Vol.32
No.04
DECEMBER 2004

Tropical Medicine and Health

Tropical Medicine
and Health

ISSN 1348-8945

Vol.32 No.04 DECEMBER 2004

ISSN 1348-8945【CONTENTS】
Original articles
Seroepidemiology of dengue and assessment of public awareness in the Dominican Republic
Makino, Y., Shichijo, A., Bello, C., Eshita, Y., Disla, M., Cesin, A., Galcia, B.,
Lora, M., Valdez, S., Aquino, J., Aono, H., Ma, S-P., and Takeshita, M.
305
Taxonomic notes on the griseifrons species-group of Simulium (Simulium)(Diptera: Simuliidae) in Northern Thailand
Takaoka, H., and Choochote, W.

Vol.32 No.04 DECEMBER 2004

311

Current status of respiratory diseases suffered by Japanese people living in Southeast Asia
Uchikoshi, A., Hamada, A., Okuzawa, E., Hondo, K., Ookubo, N., Unachak, V., Thomas, J.,
Morikawa, T., Takeuchi, K., and Basugi, N.
329
Short Communication
A pilot field trial of an in vitro drug susceptibility test using the AnaeroPack!
malaria culture system on the Thai-Myanmar border
Hatabu, T., Kawazu, S., Kojima, S., Singhasivanon, P., Krudsood, S.,
Looareesuwan, S., and Kano, S.

335

Shiga-toxin producing Escherichia coli in Vietnam
Nguyen, BM., Phung, DC., Nakasone, N., Toma, C., Higa, N., Iyoda, S., and Iwanaga, M.

339

Book reviews
保健と医療の人類学
Moji, K.
(in Japanese)343

調査研究の手引き

Tropical Medicine
and HealthApplied Health Research Manual

JAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

Trop.Med.Health日本熱帯医学会表紙!−" ＣＭＹＫ 中村 中村

背は３mm（＊前回同様）Vol.32
No.04
DECEMBER 2004

Tropical Medicine and Health

Tropical Medicine
and Health

ISSN 1348-8945

Vol.32 No.04 DECEMBER 2004

ISSN 1348-8945【CONTENTS】
Original articles
Seroepidemiology of dengue and assessment of public awareness in the Dominican Republic
Makino, Y., Shichijo, A., Bello, C., Eshita, Y., Disla, M., Cesin, A., Galcia, B.,
Lora, M., Valdez, S., Aquino, J., Aono, H., Ma, S-P., and Takeshita, M.
305
Taxonomic notes on the griseifrons species-group of Simulium (Simulium)(Diptera: Simuliidae) in Northern Thailand
Takaoka, H., and Choochote, W.

Vol.32 No.04 DECEMBER 2004

311

Current status of respiratory diseases suffered by Japanese people living in Southeast Asia
Uchikoshi, A., Hamada, A., Okuzawa, E., Hondo, K., Ookubo, N., Unachak, V., Thomas, J.,
Morikawa, T., Takeuchi, K., and Basugi, N.
329
Short Communication
A pilot field trial of an in vitro drug susceptibility test using the AnaeroPack!
malaria culture system on the Thai-Myanmar border
Hatabu, T., Kawazu, S., Kojima, S., Singhasivanon, P., Krudsood, S.,
Looareesuwan, S., and Kano, S.

335

Shiga-toxin producing Escherichia coli in Vietnam
Nguyen, BM., Phung, DC., Nakasone, N., Toma, C., Higa, N., Iyoda, S., and Iwanaga, M.

339

Book reviews
保健と医療の人類学
Moji, K.
(in Japanese)343

調査研究の手引き

Tropical Medicine
and HealthApplied Health Research Manual

JAPANESE SOCIETY OF TROPICAL MEDICINEJAPANESE SOCIETY OF
TROPICAL MEDICINE

Trop.Med.Health日本熱帯医学会表紙!−" ＣＭＹＫ 中村 中村

背は３mm（＊前回同様）Tropical Medicine and Health Vol. 32 No. 4, 2004, pp. 305-309
Copyright! 2004 by The Japanese Society of Tropical Medicine

３０
５

SEROEPIDEMIOLOGY OF DENGUE AND
ASSESSMENT OF PUBLIC AWARENESSIN THE DOMINICAN REPUBLICYOSHIHIRO MAKINO１, AKIHISA SHICHIJO２, CASTRO BELLO２, YUKI ESHITA１,MILDRE DISLA２, ANA J. CESIN２, BARBARA GALCIA２, MIGUEL LORA２,
SONIA VALDEZ２, JOSE DIAZ AQUINO１, HIROSHI AONO１,
SHAO-PING MA１ and MASAZUMI TAKESHITA２
Accepted 6, September, 2004

Abstract: Dengue fever (DF) is a major public health concern in the Dominican Republic. In recent years, several
epidemics of DF have been reported to the Pan American Health Office (PAHO), but the extent of the epidemics
has not been clearly understood yet. Therefore, we conducted a nationwide seroepidemiology of dengue (DEN) infection. At the same time, we conducted an interview survey to assess public awareness regarding the disease. The
serum samples were collected at seven main cities in the Dominican Republic and screened for DEN antibody witha commercial ELISA kit. A total of 2007 serum specimens were examined. The prevalence of DEN antibody in theseven cities varied between 43.1 and 89.7%. Neutralization (N) test carried out on the ELISA-positive serum from
Samana, one of the high antibody-prevalent cities, revealed that all the sera showed positive to at least two DEN serotypes. Geometric mean N titers against DEN-1, 2, 3 and 4 were 40.5, 463.7, 59.9 and 454.4 respectively. No difference in antibody prevalence was observed between males and females. It appeared that a high level of awarenessregarding DF did little affect DEN prevalence. Strong, concrete public health strategies that motivate the local community to combat DF are required.

INTRODUCTION
The Dominican Republic is located at the Greater Antilles in the Caribbean Sea, on the east border of Haiti. The
land area is 48,442km2 and the population about 8.7million.
In recent years, service industries have exceeded agriculture
as the main industry due to the growth of tourism and thedevelopment of free trade zones.
Control and prevention of infectious diseases are themajor public health problems in the Dominican Republic.
Dengue fever/dengue hemorrhagic fever (DF/DHF) is a
public health concern that, in severe cases, causes hemorrhagic diatheses and death due to hemorrhagic shock [1,2].
Four serotypes of dengue virus (DEN-1 to DEN-4) form asingle antigenic complex. The main vector is Aedes aegypti,
which is commonly found in tropical and subtropical areas
including the Dominican Republic [3]. The first laboratoryconfirmed dengue epidemic (DEN-3) in the Dominican Republic was in 1963 [4]. Since then, epidemics caused bymultiple serotypes of DEN virus have been reported [5]. In
1988, four cases of DHF were reported for the first time inthe country [4]. During a worldwide pandemic of DF/DHF
1
2

in 1998, a total of 2,335 cases including 176 cases of confirmed DHF with 10 deaths were reported in the Dominican
Republic [4]. In 1997, the Ministry of Public Health initiated the DEN Control Campaign through surveillance anderadication of Aedes aegypti and health education to thecommunity. Although the campaign seemed effective incombating mosquitoes, the DEN epidemic persisted. In
2000, 3, 462 cases of DF, including 58 DHF with 6 deaths
were reported. During this epidemic, all four serotypes of
DEN virus were isolated [4].
In view of background, we conducted serological andinterview surveys of DEN infection at the seven main cities
in the Dominican Republic in order to determine the extent
of DEN infection in the country and to assess public awareness regarding DF/DHF. In this paper, we report the resultsof the survey on DEN infection among a healthy population
living in the seven main cities of the Dominican Republic.
To our knowledge, this was the first nationwide survey of
DEN infection in the Dominican Republic.

Department of Infectious Diseases, Faculty of Medicine, Oita University, Japan;
Centro de Educacion Medica de Amistad Dominico-Japonesa, Dominican Republic

３
０
６

MATERIALS AND METHODSInterview survey and collection of serum specimens
During the period from September 2000 to July 2001,
we conducted a survey of DEN together with a healthcheckup for local residents of seven main cities (Santo
Domingo, S.P. de Macoris, La Romana, Bani, Samana, La
Vega, and Santiago) in the Dominican Republic with thecollaboration of the staff of local health centers, medical
students and an NPO group (Fig. 1). Before initiating thehealth checkup, we explained the DEN survey project to theparticipants and others who wished to volunteer for the project, then proceeded to the interview survey and blood collection. The teaching staff of the epidemiology section at
CEMADOJA did the interview in Spanish. The questionnaire consisted of demographic information, the location ofwater containers (including the presence or absence of lids)
in and around their residence. The knowledge on DEN was
evaluated by the questions “Have you ever heard of DF?”“Do you have any idea what transmits DF?” and “Whatwould you do to avoid DF?”
Since no ethical code had been established at this point,
the teaching staff explained the survey to the participantsverbally, and only those who consented were subjected tothe interview and blood sampling.
The serum specimens were stored at either −20 or−80C until use.

Santiago 7

HAITI

Samana 5

La Vega 6

Hispaniola

SANTO
DOMINGO

1

La Romana

2

4

3

San Pedrode Macoris

Bani

0

40 80kmFigure 1. Map of the Dominican Republic.
The survey sites are indicated by numbers.

Detection of DEN antibody
Commercial ELISA kit (Microwell ELISA Dengue
IgG, Diagnostic Automation Inc. California, U.S.A.) wasused according to the manufacture’s instructions. An antibody (Ab) index (titer) of 0.9 or over was considered positive.
Fifty-percent focus-reduction neutralization (N) test,
using Vero cells, was carried out [6,7]. An N titer of 20 orover was considered positive.
Statistics
The χ２ test was used to compare the Ab prevalence between different locations. It was also used to analyze thedifference of Ab titer/prevalence among various settings.
The unpaired-student’s- t-test was used to compare the Ab
titers between subjects with and without DEN-related
knowledge. We used the Statistical Package for Biosciences(SPBS, Winesteem Institute of Community Medicine, Toda,
Saitama, Japan).

RESULTS
Serum specimens
A total of 2007 serum specimens were collected. The
number of serum specimens collected at the seven cities isshown in Table 1. Females predominated in six cities, but
accounted for only 42.2% in one city (Bani) (overall female
proportion was 72.2%). The age distribution of the volunteers varied from 16 to 97 years with a median age between
34 and 43 years.
Prevalence of DEN Ab
The prevalence of DEN Ab is presented with demographic data from each city in Table 1. The Ab prevalence
varied from 43.1 to 89.7%, and southern cities seemed tohave a lower prevalence than eastern and northern cities(south vs. east: χ２=46.906, P<0.001; south vs. north: χ２=
33.777, P<0.001). There was no statistical difference in Ab
prevalence between males and females (P=0.097 by MantelHaenszel method).
Samana was one of the most highly prevalent areas(Ab prevalence of 86.0% by ELISA). In order to examine
which Ab to DEN serotypes was predominant in this area,
50 Ab-positive sera were randomly selected and N test was
performed. All the sera showed positive to at least two DEN
serotypes. Geometric mean N titers against DEN-1, 2, 3 and
4 were 40.5, 463.7, 59.9 and 454.4 respectively.
Interview survey
A total of 1,988 volunteers responded to the inquiry.
Most of them (1,986 individuals) stated that they had cisterns inside the house. Among these, only 255 (12.8%)
stated that they placed a lid on the cistern (Table 2). Commonly found utensils containing water were vases andplanters. When asked about DF, a majority of the volunteers(80.6%) answered “yes”, meaning that they had some DENrelated knowledge. More people living in St. Domingo, thecapital, knew about DEN (94.6%) than those in other cities(Table 3). There was no statistical correlation between DEN

３０７

Table 1. Demography and DEN antibody prevalence
Total
Population (x103)
No.specimens
Age (years)
MedianRange
GenderMale (%)
Female (%)
DEN-Ab positive
Total (%)
Regional subtotal (%)
Male (%)
Female (%)
DEN-Ab negative
Total (%)
Male (%)
Female (%)
Mean Ab index (SD)St Domongo
4,707
2,694
2,007
492

South
S.P.Macoris La Romana
262
215
274
252

223
258

East
Samana
82
250

Bani

North
La Vega
Santiago
391
840
250
231

39
16-97

41.5
19-97

34
17-71

39
18-85

36
19-80

40
19-76

43
16-85

40
19-78

624 (31.1)
1,383 (68.9)

131 (26.6)
361 (73.4)

70 (25.5)
204 (74.5)

56 (22.2)
196 (77.8)

149 (57.8)
109 (42.2)

61 (24.4)
189 (75.6)

82 (32.8)
168 (67.2)

75 (32.5)
156 (67.5)

1,369 (68.2)

327 (66.5)

226 (89.7)

115 (44.6)

85 (64.9)
242 (67.0)

48 (85.7)
178 (90.8)

60 (40.3)
55 (50.5)215 (86.0)
215 (86.0)
52 (85.2)
163 (86.2)

169 (67.6)

394 (63.1)
975 (70.5)

118 (43.1)
786 (61.6)
29 (41.4)
89 (43.6)

54 (65.9)
115 (68.5)

199 (86.1)
368 (76.5)
66 (88.0)
133 (85.3)

638 (31.8)
230 (36.9)
408 (29.5)
1.5 (1.1)

165 (33.5)
46 (35.1)
119 (33.0)
1.5 (1.0)

156 (56.9)
41 (58.6)
115 (56.4)
0.8 (0.6)

26 (10.3)
8 (14.3)
18 (9.2)
2.1 (0.8)

143 (55.4)
89 (59.7)
54 (49.5)
0.9 (0.9)

35 (14.0)
9 (14.8)
26 (13.8)
1.6 (0.7)

81 (32.4)
28 (34.1)
53 (31.5)
1.4 (0.8)32 (13.9)9 (12.0)23 (14.7)
2.4 (1.5)

Table 2. Results of interview survey on dengue
Total
Cistern inside house?
“yes”
with lid
without lid
Any utensil containing water?
vase and planter
old tire
empty bottle
other containers
no answer
Do you know DF?
“yes”
“no”

1,988
1,986
255
1,731
1,090
238
92
315
253
1,603
385

Table 3. Results of interview survey and DEN antibody prevalence
Do you know dengue?

Total

No.interviewe
Answered “yes”(%)
Serodiangosis negative(%)
Serodiagnosis positive(%)
Answered “no”(%)
Serodianosis negative(%)
Serodiagnosis positive(%)
P value (χ２-test)

1,988
1,602
(80.6)
52.5(32.8)
1,077(67.2)
386(19.4)
109(28.2)
277(71.8)
0.086

South
St Domingo sp Macoris La Romana
479
273
250
453
221
194
(94.6)(81.0)(77.6)
153
122
18(33.8)(55.2)
(9.3)
300
99
176
(66.2)(44.8)(90.7)
26
52
56
(5.4)
(19.0)(22.4)
835
8(30.8)(67.3)(14.3)
18
17
48(69.2)(32.7)(85.7)
0.752
0.112
0.280

P value: The difference of Ab prevalence between “yes”and “no” was calculated.

