Specifically, hepatitis B surface antigen(HBSAg) was revealed in the pancreatic acinar cells and histiocytes of retroperitoneal
pancreatic lymph nodes as well as in part or nearly all of the cytoplasm of scatteredhepatocytes by orcein and immunoperoxidase methods. These findings suggest that
hepatitis B viurs may replicate in the pancreas. It was not clear whether HBsAg in thelymph nodes is related to infectiveness or not. The present study further confirms theprevious reports of extrahepatic localization of HBSAg.

INTRODUCTION
The liver is thought to be the primary site of hepatitis B viral replication and synthesis.

Although hepatitis B virus antigens have been extensively revealed in the hepatocytes,
hepatitis B surface antigen (HBSAg) and/or its related immune complexes have been showed
as extrahepatic localizations in the renal glomeruli (Brzosko et al.. 1974; Nowoslawski et al..
1972) , vascular wall (Michalak, 1978) , and pancreas (Shimoda et al.. 1981; Yoshimura, et al.,1981) . Localization of HBsAg in hepatocellular carcinoma has been reported (Shikata, 1973;

Nayak and Sachdeva, 1975; Nazarewicz et al., 1977; Wu, 1979; Senba, 1981, 1982; Kawano,1983) . Thus, present evidence indicates that humoral immune mechanisms in relation to
HBsAg are involved even in extrahepatic lesiops. Histological location of HBsAg in thelymph nodes has not well been recognized.
We have observed morphologic localization of HBsAg in extrahepatic tissues by im‑

munohistochemical method. The present postmortem case may give some evidence tosuggest that hepatitis B virus replicate in extrahepatic tissues.

CASE REPORT
A 65‑year‑old woman was admitted to Nagasaki University Hospital for treatment ofaortic aneurysm. The patient died of rupture of aortic aneurysm before operation and

1 Department of Pathology, Institue of Tropical Medicine, Nagasaki University, Nagasaki 852,

Japan
2 Department of Pathology, Medical College of Oita, Oita 879‑56, Japan274

laboratory examinations of hepatitis B virus and liver functions. At autopsy, aortic aneur‑ysm. 13 cm in diameter and located at the site 2 cm from the aortic valve, with endarteritis
and periarteritis of the adventitial vessels, and vasa vasorum surrounded by lymphocytes andplasma cells of the aorta, suggesting syphilitic aortitis, was observed. The liver showedhistologically chronic hepatitis with periportal fibrosis and lymphacytic cell infiltration.

Tissue specimens of all organs inculding liver, pancreas, and lymph nodes were taken at

autopsy and fixed in 10% formalin. After ordinary methods of histological preparations,parafiin blocks of specimens were cut at 4 p , and stained with histochemical methods using

orcein (Merck, Art. 7091, Lot 8529084) (Senba, 1982) and immunoperoxidase methods(DAKO PAP KIT: K523, Lot. 063‑3) for HBSAg,

HBsAg was revealed in numerous hepatocytes by orcein and immunoperoxidase
methods. Furthermore, HBsAg was observed in the acinar cells of pancreas (Fig. l), andhistiocytes of retroperitoneal pancreatic lymph nodes (Fig. 2) by both orcein and immunoper‑oxidase methods. No particular lesions of the pancreas and lymph nodes were ohserved,
microscopically. Any organ specimen examined ather than liver, pancreas and retroper‑
itoneal pancreatic lymph nodes did not show HBSAg.

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i'c*se'**'s'*;iPi'==' "̲:'/'::s':":t:s';"I:s'{i'=.'.;';;"::::aj'(si :' i'i'i*i:1'::s*t**.{'s':*'*''iS'+̲>:,*.

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Figure 1 HBsAg is localized in the cytoplasmof some acinar cells of the pancreas(arrows) . (Immunoperoxidase method,

Figure 2 Cytoplasmic positive staining forcounterstained with hematoxylin,

method, counterstained with hematoxylin,

original magnification, x 400

original magnification, x 400)

HBsAg in the histiocytes of lymph
nodes (arrows) . Immunoperoxidase

275

DISCUSSION
HBsAg has been revealed in both intrahepatic and extrahepatic tissues. Hepatitis B
virus infection could be associated with the production of tissue damage outside the liver. In

various extrahepatic diseases, immune complex deposition appears to be responsible fordisease manifestations. It has been reported that various immunological processes may be
involved and that host immune responses to the viral antigens may determine tissue damage
according to morphological appearances and immunopathological abnormalities.
The present study showed that there is a possibility of infectiveness and replication ofhepatitis B virus or at least an affinity of HBsAg to cells not only of the hepatocytes but also

the pancreatic parenchymal cells and histiocytes of lymph nodes. However, we were not able
to get any observation of pathogenesis of the pancreas and lymph nodes in this case. Whether
or not HBsAg in the histiocytes of lymph nodes is related to infectiveness was not clear.

