J. Med. Entomol.
23 March 1979
Vol. 15, no. 3: 286-291 ©
1978 by the Bishop Museum
PHLEBOTOMINE SAND FLIES (DIPTERA: PSYCHODIDAE) FROM A PRIMARY HILL FOREST IN WEST MALAYSIA\,2 By A. B. Knudsen3, D. j. Lewis4, R. B. Tesh\ A. Rudnick3, j. jeffery6 and I. Singh7
AbJtract: A 22-month survey was conducted in primary forest in the Gunong Besout Forest Reserve, West Ma]aysia, to test methods of collecting sand Aies, to find out what species occurred, and to examine them for arboviruses. Most sand Ales were collected in tree holes, in traps baited with leaf monkeys and in light traps. Seasonal fluctuations of flies showed a negative correlation with rainfall. Altogether ]407 flies were identified; 770 of them were females. They comprised] 5 species of which
4 \\'ere
new;
1 of these
is known
to occur
only
in the
Phlebotomine sand flies have received relatively little attention in West (peninsular) Malaysia because leishmaniasis is not established there. In 1914, Phlebotomus stantoni Newstead was described from Kuala Lumpur. In 1938, O. R. Causey thought that sand flies were rare in the Malay Peninsula, and O. Theodor (1938) made taxonomic contributions. Lewis (1957) added more infor'This study was supported in part by the University of California International Center for Medical Research through research grant AI 10051 to the Department of International Health, School of Medicine, and by research grant AI 11540 to the G. W. Hooper Foundation, University of California, San Francisco; both grants from the National Inst.itute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service. 'This project was conducted in the Arbovirus Research Laboratory in the Department of Medical Microbiology at the Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. 3The G. W. Hooper Foundation, University of California and Faculty of Medicine, University of Malaya, Kuala Lumpur. Send requests for reprints to Dr A. B. Knudsen, do Editor, G. W. Hooper Foundation, University of California, San Francisco, California 94143, USA. 4Department of Entomology, British Museum (Natural History), London, England; supported by a grant from the Medical Research Council, London. sp. O. Box 1680, Pacific Research Section, National Institute of Allergy and Infectious Diseases, National Insitutes of Health, Honolulu, Hawaii 96806, USA. 6Department of Parasitology, National University Faculty of Medicine, Kuala Lumpur, Malaysia. 7Division of Medical Ecology, Insitute for Medical Research, Kuala Lumpur, Malaysia.
(1962)
predicted
that
many species
would
be
found. Lewis & Wharton (1963) reported 16 species from West Malaysia, and Rudnick et al. (1971) discussed the species of Malaysia as a whole. In 1972, mosquitoes were studied in relation to the jungle cycle of dengue virus in the Gunong Besout Forest Reserve in Perak State, West Malaysia (Knudsen & Rudnick 1975). In early 1973, work was extended to test methods of collecting sand flies, to find out what species existed in that part of the country, and to look for viruses in sand flies. This search for virus was undertaken because in some countries plebotomines appear to be reservoir hosts of viruses of the phlebotomus fever group (Tesh & Chaniotis 1975), and because species of both the Old World genera Phlebotomus and Sergentomyia have been reported to harbor such viruses (Barnett & Suyemoto 1961). The study area, in tropical hill rain forest about 198 m above sea level and 156 km NNW of Kuala Lumpur, is 3°54.2'N, 101°19.5'E, in map grids 472,600 X 404,700 (Map sheet 75, ChangkatJong, Series L7010, Edition 2-DNMM, Malaysian National Mapping Service, Kuala Lumpur). Surveys were conducted along the NE slope of the Gunong Besout main ridge, which reaches a summit of 417.6 m. The local rock is Paleozoic (Devonian) metaquartzite and phyllite (information from the Geological Survey of Malaysia Headquarters, Kuala Lumpur). Meteorological stations established in the study area showed a mean annual temperature of 25.6°C at 1.2 m above ground, with average monthly means of 22.6° to 29.1 °c. Monthly rainfall averaged 253.0 mm (TABLEI). The forest reserve comprises 82,983 ha of primary and secondary jungle. Timber was cut between 1958 and 1970 in some areas which are rec-
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study area. Sergentomyia gombaki was the commonest species and sometimes bit man. S. gombaki and S. iyengm~ were taken in traps baited with leaf monkeys. A total of 23,569 sand flies. in 165 pools, were processed for virus isolation with negative results. The sand fly fauna, the apparentl)' negative role of Sergentomyia in virus transmission in the Old World generally, and the absence of Phlebotomus mgentiPes, the vector of kala azar in India, are discussed.
mation and reported R. H. Wharton's discovery, in 1956, of many Phlebotomus (lTgentipes Annandale & Brunetti in Lamir village oh the east coast, which stimulated interest in sand flies. Quate & Fairchild (1961) discovered that they were fairiy numerous in West Malaysia and Sabah and they and Quate
197R
Knudsen
et al.:
Forest
phlebotomines
METHODS
Twelve tree holes and 3 hollow logs were chosen for standardized weekly collecting during the 22month period from May 1973 to February 1975. Resting flies were agitated by hitting the insides of the holes with a slender stick and were aspirated at the entrance for several minutes. During the ongoing mosquito survey, sand flies were occasionally collected in vegetation sweepings along streams, in dry ice-baited light traps in a vertical transect 1.8-32 m above the forest floor, on human bait on the ground, in monkey-baited traps in the canopy at 17, 22 and 45 m, and in a trap baited with hamsters 3 m above ground. All flies caught in animal bait traps were collected alive with a battery-operated aspirator and fell undamaged into cloth holding bags placed above the suction fan. The flies were kept in a cool, shaded place until they could be anesthetized with chloroform, sorted by sex, sealed in glass ampuls, and stored on dry ice pending transport to the laboratory where they were held at -65°C. Represen-
Malaysia
287
TABLE I. Mean monthly rainfall and air temperatures* compared to number of phlebotomines collected from tree hole I, Gunong Besout Forest Reserve, Perak, Malaysia (March 1973-December 1974).
MONTH
RAINFALL
TEMPERATURE °C Max.