Bani
257
207
(80.5)
119(57.5)
88(42.5)
50(19.5)
23(46.0)
27(54.0)
0.143

East
Samana249
177(71.1)
25(14.1)
152(85.9)
72(28.9)
10(13.9)
62(86.1)
0.961

North
La Vega
Sontiago
249
231
193
157
(77.5)(68.0)
66
22(34.2)(14.0)
127
135(65.8)(86.0)
5674(22.5)(32.0)
1510(26.8)(13.5)
41
64(73.2)(86.5)
0.297
0.918

３
０
８

-related knowledge and Ab prevalence. The difference inmean Ab index between those who answered “yes” and“no” was also not statistically different (1.5 vs. 1.6; P:
0.094 by unpaired- student’s-t-test).

DISCUSSION
The Dominican Republic has experienced several DEN
epidemics in recent years. However, few virological or seroepidemiological analyses have been carried out. Therefore,
we conducted a nationwide serological survey of DEN infection, together with virus isolation from DEN suspectedcases. Since our survey was carried out during office hours
on weekdays, more female (mainly homemakers) could participate in it. The Ab-positive rate varied from 43.1 to89.7% with an overall rate of 68.2%. Bani and S.P. de Macoris appeared to be relatively low DEN endemic areas.
Bani is well known for its mango plantations and the contributions of these make the city’s financial stability. The cityof S.P. de Macoris, meanwhile, is the eastern center of thecountry and has a medical school and international baseballstadium where many foreign athletes gather. The healthadministration systems of these cities should be evaluated in
more detail.
The N titers to DEN-2 and DEN-4 appeared higher
than DEN-1 and DEN-3, although it is not clear how manyof these Abs represent prior DEN infection or cross-reactive
response. It should be noted that during this period, DEN-2
was isolated from patients with unknown fever. These results were consistent with the report that multiple serotypesof DEN virus had circulated in the Dominican Republic forthe past few years [4].
The government conducted community programs topromote DEN-related knowledge through televised public
service announcements, posters, health education, etc. Ourinterview survey also showed that a considerable proportionof the volunteers (68.0-94.6%) had some knowledge of DF.
Despite good public awareness, DF was still endemic inthese areas. Our data showed that there was no direct association between the knowledge of DF and the prevalence of
DEN antibody. In 2003, another DF/DHF epidemic occurred and 5,170 cases with 107 deaths were registered [8].
In nearby Puerto Rico, where DF is also a major healthproblem, a variety of programs have been conducted. These
have resulted in an elevation of awareness, some behavioral
changes, and a limited change in larval indices [9]. Thesefindings indicate that more specific information about the
behaviors that need to be changed should be provided alongwith political and financial support.
In the present study, an interview survey among adultand young adult populations was conducted to determine

the level of the public awareness regarding DF prevention
together with a serological survey. A serological survey onchildren, who are more susceptible to DEN but who were
not included in the present study, will be conducted in a
subsequent study. Recently, Ab to West Nile virus (WNV)
was detected from domestic birds in the Dominican Republic [10]. Although no human case has been reported, allmembers of the genus Flavivirus, including WNV and DEN,
share common antigenic sites [11], and thus care should betaken in interpreting the antibody-positive results of serological tests.

ACKNOWLEDGEMENT
The main part of this study was carried out as part of
the training course of epidemiology in the Centro de Educacion Medica de Amistad Dominico-Japonesa (CEMADOJA) supported by Japan International Cooperation
Agency (JICA).

REFERENCES
１．Hammon, W.McD., Rudnick, A. and Satlier, G.E. (1960):
Viruses associated with hemorrhagic fevers of the Philippines and Thailand. Science, 131, 1102-1103
２．Halstead, S.B. (1965): Dengue and hemorrhagic fevers inSoutheast Asia. Yale J. Biol. Med., 37, 434-454
３．World Health Organization. (1997): Dengue hemorrhagic
fever - Diagnosis, treatment, prevention and control-, 2nded., 1-11, World Health Organization, Geneva
４．Schneider, J. and Droll, D. (2001): A timeline for dengue
in the Americas to December 31, 2000 and noted first occurrences. In: The History of Dengue and Dengue Hemorrhagic Fever (DHF) in the Region of the Americas,
1635-2001, 1-20, Pan American Health Office, Washington, DC
５．Lotono-Pino, M.A., Cropp, C.B., Farfan, J.A., Vorndam,
A.V., Rodriguez-Angulo, E.M., Rosado-Paredes, E.P.,
Flores-Flores, L.F., Beaty, B.J. and Gubler, D.J. (1999):
A common occurrence of concurrent infections by multiple dengue virus serotypes. Am. J. Trop. Med. Hyg., 61,
725-730
６．Okuno, Y., Igarashi, A. and Fukai, K. (1978): Neutralization tests for dengue and Japanese encephalitis viruses by
the focus reduction method using peroxidase-antiperoxidase staining. Biken J., 21, 137-147
７．Ishimine, T., Tadano, M., Fukunaga, T. and Okuno, Y.
(1987): An improved micromethod for infectivity assays
and neutralization tests on dengue viruses. Biken J., 30,
139-144
８．Capitan, M.A. (2003): UASD dice dengue es epidemia dehecho. In: Hoy Digital,10del diciembre, 152, Santo Domingo

３０
９９．Winch, P.J., Eeontsini, E., Rigau-Perez, J.G., Ruiz-Perez,
M., Clark, G.G. and Gubler, D.J. (2002): Communitybased dengue prevention programs in Puerto Rico: Impact on knowledge, behavior, and residential mosquito
infestation. Am.J.Trop. Med. Hyg., 67, 363-370
１０．Komar O. Robbins MB. Klenk K. Blitvich BJ. Marlenee
NL. Burkhalter KL. Gubler DJ. Gonzalvez G. Pena CJ.

Peterson AT. Komar N. (2003): West Nile virus transmission in resident birds, Dominican Republic. Emerging Infectious Diseases, 9, 1299-302
１
１．Burke, D.S. and Monath, T.P.(2001):Fields Virology 4thed., 1043-1125, Lippincott Williams & Wilkins co., NewYork

Tropical Medicine and Health Vol. 32 No. 4, 2004, pp. 311-327
Copyright! 2004 by The Japanese Society of Tropical Medicine

３
１
１

TAXONOMIC NOTES ON THE GRISEIFRONS SPECIES-GROUP
OF SIMULIUM (SIMULIUM ) (DIPTERA: SIMULIIDAE)
IN NORTHERN THAILAND
HIROYUKI TAKAOKA１ and WEJ CHOOCHOTE２
Accepted 15, September, 2004

Abstract: Two known and two new species of the griseifrons species-group of the subgenus Simulium (Simulium)
in Northern Thailand are reported. The male, pupa and mature larva of S. (S.) rudnicki Takaoka and Davies, andthe pupa and mature larva of S. (S.) suchariti Takaoka and Choochote, are described for the first time. Furthermore,
two new simuliid species, S. (S.) mediocoloratum sp. nov. and S. (S.) crocinum sp. nov., both of which are very
similar to S. (S.) rudnicki, are also described.
Key words: black fly, Simuliidae, Simulium, Thailand, new species, griseifrons species-group

In Thailand, the griseifrons species-group of the subgenus Simulium (Simulium) is represented by eight species[1]. During recent surveys on the immature stages of black
flies in Northern Thailand, we collected S. rudnicki Takaoka
and Davies [2], S. suchariti Takaoka and Choochote [3],and two new species, all of which are assigned to thisspecies-group. The male, pupa and mature larva of S. rudnicki, and the pupa and mature larva of S. suchariti (onlyfemales of both species were so far known) are describedhere for the first time. The two new species are also described based on adult, pupal and larval specimens.
The terms for morphological features used here follow
those of Takaoka [4]. Holotype and most paratype specimens of the new species will be deposited at the Department of Infectious Disease Control, Oita University.
Simulium (Simulium) rudnicki Takaoka and Davies,
1995
Simulium (Simulium) rudnicki Takaoka and Davies,
1995: 155 (female only).
DESCRIPTION. Male. Body length 3.8―4.0 mm. Head.
Slightly wider than thorax. Upper eye consisting of large
facets in 24 horizontal and 22 or 23 vertical rows. Clypeus
black, white pruinose, iridescent when illuminated, sparsely
covered with dark brown hairs. Antenna composed of 2+9
segments, pale yellow on scape, pedicel, and base of 1stflagellar segment, dark yellow on the rest except 3 or 4 apical flagellar segments dark brown, (in 1 male, pale yellow
on scape, pedicel, and base of 1st flagellar segment, light tomedium brown on the rest of 1st flagellar segment, 2nd and3rd flagellar segments, and dark brown on 4th to 9th flagellar segments); 1st flagellar segment elongate, about twice aslong as 2nd one. Maxillary palp composed of 5 segments,
proportional lengths of 3rd, 4th, and 5th segments 1.0:1.2:
2.8; 3rd segment (Fig. 1A) of normal size, with a small
globular sensory vesicle having a very small opening. Thorax. Scutum black, with silvery iridescent pattern differingwith angles of light: when illuminated anteriorly and viewed
dorsally scutum shows subanteriorly a transverse pair ofnarrow silvery iridescent spots widely spaced in middle(distance between spots about one-third that of scutum);when illuminated laterally or posterolaterally and viewed
dorsolaterally, the subanterior paired spots fade and are replaced by an anterior pair of narrow iridescent spots onshoulders which extend posteriorly along lateral margins ina wide band and connect to a large transverse posterior spot
on prescutellar area; when illuminated anterolaterally andviewed dorsolaterally, scutum on each shoulder has a large
anterior iridescent spot including anterior and subanterior
narrow spots mentioned above, which is also contiguous toa broad lateral iridescent band along lateral border; scutum
uniformly covered with yellow recumbent short hairs interspersed with dark long upright hairs on prescutellar areaand dark short and medium-long hairs near anterior margin.
Scutellum black except posterior margin brownish, withseveral dark long upright hairs as well as yellow short hairs.
Postnotum black, silvery iridescent when illuminated, andbare. Pleural membrane bare. Katepisternum longer thandeep, and bare. Legs. Foreleg: coxa pale yellow; trochanter

1. Department of Infectious Disease Control, Faculty of Medicine, Oita University, Hasama, Oita, 879-5593 Japan
2. Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand

３
１
２

and femur medium or dark yellow with narrow apical portion light brown on outer surface; tibia brownish black withbasal 3/4 narrowly white on outer surface (white sheeny
when illuminated); tarsus black, with moderate dorsal haircrest; basitarsus somewhat dilated, about 6.4 times as longas its greatest width. Midleg: coxa dark brown or blackish
brown; trochanter pale yellow with apical 1/2 or a little
more dark yellow to light brown; femur entirely yellow;
tibia entirely yellow, with a broad white sheen on posterior
surface when illuminated; tarsus brownish black except basal 1/2 or 2/3 yellow, though its border not well defined.
Hind leg: coxa dark brown or brownish black; trochanter
yellow; femur yellow except apical cap dark brown; tibiabrownish black with base narrowly whitish yellow; tarsus
brownish black except basal 1/2 of basitarsus and basal 1/2
of 2nd tarsal segment whitish yellow; basitarsus (Fig. 1B)
somewhat widened from base toward apical 3/4, then somewhat narrowed toward apex, 4.5 times as long as its greatest

width, and 0.8 times as wide as the greatest width of hindtibia, which is as wide as that of femur; calcipala of medium size, nearly as long as wide; pedisulcus distinct. Wing.
Length 2.9―3.0 mm; costa with spinules and hairs; subcosta
bare; basal section of vein R bare; R１ with spinules andhairs; R２ with hairs only; hair tuft at base of stem vein darkbrown; basal cell absent. Abdomen. Basal scale blackishwith a fringe of long dark hairs. Abdomen brownish blackwith dark hairs; segments 2, 5―7, each with a pair of silvery
iridescent spots dorsolaterally, those on segment 2 broadly
connected in middle. Genitalia (Fig. 1C―L). Coxite in ventral view nearly quadrate. Style elongate, spatulate ventrodorsally, 2.2 times as long as coxite, gradually narrowed
from base to apical 1/3, then widened to rounded apex
(widths at base and near apex subequal to each other andabout 1.4 times as wide as the narrowest width at middle),
with a slender subterminal spine; style with a prominent basal protuberance pointed dorsally and furnished at and nearFig. 1. Adult male of Simulium (Simulium) rudnicki Takaoka and Davies. A, 3rd segment of maxillary palp (right side, frontalview); B, hind basitarsus and 2nd tarsal segment (left side, outer view); C, coxite and style (right side, ventral view); D,
coxite (left side, lateral view); E, style (left side, outer view); F, basal portion of style (right side, inner view); G, style (leftside, end view); H―J, ventral plates (H, ventral view; I, lateral view; J, end view); K, median sclerite; L, dorsal plate. Scales.
0.02 mm for A, C―L; 0.1 mm for B.

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apex with many conical spines on anterior and inside surfaces. Ventral plate in ventral view transverse, somewhat
wider than its length, rounded posterolaterally, with posterior margin untoothed and somewhat concave medially, andcovered with fine setae centrally on ventral surface; basalarms long and stout, curved outwardly and forwardly.
Parameres wide basally, each with numerous parameral
hooks. Median sclerite narrow, moderately widened toward
apex. Aedeagal membrane densely covered with needlelike setae and with well-sclerotized narrow dorsal plate.
Segment 10 without any distinct hair. Cercus rounded with
9―13 distinct hairs.
Pupa. Body length (excluding gill filaments) ca. 4.0
mm. Head. Integument yellowish brown, bare; face with apair of long simple trichomes; frons with 2 pairs of longsimple and bifid trichomes; antennal sheath bare. Thorax.
Integument yellowish brown, bare on anterior 1/2 includinga small raised area at base of gill, moderately covered withsmall cone-shaped tubercles dorsally on posterior 1/2; thorax anteriorly with 3 dorsal and 2 lateral pairs of long
branched trichomes (split into 2―4), posteriorly with 1 lateral pair of long bifid trichomes, and ventrolaterally with 3
pairs of long simple or bifid trichomes. Gill (Fig. 2A) with
6 filaments arranged in sessile pairs; all filaments darkbrown, subequal in length (1.5―2.2 mm) and thickness except inner filament of lower pair somewhat thinner than others; all filaments gradually tapered toward apical tip, withannular ridges and furrows throughout their length (though

not marked near base), and densely covered with minute tubercles. Abdomen. Dorsally, segment 1 yellow with darkgrey narrow portion along posterior margin, with 1 simple
slender hair-like seta on each side; segment 2 dark grey onanterior 2/5, transparent on posterior 3/5, with 1 long simple hair-like seta (Fig. 2B), 4 short simple hooked spines of
equal size (Fig. 2C) (much smaller than those on segments
3 and 4) and 1 short spinous seta (Fig. 2D) on each side;
segments 3 and 4 each with 4 hooked spines along posterior
margin on each side; segment 5 bare; segments 6, 7 and 9each with a transverse row of comb-like groups of minutespines but lacking spine-combs on each side; segment 8
with a transverse row of spine-combs as well as a transverse
row of comb-like groups of minute spines on each side; segment 9 lacking terminal hooks. Ventrally, segment 4 with 1
bifid hook, 1 slender medium-long seta and 2 short setae oneach side; segment 5 with a pair of bifid hooks submediallyon each side; segments 6 and 7 each with a pair of bifid inner and simple outer hooks somewhat separated from eachother, on each side. Grapnel-like hooklets absent. Cocoon(Fig. 2E). Wall-pocket-shaped, with a large anterolateralwindow on each side; thin, film-like, individual threads not
visible; anterior margin not well defined, often broken;
about 4.5 mm long by 2.5 mm wide.
Mature larva. Body length 8.5―11.0 mm. Body color
dark grey to greyish black. Abdomen gradually widened toward posterior tip, with the widest portion on segment 8,
and abruptly narrowed to posterior tip. Cephalic apotome

Fig. 2. Pupa and mature larva of Simulium (Simulium) rudnicki Takaoka and Davies. A, pupal gill filaments; B―D, hair-like seta,
hooked seta and minute seta on the dorsal surface of the 2nd abdominal segment of pupa, respectively; E, cocoon (dorsalview); F, larval antenna (left side, dorsal view); G, larval mandible; H, larval hypostomium. Scales. 0.01 mm for B―D; 0.02
mm for G and H; 0.1 mm for F; 0.2 mm for A; 1.0 mm for E.