REFERENCES
1 ) Brozosko, W.J., Krawczynski. K., Nazarewicz, T., Morzycka, M. and Nowoslawski, A. (1974) :Glomerulonephritis associated with hepatitis‑B surface antigen immune complexes in children,
Lancet II, 477‑482
2 ) Kawano, Y. (1983): Localization of hepatitis B surface antigen in hepatocellular carcinoma,
Acta Pathol. Jpn., 33, 1087‑l093
3 ) Michalak, T. (1978) : Immune complexes of hepatitis B surface antigen in the pathogenesis ofperiarteritis nodosa. Am. J. Pathol., 90, 619‑632

4 ) Nayak, N.C. and Sachdeva. R. (1975): Localization of hepatitis B surface antigen in conven‑tional paraffin sections of the liver, Am. J. Pathol., 81, 479‑492

5 ) Nazarewicz, T., Krawczynski, K., Slusarczyk, J. and Nowoslawski, A. (1977): Cellular
localization of hepatitis B virus antigens in patients with hepatocellular carcinoma coexistingwith liver cirrhosis, J. Infect. Dis., 135, 298‑302

6 ) Nowoslawski, A., Krzysztof. K.. Brzosko, W.J. and Madalinski. K. (1972) : Tissue localizationof Australia antigen immune complexes in acute and chronic hepatitis and liver cirrhosis, Am.
J. Pathol., 68, 31‑56

7 ) Senba, M. (1981): Localization of hepatitis B surface antigen in the pancreas and hepatocel‑lular carcinoma, Kyushu Clin. Pathol., 18, 1068 ) Senba, M. (1982): Staining method for hepatitis B surface antigen (HBSAg) and its mecha‑
nism, Am. J. Clin. Pathol., 77, 312‑315
9 ) Shikata. T. (1973): Australia antigen in hepatoma and cirrhosis tissue. Tumor Res., 8, 127‑130
10) Shimoda, T., Shikat. a, T., Karasawa. T., Tsukagoshi, S., Yoshimura, M. and Sakurai, I. (1981):

Light microscopic localization of hepatitis B virus, antigens in the human pancreas, Gas‑
troenterology, 81, 998‑1005
11) Yoshimura, M., Sakurai, I.. Shimoda, T., Abe, K., Okano. T. and Shikata. T. (1981): Detectionof HBsAg in the pancreas, Acta Pathol. Jpn., 31, 711‑717
12) Wu, P.C. (1979) : Patterns of hepatitis B surface antigen, Arch. Pathol. Lab. Med., 103, 165‑168276

膵臓およびリンパ節内に存在するB型肝炎ウイルス表層抗原

千馬 正敬1・山下

裕人2・板倉英普

梅毒性大動脈炎に起因したと考えられる，大動脈瘤破裂により死亡した65歳女性の剖検例において慢性肝炎が見出され，オルセイン組織化学染色および酵素抗体法により，肝実質細胞の胞体のほかに，膵臓の腺房細胞胞体やリンパ節の組織球内にも，B型肝炎ウイルス表層抗原（HBsAg）が観察された。HBsAgの存在する膵臓およびリンパ節の組織内において，病原体や異物などに起因する反応性所見は，見出せなかった。このことはB型肝炎ウイルスによる感染は，肝臓と肝臓以外の組織において，生体防御が異なるものと思われるが，肝臓以外におけるHBsAgの病態学的意義は，今後検討しなければならない。B型肝炎ウイルスは，肝実質細胞のほかに膵臓実質細胞にも存在しうることが，今までにも示唆されているが，本症例はこのことをいっそう裏付けるものである。なおHBsAgがリンパ節内に明らかに存在したという剖検例の報告は，我々が調べた範囲ではこれまでのところ見当らない。

長崎大学熱帯医学研究所病理学部門
大分医科大学病理学教室

JAPANESE JOURNALOF
TROPICAL MEDICINE AND HYGIENE
Vol. 17 No. 3

September 1989

CONTENTS
Original article

Tsukamoto, M.
Two New Mosquito Species from a Pitcher Plant of Mt. Kinabalu,
Sabah, Malaysia: Culex rajah and Tonorhynchites rajah(Diptera: Culicidae)

215‑228

Tanabe, K., Otsuji, Y., Nakabayashi, T., Ohtomo, H. and Tanaka, H.

Management and Utilization of Orphan Drugs against Parasitic
Infections in Recent 7 Years in Japan229‑236

Maki, J. and Yanagisawa, T.
Studies on Effects of Paromomycin Sulphate, Bithionol,

Mebendazole and Flubendazole in the Treatment of Mice Infectedwith Plerocercoids of Diphyllobothrium erinacei

237‑241

Takaoka, H.
Notes on Blackflies (Diptera: Simuliidae) from Myanmar(formerly Burma) """""'

243‑257

Yano, K., Maramorosch, K. and Kozlowska, A.
Effects of Environmental Temperature and Membrane Feeding
Solutions of Purified Ookinetes on the Sporogonic Development of
259‑267

Plasmodium berghei ･･･････‑

Research note
John, A.

Survey of Plasmodium falciparum in Makurdi, Nigeria

269‑271

Case report

Senba, M., Yamashita, H. and Itakura, H.
Localization of Hepatitis B Surface Antigen in the Pancreas andLymph Nodes"""""""""""""""""""""""""""""･""""""'
Published byJAPANESE SOCIETY OF TROPICAL MEDIClNE
c/o Institute of Tropical Medicine, Nagasaki University

12‑4 Sakamoto‑machi, Nagasaki 852, Japan273‑276