(MM)
Min.
NO.
8.5 9.5
March 1973** April** May**
June
AVG.
COLLECT~:[) PER MONTH
79.0
July Aug.
64.0*** 103.5 192.6
30.3 29.9 30.3
23.7 22.7 22.7
16.5 159.0 239.3
Sept. Oct. Nov. Dec.
157.1 674.5 312.6 371.3
30.2 29.7 28.9 27.4
22.4 22.6 22,7
22.3
91.4 36.0 43.2 i32.4
Jan. 1974 Feb. March
117.2 254.6 138.1
28.2 28.8 29.8
21.8 21.9 21.9
170.3 189.7 212.6
April
369.6 317.3 282.7
29,1 28.9 28.4
23.0 22.8
146.8 309.3 277.1
28.2 29.1 28.1
22.5
253.0
22.7
508.0
Sept.
146.6 71.4 410.8
22.4
78.0
Oct. Nov. Dec.
148.5 225.0 261.4
28.4 29.0 29.9
23.0 22.6 22.3
49.8 150.0 24.3
Mean
253.0
29.1
22.6
150.7
May June July
Aug.
* Weather station ** No reading. *** Ten days only.
located
2~.6
0.8 km from site.
tative samples were kept in vials of alcohol for identification at the British Museum (Natural History), London. The flies processed for virus isolation in Kuala Lumpur were sorted by sex and genus or species into pools of variable size, and triturated in 33% normal heat-inactivated rabbit-serum beef-heart infusion broth containing 500 units of penicillin and 500 I-Lgof streptomycin per ml. After centrifugation at 2500 g, the supernatant was inoculated into 12 two-day-old Swiss white mice, 0.01 ml intracerebrally and 0.03 intraperitoneally. Mice were observed for signs of illness for 21 days. If no illness was observed by the 8th day postinoculation, brains of 2 mice were harvested and passed to new litters. Three such blind passages were made before concluding that a pool was negative for presence of virus. The majority of the flies were flown, on dry ice, to Honolulu, Hawaii, where they were pooled in groups of 50-250 by sex. Each pool was triturated with 3.0 ml of diluent comprising buffered saline
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ognizable by the presence of many secondary plant-community specIes. Many pnmary forest trees are still evident, and the original flora of the main study area retains its virgin state as a primary hill forest. The trees of the primary forest belong to the families Burseraceae (Canarium spp.), Myrtaceae (Eugenia spp.) and Dipterocarpaceae (a few species of Shorea). Some typical undergrowth plants are Eugeissona and Arenga palms and species of Randia, Globba and Pandanus. Malaysian hill forests have 2 heterogeneous canopy layers representing many families. The lower layer, reaching a height of 9-12 m, consists mainly of Euphorbiaceae, Meleaceae, various palms, Dilleniaceae and Myristicaceae. The upper, primary layer rises to 30.540.7 m and includes species of Leguminosae, Burseraceae, Dipterocarpaceae and Rhizophoraceae. Tall trees like Shorea reach 61 m to form an emergent canopy. The numerous large vertebrates include elephant, tiger, Malayan sun bear, civet, bear cat (binturong), wild boar, deer, monitor lizard, porcupine, scaly anteater, 2 species of gibbons, and 4 species of cercopithecid monkeys. Many small vertebrates occur, such as lizards, squirrels, rats, vipers, coral snakes, cobras, frogs and toads. The numerous species of birds range from small bulbuls to the great rhinoceros hornbill.
from
288
J.
Mcd. Entomol.
Vol. 15, no. 3
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Reid) and at times Mac'aca species, suspended 22 m above the forest floor, yielded 7.9% of the total, with 2149 flies caught in 1260 twelve- and 24-h collections (average 1.7). CDC-type light traps with dry ice, at 1.8-32 m, used 958 times, twice weekly, produced 659 flies (2.4%, average 0.7). In incidental human bait catches, 51 flies were caught (0.2%). Vegetation sweepings conducted 516 times resulted in 52 flies (0.2%). A small Magoon-type trap housing 2 Silvered Leaf Monkeys (P. cristata) was operated 45 m above the forest floor in emergent canopy. It yielded 33 flies in 239 daily collections (0.1%). Pig-tailed Monkeys, Macaca nemestrina Linnaeus (4-6 animals), placed at ground level in a large Magoon-type trap produced only 8 sand flies in 1340 twelve- and 24-h collections. Hamsters, used in small Magoon-type traps, attracted only 20 flies in 64 twice-weekly collections. A single Long-tailed Monkey, M. fascicularis Raffles, placed at 17 m above ground yielded only 1 sand fly in 730 collections. Only 51 (0.2%) of the phlebotomines collected from tree holes showed evidence of a blood meal. We collected 432 engorged phlebotomines in the monkey-baited trap at 22 m during 1973 and 1974. Of these, 3 specimens were identified as Sergentomyia gombaki but the majority of the engorged specimens appeared to be S. gombaki as well, although they were not precisely identified. Ten of 20 from FIG. I. Tree hole I from which the majority of sand Aies were collected. a hamster-baited trap positioned at tree hole 1 were engorged; these engorged specimens also appeared to be S. gombaki. Thirteen were taken in (pH 7.4) with 0.5% gelatin, 25% heat-inactivated human bait collections. Seventeen engorged speccalf serum, and antibiotics. The suspension was imens were taken in light traps. centrifuged at 8800 g for 30 min at 5°C, and 0.1 The fluctuations in numbers of sand flies colml of supernatant was inoculated into each of 3 lected at the best site, tree hole 1, were correlated tube cultures of Vero cells. After absorption for 1 with the amount of rainfall at the camp about 0.8 h at 37°C, maintenance medium was added and km away. The hole, open to rain but permanently changed every 3-4 days thereafter (Tesh et al. shaded in a tree leaning at about 60°, was 2.86 m 1977). Cultures were incubated at 37°C for 14 days above ground and measured 63.5 cm high, 25.4 and were examined regularly for cytopathic effect. em wide, and 134.6 cm deep (FIG. 1). In 1974, measurable rain fell on 184 (55%) of 334 days meaRESULTS sured. The mean annual rainfall exceeded 3000 mm, with peaks in October and December (1973) During the 22-month period 27,084 phlebotoand in April and September (1974). mines, 41.3% of them fema~es, were collected by all methods. The species of phlebotomines. A total of 1407 sand From tree holes (including hollow logs) 869 col- flies were identified at the British Museum (Natlections yielded 24, III flies (89.0%), with an av- ural History); 770 were females and 637 males. Identifications of the females are shown in TABLE erage catch of 27.7. A large Magoon-type trap housing 6-8 leaf 2. The males are not listed, since specific identifimonkeys (Presby tis cristata Raffles and P. obscum cations of most were not made.