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４

almost entirely medium to dark brown except narrow area
along each lateral margin of anterior 1/3 pale; head spots
distinctively positive except posterolateral ones usuallymerged into ground color (Plate 1A); lateral and ventral surfaces medium to dark brown except eye-spot region pale;spots around eye-spot region, and those on both sides of
postgenal cleft faintly negative or merged into ground color(Plate 1E). Antenna (Fig. 2F) composed of 3 segments andapical sensillum, much longer than stem of labral fan;length ratio of segments (from base to tip) 1.0:1.1―1.2:0.5;
all segments pale white to yellow except 3rd segment entirely brownish black, and apical small portion of 2nd segment somewhat darkened. Labral fan with 60―64 main rays.
Mandible (Fig. 2G) with usual mandibular serration of 1
medium-sized tooth and 1 small one; main tooth at an obtuse angle apically to mandible; supernumerary serrations
absent; comb-teeth decreased in length from 1st to 3rd. Hypostomium (Fig. 2H) with 9 apical teeth, of which median
tooth slightly longer than each corner tooth; 3 intermediate
teeth on each side shorter than corner teeth; lateral margins
moderately serrate apically; 7―9 hypostomal bristles diverging posteriorly from lateral border on each side. Postgenal
cleft (Plate 1E) narrow, subtriangular, 1.7―1.9 times as longas postgenal bridge; lateral margins on posterior 1/2 nearlyparallel-sided or slightly converged at base, and pointed apically; subesophagial ganglion well pigmented, wine-glass
shaped. Thoracic cuticle almost bare. Abdominal cuticle
bare except last segment moderately covered with short,colorless setae on each side of anal sclerite. Rectal scales
present. Rectal organ of 3 lobes, each with 14―19 fingerlike secondary lobules. Anal sclerite X-shaped, with broadened anterior arms about 0.7 times as long as posterior
ones; 3―5 sensilla on the basal juncture area, and 6―12 sensilla just posterior to posterior arms. Ventral papillae absent.
Posterior circlet with 144―160 rows of hooklets, with up to
20―22 hooklets per row.
SPECIMENS EXAMINED. 5 females, 5 males (all reared
from pupae), 10 pupae, 5 pupal exuviae and 50 mature larvae, collected from a stream, Mae Klang Waterfall, Doi Inthanon National Park, Chiang Mai, Thailand, 31. XII. 2003,
by W. Choochote.
ECOLOGICAL NOTES. The pupae and larvae of S. rudnicki were found in great numbers on the surface of the
rocky stream-bed of a stream 5.0 m wide, with moderate to
rapid flows, exposed to the sun. Water temperature was 19.5°
C. Altitude was ca. 400 m. No other species was collected
together with S. rudnicki.
DISTRIBUTION. Peninsular Malaysia and Thailand.

REMARKS. The male of S. rudnicki is very similar to S.
yongi Takaoka and Davies, originally described from Malaysia [5] in most features including the genitalia, but isreadily distinguished from the latter by the entirely yellow
femur and tibia of the midleg, which are almost brownish
black in S. yongi [5].
The pupa of S. rudnicki is also very similar to that of S.
yongi [5] but differs in that it has the inner filament of theventral pair not narrowed basally, and the small raised areaof the thoracic integument near the base of the gill filaments
without tubercles (Fig. 2A), and the cocoon with a large anterolateral window on each side (Fig. 2B). The larva of S.
rudnicki has a dark cephalic apotome (Plate 1A) while thatof S. yongi has a pale one [5].
Simulium (Simulium) mediocoloratum sp. nov.
DESCRIPTION. Female. Body length 3.0-3.4 mm. Head.
Narrower than width of thorax. Frons brownish black, shiny,
not pruinose, with several dark stout hairs along lateral margins; frontal ratio 1.2―1.3:1.0:1.2; frons-head ratio1.0:4.0―
4.3. Fronto-ocular area (Fig. 3A) well developed, triangular.
Clypeus brownish black, shiny, with silvery iridescent pruinosity, moderately covered with dark stout hairs except medial portion of upper part bare. Labrum 0.62―0.67 times aslong as clypeus. Antenna composed of 2+9 segments,
brownish black except scape, pedicel, and base of 1st flagellar segment yellow when viewed posteriorly, or brownish
black except scape, pedicel, and a few basal flagellar segments yellow to dark yellow when viewed anteriorly; eachflagellar segment with a distinct pit apically on each lateralsurface gradually becoming smaller in size toward apical tip
except those on segment 9 located medially; each pit provided with numerous minute sensilla; each flagellar segment also with a small pit apically on dorsal surface exceptthat on segment 9 located medially. Maxillary palp brownish
black, with 5 segments, proportional lengths of 3rd, 4th and5th segments 1.0:1.2―1.3:2.8―2.9; 3rd segment (Fig. 3B)
not enlarged; sensory vesicle ellipsoidal, with rugged surface, 0.36―0.39 times length of 3rd segment, with medium
to large round opening apically. Maxillary lacinia with 13
inner and 13 or 14 outer teeth. Mandible with 28―31 innerand 12―14 outer teeth. Cibarium (Fig. 3C) with a cluster ofca. 90 conical processes medially. Thorax. Scutum brownish
black, shiny, whitish-grey pruinose (there appear to be faintscutal patterns formed by pruinose and non-pruinose portions differing by the angles of light: e.g. when illuminateddorsally and viewed laterally, 1 medial and 2 submedial narrow longitudinal whitish-grey pruinose vittae and 2 widelongitudinal whitish-grey pruinose bands along lateral margins connected to large whitish-grey pruinose portions on

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５

shoulders), moderately covered with yellow recumbent
short hairs as well as dark brown ones interspersed withlong upright dark hairs on prescutellar area. Scutellum
brownish black, with long dark hairs as well as yellow shorthairs. Postnotum brownish black, shiny, whitish-grey pruinose, and bare. Pleural membrane bare. Katepisternum
longer than deep, and bare. Legs. Foreleg (Fig. 3D): coxa
pale yellowish-white; trochanter pale yellowish-white withouter surfaces somewhat darkened; femur yellowish on inside surface but light yellowish-brown to light brown onouter surface; tibia largely white except apical 1/5 darkbrown and inner surface of apical 1/2 light to dark brown,
and with a large white sheen on outer surface when illuminated; tarsus dark brown; basitarsus dilated, 5.5 times aslong as its greatest width. Midleg (Fig. 3E): coxa darkbrown; trochanter dark yellow or light yellowish-brownwith base yellowish white; femur yellowish white to dark
yellow (light brown in some females); tibia yellowish white
except apical cap light to medium brown, with a large white
sheen on posterior surface when illuminated; tarsus medium
to dark brown except basal 3/5 or 3/4 of basitarsus yellowish white. Hind leg (Fig. 3F): coxa medium brown; trochanter yellowish white; femur light to medium brown withbase pale yellowish-white and apical cap medium to darkbrown; tibia light to dark brown with basal 1/3 and posterior surface of basal 2/3 white (its margin obliquely demarcated when viewed laterally), and with a large white sheen
on posterior surface when illuminated; tarsus dark brown
except a little more than basal 1/2 of basitarsus yellowish
white and basal 1/2 of 2nd tarsal segment yellow; basitarsus

Fig. 3. Adult female of Simulium (Simulium) mediocoloratum sp. nov. A, fronto-ocular area (right side, frontal view); B, 3rd segment of maxillary palp (right side, frontal view); C, cibarium; D―F, foreleg, midleg and hind leg, respectively (all left side); G,
8th sternite, ovipositor valves, genital fork and spermatheca (ventral view); H, paraproct (right side, ventral view); I, paraproct and cercus (right side, lateral view). Scales. 0.02 mm for C and G―I; 0.05 mm for A and B; 0.2 mm for D―F.

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６

nearly parallel-sided, 6.0 times as long as wide, and 0.8 and0.7 times as wide as the greatest widths of hind tibia and femur, respectively; calcipala short, 0.9 times as long as its
basal width and 0.4 times as wide as the greatest width ofbasitarsus; pedisulcus distinct at basal 1/3 of 2nd tarsal segment. Tarsal claws simple without any tooth. Wing.
Length 2.8―3.0 mm; costa with dark spinules and hairs;
subcosta fully haired; basal section of vein R bare; R１ with
spinules and hairs; R２ with hairs only; hair tuft at base ofstem vein dark brown; basal cell absent. Abdomen. Basal
scale light to medium brown, with a fringe of pale hairs;
segment 2 yellowish white except posterior 1/3 or 2/5 medium brown dorsally, with a dorsolateral pair of shiny whitish spots broadly connected in middle; remaining segments
medium to dark brown dorsally, with dark hairs; tergites 6-8
shiny. Genitalia (Fig. 3G-I). Ventral surface of abdominal
segment 7 without sternal plate. Sternite 8 well sclerotized,
bare medially, with 3―9 pale short setae submedially nearposterior border, and with 13 or 14 dark long hairs laterally
on each side; ovipositor valve triangular in shape, membranous, expanded ventrally, each covered with 18―28 short setae as well as numerous microsetae except narrow portion
along inner margin and rather wide portion along posterior
margin bare and transparent; inner borders not sclerotized,
narrowly separated from each other. Genital fork of
inverted-Y form, with well sclerotized stem; arms rather
wide, each with strongly sclerotized distal ridge having a
distinct projection directed anterodorsally. Paraproct in ventral view rounded posterolaterally, with anteromedial surface strongly sclerotized, having about 20 short spinous setae on it; paraproct slightly produced ventrally along medial
margin, covered with numerous stout hairs laterally andventrally. Cercus rounded posteriorly, about half as long as
wide, covered with numerous stout hairs. Spermatheca
globular, well sclerotized (except small area of its juncturewith duct unsclerotized), with reticulate surface pattern atleast near base, with minute internal setae; both accessory
ducts subequal in width to main one.
Male. Body length 3.0―3.2 mm. Head. Slightly
wider than thorax. Upper eye consisting of large facets in
22 or 23 horizontal and 22 vertical rows. Clypeus black,white pruinose, iridescent when illuminated, covered withdark-brown long hairs along lateral margins (median large
portion nearly bare). Antenna composed of 2+9 segments,
dark brown except base of 1st flagellar segment pale; 1stflagellar segment elongate, 1.8 times as long as 2nd one;flagellar segments 1-8 each with 1 or 2 small pits apicallyon each lateral surface, which are not so developed as compared to those of female. Maxillary palp composed of 5
segments, proportional lengths of 3rd, 4th, and 5th segments 1.0:1.3:3.2; 3rd segment (Fig. 4A) of normal size,with a small, ellipsoidal sensory vesicle, 0.24―0.27 times
length of 3rd segment, and with a small opening medially.
Thorax. Scutum brownish black (3 narrow longitudinal
black vittae visible in alcoholic specimen), with silvery iridescent pattern differing with angles of light, as in male ofS. rudnicki; scutum uniformly covered with yellow (orbright brassy in some males) recumbent short hairs interspersed with dark long upright hairs on prescutellar area.
Scutellum brownish black, silvery iridescent when illuminated, with several upright dark hairs as well as yellow orbright-brassy short hairs. Postnotum brownish black, silvery iridescent when illuminated, and bare. Pleural membrane bare. Katepisternum longer than deep, and bare. Legs.
Foreleg: coxa whitish yellow; trochanter and femur medium
brown with apical cap of femur dark brown; tibia darkbrown to brownish black except outer surface of basal 3/4
white, and with a large white sheen on most of outer surface
when illuminated; tarsus brownish black; basitarsus somewhat dilated, 6.4 times as long as its greatest width. Midleg:coxa brownish black; trochanter and femur medium brown;
tibia light to medium brown except posterior surface of basal 1/2 white (its margin obliquely defined when viewed laterally) and with a white sheen on posterior surface when illuminated; tarsus dark brown to brownish black except basal 3/5 whitish yellow (though its border not well defined).
Hind leg: coxa dark brown; trochanter yellow; femur medium brown except extreme base yellow and apical cap darkbrown; tibia dark brown except extreme base yellowish
white, and with a whitish sheen basally on posterior surface
when illuminated; tarsus dark brown except a little less thanbasal 1/2 of basitarsus and basal 1/3 of 2nd tarsal segment
yellowish; basitarsus (Fig. 4B) somewhat enlarged, spindleshaped, 4.8 times as wide as its greatest width, and 0.8
times as wide as the greatest width of hind tibia, which isnearly as wide as that of hind femur; calcipala small, 0.9
times as long as wide and 0.3 times as wide as the greatest
width of hind basitarsus; pedisulcus distinct. Wing. Length
2.8―2.9 mm; other characters as in female except subcosta
completely bare. Abdomen. As in male of S. rudnicki.
Genitalia (Figs. 4C―K). Coxite in ventral view nearly quadrate. Style elongate, spatulate ventrodorsally, 2.2 times aslong as coxite, gradually narrowed from base to apical 1/2,
then widened to rounded apex (width at base about 1.2
times that near apex and about 1.7 times the narrowest
width at middle), with a slender subterminal spine; stylewith a prominent basal protuberance pointed dorsally andfurnished at and near apex with many conical spines on anterior and inside surfaces. Ventral plate in ventral viewtransverse, somewhat wider than its length, rounded posterolaterally, with posterior margin untoothed and somewhat concave medially, and covered with fine setae cen-

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７

trally on ventral surface; basal arms long and stout, curved
outwardly and forwardly. Parameres wide basally, each withnumerous parameral hooks. Median sclerite narrow, moderately widened from base to basal 1/3, then nearly parallelsided, with small median incision on apex. Aedeagal membrane densely covered with needle-like setae, with wellsclerotized narrow dorsal plate. Segment 10 without any
distinct hair. Cercus rounded with 10―16 distinct hairs.
Pupa. Body length (excluding gill filaments) 3.0―3.5
mm. Head. Integument yellowish brown, bare, with 1 facial
pair of long, simple or bifid trichomes and 2 frontal pairs oflong, bifid or trifid trichomes; antennal sheath bare. Thorax.
Integument yellowish brown, bare on anterior 1/2 except asmall raised area at base of gill densely covered with tubercles of irregular shapes, moderately covered with small
cone-shaped tubercles on posterior 1/2; thorax anteriorly
with 3 dorsal and 2 dorsolateral pairs of long branchedtrichomes (split into 2―5), posteriorly with 1 lateral pair ofbranched trichomes (split into 2―4) and ventrolaterally with
3 pairs of long trichomes (simple, bifid or trifid). Gill (Fig.
5A) with 6 filaments arranged in sessile pairs, and withmedium-sized transparent protuberance at base; outer filaments of all pairs dark brown, subequal in thickness to one
another but somewhat differing in length (1.5―2.0 mm), allgradually tapered from base to apex; inner filaments of allpairs medium brown, somewhat differing in length (1.0―1.5mm), inner filaments of dorsal and middle pairs subequal in
thickness to each other and gradually tapered toward apex
after running with the same width or very slightly widened
from base to basal 1/5 or 1/4 of their length, inner filamentof lower pair apparently increasing its width from base tobasal 1/5 or 1/4 of its length, then tapered toward apical tip;
inner filament of each pair usually somewhat paler andshorter than its outer counter filament; all filaments with annular ridges and furrows throughout their length except nearbase, densely covered with minute tubercles. Abdomen.
Dorsally, segment 1 light brown, with 1 simple long hairlike seta on each side; segment 2 light brown on anterior 1/2
or 1/3, with 1 simple long hair-like seta, 1 short spinous seta
and 4 short simple hooked spines of equal size on each side;
segments 3 and 4 each with 4 hooked spines along posterior
margin on each side; segment 8 with a transverse row ofFig. 4. Adult male of Simulium (Simulium) mediocoloratum sp. nov. A, 3rd segment of maxillary palp (right side, frontal view); B,
hind basitarsus and 2nd tarsal segment (left side, outer view); C, coxite and style (right side, ventrolateral view); D and E,
styles (both left side; D, inner view; E, outer view); F―H, ventral plates (F, ventral view; G, lateral view; H, end view); I, median sclerite; J, dorsal plate; K, abdominal tip showing cercus (lateral view). Scales. 0.02 mm for C―K; 0.05 mm for A; 0.1
mm for B.

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spine-combs on each side; segment 5 bare; segments 6, 7
and 9 each lacking spine-combs, with a transverse row of
comb-like groups of minute spines on each side; segment 9
lacking terminal hooks. Ventrally, segment 4 with 1 simple
hook and a few short setae on each side; segments 5―7 eachwith a pair of simple or bifid inner and simple outer hooks
on each side. Grapnel-like hooklets absent. Cocoon. Wallpocket-shaped, pale yellow, thin, with anterior margin irregular in form, usually appearing to have a broad anterodorsal projection when viewed dorsally (Fig. 5B,C); some
cocoons have a fragment of thread of various lengths directed downward from each side of the apex of the anterodorsal projection (Fig. 5D), suggesting that there may be alarge anterolateral window on each side; cocoon usuallysomewhat extending ventrolaterally; 3.0―3.5 mm long by
2.0―2.2 mm wide.
Mature larva. Body length 7.0―8.0 mm. Body color
usually dark greyish on thorax and light to dark brown or
blackish brown on abdomen (darkened posteriorly). Abdomen gradually widened toward segment 7, then narrowed toposterior tip when viewed laterally; segments 6 and 7 appear to be equal in width to each other when viewed dorsally. Cephalic apotome (Plate 1B) pale yellow on anterior
1/2 or less, light to medium brown on the rest (except narrow portion along each lateral margin usually pale down tolevel of posterolateral spots), with a small dark area in middle along posterior border; anteromedial longitudinal spot
faintly positive or negative, or sometimes merged into
ground color, posteromedial spot usually positive or rarely
merged into ground color; anterior 1/2 of mediolateral spoton each side usually negative or merged into ground color,and posterior 1/2 usually positive; posterolateral spots usually merged into ground color, and rarely anterior one ofthese spots negative; lateral and ventral surfaces variable incolor by populations; eg. in most larvae collected fromMonthatharn Waterfall, nearly yellowish with spots merged
into ground color, or in most larvae collected from Siribhume Waterfall, light to medium brown except eye-spot region yellowish and medial narrow area of postgenal bridge
somewhat paler than ground color, with spots faintly ormoderately negative (Plate 1F). Antenna composed of 3
segments and apical sensillum, much longer than stem oflabral fan; length ratio of segments (from base to tip) 1.0:
1.2:0.4―0.5; all segments pale white to yellow except 3rd
segment entirely brownish black and apical small portion of
2nd segment somewhat darkened, as in S. rudnicki. Labral
fan with about 52 main rays. Mandible (Fig. 5E) with usual
mandibular serration of 1 medium-sized tooth and 1 small
one; main tooth at an obtuse angle to mandible on apicalside: supernumerary serrations absent; comb-teeth decreased in length from 1st to 3rd. Hypostomium (Fig. 5F)
with 9 apical teeth, of which median tooth and each corner
tooth subequal in length to each other, and longer than others; lateral margins moderately serrate apically; 8 or 9 hypostomal bristles diverging posteriorly from lateral borderon each side. Postgenal cleft (Plate 1F) narrow, subtriangular or bullet-shaped, 1.6―2.3 times as long as postgenal
bridge; lateral margins on posterior 1/2 nearly parallel-sided
or slightly diverged at base, and pointed apically; subesophagial ganglion well pigmented, wine-glass-shaped. Thoracic

Fig. 5. Pupa and mature larva of Simulium (Simulium) mediocoloratum sp. nov. A, pupal gill filaments; B―D, cocoons (B and C,
dorsal view; D, lateral view); E, larval mandible; F, larval hypostomium. Scales. 0.01 mm for E; 0.05 mm for F; 0.2 mm for
A; 1.0 mm for B―D.

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９cuticle almost bare. Abdominal cuticle bare except last segment moderately covered with short colorless setae on eachside of anal sclerite. Rectal scales present. Rectal organ of 3
lobes, each with 13―17 finger-like secondary lobules. Analsclerite X-shaped, with broadened anterior arms about 0.7
times as long as posterior ones; 5―11 sensilla on basal areaof anal sclerite, and 8―13 sensilla just posterior to posteiorarms. Ventral papillae absent. Posterior circlet with 146―
150 rows of hooklets, with up to 20 hooklets per row.
TYPE SPECIMENS. Holotype female, reared from pupa
collected from a cascading stream, Monthatharn Waterfall,
Chiang Mai, Thailand, 31. XII. 2001, by W. Choochote.
Paratypes: 8 females, 11males, reared from pupae, 5maturelarvae, in alcohol, same data as holotype; 5 pupae and 20
mature larvae, collected from a small stream, Siribhume
Waterfall, Doi Inthanon National Park, Chiang Mai, 15. II.
2004, by W. Choochote.
ECOLOGICAL NOTES. The pupae and larvae of S. mediocoloratum were found on the surface of the stream-bed rockin two streams: one in Monthatharn Waterfall (ca. 750 m inaltitude) was a cascading stream, exposed to the sun or partially shaded, and water temperature was 17°
C, and theother in Siribhume Waterfall (ca. 1,500 m in altitude) was asmall, moderately-flowing stream 0.5m wide, exposed tothe sun and its water temperature was 18.5°
C.
Simulium mediocoloratum was collected together withmany other species in the two streams.
Mermithid and microsporidan infections were found in
five and one of the 47 immature larvae of S. mediocoloratum collected from Monthatharn Waterfall, respectively.
ETYMOLOGY. The specific name mediocoloratum refers
to the female of this species which is intermediate in the leg
color between S. rudnicki and S. yongi.
DISTRIBUTION. Thailand.
REMARKS. Simulium mediocoloratum sp. nov. is assigned
to the griseifrons species-group by the combination of thefollowing characters: tarsal claws simple in the female;style with a prominent basal protuberance (Fig. 4D,E) andventral plate transverse, without toothed margins (Fig. 4F)
in the male; and gill with six filaments per side in the pupa
(Fig. 5A).
The female of S. mediocoloratum resembles that of S.
rudnicki in many characters including the antenna with pits
and the ovipositor valve with transparent inner and posterior
margins and is barely differentiated from the latter by the
mid tibia yellowish white except its apical cap light to me-dium brown (Fig. 3E) (cf. entirely yellow in S. rudnicki).
On the other hand, this new species is readily distinguishedfrom S. rudnicki in the male by the medium-brown mid femur and the style with the narrowest portion medially (Fig.
4C), in the pupa by the inner filament of the ventral pairnarrowed basally as well as the small raised area near thegill base tuberculate (Fig. 5A), and in the larva by the cephalic apotome darkened only on the posterior 1/2 (Plate 1
B).
This new species is also very similar to S. yongi [5]but differs from the latter in that it has the more slender fore
basitarsi in both sexes, the cibarium with about 90 conicalprocesses (Fig. 3C), the mid femur entirely yellow (Fig. 3E)
in the female, the ventral plate somewhat bent ventrally (Fig.
4G) in the male, the cocoon appearing to have an anterodorsal projection (Fig. 5B,C), and the larval cephalic apotome
pale on the anterior 1/2 (Plate 1B).
The male genitalia suggest that this new species isclosely related to S. ufengense Takaoka, described from Taiwan [6], and S. fuzhouense Zhang and Wang, from Fujian,south China [7]. However there are differences in the leg
colors, the arrangement of the pupal gill filaments, and thecocoon. In the latter two species, the femora of the malelegs are largely yellowish, four of six pupal gill filaments
are distinctively more slender than the remaining two filaments, and the cocoon has a thick anterodorsal margin.
Simulium taipokauense Takaoka, Davies and Dudgeon(only the male adult is known) from Hong Kong [8] showssome similarities to S. mediocoloratum but differs in that it
has the much longer basal protuberance of the style and thesaddle-shaped ventral plate with a nipple-like median process.
Simulium (Simulium) crocinum sp. nov.
DESCRIPTION. Female. Body length 3.0 mm. Head.
Nearly as in female of S. mediocoloratum except following
characters. Frontal ratio 1.3:1.0:0.9; frons-head ratio 1.0:3.7.
Fronto-ocular area (Fig. 6A) well developed, triangular.
Clypeus moderately covered with dark long hairs except
median portion largely bare. Labrum 0.56 times as long as
clypeus. Maxillary palp brownish black, with 5 segments,
proportional lengths of 3rd, 4th, and 5th segments 1.0:1.1:
2.6; 3rd segment (Figs. 6B, C) not enlarged; sensory vesicle
ellipsoidal or oblong, with rugged surface, 0.38―0.43 times
length of 3rd segment, with a large opening. Maxillary lacinia with 12 or 13 inner and 15 or 16 outer teeth. Mandiblewith 28―30 inner and 15 outer teeth. Cibarium (Fig. 6D)with a cluster of about 56 conical processes medially. Thorax. Nearly as in female of S. mediocoloratum except
scutum almost black and scutellum dark brown. Legs. Fore-３２０leg (Fig. 6E): coxa pale yellowish-white; trochanter paleyellowish-white with outer surfaces somewhat yellowish;
femur yellow with extreme apex somewhat darkened; tibia
pale yellowish-white except apical 1/5 brownish black, witha large white sheen on outer surface when illuminated; tarsus black; basitarsus dilated, 5.4 times as long as its greatest
width. Midleg (Fig. 6F): coxa dark brown; trochanter yellow with apical 1/2 somewhat darkened; femur entirely yellow; tibia yellowish-white to yellow, with a large white
sheen on posterior surface when illuminated; tarsus medium
to dark brown except basal 2/3 of basitarsus and base of 2nd
segment yellowish white. Hind leg (Fig. 6G): coxa darkbrown; trochanter yellow; femur yellow with apex medium
brown narrowly; tibia yellowish-white to yellow with apicalcap dark brown on inner surface (Fig. 6H) but there is abroad light brown area extending forwardly from dark apical cap to a little less than basal 1/2 on outer surface (Fig. 6G), and with a large white sheen on posterior surface whenilluminated; tarsus brownish black except basal 3/5 of basi-

tarsus and basal 1/3 of 2nd tarsal segment yellowish white;basitarsus (Fig. 6I) nearly parallel-sided, 5.7 times as longas wide, and 0.7 and 0.6 times as wide as the greatest
widths of hind tibia and femur, respectively; calcipala short,
0.8 times as long as its basal width and 0.4 times as wide as
the greatest width of basitarsus; pedisulcus distinct at basal
1/3 of 2nd tarsal segment. Tarsal claws simple without any
tooth. Wing. Length 2.8 mm; other characters as in femaleof S. mediocoloratum. Abdomen. Basal scale medium
brown with a fringe of pale hairs; dorsal surface of abdomen dark brown to brownish black except anterior 1/2 medium brown and with a dorsolateral pair of shiny whitish
spots broadly connected in middle, tergites 6―8 shiny, covered with dark short hairs. Genitalia (Fig. 6J―L). Ventral
surface of abdominal segment 7 with a pair of weaklydeveloped sternal plates. Sternite 8 well sclerotized, baremedially, with 5―8 pale short setae submedially near posterior border, and with 24 or 25 dark long hairs laterally oneach side; ovipositor valve triangular in shape, membranous,

Fig. 6. Adult female of Simulium (Simulium) crocinum sp. nov. A, fronto-ocular area (right side, frontal view); B and C, 3rd segments of maxillary palp (B, right side; C, left side; both frontal view); D, cibarium; E-G, foreleg, midleg and hind leg, respectively (all left side); H, hind tibia (left side, inside view); I, hind basitarsus and 2nd tarsal segment (outer view); J, 8th sternite,
ovipositor valves, genital fork and spermatheca (ventral view); K, paraproct (right side, ventral view); L, paraproct and cercus(right side, lateral view). Scales. 0.02 mm for D and J―L; 0.05 mm for A―C; 0.1 mm for I; 0.2 mm for E―H.