Knudsen et a!.:
1978
TAHLE
2.
Forest phlebotomines
Identification of 770
'l'
from Malaysia
289
sand flies collected by different methods.
TREE
LIGHT
ROOT
LEAF
BITING
HOLES
TRAPS
HOLES
MONKEYS
MAN
Phlebotolllus (ldiophlebotollltL\ ) frolldifer Lewis & Lane (A naphlebotoll/us) stalltoni Newstead
TOTALS OTHERS*
%
0.4
3 7
0.9
138 61 8 31 19 1 2 4
46.3 10.8 3.5 4.0 4.6 0.4 0.5 0.8
19
5.4
14 57 17 2
2.2 11.4 6.2 2.6
(ungrouped) pach)'slollla (Quate & Fairehild) t'eidi (Lewis) cheongi Lewis & jeffery klltl/Lwli Lewis & jeffery
Total %
* Separate
49.5 general collections.
P. frondifeT is known only from the study area. It belongs to the subgenus Idiophlebotomus, a peculiar group whose members are known from a few isolated bat caves scattered through the Oriental Region. Three females and 5 males of P. frondifer were found in a cave like shelter formed by a fallen
100
tree.
No true caves were
found
in t.he
vicinity, merely narrow cracks in outcrops of rock. The only other specimen, a male, came from a light trap located approximately 250 m away. S. gombaki fed to repletion on the face and hands of collectors, in camp or near a tree hole, and were attracted to humans occasionally. Two larval mites were found on females of S. gombaki and S. malayae. They are related to the trombidiid genus Neotrombidium Leonardi (Neotrombidiinae), species of which parasitize beetles living under bark (Lindquist & VercammenGrandjean 1971). Search for viruses. A total of 14,636 sand Aies (5308 females and 9328 males), grouped into 138 pools, were processed for virus isolation in Honolulu. An additional 8933 sand Aies were processed for virus isolation in 27 pools in Kuala Lumpur. No viruses were isolated. DISCUSSION
Sand flies. The genus Sagentomyia is widespread in many parts of the Old World tropics, so
its preponderance in the Gunong Besout area is not unexpected. The small number of man-bitings recorded are in line with the known preference for reptiles of many species of Sergentomyia, although in some countries several habitually reptile-biting species are known
to attack
man occasionally.
S. gombaki was by far the commonest species and the only known man-biter. Its numbers in leafmonkey traps, coupled with the total catch of sand flies in these traps, suggest a definite attraction to these animals. S. gombaki has a long proboscis, which is a normal feature of mammal-biting sand Aies. The numbers of sand Aies coming to traps baited with different monkeys suggest a host-specific attraction, like that reported by Chaniotis et al.(l971). The number of S. iyengari in the leaf-monkey trap, compared with its numbers in light traps and in the area generally, suggests a definite attraction to this animal. One S. iyengari was taken 45 m above ground. The collections show many sand Aies occur in the canopy. The detection of species at various heights above ground may be inAuenced by the collecting methods. All or most P. frondifer, S. malayae, S. barraudi, S. reidi and S. knudseni were taken at or near ground level, S. gombaki at various levels, and S. iyengari in the canopy. During months with less than average rainfall,
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SergelltulI/)'ia (I? olldatlOlIl)'ia) gumbaki (Lewis & Wharton) i)'engari (Sinton) lIla/a)'(l/! (Lewis) wharloni (Lewis) jeffetyi Lewis lambotl Lewis & jeffery gl'lllmea Lewis & jeffery hamidi Lewis & jeffer)' (Pan-ulolll)'ia) ban"01uli (Sinton)
290
J.
Med. Entomol.
the number of flies increased. When monthly rainfall exceeded the yearly mean, the number of flies decreased. Fly numbers and rainfall showed a negative correlation; it is likely that rainfall of more than 250 mm in a month prevented emergence of adults. These findings are similar to those reported by Chaniotis et a!. (1971).
P. argentiPes extends from Iran to Bali and is an important vector of kala azar in India, where it is peridomestic and bites man but prefers cattle. Its biology and distribution in Southeast Asia are of interest in relation to the possible eastward limit of kala azar and the possible effect of large-scale deforestation (Lewis 1973, 1974). It has been found sporadically in West Malaysia and was temporarily abundant in 1 village (Lewis & Killick-Kendrick 1973), and it bites man occasionally (Lewis & Wharton 1963). The absence of P. argentiPes in the Gunong Besout study area shows that it is by no means evenly distributed in West Malaysia, even as a wild forest species. There is need for further study of its distribution. Acknowledg-rnf'11LI".·
We thank
Mr Samuel
W. James
and othel'
members of the Arbovirus Research Laboratory field staff for their invaluable assistance in making field collections. We are indebted to the staff of the Department of Medical Microbiology, University of Malaya, and to the Director of the Institute for Medical Research. Our gratitude and app"eciation go to Dr E. Soepadmo and Encik Malmud bin Sider of the Department of Botany, Faculty of Science, University of Malaya, for classification and identification of the Aora in the study area; to Mr Mah Weng Hong and his staff of the Geological Survey of Malaysia Headquarters for providing information on the geology of the area; to Mr Chan Wah Sun of our laboratory for ingeniously constructing cages, light traps and other devices; and to Mr D. Macfarlane of the Commonwealth Institute of Entomology, who kindly examined the mites.