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１

expanded ventrally, each covered with about 20 short setae
as well as numerous microsetae except narrow portion
along inner margin and rather wide portion along posterior
margin bare and transparent; inner borders not sclerotized,
moderately separated from each other. Genital fork of
inverted-Y form, with well sclerotized stem; arms rather
wide, each with strongly sclerotized distal ridge having a
distinct projection directed anterodorsally. Paraproct in ventral view rounded posterolaterally, with anteromedial surface moderately sclerotized, having about 10 short spinous
setae as well as numerous microsetae on it; paraproctslightly produced ventrally along medial margin, covered
with numerous stout hairs laterally and ventrally. Cercus
rounded posteriorly, about 0.6 times as long as wide, covered with numerous stout hairs. Spermatheca ellipsoidal,
well sclerotized (except small area of its juncture with duct
unsclerotized), with reticulate surface pattern at least nearbase, with minute internal setae; both accessory ducts subequal in width to main one.
Male. Body length 3.0―3.3 mm. Head. Slightly wider
than thorax. Upper eye consisting of large facets in 20 horizontal and 22 vertical rows. Clypeus black, white pruinose,
iridescent when illuminated, sparsely covered with darkbrown hairs (median portion largely bare or with a few
hairs). Antenna composed of 2+9 segments, dark yellow tolight yellowish-brown on scape, pedicel, and a few basal
flagellar segments, and the rest medium to dark brown; 1stflagellar segment elongate, 1.8 times as long as 2nd one.
Maxillary palp composed of 5 segments, proportional
lengths of 3rd, 4th, and 5th segments 1.0:1.1:2.3; 3rd segment (Fig. 7A) of normal size, with an ellipsoidal sensory
vesicle having a small opening apically; sensory vesicle
0.25 times as long as 3rd segment. Thorax. Nearly as inmale of S. mediocoloratum. Legs. Foreleg (Fig. 7B): coxa
pale yellow; trochanter yellow with outer and posterior portions somewhat darkened; femur yellow on inside surface
but dark yellow or light brown on outer surface; tibia medium brown to brownish black with basal 3/4 narrowly
white on outer surface (white sheeny when illuminated);tarsus black, with moderate dorsal hair crest; basitarsus
somewhat dilated, about 5.3 times as long as its greatest
width. Midleg (Fig. 7C): coxa dark brown or blackish; trochanter medium brown with base yellow; femur entirely
yellow; tibia entirely yellow, with a white sheen widely on
posterior surface when illuminated; tarsus brownish black
except basal 2/3 of basitarsus yellow (though its border not
well defined) and extreme base of 2nd segment pale yellow.
Hind leg (Fig. 7D): coxa dark brown; trochanter yellow; femur yellow except apical cap medium brown; tibia darkbrown to brownish black with base whitish yellow (in another male, anterior surface of tibia pale more extentively

from base to near apical cap); tarsus brownish black except
a little more than basal 1/2 of basitarsus whitish yellow andbasal 1/3 of 2nd tarsal segment dark yellow; basitarsus (Fig.
7E) somewhat widened from base toward apical 3/4, thensomewhat narrowed toward apex, 4.3 times as long as its
greatest width, and 0.8 times as wide as the greatest widthof hind tibia which is as wide as that of femur; calcipala ofmedium size, nearly as long as wide; pedisulcus distinct.
Wing. Length 2.6 mm; other characters as in male of S.
rudnicki. Abdomen. As in male of S. rudnicki. Genitalia(Fig. 7F―N). Coxite in ventral view nearly quadrate. Styleelongate, spatulate ventrodorsally, 1.7 times as long as
coxite, gradually narrowed from base to apical 1/3, thenslightly widened to rounded apex, width near apex 0.75
times as wide as basal width, and about 1.4 times as wide as
the narrowest width (in another male, style gradually narrowed from base to middle, then slightly widened toward
apex, width near apex 0.85 times as wide as basal width,and about 1.3 times as wide as the narrowest width, asshown in Fig. 7G), with no subterminal spine (though rightstyle of 1 male bears a slender hair-like spine in place ofusual needle-like subterminal spine, as shown in Fig. 7G);
style with a prominent basal protuberance pointed dorsallyand furnished at and near apex with several conical spines
mostly on anterior surface. Ventral plate in ventral viewtransverse, somewhat wider than its length, rounded posterolaterally, with posterior margin untoothed and slightly
concave medially, and covered with fine setae on anterior
1/2 of ventral surface; basal arms long and stout, directed
forwardly and somewhat outwardly. Parameres wide basally, each with numerous parameral hooks. Median scleritein end view widened from base to basal 1/3, somewhat narrowed to apical 1/3, then widened toward apex, plate-like,
and well sclerotized except apical portion. Aedeagal membrane densely covered with needle-like setae and with wellsclerotized narrow dorsal plate. Segment 10 with 2 distinct
hairs on each lateral side (in another male, 1 distinct hair onright lateral side and no hair on left lateral side). Cercus
rounded with 7―10 distinct hairs.
Pupa. Body length (excluding gill filaments) about 3.0
mm. Head. Integument medium to dark brown, bare; facewith a pair of long simple or bifid trichomes; frons with 2
pairs of long bifid trichomes somewhat separated from eachother; antennal sheath bare. Thorax. Integument medium todark brown, bare on anterior 1/2 (except a few small raised
areas at base of gill densely covered with tubercles), moderately covered with small cone-shaped tubercles dorsally on
posterior 1/2; thorax anteriorly with 3 dorsal and 2 lateralpairs of long simple and/or branched trichomes (split into
2―4), posteriorly with 1 lateral pair of long bifid or trifid
trichomes, and ventrolaterally with 3 pairs of long simple

３
２
２

Fig. 7. Adult male of Simulium (Simulium) crocinum sp. nov. A, 3rd segment of maxillary palp (right side, frontal view); B―D, foreleg, midleg and hind leg, respectively (all left side); E, hind basitarsus and 2nd tarsal segment (left side, outside view); F,
coxite and style (right side, ventrolateral view); G and H, styles (G, right side, of different male, ventrolateral view; H, left
side, outer view); I―K, ventral plates (I, ventral view; J, lateral view; K, end view); L, median sclerite; M, dorsal plate; N, abdominal tip showing cercus (lateral view). Scales. 0.02 mm for F―N; 0.05 mm for A; 0.1 mm for E; 0.2 mm for B―D.

and/or bifid trichomes. Gill (Fig. 8A) with 6 filaments arranged in sessile pairs, shortened in length from dorsal toventral, outer filament of dorsal pair longest of all (1.0―1.5
mm long); usually outer filament of each pair somewhat
longer than its counter inner filament; outer filament of dorsal pair slightly thicker than outer filament of middle pair,which is much thicker than the other filaments; inner filament of dorsal pair usually somewhat thicker than inner
filament of middle pair and both inner and outer filamentsof ventral pair; outer filaments of dorsal and middle pairs
dark brown to brownish black at least basal 1/3, then gradually become paler toward apex, basal portions of these two
filaments much darker than the other filaments which are
uniformly light brown; outer filaments of dorsal and middle
pairs gradually tapered toward apical tip, all inner filaments

and outer filament of ventral pair nearly parallel-sided along
basal 1/3 or 1/4, then tapered toward apical tip; all filamentswith annular ridges and furrows throughout their length,and densely covered with minute tubercles; gill with mediumsized transparent protuberance at base. Abdomen. Dorsally,
segment 1 greyish light-brown, with 1 medium-long simple
hair-like seta on each side; segment 2 grey on anterior 2/5,transparent on posterior 3/5, with 1 medium-long simple
hair-like seta, 4 short simple hooklets of equal size (muchsmaller than those on segments 3 and 4) and 1 short spinous
seta on each side; segments 3 and 4 each with 4 hooked
spines and 1 short simple hooklet along posterior margin oneach side; segments 5―9 each with a transverse row of
comb-like groups of minute spines (though those on segment 5 very weakly developed) and lacking spine-combs on

３
２
３

each side except segment 8 with a transverse row of distinct
spine-combs; segment 9 lacking terminal hooks. Ventrally,
segment 4 with 1 simple hook, 1 simple hooklet and 2 shortsetae on each side; segment 5 with a pair of simple hooks
submedially on each side; segments 6 and 7 each with a pairof simple (or bifid) inner and simple outer hooks somewhat
separated from each other, on each side. Grapnel-like hooklets absent. Cocoon (Fig. 8B,C). Wall-pocket-shaped, witha medium to large anterolateral window on each side; thick,individual threads not visible; anterior margin well defined;cocoons attached on sticks and grass leaves thickly woven
and well pigmented but those on rock surfaces usuallythinly woven, less pigmented and sometimes nearly transparent; 3.2―3.6 mm long by 1.7―2.0 mm wide.
Mature larva. Body length 6.5―7.0 mm. Body color
light to medium brown. Abdomen gradually widened toward segment 7, then narrowed to posterior tip whenviewed laterally; segments 6 and 7 appear to be equal inwidth to each other when viewed dorsally. Cephalic apotome (Plate 1C) pale white on anteior 1/2 and pale to dark
yellow on posterior 1/2 with a small dark area in middle
along posterior border, with mediolateral spots and posteromedial longitudinal spot always distinctively positive(and rarely anteromedial longitudinal spot and posterolateral spots faintly positive); lateral surface yellowish
brown to dark brown except eye-spot region and its dorsal
area pale white to yellow; spots around eye-spot region
faintly to distinctively negative; ventral surface (Plate 1G)
light to dark brown with elongate spot on each side of postgenal cleft negative. Antenna composed of 3 segments andapical sensillum, much longer than stem of labral fan;length ratio of segments (from base to tip) 1.0:1.2:0.4; allsegments pale white to yellow except 3rd segment entirely

brownish black and apical small portion of 2nd segment
darkened to some extent. Labral fan with 40―44 main rays.
Mandible (Fig. 8D) with usual mandibular serration of 1
medium-sized tooth and 1 small one, without supernumerary serrations; main mandibular tooth at an obtuse angle tothe mandible on apical side; comb-teeth decreasing inlength from 1st to 3rd. Hypostomium (Fig. 8E) with 9 apical teeth, of which median tooth and each corner tooth subequal in size, longer than others; lateral margins moderately
serrate apically; 8―10 hypostomal bristles diverging posteriorly from lateral border on each side. Postgenal cleft (Plate
1G) bullet-shaped, somewhat pointed apically, 1.9―2.3 times
as long as postgenal bridge; subesophagial ganglion wellpigmented, wine-glass shaped. Cervical sclerite composedof a pair of small rod-like pieces, widely separated in middle from each other, not fused to occiput. Thoracic cuticle
almost bare. Abdominal cuticle bare except last segment
moderately covered with short colorless setae on each sideof anal sclerite; rectal scales present (indistinct, then often
overlooked). Rectal organ of 3 lobes, each with 19―22
finger-like secondary lobules. Anal sclerite X-shaped, withbroadened anterior arms about 0.7 times as long as posterior
ones; 16―18 sensilla just posterior to posterior arms. Ventral
papillae absent. Posterior circlet with about 136 rows ofhooklets, with up to 18―22 hooklets per row.
TYPE SPECIMENS. Holotype female, with associated pupal exuvia and cocoon, collected from a small stream, Siribhume waterfall, Doi Inthanon National Park, Chiang Mai,
Thailand, 28. II. 2004, by W. Choochote. Paratypes: 2
males (reared from pupae), and 13 pupae, same data anddate as holotype; 11 pupae and 6 mature larvae, same data
as holotype but date, 15. II. 2004.

Fig. 8. Pupa and mature larva of Simulium (Simulium) crocinum sp. nov. A, pupal gill filaments; B and C, cocoons (B, dorsal view;C, lateral view); D, larval mandible; E, larval hypostomium. Scales. 0.02 mm for D; 0.05 mm for E; 0.1 mm for A; 1.0 mm
for B and C.

３２
４

ECOLOGICAL NOTES. The pupae and larvae of S. crocinum sp. nov. were found on the rock surface of a moderately flowing mountainous stream 0.5 m wide, exposed tothe sun. A few pupae were also collected on trailing grasses
and slender sticks in the fast flowing water. Water temperature was 18.5°
C. Altitude was ca. 1,500 m.
This species was collected together with S. (S.) rufibasis Brunetti, S. (S.) mediocoloratum sp. nov., S. (Gomphostilbia) inthanonense Takaoka and Suzuki, S. (Nevermannia) feuerborni Edwards and S. (Montisimulium) sp.
ETYMOLOGY. The specific name crocinum refers to theyellowish legs of both sexes of this new species. The Latin
word crocinus means yellow.
DISTRIBUTION. Thailand.
REMARKS. Simulium crocinum sp. nov. is assigned to thegriseifrons species-group, as in the previous species.
The female of this new species resembles those of S.
rudnicki and S. mediocoloratum in many characters including the antenna with pits and the ovipositor valve withtransparent inner and posterior margins (Fig. 6J), but is differentiated from the latter two species by the dorsal surfaceof the anterior 1/2 of the second abdominal segment medium brown (cf. white or yellowish white in S. rudnicki and
S. mediocoloratum), the fore tibia pale yellowish-white except apical 1/5 brownish black (cf. usually dark portion extending from the apical cap to basal 1/2 or more along theinside margin in S. rudnicki and S. mediocoloratum), thehind tibia yellowish-white to yellow except the apical cap
dark brown at least on the inside surface though the outersurface with an excess light brown portion extending fromthe apical cap to a little less than basal 1/2 (Fig. 6G) (cf.
hind tibia light to dark brown or brownish black except basal 1/3 and posterior surface of basal 2/3 white in S. rudnicki and S. mediocoloratum), and the ellipsoidal spermatheca (Fig. 6J) (cf. globular in the other two species).
The female of this species is also distinguished from S. mediocoloratum by the entirely yellowish-white mid tibia.
The male of S. crocinum is very similar to that of S.
rudnicki, but is barely distinguished by the hind tibia darkbrown to brownish black with basal 1/4 or 1/5 whitish yellow (Fig. 7D) (cf. only the extreme base of hind tibia white
in S. rudnicki) and the style without subterminal spine (Fig.
7F) (subterminal spine present in S. rudnicki). On the other
hand, this new species is readily separated in the immature
stages from S. rudnicki and S. mediocoloratum: in the pupa
by the gill with two filaments much thicker than the otherfour filaments (Fig. 8A) and the light to medium brown,
thickly-woven cocoon with an anterolateral window oneach side (Fig. 8B,C), and in the larva by the cephalic apotome with its ground color mostly pale (Plate 1C).
The male genitalia, leg colors and the pupal gill filaments of S. crocinum are very similar to those of S. ufengense Takaoka, described from Taiwan [6], and of S. fuzhouense Zhang and Wang, from Fujian, south China [7].
However, this new species is easily separated from these
two species by the fenestrated cocoon (Fig. 8B,C) (cf. cocoons simple without lateral windows in S. ufengense and S.
fuzhouense [6, 7]).
Simulium taipokauense from Hong Kong [8] showssome similarities to S. crocinum but differs in that it has themuch longer basal protuberance of the style and the saddleshaped ventral plate with a nipple-like median process.
Simulium (Simulium) suchariti Takaoka and Choochote,2004
Simulium (Simulium) suchariti Takaoka and Choochote,2004: 31―33 (female only).
DESCRIPTION. Pupa. Body length (excluding gill filaments) 3.5―4.0 mm. Head. Integument yellowish brown,densely and elaborately covered with tubercles of various
sizes (Fig. 9A); all tubercles but small ones with several
minute projections on their surface; antennal sheath also
densely covered with tubercles; face with a pair of mediumlong trifid or quadrifid trichomes (Fig. 9B); frons with 2
pairs of medium-long branched (split into 4―6) trichomesslightly shorter than facial ones (Fig. 9C); 2 frontal
trichomes on each side arising close together. Thorax. Integument yellowish brown, moderately covered with tubercles of various sizes (Fig. 9D) (similar to those on head but
most are much larger) on anterior 1/2 including areas nearbase of gill, and moderately covered with small coneshaped tubercles dorsally on posterior 1/2; thorax anteriorly
with 3 dorsal and 2 lateral pairs of very long fan-liketrichomes each with 20―31 branches (Fig. 9E) (about twice
as long as facial trichomes), posteriorly with 1 lateral pairof medium-long trichomes each with 7-10 branches (Fig. 9F), and ventrolaterally with 3 pairs of long and mediumlong branched (split into 2―7) trichomes. Gill (Fig. 9G)
with 6 filaments arranged in pairs; all pairs almost sessile
except dorsal pair with very short stalk; all filaments usuallypale, gradually decreasing in length and thickness from dorsal to ventral; outer filament of dorsal pair longest (1.8―2.0mm) and thickest of all, and inner filament of lower pairshortest (0.5―0.8 mm) and thinnest (about half times as
thick as the thickest dorsalmost filament); all filaments
gradually tapered toward apical tip, with annular ridges andfurrows throughout their length, and densely covered withminute tubercles. Abdomen. Dorsally, segment 1 light yel-３２
５

low with narrow pale portion along posterior margin, with 1
bifid or trifid (or quadrifid) medium-long seta on each side;
segment 2 almost pale, nearly transparent, with 1 simple orbifid medium-long seta, 1 short minute seta and 4 short simple spines of equal size on each side; segments 3 and 4 eachwith 4 hooked spines along posterior margin on each side;
segment 5 bare; segments 6―9 each with a transverse row of
comb-like groups of minute spines on each side; segment 8
with a transverse row of distict spine-combs on each side;
segment 7 sometimes with a transverse row of spine-combs,
though much fewer in number and smaller in size thanthose on segment 8; segment 9 lacking terminal hooks. Ventrally, segment 4 with 1 simple hook (similar in size tothose on segments 5―7) and a few simple or bifid short setaeon each side; segment 5 with a pair of simple or bifid hooks
submedially and a few simple or bifid hooklet-like setae oneach side; segments 6 and 7 each with a pair of bifid innerand simple or bifid outer hooks somewhat separated fromeach other and a few simple or bifid setae on each side.

Grapnel-like hooklets absent. Cocoon (Fig. 9H―J). Wallpocket-shaped, with a large anterolateral window (in some
cocoons 1 of 2 windows is small, or even absent, though thewindow on the other side is large; in other cocoons, window
is divided into 2 or 3 spaces of various sizes) on each side;
ventolateral tips of anterior margin approaching to eachother anteroventrally, sometimes connected to each other;cocoon thin and usually pale; individual threads partiallyvisible; 5.0―5.2 mm long by 1.8―2.0 mm wide.
Mature larva. Body length 7.5―8.0 mm. Body color
greyish. Abdomen gradually widened toward posteriorly,
with the widest portion on segment 7. Cephalic apotome(Plate 1D) almost entirely pale yellow except a small somewhat dark area in middle along posterior border (though
mediolateral spots and anterior ones of posterolateral spots
very faintly positive in 1 of 3 larvae examined); lateral surface entirely pale yellow without any dark spot; ventral surface (Plate 1H) somewhat darkened with elongate spot oneach side of postgenal cleft usually faintly negative. An-

Fig. 9. Pupa and mature larva of Simulium (Simulium) suchariti Takaoka and Choochote. A―G, pupa; K―M, larva. A, tubercles onfrons; B, trichome on face; C, 2 trichomes on frons (right side); D, tubercles on dorsal surface of thorax; E, trichome and tubercles near its base on dorsal surface of thorax; F, trichome on posterolateral surface of thorax; G, gill filaments; H―J, cocoons (H and I, dorsal view; J, lateral view); K, antenna (left side, dorsal view); L, mandible; M, hypostomium. Scales. 0.01
mm for A and D; 0.02 mm for B, C, F and L; 0.05 mm for E, K and M; 0.1 mm for G; 1.0 mm for H―J.

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６

tenna (Fig. 9K) composed of 3 segments and apical sensillum, much longer than stem of labral fan; length ratio ofsegments (from base to tip) 1.0:1.6:0.7; all segments pale or
yellow. Labral fan with about 40 main rays. Mandible (Fig.
9L) with usual mandibular serration of 1 medium-sized
tooth and 1 small one, without supernumerary serrations(though left mandible of 2 larvae with 1 additional small
tooth); main mandibular tooth at an obtuse angle to themandible on apical side; 1st comb-tooth longest and thickest, followed by 2nd and 3rd teeth of similar size. Hypostomium (Fig. 9M) with 9 apical teeth, of which median
tooth subequal in size to, or a little larger than, each corner
tooth, and intermediate teeth small; lateral margins moderately serrate apically; 7―10 hypostomal bristles diverging
posteriorly from lateral border on each side. Postgenal cleft(Plate 1H) bullet-shaped, rounded apically, 1.8―2.0 times aslong as postgenal bridge; subesophagial ganglion not visible. Cervical sclerite composed of a pair of small rod-likepieces, widely separated in middle from each other, notfused to occiput. Thoracic cuticle almost bare. Abdominal
cuticle bare except last segment moderately covered withshort colorless setae on each side of anal sclerite; rectal
scales present. Rectal organ of 3 lobes, each with about 17
finger-like secondary lobules. Anal sclerite X-shaped, withbroadened anterior arms about 0.64 times as long as posterior ones; 10―20 sensilla just posterior to posterior arms.
Ventral papillae absent. Posterior circlet with about 120

Plate 1.

rows of hooklets, with up to 18―20 hooklets per row.
SPECIMENS EXAMINED. 2 pharate females, 8 pupae, 1
pupal exuvia and 5 mature larvae, all collected from a small
forest stream, Ang Ka, Doi Inthanon National Park, Chiang
Mai, Thailand, 10. XII. 2003, by W. Choochote.
ECOLOGICAL NOTES. The pupae and larvae of S.
suchariti were found on the surface of the rocky stream-bed
of a shaded stream 0.3 m wide, slow-flowing in natural forest. Water temperature was 6.5°
C. Altitude was 2,565 m.
This species was collected together with S. (S.) setsukoae Takaoka and Choochote, S. (Montisimulium) sp.
and S. (Nevermannia) caudisclerum Takaoka and Davies.
DISTRIBUTION. Thailand.
REMARKS. Simulium suchariti differs from the three related species treated above in the pupa by the head and thoracic integuments moderately or densely covered with tubercles as well as the arrangement of the gill filaments (Fig.
9A,G), and in the larva by the pale cephalic apotome (Plate
1D), the antenna with the third segment not darkened compared to the first and second segments (Fig. 9K) and thesubesophageal ganglion not visible (Plate 1H).
The pupa and larva of S. suchariti are similar to thoseof S. maenoi Takaoka and Choochote, described from Thai-

Larval head capsules of four Simulium (Simulium) species. A and E, S. rudnicki Takaoka and Davies; B and F, S. mediocoloratum sp. nov.; C and G, S. crocinum sp. nov.; D and H, S. suchariti Takaoka and Choochote. A―D, dorsal view; E―H,
ventral view.

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７

land [9] in the arrangement of the pupal gill filaments (Fig.
9G), and the pale larval cephalic apotome (Plate 1D) andthe shape of the larval body. However S. suchariti is readily
distinguished from S. maenoi in the pupa by the absence ofthe terminal hooks, head integument with tubercles of different sizes (Fig. 9A), and dorsal trichomes of the thorax
with 20-31 branches (Fig. 9E), and the cocoon with a large
anterolateral window on each side (Fig. 9J), and in the larva
by the pale ventral surface of the head capsule (Plate 1H).

ACKNOWLEDGEMENTS
We are grateful to Mr. Surachai Tuamsomboon, Chief,
Headquarters of Doi Inthanon National Park, Wildlife andPlant Conservation Department, Ministry of National Resources and Environment, for his permission to conduct thissurvey inside the park. Thanks are due to Dr. Y. Otsuka and
Ms. C. Aoki, Department of Infectious Disease Control,
Faculty of Medicine, Oita University, who kindly took photographs of the larval heads needed for this paper. We are
grateful to Dr. R.W. Crosskey, Natural History Museum,London, U.K. for valuable suggestions on names of the new
species.
This work was financially supported by the Grant-inAid of Japan-US Medical Research Cooperation Program(the fiscal year 2003).

LITERATURE CITED
１．Takaoka H, Choochote W. A list of and keys to black flies(Diptera: Simuliidae) in Thailand. Trop. Med. Hlth. 2004;
32 (2): 189―197.
２．Takaoka H, Davies DM. The black flies (Diptera: Simuliidae) of West Malaysia. viii + 175p. Kyushu University
Press, Fukuoka; 1995.
３．Takaoka H, Choochote W. Two new species of Simulium(Simulium) (Diptera: Simuliidae) from Thailand. Trop.
Med. Hlth. 2004; 32 (1): 31―36.
４．Takaoka H. The black flies (Diptera: Simuliidae) of Sulawesi, Maluku and Irian Jaya. xxii + 581p., Kyushu University Press, Fukuoka; 2003.
５．Takaoka H, Davies DM. Simulium (Simulium) yongi sp.
nov. (Diptera: Simuliidae) from Peninsular Malaysia. Jpn.
J. Trop. Med. Hyg. 1997; 25 (1): 11―16.
６．Takaoka H. The black flies of Taiwan (Diptera: Simuliidae). Pac. Insects 1979; 20: 365―403.
７．Zhang T, Wang DQ. A new species of Simulium from Fujian, China (Diptera: Simuliidae). Acta Zootax. Sin.
1991; 16: 109―113.
８．Takaoka H, Davies DM, Dudgeon D. Black flies (Diptera: Simuliidae) from Hong Kong: Taxonomic notes
with descriptions of two new species. Jpn. J. Trop. Med.
Hyg. 1995; 23: 189―196.
９．Takaoka H, Choochote W. Taxonomic notes on thegriseifrons species-group in Simulium (Simulium) (Diptera: Simuliidae) from Thailand: Descriptions of two new
species and description of the male, pupa and larva of S.
(S.) digrammicum Edwards. Jpn. J. Trop. Med. Hyg.
2002; 30: 115―132.

Tropical Medicine and Health Vol. 32 No. 4, 2004, pp. 329-333
Copyright! 2004 by The Japanese Society of Tropical Medicine

３２
９

CURRENT STATUS OF RESPIRATORY DISEASESSUFFERED BY JAPANESE PEOPLE
LIVING IN SOUTHEAST ASIA
AKIRA UCHIKOSHI１, ATSUO HAMADA１, EIICHI,OKUZAWA１, KIYOMI HONDO１,
３
４
５
NORIHIKO OOKUBO２, VARPHAN UNACHAK , JACOB THOMAS , TETSUYUKI MORIKAWA ,５
１
KOUICHIRO TAKEUCHI , NORIHIKO BASUGIAccepted 17, september, 2004
Abstract: We gathered and analyzed date on respiratory diseases suffered by Japanese adults treated in Ram Hospital in Chiang Mai and Subang Jaya Medical Center in Kuala Lumpur. In both hospitals, the percentages of patients undergoing treatment for respiratory diseases was the greatest. Of these, relatively mild symptoms such as
upper respiratory tract infection account for the majority of the diseases, but also included are instances of lower
respiratory tract infection or chronic respiratory diseases such as chronic sinusitis and COPD/bronchial asthma. In
Kuala Lumpur, we conducted a questionnaire-based survey targeted on Japanese people living there, in order to detemine the currnt status of respiratory deseases. The date showed that many Japanese had symptoms of respiratory
diseases and felt that air pollution was serious.
It is important for Japanese people living in Asia to be aware of preventative measures to prevent respiratory
diseases, such as those caused by air pollution and infection.
Key words: Japanese people living abroad;Respiratory diseases; Air pollution

INTRODUCTION
During the post-war era, the number of Japanese traveling and living abroad long-term increased dramatically.
According to statistics from the Ministry of Justice, those
who went abroad in 2000 exceeded 17 million; roughly
double that of 10 years ago. Also, according to statistics
from the Ministry of Foreign Affairs, long-term overseas
residents (three months or more) reached an all-time high of
540,000 in 2001. Particularly, the percentage of Japanesestaying long-term in the Asian region has reached about
20% of the world total, and is increasing yearly.
In the past, health problems for Japanese people living
abrond consisted mainly of infection, such as malaria anddiarrhea. Recently, however, respiratory diseases have been
increasing. Respiratory organs are sensitive to external influences, especially to changes in temperature and humidity.
These are the main causes of respiratory diseases.
Also, rapid economic development, with its accompanying air pollution, is becoming serious in Asia. Reports
from WHO rank cities in Asia, including those in China, In123
45

dia ets., as having the worst air pollution problems [1].
Many reports have alreadey linked the air pollution problem
to respiratory diseases [2]. An effective response to thisproblem is needed because increasing numbers of Japanesepeople are going to live in the Asian region.
To date, research on diseases suffered by Japaneses,
European and American people living abroad has been conducted frequently [3] [4].As a result, it is clear that there aremany cases of respiratory diseases among those treated inlocal medical facilities. For example, research done by us in2003 at two medical facilities in Southeast Asia (Thailand
and Malaysia) on Japanese adults showed that the largest
number of cases encountered were respiratory diseases [5].
Therefore, this time we concentrated on analyzing the datafrom these two facilities in order to learn more clearly theextent of respiratory diseases. We also analyze date fromquestionnaires given in Kuala Lumpure, Malaysia to learn
the extent of respiratory diseases suffered by Japanese living there.
On the basis of these analyses, we describe the current
status of respiratory diseases afflicting Japanese people liv-

Japan Overseas Health Administration Center, Japan Labour Health and Welfare Organization
Japan Overseas Medical Fund
Chiang Mai Ram Hospital
Subang Jaya Medical Center
Respiratory Disease Section,Yokohama Rosai Hospital３
３０

ing in Southeast Asia and the preventative measures used tocombat these health problems.

METHODS(1)Survey on diseases under medical treatment in Kuala
Lumpure, Malaysia and Chiang Mai, Thailand.
The medical facilities covered by this research are:Ram Hospital in Chiang Mai, Thailand and Subang Jaya
Medical Center in Kuala Lumpur, Malaysia. Ram Hospital,
opened in 1993, is a private hospital with 300 beds, used bymost of the Japanese people living in the city of Chiang Mai(Japanese population approximately 1,000). Subang Jaya
Medical Center, opened in 1985, is a private hospital with400 beds, located in the Subang Jaya area in the suburbs ofKuala Lumpur (Japanese population approximately 6,000).
There are other hospitals in the city, such as Pantai Medical
Center, Japan Medicare Clinic and Gleneagles Medical
Center, each of which is used by many Japanese people.
The research spanned two years from January 2000 to
December 2001. We gathered information including age,gender and diseases of Japanese being treated as out patients. To protect privacy, we gave patients ID numbers instead of using names. The number shown for each month
represents the total number of treatments (some patientswere counted more than once). The diseases treated arenumbered 1 to 21 in accordance with the International Classification of Diseases (ICD-10). While most of patientscoverd are long-term residents, the date also includes anumber of short-stay tourists.
(2)Questionnaire survey in Kuala Lumpur, Malaysia
Questionnaires were given to adults in Kuala Lumpur,
Malaysia when they came for a health consultation conducted in February 2003 by us. The contents of the ques-tionnaire consisted of the followin items: ! Do you havesymptoms of respiratory disease? " If so, what are thesymptoms? # When did you begin to feel the symptoms?
④ Do you feel that air pollution is serious? $ Do you feelthat air pollution is harmful to your health? % What do you
think is the cause of the air pollution?

RESULTS
１）Diseases suffered by Japanese patients treated in Ram
Hospital
Out of a total of 5,379 Japanese patients, 4,315 were
adults (16 and over). The 30 to 39 age group was the largest,with 1,217 patients, followed by 20 to 29 with 937, and 40
to 49 with 773. By gender, 2,482 were male and 1,332 werefemale (1unknown). The average monthly number of treatments was 180, with more during the dry season from November to February and the rainy season from July to August. The fewest number of treatments occurred durting thehottest month of April.
Over the two-year period of the study, we noted thatthe largest number (739 patients representing 17.1%) suffered respiratory diseases, followed by digestive tract diseases, infections, injuries and skin diseases.
Out of these respiratory diseases, upper respiratory
tract infection such as common cold and laryngopharyngitis
accounted for more than half, (59.4%) of the total, followed
by bronchitis (13.9%), tonsilitis (10.8%), chronic sinusitis(7.4%), chronic obstructive pulmonary disease (COPD) /bronchial asthma (4.2%) (Table 1). The number of patientstreated on a monthly basis shows certain unevenness between 2000 and 2001, though the trend was high in the dry
season from November to February (Figure 1).