LITERATURE
CITED
Abonnenc, E. 1972. Les phlebotomes de la region ethiopienne (Diptera, Psychodidae). Mem. Off. Reck. Sci. Tech. Outre-Mer. 55: 1-289. Aitken, T. H. C., J. P. Woodall, A. M. P. Andrade, G. Bensaboth &: R. E. Shope. 1975. Pacui virus, phlebotomine Aies, and small mammals in Brazil: an epidemiological study. Am. J. Trap. Med. Hyg. 24: 358-68. Barnett, H. C. &: W. Suyemoto. 1961. Field studies on sandAy fever and kala-azar in Pakistan, in Iran, and in Baltistan (Little Tibet) Kashmir. Trans. N. Y. A cad. Sci. 23: 609-17. Causey, O. R. 1938. Phlebotomw; of Siam with a description of a new variety. Am. J. Hyg. 28: 487-89. Chaniotis, B. N., J. M. Neely, M. A. Correa, R. B. Tesh &: K. M. Johnson. 1971. Natural population dynamics of ph lebotomine sandAies in Panama. J. Med. Entomot. 8: 339-52. Knudsen, A. B. &: A. Rudnick. 1975. The silent jungle cycle of dengue virus and its tenable relationship to endemic dengue in Malaysia. (Abstract.) Proc. J 3th Pac. Sci. Congr. 1: 334. Lewis, D.1- 1957. Some sandAies (Phlebotominae) of Malaya. Proc. R. Entomol. Soc. London Ser. B 26: 165-71. 1973. Absence of leishmaniasis in South-East Asia. Absl. 9th In/. Congr. Trap. Med. Malar.: 147-48. 1974. The biology of Phlebotomidae in relation to leishmaniasis. p. 363-84. In: Smith, R. F., T. T. Mittler & C. N. Smith, eds., Annu. Rev. Entomol. Vol. 19. Annual Reviews, Inc.
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Viruses. Viruses of the phlebotomus fever arbovirus group have been isolated in southern Europe, Africa, central Asia and Central and South America, and at least 25 different serotypes are now known (Tesh et a!. 1977). In view of the relatively high virus infection rates observed among sand flies in other areas (Tesh et a!. 1974, 1977, Aitken et a!. 1975), one might expect these agents also to be present in Malaysia. However, no virus isolates were obtained from the 23,569 sand flies processed from Gunong Besout. These results suggest that mammal-adapted phlebotomus fever viruses are absent from the study area. At least one virus isolate has been reported from Sergentornyia species in Iran (Barnett & Suyemoto 1961). This apparent absence of phlebotomus fever viruses may well be related to the small number of species and individuals of the genus Phlebotomus, which includes all of the habitual mammal biters of the Old World. In the Gunong Besout area Phlebotomus made up only 1% of the total catch and was represented by 1 obscure aberrant species that probably bites bats and is not known to attack man. The findings contribute something to the world picture of phlebotomus fever viruses and their vectors (Tesh et a!. 1976), by demonstrating the apparent absence of the viruses in an area dominated by Sergentomyia. In contrast, in some tropical American forests (Tesh & Chaniotis 1975) sand fly fever viruses occur in the presence of many mammal-biting sand flies of the genus Lutzomyia. The negative results in the Gunong Besout area do not support the idea of Sergentomyia as a reservoir of mammal-adapted virus. Possibly the reports of infection in Sergentomyia may have resulted from sporadic feeds on mammals in an area where many Phlebotomus flies maintained the infection. However, Sergentomyia may harbor reptile-adapted viruses. Our findings, together with the absence of sand fly fever from vast areas of the world where Sergentomyia predominates, suggest that sand flies of this genus play no significant role in the epidemiology of mammal-adapted phlebotomus fever viruses.
Vol. 15, no. 3
1978
Knudsen et al.:
Forest phlebotomines from Malaysia
Quate, L. W. Be C. B. Fairchild. ]961. Phlebotomus sandflies of Malaya and Borneo (Diptera: Psychodidae). Pac 1m. 3: 203-22. Rudnick, A., R. Garcia, J. Jeffery, N. J. Marchette Be D. W. MacVean. 1971. The phlebotomine sand flies of Malaysia. Southeast Asian}. Trop. Med. Public Health 2: 86. Tesh, R. B. Be B. N. Chaniotis. 1975. Transovarial transmission of viruses by phlebotomine sand flies. Ann. N. Y. Acari. Sci. 266: ]25-34. Tesh, R. B., B. N. Chaniotis, P. H. Peralta Be K. M. Johnson. 1974. Ecology of viruses isolated from Panamanian phlebotomine sandflies. Am.}. Tmp. Merl. Hyg. 23: 258-69. Tesh, R. B., S. Saidi, S. Ja. Gaidamovic, P. Rodbain Be J. Vesenjak-Hirfan. ]976. Serological studies on the epidemiology of Phlebotomus fever in the Old World. Bull. World Health Organ. 54: 663-74. Tesh, R. B., S. Saidi, E. Javadian Be A. Nadim. 1977. Studies on the epidemiology of sandfly fever in Iran. I. Virus isolates obtained from Phlebotomus. Am). Tmp. Merl. Hyg. 26: 282-87. Theodor, O. 1938. On sand fly (Phh,botornus) from Ceylon, Siam and Malay. Indian). Merl. Res. 26: 261-69.
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1978. The phlebotomine sandflies (Diptera: Psychodidae) of the Oriental Region. Bull. Br. Mw. Nat. Hist. Entomol. 37: 2]7-343. Lewis, D. J. Be R. KilIiek-Kendrick. 1973. Some phlebotomid sandflies and other Diptera of Malaysia and Sri Lanka. TranI. R. Soc. Trop. Med. Hyg. 67: 4-5. Lewis, D. J. Be R. P. Lane. 1976. A taxonomic review of Phlebotolllw (Idiophiebotolllus) (Psychodidae). Syst. Entomot. I: 53-60. Lewis, D. J. Be R. H. Wharton. 1963. Some Malayan sand flies (Diptera: Psychodidae). Proc. R. Entomot. Soc. London Ser. B 32: 117-24. Lindquist, E. E. Be P. H. Vercammen-Grandjean. 1971. Revision of the chigger-like larvae of the genera NeotmmbirliulIl Leonardi and Mopunguis Wharton, with a redefinition of the subfamily Neotrombidiinae Feider in the Trombidiidae (Acarina: Prostigmata). Can. Entomot. 103: 1557-90. Newstead, R. ]914. Notes on Phlebotomll.S with descriptions of new species. Part II. Bull. Entomol. Res. 5: ]79-92. Quate, L. W. 1962. A review of the Indo-Chinese Phlebotominae (Diptera: Psychodidae). Pac. Ins. 4: 251-67.
29]
23 March 1979
Vol. 15, no. 3: 286-291 ©
1978 by the Bishop Museum
PHLEBOTOMINE SAND FLIES (DIPTERA: PSYCHODIDAE) FROM A PRIMARY HILL FOREST IN WEST MALAYSIA\,2 By A. B. Knudsen3, D. j. Lewis4, R. B. Tesh\ A. Rudnick3, j. jeffery6 and I. Singh7
AbJtract: A 22-month survey was conducted in primary forest in the Gunong Besout Forest Reserve, West Ma]aysia, to test methods of collecting sand Aies, to find out what species occurred, and to examine them for arboviruses. Most sand Ales were collected in tree holes, in traps baited with leaf monkeys and in light traps. Seasonal fluctuations of flies showed a negative correlation with rainfall. Altogether ]407 flies were identified; 770 of them were females. They comprised] 5 species of which
4 \\'ere
new;
1 of these
is known
to occur
only
in the
Phlebotomine sand flies have received relatively little attention in West (peninsular) Malaysia because leishmaniasis is not established there. In 1914, Phlebotomus stantoni Newstead was described from Kuala Lumpur. In 1938, O. R. Causey thought that sand flies were rare in the Malay Peninsula, and O. Theodor (1938) made taxonomic contributions. Lewis (1957) added more infor'This study was supported in part by the University of California International Center for Medical Research through research grant AI 10051 to the Department of International Health, School of Medicine, and by research grant AI 11540 to the G. W. Hooper Foundation, University of California, San Francisco; both grants from the National Inst.itute of Allergy and Infectious Diseases, National Institutes of Health, U.S. Public Health Service. 'This project was conducted in the Arbovirus Research Laboratory in the Department of Medical Microbiology at the Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. 3The G. W. Hooper Foundation, University of California and Faculty of Medicine, University of Malaya, Kuala Lumpur. Send requests for reprints to Dr A. B. Knudsen, do Editor, G. W. Hooper Foundation, University of California, San Francisco, California 94143, USA. 4Department of Entomology, British Museum (Natural History), London, England; supported by a grant from the Medical Research Council, London. sp. O. Box 1680, Pacific Research Section, National Institute of Allergy and Infectious Diseases, National Insitutes of Health, Honolulu, Hawaii 96806, USA. 6Department of Parasitology, National University Faculty of Medicine, Kuala Lumpur, Malaysia. 7Division of Medical Ecology, Insitute for Medical Research, Kuala Lumpur, Malaysia.
(1962)
predicted
that
many species
would
be
found. Lewis & Wharton (1963) reported 16 species from West Malaysia, and Rudnick et al. (1971) discussed the species of Malaysia as a whole. In 1972, mosquitoes were studied in relation to the jungle cycle of dengue virus in the Gunong Besout Forest Reserve in Perak State, West Malaysia (Knudsen & Rudnick 1975). In early 1973, work was extended to test methods of collecting sand flies, to find out what species existed in that part of the country, and to look for viruses in sand flies. This search for virus was undertaken because in some countries plebotomines appear to be reservoir hosts of viruses of the phlebotomus fever group (Tesh & Chaniotis 1975), and because species of both the Old World genera Phlebotomus and Sergentomyia have been reported to harbor such viruses (Barnett & Suyemoto 1961). The study area, in tropical hill rain forest about 198 m above sea level and 156 km NNW of Kuala Lumpur, is 3°54.2'N, 101°19.5'E, in map grids 472,600 X 404,700 (Map sheet 75, ChangkatJong, Series L7010, Edition 2-DNMM, Malaysian National Mapping Service, Kuala Lumpur). Surveys were conducted along the NE slope of the Gunong Besout main ridge, which reaches a summit of 417.6 m. The local rock is Paleozoic (Devonian) metaquartzite and phyllite (information from the Geological Survey of Malaysia Headquarters, Kuala Lumpur). Meteorological stations established in the study area showed a mean annual temperature of 25.6°C at 1.2 m above ground, with average monthly means of 22.6° to 29.1 °c. Monthly rainfall averaged 253.0 mm (TABLEI). The forest reserve comprises 82,983 ha of primary and secondary jungle. Timber was cut between 1958 and 1970 in some areas which are rec-
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study area. Sergentomyia gombaki was the commonest species and sometimes bit man. S. gombaki and S. iyengm~ were taken in traps baited with leaf monkeys. A total of 23,569 sand flies. in 165 pools, were processed for virus isolation with negative results. The sand fly fauna, the apparentl)' negative role of Sergentomyia in virus transmission in the Old World generally, and the absence of Phlebotomus mgentiPes, the vector of kala azar in India, are discussed.
mation and reported R. H. Wharton's discovery, in 1956, of many Phlebotomus (lTgentipes Annandale & Brunetti in Lamir village oh the east coast, which stimulated interest in sand flies. Quate & Fairchild (1961) discovered that they were fairiy numerous in West Malaysia and Sabah and they and Quate
197R
Knudsen
et al.:
Forest
phlebotomines
METHODS
Twelve tree holes and 3 hollow logs were chosen for standardized weekly collecting during the 22month period from May 1973 to February 1975. Resting flies were agitated by hitting the insides of the holes with a slender stick and were aspirated at the entrance for several minutes. During the ongoing mosquito survey, sand flies were occasionally collected in vegetation sweepings along streams, in dry ice-baited light traps in a vertical transect 1.8-32 m above the forest floor, on human bait on the ground, in monkey-baited traps in the canopy at 17, 22 and 45 m, and in a trap baited with hamsters 3 m above ground. All flies caught in animal bait traps were collected alive with a battery-operated aspirator and fell undamaged into cloth holding bags placed above the suction fan. The flies were kept in a cool, shaded place until they could be anesthetized with chloroform, sorted by sex, sealed in glass ampuls, and stored on dry ice pending transport to the laboratory where they were held at -65°C. Represen-
Malaysia
287
TABLE I. Mean monthly rainfall and air temperatures* compared to number of phlebotomines collected from tree hole I, Gunong Besout Forest Reserve, Perak, Malaysia (March 1973-December 1974).