Table 1. Number of respiratory diseases
Ram Hospital
2000

2001

Total (%)

Subang Jaya Medical Center
2000

2001

Total (%)

&Upper Respiratory Tract Infection
Common Cold
Laryngopharyngitis
Tonsilitis

132
98
36

116
94
44

248 (33.5%)
192 (25.9%)80 (10.8%)

120
45
7

144
37
7

264 (51.3%)
82 (15.9%)
14 (2.7%)'Lower Respiraory Tract Infection
Bronchitis
Pneumonia

44
0

59
0

103 (13.9%)
0

8
0

1
0

9 (1.2%)0(Chronic Sinusitis

41

14

55 (7.4%)

15

15

30 (5.8%))COPD/Bronchial asthma

15

16

31 (4.2%)

21

13

34 (6.6%)

３
３
１
（n）
50
40
30
20
10
0
2000.1 2

3

4

5

6

7

8

9

10 11 12 2001.1 2

3

4

5

6

7

8

9

10 11 12Figure 1. Ram Hospital
The monthly trend of respiratory disease patients

２）Diseases suffered by Japanese patients treated in
Subang Jaya Medial Center
Out of a total of 7,313 patients, 4,570 were adults. The
30 to 39 age group was the largest, with 2,066 patients, followed by 40 to 49 with 1,066 and 20 to 29 with 630. By
gender, 2,403 were male and 2,165 were female (two unknown). The average monthly number of treatments was
190, with more during the relatively dry season from May
to July.
Over the two-year period of the study, we noted thatthe largest nuver (514 patients representing 11.2%) sufferedrespiretory diseases, followed by occular diseases, various
clinical symptoms, skin diseases and muscle and skeletal
diseases.
Out of these respiratory deiseases, acute upper respiratory tract infection such as common cold and laryngopharyngitis accounted for about 67% of the total, follwed by
COPD / bronchial asthma (6.6%), chronic sinustitis (5.8%),
tonsilitis (2.7%), bronchitis (1.2%) (Table 1). The monthly
trend was high for treatments occurring during the dry season from May to July and in December (Figure 2).

Table 2. Result of questionnaire survey in Kuala Lumpur!Do you have symptoms of respiratory disease?
yesno"If so, what are the symptoms?
sticking sputum
coughing
shortness of breathnasal secretion and congestion
sore throat
hoarse voice
wheezing#When did you begin to feel the symptoms?
before arrival
after arrival
not sure or did not answer$Do you feel that air pollution is serious?
yesno%Do you feel that air pollution is harmful to your health?
yesno&What do you think is the cause of the air pollution?
industrial gas
automobile exaust
climateEnviromental sanitation
Haze (smog from forest fire)

（n）
35
30
25
20
15
10
5
0
2000.1 2

3

4

5

6

7

8

9

10 11 12 2001.1 2

Figure 2. Subang Jaya Medical Center
The monthly trend of respiratory disease patients

3

4

5

6

7

8

9

10 11 1227 (42.9%)
36 (57.1%)
19 (30.2%)
18 (28.6%)
17 (27.0%)
10 (15.9%)
7 (11.1%)
6 (9.5%)3 (4.8%)9 (33.3%)
4 (14.8%)
5 (18.5%)
36 (57.1%)
24 (38.1%)
18 (28.6%)
38 (60.3%)
8 (12.7%)
26 (41.3%)
6 (9.5%)2 (3.2%)27 (42.9%)

３３２３）Results of the questionnaire survey in Kuala Lumpure(Table2)
The total number of responders was 63, of whom 37
were male and 26 female. The average age was 55 and 47 ofthese had live there for two years or more.
Those who had symptoms of respiratory disease numbered 27, a high proportion of the total (42.9%). Of these27, many suffered from light symptoms of upper tract infection, more specifically, 19 from sticking sputum, 18 fromcoughing, 17 from shortness of breath, 10 from nasal secreation and congestion, seven from sore throat, six fromhoarse voice and three from wheezing or stridor. Also
among the same 27, four indicated that symptoms began after their arrival and nine before, while 14 were not sure or
did not answer at all.
More than half (36 responders, representing 57.1%) responded that air pollution was serious. On the other hand,
only 18 responders (28.6%) indicated awareness that air
pollution causes health problems. As to the causes of air
pollution, eight mentioned industrial gas emission (12.7%),
26 automobile exhaust (41.3%), 27 haze (42.9%), and six
climate (9.5%).

DISCUSSION
Since respiratory organs are in direct contact with theoutside, they are affected by factors such as climate and pollution and can also be the entry point for viruses and bacteria. In 2003, the SARS epidemic ravaged Asia, and other infectious respiratory disease epidemics like influenza havebeen occurring continuously. Clearly, we must stress theimportance of measures to prevent respiratory diseases
among Japanese people living abroad.
The occurrence of respiratory diseases is common
among travelers [3]. Steffen et al. estimated the rate of acuterespiratory tract infections with fever to be 1.261 per
100,000 travelers for a stay of one month in a developing
country [6].
Also, we see that many Japanese living abroad are being treated for respiratory diseases at overseas medical facilities [4]. Medical personnel of the Japanese Ministry ofForeign Affairs analyzed diseases being treated at Japanese
embassies (178,014 cases from 1989 to 1998). The results
indicated that respiratory diseases were the most prevalent,
accounting for 31.7% of the total. Moreover, research conducted by the Japan Overseas Cooperation Volunteers on
Japanese people living in developing countries indicatesthat respiratory diseases rank high among diseases under
medical treatment[7]. In our previousu study on two medical facilities in Southeast Asia, we saw that respiratory diseases were the most prevalent, far exceeding infectious dis-eases and digestive tract diseases [5].
From the results of this latest research, we can see that
instances of respiratory diseases increase during the dry season. At Ram Hospital in Chiang Mai, Thailand, the climate
is tropical monsoon. The seasons are broadly divided into
the dry season mainly from November to May, and therainy season from June to October. The cold season is from
November to February, while the hot season is from March
to May. The average yearly temperature is around 25 degrees, and sometimes exceeds 40°
C during the hottest season.
Subang Jaya Medical Center is located in Kuala Lumpur,
the capital of Malaysia. The climate of Malaysia, thoughdifferent on the east and west sides of the Malay Peninsula,is generally that of a tropical rain forest. While temperatureand humidity are high all year, the dry season is from May
to September when precipitation is relatively low and therainy season is from October to February when there is anabundant precipitation.
The number of cases of respiratory diseases at both facilities tends to increase during the dry season, though somedifference is evident between 2000 and 2001. Relatively
mild symptoms such as upper respiratory tract infection account for the majority of the respiratory diseases, but these
also include instances of lower respiratory infection andchronic respiratory diseases such as COPD/bronchialasthma.
When the air is dry, the effectiveness of mucous membranes in the air passages diminishes. Also, dust and automobile exhaust are stirred up, causing an increase in respiratory diseases, mainly in the respiratory tract. We also
know that many infectious viral respiratory diseases spreadwhen temperature and humidity are low. Even in Japan, respiratory diseases caused by influenza or RS virus spread
more readily during the winter dry period, and there aremany cases of upper respiratory tract infection. Therefore,
we feel that climactic factors, such as changes in temperature and humidity greatly affect the development of respiratory diseases in Japanese people living abroad.
Air pollution is another serious factor recently in the
Asian region. Air pollutants such as sulfur oxides, nitrogenoxides, and suspended particulate matter (SPM) are known
to cause damage to health. These substances can cause
bronchitis, bronchial asthma, COPD and other respiratory
diseases. SPM is a particle of 10 µm (PM 10) or less thatfloats in the atmosphere. Recent studies have reported a correlation between particulate air pollution and daily mortality
rate. Especially particles of 2.5 µm (PM 2.5) contribute to
the mortality related to respiratory diseases [2]. According
to WHO, Asian cities such as those in China and India haveacute air pollution levels, with the density of air pollutants

３
３
３

greatly exceeding Japanese standards. In 2002 during thehealth consultation in India, our questionnaire survey
showed that many Japanese people living in India were
aware of the health problems associated with air pollution,
respiratory diseases in particular [8].
Near Malaysia, where Subang Jaya Medical Center islocated, large-scale forest fires create smoke pollution(called “haze”) during the dry season, causing respiratory
diseases to residents of the surrounding areas. Every year inthis region, fires are set to clear forests for agriculture. During abnormally dry weather, fires can rage out of control.
Large-scale smoke pollution occurred in 1991 and 1997,
causing health problems for many inhabitants. Reports by
Takeuchi in 2000 showed that many Japanese people livingin Malaysia suffered from symptoms of respiratory disease,mainly those of upper respiratory tract infection. And many
were concerned about the lack of information and effectiveness of local medical treatment [9].
In Kuala Lumpur our questionnaire survey showed thatmany Japanese living there had symptoms of respiratory
diseases. Most Japanese people knew of haze, and many feltthat the air pollution problem was serious even though thesurvey showed that relatively few of them were aware of thehealth problems caused by air pollution.
As more and more Japanese people migrate to Asian
regions, the need for education in prevention of environmental diseases is becoming critical. Air pollution, temperature and humidity fluctuations, newly emerging infections
and other factors are increasing. Although respiratory diseases caused by changes in weather and air pollution are apt
to be overlooked because the early-stage symptoms are relatively light, adequate preventative measures are required inview of the fact that irreversible damage to health can occur
with long-term exposure.
Based on the results of this research, we will continue
to develop and issue preventative measures, and continue
our research efforts in the Asian region.

ACKNOWLEDGEMENTS
This work was supported in part by International Cooperation Research Grant (13P2) from International Medical
Center of Japan.

REFERENCES
１．UNEP/WHO: Urban Air Pollution in Megacities of theWorld, 1992
２．Dockey DW, Pope CA, Xu X, et al: An association between air pollution and mortality in six U.S. cities. N.
Engl. J. Med. 1993; 329: 1753-1759
３．Steffen R, Dupont H: Respiratory tract infections in Textbook of Travel Medicine and Health, 2nd ed. Hamilton,
Ontario: C. Decker, 2001.
４．Hamada A, Current situation of diseases among the Japanese living in foreign countries. Biomedical Perspectives.
1998; 8: 282-289
５．Uchikoshi A, Hamada A. Diseases Trends among Japanese expatriates living in southeast asia. Japanese J. Occupational Medicine and Traumatology. 2003; 51: 432436
６．Steffen et al. Health problems after travel to developing
countries. J. infect. Dis. 1987; 156: 84-91
７．Hiroshi Ohara, Kyoko Ito: Health management of Japan
Overseas Cooperation Volunteers leading diseases andpreventive measures. Workshop on Health Care Issues forOverseas Workers, Atlanta, Georgis, 1991
８．Uchikoshi A, Iizuka T. The effect of air pollution in India
on the health of Japanese community. Japanese J Occupational Medicine and Traumatology. 2002; 50: 440-444
９．Takeuchi K, Hiroshige Y. The role of air pollution of forest fires in Indonesia called “Haze” on the health of Japanese community in Malaysia and health problems ofoverseas workers and their families. Japanese J Occupational Medicine and Traumatology. 2000; 48: 118-122

Tropical Medicine and Health Vol. 32 No. 4, 2004, pp. 335-337
Copyright! 2004 by The Japanese Society of Tropical Medicine

３
３
５

A PILOT FIELD TRIAL OF AN IN VITRODRUG SUSCEPTIBILITY TEST USING
THE ANAEROPACK MALARIA CULTURE SYSTEM
ON THE THAI-MYANMAR BORDER"TOSHIMITSU HATABU１，２, SHIN-ICHIRO KAWAZU１, SOMEI KOJIMA３, PRATAP SINGHASIVANON４,SRIVICHA KRUDSOOD４, SORNCHAI LOOAREESUWAN４, SHIGEYUKI KANO１，＊
Accepted 10, September, 2004

Abstract: The AnaeroPack" malaria culture system with a portable thermostat incubator was evaluated in a field
laboratory on the Thai-Myanmar border conducting in vitro drug susceptibility tests on blood samples from 5
Karen children infected with P. falciparum. Only one isolate was susceptible to chloroquine; the others were highly
resistant. The IC５０ value of an isolate was only resistant to mefloquine, whereas the values of the 3 patients who
presumably showed recrudescence were slightly elevated in the susceptible ranges. These results suggested thatchloroquine should no longer be used for P. falciparum malaria in this geographic area, and that mefloquine should
be carefully monitored for its in vivo effectiveness. In this study, the AnaeroPack" malaria culture system withportable thermostatic incubator is a powerful and useful mobile tool, which aids in providing detailed evidencebased distribution data concerning of drug resistant malaria in the field.
Key words: AnaeroPack", Drug susceptibility test, Plasmodium falciparum

INTRODUCTION
Since chloroquine-resistant Plasmodium falciparum
was first reported in 1959 in Thailand, it has developed resistance to all commonly used drugs [1]. The spread ofmulti-drug resistant P. falciparum is now a major publichealth problem worldwide, with prophylactic and therapeutic implications [2]. Evidence-based detection of drugresistant parasites is important for the accurate evaluation ofsusceptibility to antimalarial drugs. However, isolation of
fresh parasites for in vitro drug susceptibility testing isoften difficult in the field because of the precise experimental conditions needed for the test. In vivo susceptibility tests
are frequently conducted, but they sometimes fail becauseof difficulties in following up the patients. In fact, it issometimes difficult to determine whether an adequate drugconcentration has been successfully achieved in the patients’ blood. Therefore, in vitro tests are indispensable todetermine the exact degree of resistance acquired by theparasites.
1

We previously reported that the AnaeroPack" gas system can be used for the continuous cultivation of both laboratory strains and fresh isolates of P. falciparum from patients [3]. This gas system is safer, simpler, and easier to use
than the candle jar method. In this study, we evaluated theAnaeroPack" malaria culture system with a portable thermostat incubator in a field laboratory on the Thai-Myanmar
border to conduct in vitro drug susceptibility tests on P. falciparum. The feasibility of the system and the results of thetests are described in this report.
The study was conducted at Rajanagarindra Tropical
Disease International Center, a field laboratory center of theFaculty of Tropical Medicine, Mahidol University, locatedin Suan Phung, Rachaburi, about 200 km west of Bangkok.
The area was inhabited by Karen as well as Thai people,
and in 2001 more than 6,000 people were subjected to microscopic diagnosis of malaria in the center with about
1,000 positive cases found. Malaria is endemic throughout
the year in this area with the peak season occurring aroundMay and June. Mefloquine is currently the first drug of

Research Institute, International Medical Center of Japan, 1-21-1 Toyama, Shinjuku, Tokyo 162-8655, Japan.
Gunma University School of Health Sciences, Maebashi, Gunma 371-8511, Japan.
3
Asian Centre of International Parasite Control, Bangkok 10400, Thailand.
4
Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand*Corresponding author:
Shigeyuki Kano,
Department of Appropriate Technology Development and Transfer, Research Institute, International Medical Center of Japan, 1-21-1 Toyama,
Shinjuku, Tokyo 162-8655, Japan.
Tel: +81-3-3202-7181 (ext. 2877), Fax: +81-3-3202-7364, E-mail: kano@ri.imcj.go.jp
2

３
３
６

choice for the treatment of uncomplicated malaria, and artesunate, a qinghaosu derivative, is administered in complicated cases.
The AnaeroPack! CO２ (Mitsubishi Gas Co., Tokyo, Japan) is a foil-packed paper sachet that on exposure to air
immediately absorbs atmospheric O２ and simultaneously
generates CO２ until a condition of 15% O２ and 5% CO２ isattained. The microaerophilic atmosphere produced within asealed jar (AnaeroPack! Kakugata jar, SUGIYAMA-GEN
Co., Ltd., Tokyo, Japan) can be maintained for at least 24
hours. A portable thermostat incubator (SUGIYAMA-GEN
Co., Ltd.) was carried to the laboratory, and the temperatureinside the incubator was adjusted to 37°
C. During P. falciparum cultivation, the sachet inside the jar was replaced witha new sachet every day when the culture medium waschanged (Fig. 1).
The in vitro drug susceptibility test used in this study
was a modified semi-micro test described previously [4, 5].
The following procedures were conducted right after the
sampling in the field laboratory center. Briefly, blood samples were washed 3 times with RPMI 1640 and resuspendedin RPMI 1640 (GIBCO BRL), pH 7.4, supplemented with

(A)

(B)
!