MONTH
RAINFALL
TEMPERATURE °C Max.
(MM)
Min.
NO.
8.5 9.5
March 1973** April** May**
June
AVG.
COLLECT~:[) PER MONTH
79.0
July Aug.
64.0*** 103.5 192.6
30.3 29.9 30.3
23.7 22.7 22.7
16.5 159.0 239.3
Sept. Oct. Nov. Dec.
157.1 674.5 312.6 371.3
30.2 29.7 28.9 27.4
22.4 22.6 22,7
22.3
91.4 36.0 43.2 i32.4
Jan. 1974 Feb. March
117.2 254.6 138.1
28.2 28.8 29.8
21.8 21.9 21.9
170.3 189.7 212.6
April
369.6 317.3 282.7
29,1 28.9 28.4
23.0 22.8
146.8 309.3 277.1
28.2 29.1 28.1
22.5
253.0
22.7
508.0
Sept.
146.6 71.4 410.8
22.4
78.0
Oct. Nov. Dec.
148.5 225.0 261.4
28.4 29.0 29.9
23.0 22.6 22.3
49.8 150.0 24.3
Mean
253.0
29.1
22.6
150.7
May June July
Aug.
* Weather station ** No reading. *** Ten days only.
located
2~.6
0.8 km from site.
tative samples were kept in vials of alcohol for identification at the British Museum (Natural History), London. The flies processed for virus isolation in Kuala Lumpur were sorted by sex and genus or species into pools of variable size, and triturated in 33% normal heat-inactivated rabbit-serum beef-heart infusion broth containing 500 units of penicillin and 500 I-Lgof streptomycin per ml. After centrifugation at 2500 g, the supernatant was inoculated into 12 two-day-old Swiss white mice, 0.01 ml intracerebrally and 0.03 intraperitoneally. Mice were observed for signs of illness for 21 days. If no illness was observed by the 8th day postinoculation, brains of 2 mice were harvested and passed to new litters. Three such blind passages were made before concluding that a pool was negative for presence of virus. The majority of the flies were flown, on dry ice, to Honolulu, Hawaii, where they were pooled in groups of 50-250 by sex. Each pool was triturated with 3.0 ml of diluent comprising buffered saline
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ognizable by the presence of many secondary plant-community specIes. Many pnmary forest trees are still evident, and the original flora of the main study area retains its virgin state as a primary hill forest. The trees of the primary forest belong to the families Burseraceae (Canarium spp.), Myrtaceae (Eugenia spp.) and Dipterocarpaceae (a few species of Shorea). Some typical undergrowth plants are Eugeissona and Arenga palms and species of Randia, Globba and Pandanus. Malaysian hill forests have 2 heterogeneous canopy layers representing many families. The lower layer, reaching a height of 9-12 m, consists mainly of Euphorbiaceae, Meleaceae, various palms, Dilleniaceae and Myristicaceae. The upper, primary layer rises to 30.540.7 m and includes species of Leguminosae, Burseraceae, Dipterocarpaceae and Rhizophoraceae. Tall trees like Shorea reach 61 m to form an emergent canopy. The numerous large vertebrates include elephant, tiger, Malayan sun bear, civet, bear cat (binturong), wild boar, deer, monitor lizard, porcupine, scaly anteater, 2 species of gibbons, and 4 species of cercopithecid monkeys. Many small vertebrates occur, such as lizards, squirrels, rats, vipers, coral snakes, cobras, frogs and toads. The numerous species of birds range from small bulbuls to the great rhinoceros hornbill.
from
288
J.
Mcd. Entomol.
Vol. 15, no. 3
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Reid) and at times Mac'aca species, suspended 22 m above the forest floor, yielded 7.9% of the total, with 2149 flies caught in 1260 twelve- and 24-h collections (average 1.7). CDC-type light traps with dry ice, at 1.8-32 m, used 958 times, twice weekly, produced 659 flies (2.4%, average 0.7). In incidental human bait catches, 51 flies were caught (0.2%). Vegetation sweepings conducted 516 times resulted in 52 flies (0.2%). A small Magoon-type trap housing 2 Silvered Leaf Monkeys (P. cristata) was operated 45 m above the forest floor in emergent canopy. It yielded 33 flies in 239 daily collections (0.1%). Pig-tailed Monkeys, Macaca nemestrina Linnaeus (4-6 animals), placed at ground level in a large Magoon-type trap produced only 8 sand flies in 1340 twelve- and 24-h collections. Hamsters, used in small Magoon-type traps, attracted only 20 flies in 64 twice-weekly collections. A single Long-tailed Monkey, M. fascicularis Raffles, placed at 17 m above ground yielded only 1 sand fly in 730 collections. Only 51 (0.2%) of the phlebotomines collected from tree holes showed evidence of a blood meal. We collected 432 engorged phlebotomines in the monkey-baited trap at 22 m during 1973 and 1974. Of these, 3 specimens were identified as Sergentomyia gombaki but the majority of the engorged specimens appeared to be S. gombaki as well, although they were not precisely identified. Ten of 20 from FIG. I. Tree hole I from which the majority of sand Aies were collected. a hamster-baited trap positioned at tree hole 1 were engorged; these engorged specimens also appeared to be S. gombaki. Thirteen were taken in (pH 7.4) with 0.5% gelatin, 25% heat-inactivated human bait collections. Seventeen engorged speccalf serum, and antibiotics. The suspension was imens were taken in light traps. centrifuged at 8800 g for 30 min at 5°C, and 0.1 The fluctuations in numbers of sand flies colml of supernatant was inoculated into each of 3 lected at the best site, tree hole 1, were correlated tube cultures of Vero cells. After absorption for 1 with the amount of rainfall at the camp about 0.8 h at 37°C, maintenance medium was added and km away. The hole, open to rain but permanently changed every 3-4 days thereafter (Tesh et al. shaded in a tree leaning at about 60°, was 2.86 m 1977). Cultures were incubated at 37°C for 14 days above ground and measured 63.5 cm high, 25.4 and were examined regularly for cytopathic effect. em wide, and 134.6 cm deep (FIG. 1). In 1974, measurable rain fell on 184 (55%) of 334 days meaRESULTS sured. The mean annual rainfall exceeded 3000 mm, with peaks in October and December (1973) During the 22-month period 27,084 phlebotoand in April and September (1974). mines, 41.3% of them fema~es, were collected by all methods. The species of phlebotomines. A total of 1407 sand From tree holes (including hollow logs) 869 col- flies were identified at the British Museum (Natlections yielded 24, III flies (89.0%), with an av- ural History); 770 were females and 637 males. Identifications of the females are shown in TABLE erage catch of 27.7. A large Magoon-type trap housing 6-8 leaf 2. The males are not listed, since specific identifimonkeys (Presby tis cristata Raffles and P. obscum cations of most were not made.
Knudsen et a!.:
1978
TAHLE
2.
Forest phlebotomines
Identification of 770
'l'
from Malaysia
289
sand flies collected by different methods.
TREE
LIGHT
ROOT
LEAF
BITING
HOLES
TRAPS
HOLES
MONKEYS
MAN
Phlebotolllus (ldiophlebotollltL\ ) frolldifer Lewis & Lane (A naphlebotoll/us) stalltoni Newstead
TOTALS OTHERS*
%
0.4
3 7
0.9
138 61 8 31 19 1 2 4
46.3 10.8 3.5 4.0 4.6 0.4 0.5 0.8
19
5.4
14 57 17 2
2.2 11.4 6.2 2.6
(ungrouped) pach)'slollla (Quate & Fairehild) t'eidi (Lewis) cheongi Lewis & jeffery klltl/Lwli Lewis & jeffery
Total %
* Separate
49.5 general collections.
P. frondifeT is known only from the study area. It belongs to the subgenus Idiophlebotomus, a peculiar group whose members are known from a few isolated bat caves scattered through the Oriental Region. Three females and 5 males of P. frondifer were found in a cave like shelter formed by a fallen
100
tree.
No true caves were
found
in t.he
vicinity, merely narrow cracks in outcrops of rock. The only other specimen, a male, came from a light trap located approximately 250 m away. S. gombaki fed to repletion on the face and hands of collectors, in camp or near a tree hole, and were attracted to humans occasionally. Two larval mites were found on females of S. gombaki and S. malayae. They are related to the trombidiid genus Neotrombidium Leonardi (Neotrombidiinae), species of which parasitize beetles living under bark (Lindquist & VercammenGrandjean 1971). Search for viruses. A total of 14,636 sand Aies (5308 females and 9328 males), grouped into 138 pools, were processed for virus isolation in Honolulu. An additional 8933 sand Aies were processed for virus isolation in 27 pools in Kuala Lumpur. No viruses were isolated. DISCUSSION
Sand flies. The genus Sagentomyia is widespread in many parts of the Old World tropics, so
its preponderance in the Gunong Besout area is not unexpected. The small number of man-bitings recorded are in line with the known preference for reptiles of many species of Sergentomyia, although in some countries several habitually reptile-biting species are known
to attack
man occasionally.
S. gombaki was by far the commonest species and the only known man-biter. Its numbers in leafmonkey traps, coupled with the total catch of sand flies in these traps, suggest a definite attraction to these animals. S. gombaki has a long proboscis, which is a normal feature of mammal-biting sand Aies. The numbers of sand Aies coming to traps baited with different monkeys suggest a host-specific attraction, like that reported by Chaniotis et al.(l971). The number of S. iyengari in the leaf-monkey trap, compared with its numbers in light traps and in the area generally, suggests a definite attraction to this animal. One S. iyengari was taken 45 m above ground. The collections show many sand Aies occur in the canopy. The detection of species at various heights above ground may be inAuenced by the collecting methods. All or most P. frondifer, S. malayae, S. barraudi, S. reidi and S. knudseni were taken at or near ground level, S. gombaki at various levels, and S. iyengari in the canopy. During months with less than average rainfall,
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SergelltulI/)'ia (I? olldatlOlIl)'ia) gumbaki (Lewis & Wharton) i)'engari (Sinton) lIla/a)'(l/! (Lewis) wharloni (Lewis) jeffetyi Lewis lambotl Lewis & jeffery gl'lllmea Lewis & jeffery hamidi Lewis & jeffer)' (Pan-ulolll)'ia) ban"01uli (Sinton)
290
J.
Med. Entomol.
the number of flies increased. When monthly rainfall exceeded the yearly mean, the number of flies decreased. Fly numbers and rainfall showed a negative correlation; it is likely that rainfall of more than 250 mm in a month prevented emergence of adults. These findings are similar to those reported by Chaniotis et a!. (1971).
P. argentiPes extends from Iran to Bali and is an important vector of kala azar in India, where it is peridomestic and bites man but prefers cattle. Its biology and distribution in Southeast Asia are of interest in relation to the possible eastward limit of kala azar and the possible effect of large-scale deforestation (Lewis 1973, 1974). It has been found sporadically in West Malaysia and was temporarily abundant in 1 village (Lewis & Killick-Kendrick 1973), and it bites man occasionally (Lewis & Wharton 1963). The absence of P. argentiPes in the Gunong Besout study area shows that it is by no means evenly distributed in West Malaysia, even as a wild forest species. There is need for further study of its distribution. Acknowledg-rnf'11LI".·
We thank
Mr Samuel
W. James
and othel'
members of the Arbovirus Research Laboratory field staff for their invaluable assistance in making field collections. We are indebted to the staff of the Department of Medical Microbiology, University of Malaya, and to the Director of the Institute for Medical Research. Our gratitude and app"eciation go to Dr E. Soepadmo and Encik Malmud bin Sider of the Department of Botany, Faculty of Science, University of Malaya, for classification and identification of the Aora in the study area; to Mr Mah Weng Hong and his staff of the Geological Survey of Malaysia Headquarters for providing information on the geology of the area; to Mr Chan Wah Sun of our laboratory for ingeniously constructing cages, light traps and other devices; and to Mr D. Macfarlane of the Commonwealth Institute of Entomology, who kindly examined the mites.