Figure 1. AnaeroPack culture system. (A) the portable incubator and (B) the AnaeroPack! Kakugata jar with aculture plate inside.

10% human serum (from non-immune Japanese donors
without a previous history of malaria; Blood Type A) and25 µg/ml gentamicin (Sigma, Saint Louis, Mo), 25 mM
HEPES, and sodium bicarbonate at a hematocrit of 5%.
Five hundred microliters of the erythrocyte suspension wereplaced in each well of a tissue culture plate (24-well flatbottom, Corning Costar, NY). Chloroquine diphosphate waspurchased from Sigma. Mefloquine hydrochloride waskindly provided by Hoffman-La Roche Ltd., Basel. Twenty
microliters of chloroquine diphosphate or mefloquine wasadded to each well (for chloroquine to create a series ofdoubling dilutions from 20 to 10240 nM, and for mefloquine to create a series of 10 times dilutions from 0.01 to
1000 nM dissolved in distilled water). To monitor parasite
growth, 6 wells per plate served as controls without antimalarials. When the schizonts were fully grown in the control
wells, the culture plate was removed from the incubator.
Thin-smear specimens stained with Giemsa solution weremade from each well, and we counted the number of erythrocytes microscopically in the control smears until encountering 50 schizonts. We defined the schizonts as parasitesthat have both dark brown pigment and more than 3 nuclei[6]. The effect of antimalarials on parasite growth wasevaluated by observing the decreased number of schizonts
per equal number of erythrocytes counted previously in thecontrol cultures. The percentage of growth inhibition effectwas calculated as follows: test well schizont count/control
well schizont count (50) x 100. Fifty percent growth inhibitory concentration (IC５０) was calculated from the inhibition
curve obtained from the values by a statistical probit
method.
Blood samples were obtained from 5 Karen children
age 5 to 13 years (Table 1). The guardians gave written consent to this study, which had been approved by the EthicalCommittee of Mahidol University. This survey research also
followed the ethical guidelines for epidemiological studies
by the Ministry of Education, Culture, Science and Technology and Ministry of Health, Labour and Welfare of Japan. Three of the 5 patients had a history of P. falciparum
malaria within 1 month, and they had received mefloquine
at that time. The parasitemias of the patients were relatively
low, 0.05-0.20%. The incubation period (the time needed
for the tested isolates to grow to schizonts) ranged from 48
to 96 hrs. The IC５０ values of the isolates for chloroquinevaried from 91 to 402 nM, with a geometric mean of 273
nM and SD of 90 nM. Only one isolate, from patient B, wassusceptible to chloroquine; the others were highly resistant.
The IC５０ values of the isolates for mefloquine varied from 452 nM, with a geometric mean of 24 nM and SD of 13 nM.
The IC５０ value of the isolate from patient A was only resistant to mefloquine, whereas the values of the 3 patients who

３３７

Table 1

Profiles of the P. falciparum isolates from 5 patients in Suan Phung

No. Age

Sex% Parasitemia

A 12
B 13
C 8
D 12
E 5

female
male
male
male
female

0.30
0.05
0.20
0.11
0.11

Day ofrecrudescence
none
D29
D24
none
D22

Chloroquine
IC５０ (nM)
Judgment＊
Resistant
231
Susceptible
91
Resistant
358
Resistant
285
Resistant
402

Mefloquine
IC５０ (nM)
Judgment＊＊
Resistant
52
Susceptible
18
Susceptible
27
Susceptible
4
Susceptible
17* Threshold of IC５０ for chloroquine resistance is 114 nM [5].
** Threshold of IC５０ for mefloquine resistance is 40 nM [9].

presumably showed recrudescence were slightly elevated in
the susceptible ranges. These results suggested that chloroquine should no longer be used to treat P. falciparum malaria in this geographic area, and that mefloquine should be
carefully monitored for its in vivo effectiveness.
The World Health Organization’s test with the candle
jar is the standard method used to cultivate parasites for
drug susceptibility testing in the field [7]. This method issometimes difficult to carry out under field conditions. Our
study found the AnaeroPack! system to be an effective alternative method for cultivation of malaria parasites and forin vitro drug susceptibility testing in the field. The AnaeroPack! gas system does not need a catalyst, water, or hydrogen for the activation of gas production, and the gas jars
or incubator chambers are lightweight and portable. Ourfindings were consistent with other reports that the AnaeroPack! culture system produced acceptable results forthe growth of malaria parasites in the laboratory application.
The IC５０ values attained with the AnaeroPack! CO２ weresimilar to those attained by the candle jar method [3, 4, 8].
In addition, the thermostatic incubator of AnaeroPack! malaria culture system can now be powered by a car battery.
The AnaeroPack! malaria culture system with portable thermostatic incubator is a powerful and useful mobile tool thathelps to provide detailed evidence-based distribution data
concerning drug resistant malaria in the field.

ACKNOWLEDGEMENTS
The authors thank the laboratory staff in the Suan
Phung field station for their kind assistance and support.
This study was supported by a Grant-in-Aid for Scientific
Research (B) (International Research) (13576012,
16406012) from the Japan Society for the Promotion of Science, by Asian Centre of International Parasite Control(ACIPAC) project (Hashimoto Initiative) under the Japan
International Cooperation Agency (JICA) program, and by
a grant from Mahidol University.

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Tropical Medicine and Health Vol. 32 No. 4, 2004, pp. 339-341
Copyright! 2004 by The Japanese Society of Tropical Medicine

３
３
９

SHIGA-TOXIN PRODUCING ESCHERICHIA COLI IN VIETNAM

BINH MINH NGUYEN１, DAC CAM PHUNG１, NOBORU NAKASONE２, CLAUDIA TOMA２,NAOMI HIGA２, SUNAO IYODA３, and MASAAKI IWANAGA２.
Accepted 12, October, 2004

Abstract: Shiga-toxin producing Escherichia coli isolated in Vietnam were examined. The 9 isolates included 5
from 400 diarrheal patients and 4 from 42 cows in 2003. No isolate carried eae gene. All human isolates except
one and one of four animal isolates carried both stx1 and stx2. One of the human isolates could not be definitely
identified as a Shiga-toxin producing Escherichia coli, because it showed positive for PCR using a common primer
set for stx gene family and also positive for stx2d subtype, but negative for stx1, stx2 and for expression of thetoxin. The serotypes of the isolates were various, but major serogroups such as O157, O26 and O111 were notfound.

Enterohemorrhagic Escherichia coli(EHEC) has beenrecognized as a distinct class of diarrheagenic E. coli since
1982, when outbreaks of hemorrhagic colitis were observedin the United States and Canada [1, 2]. Prior to these outbreaks, in 1980, verocytotoxin-producing E. coli was reported in classical enteropathogenic E. coli, EPEC [3]. In
the early 1980s, O’Brien et al . investigated an E. coli O26
strain and purified a toxin closely related to Shiga-toxin of
Shigella dysenteriae type1 [4, 5]. EHEC was once called
Verotoxin producing E. coli(VTEC), and recently it is commonly called Shiga-toxin-producing E. coli(STEC). Readers in the other research fields thus have to keep in mind
that the terms EHEC, VTEC and STEC are basically thesame. We use the term STEC hereafter. After the initialoutbreaks of STEC infection in 1982, STEC attracted
worldwide attention because the illness is clinically severe
and can be followed by serious sequels such as hemolytic
uremic syndrome (HUS) and central nervous disorder. Outbreaks involving hundreds of individuals cause a sensation,
but sporadic infections are frequently overlooked unless theillness is serious. Nevertheless, the sporadic infection represents the major impact of this pathogen. The epidemiologyof the illness and the distribution of STEC are well studied

in industrialized countries [6], but there are not many reports from the developing world. STEC strains were actively sought in a study on Bangladeshi children, but nochild was infected with STEC [7]. In Lao People’s Democratic Republic, 880 diarrheal patients randomly collected
in1996 and 1997 were examined, but STEC with O111 serogroup was detected in only1 patient. Also, 1 strain without eae gene was detected among 278 patients examined in
2002 [8, 9]. Detection of STEC in Thailand was also reported but serotype O157:H7 strains were not found [10]. In
the present study, we intensively examined human diarrheal
stools and cattle stools for STEC in Vietnam.
E. coli strains were isolated from human diarrheal
stools and cow’s stools. The human diarrheal stools werecollected from 400 patients in Nam Dinh province, and thecow stools were collected from 42 animals at a stock farmin a suburb of Hanoi in 2003. The isolated E. coli were firstexamined by multiplex PCR as described by Toma et al .
[11]. stx positive strains were examined again using a single primer set (v1/v5 and v3/v4) to determine the toxin type.
Primer set v1 (5’AGTTAATGTGGTGGCGAA3’) and v5(5’GACTCTTCCATCTGCCG3’) was used to amplify 811 basepairs of stx１. Primer set v3 (5’TTCGGTATCCTATTCCCG

1. National Institute of Hygiene and Epidemiology, 1 Yersin Street, Hanoi, Vietnam
2. Division of Bacterial Pathogenesis, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan.
3. Department of Bacteriology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan
Correspondence:
Masaaki Iwanaga
Division of Bacterial Pathogenesis, Graduate School of Medicine,
University of the Ryukyus. 207 Uehara, Nishihara, Okinawa 903-0215, Japan
TEL: 098-895-1125 FAX: 098-895-1408
E-mail <iwanaga@med.u-ryukyu.ac.jp>

３４０3’) and v4 (5’TCTCTGGTCATTGTATTA3’) was used toamplify 471 base pairs of stx２ and its variants [12]. Further
classification of the stx２ gene family was performed using
primers reported by Wang et al. [13]. DNAs were extracted
from the organisms as described by Yokoyama [14]. The
PCR mixture used with a single primer set consisted of a total volume of 30 µl containing 10 mM Tris-HCl (pH8.3), 50
mM KCl, 0.1% Triton X-100, 1.5 mM MgCl２, 0.75 U of
Taq DNA polymerase (Toyobo, Osaka, Japan), 0.2 mM deoxynucleoside triphosphate, a 0.25 µM concentration ofeach primer and 3 µl of the DNA template. The PCR program was 95°
C for 1 min, 52°
C for 1 min and 72°
C for 1
min, for 25 cycles. stx gene positive E. coli were examinedfor production of Shiga toxin using reversed passive latex
agglutination kit VTEC-RPLA (DENKA-SEIKEN Co.
LTD., Tokyo). The organisms were cultured in CAYE medium as indicated in the manual attached to the kit. O:H serotyping of the strains was performed by bacterial agglutination as described previously by Orskov F., and Orskov I.
[15].
STEC was detected in 5 of the 400 patients examined(1 of the 5 was questionable). Therefore, the frequency ofthe definite isolation of STEC was 4/400 (1.0%). STEC
was also detected in 4 of the 42 cows examined (9.5%). Se-

rotype O157:H7 was not found. Three of the 9 isolates belonged to serotype O44:H16. In one isolate, O-antigen was
untypable but H8; the other 5 isolates belonged to serotypes
O8:H8, O100:H40, O159:H21, O55:H11 and O2:H42 (Table1). The toxin type was variable and all 9 isolates were
negative for the eae gene. PCR for toxin typing showedthat the 4 human isolates (O1-A, O3-D, O4-C, O6-B) carried both stx１ and stx２ and that the one questionable isolate
was positive for primer set VTcom-u / VTcom-d, common
to stx1 and stx2, but did not react with the primer set v1/v5
and v3/v4 specific to stx1 and stx2, respectively. Nevertheless, using the primers for detecting various genes belonging to the stx2 family reported by Wang et al., this isolate(O2-C) produced a relevant amplicon compatible with stx2d.
Moreover, it did not produce the toxin when examined by
RPLA. Since the details are not available at present, it isquestionable whether this isolate can be regarded as STEC.
Among the animal isolates, one (93-6) carried both stx１ andstx２, and the other three carried one of them (Table1 andFig.1). The production of toxins was compatible with the
genotype, except for the one questionable isolate.
To the best our knowledge, this is the first study on
STEC isolated in Vietnam and the first to suggest that
STEC strains have already spread in this country. However,

Figure 1

Lanes, M:100-bp DNA ladder (upper broad band indicates 1000 bp and lower
broad band indicates 500 bp), 1:O1-A, 2:O2-C, 3:O3-D, 4:O4-C, 5:O6-B, 6:5-5,
7:18a., 8:93-6, 9:2245-2 Primer sets v1/v5 and v3/v4 were used to detect 811
bp product of stx1 and 471 bp product of stx2, respectively. Lane2 (questionable strain O2-C as mentioned in the text)

３
４
１

Table 1
No. strains
1

O1-A

stx

PCR
stx1 stx2

Sub.

eae

+

+

2c, 2d−+RPLA
Stx-1 Stx-2+(2)＊ (O2-C)＊ (+)＊ (−)＊(−)＊ (2d)＊ (−)＊ (−)＊+

serotype
O44:H16(−)＊ (O8:H8)＊

3

O3-D

+

+

+

2

−

+

+

4

O4-C

+

+

+

2

−

+

+

OUT:H8

5

O6-B

+

+

+

2c

−

+

+

O159:H21

6

5-5

+

−

+

2c, 2d−−+O44:H16

7

18a

+

+

−

−

−

+

−

O55:H11

8

93-6

+

+

+

2c

−

+

−

O2:H42

92245-2

+

−

+

2d

−

−

+

O44:H16

O100:H40

No. 1-5: human isolates, No. 6-9: cattle isolates, OUT: O-antigen untypable,
Products from the primer set VTcom-u and VTcom-d (stx), v1 and v5 (stx1),
v3 and v4 (stx2), subtypes of stx2 family identified using primer sets described by Wang et al . (12). ( )＊: to be discussed

it is unknown whether these STEC originally developed in
Vietnam or came from somewhere else. Serotypes of STEC
in Vietnam are different from those in neighboring countries[8, 9, 10], and the organisms did not carry eae gene. Since
the clinical data were not obtained in the present study, thepathogenicity of these isolates remains unclear. But the factthat the isolates lacked an important pathogenic gene (eae)indicates the necessity for further studies on the clinicalfindings and pathogenicity of these organisms. The adhesive factors encoded outside of the locus enterocyte effacement are now under intensive investigation in our laboratory.

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