LITERATURE
CITED
Abonnenc, E. 1972. Les phlebotomes de la region ethiopienne (Diptera, Psychodidae). Mem. Off. Reck. Sci. Tech. Outre-Mer. 55: 1-289. Aitken, T. H. C., J. P. Woodall, A. M. P. Andrade, G. Bensaboth &: R. E. Shope. 1975. Pacui virus, phlebotomine Aies, and small mammals in Brazil: an epidemiological study. Am. J. Trap. Med. Hyg. 24: 358-68. Barnett, H. C. &: W. Suyemoto. 1961. Field studies on sandAy fever and kala-azar in Pakistan, in Iran, and in Baltistan (Little Tibet) Kashmir. Trans. N. Y. A cad. Sci. 23: 609-17. Causey, O. R. 1938. Phlebotomw; of Siam with a description of a new variety. Am. J. Hyg. 28: 487-89. Chaniotis, B. N., J. M. Neely, M. A. Correa, R. B. Tesh &: K. M. Johnson. 1971. Natural population dynamics of ph lebotomine sandAies in Panama. J. Med. Entomot. 8: 339-52. Knudsen, A. B. &: A. Rudnick. 1975. The silent jungle cycle of dengue virus and its tenable relationship to endemic dengue in Malaysia. (Abstract.) Proc. J 3th Pac. Sci. Congr. 1: 334. Lewis, D.1- 1957. Some sandAies (Phlebotominae) of Malaya. Proc. R. Entomol. Soc. London Ser. B 26: 165-71. 1973. Absence of leishmaniasis in South-East Asia. Absl. 9th In/. Congr. Trap. Med. Malar.: 147-48. 1974. The biology of Phlebotomidae in relation to leishmaniasis. p. 363-84. In: Smith, R. F., T. T. Mittler & C. N. Smith, eds., Annu. Rev. Entomol. Vol. 19. Annual Reviews, Inc.
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Viruses. Viruses of the phlebotomus fever arbovirus group have been isolated in southern Europe, Africa, central Asia and Central and South America, and at least 25 different serotypes are now known (Tesh et a!. 1977). In view of the relatively high virus infection rates observed among sand flies in other areas (Tesh et a!. 1974, 1977, Aitken et a!. 1975), one might expect these agents also to be present in Malaysia. However, no virus isolates were obtained from the 23,569 sand flies processed from Gunong Besout. These results suggest that mammal-adapted phlebotomus fever viruses are absent from the study area. At least one virus isolate has been reported from Sergentornyia species in Iran (Barnett & Suyemoto 1961). This apparent absence of phlebotomus fever viruses may well be related to the small number of species and individuals of the genus Phlebotomus, which includes all of the habitual mammal biters of the Old World. In the Gunong Besout area Phlebotomus made up only 1% of the total catch and was represented by 1 obscure aberrant species that probably bites bats and is not known to attack man. The findings contribute something to the world picture of phlebotomus fever viruses and their vectors (Tesh et a!. 1976), by demonstrating the apparent absence of the viruses in an area dominated by Sergentomyia. In contrast, in some tropical American forests (Tesh & Chaniotis 1975) sand fly fever viruses occur in the presence of many mammal-biting sand flies of the genus Lutzomyia. The negative results in the Gunong Besout area do not support the idea of Sergentomyia as a reservoir of mammal-adapted virus. Possibly the reports of infection in Sergentomyia may have resulted from sporadic feeds on mammals in an area where many Phlebotomus flies maintained the infection. However, Sergentomyia may harbor reptile-adapted viruses. Our findings, together with the absence of sand fly fever from vast areas of the world where Sergentomyia predominates, suggest that sand flies of this genus play no significant role in the epidemiology of mammal-adapted phlebotomus fever viruses.
Vol. 15, no. 3
1978
Knudsen et al.:
Forest phlebotomines from Malaysia
Quate, L. W. Be C. B. Fairchild. ]961. Phlebotomus sandflies of Malaya and Borneo (Diptera: Psychodidae). Pac 1m. 3: 203-22. Rudnick, A., R. Garcia, J. Jeffery, N. J. Marchette Be D. W. MacVean. 1971. The phlebotomine sand flies of Malaysia. Southeast Asian}. Trop. Med. Public Health 2: 86. Tesh, R. B. Be B. N. Chaniotis. 1975. Transovarial transmission of viruses by phlebotomine sand flies. Ann. N. Y. Acari. Sci. 266: ]25-34. Tesh, R. B., B. N. Chaniotis, P. H. Peralta Be K. M. Johnson. 1974. Ecology of viruses isolated from Panamanian phlebotomine sandflies. Am.}. Tmp. Merl. Hyg. 23: 258-69. Tesh, R. B., S. Saidi, S. Ja. Gaidamovic, P. Rodbain Be J. Vesenjak-Hirfan. ]976. Serological studies on the epidemiology of Phlebotomus fever in the Old World. Bull. World Health Organ. 54: 663-74. Tesh, R. B., S. Saidi, E. Javadian Be A. Nadim. 1977. Studies on the epidemiology of sandfly fever in Iran. I. Virus isolates obtained from Phlebotomus. Am). Tmp. Merl. Hyg. 26: 282-87. Theodor, O. 1938. On sand fly (Phh,botornus) from Ceylon, Siam and Malay. Indian). Merl. Res. 26: 261-69.
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1978. The phlebotomine sandflies (Diptera: Psychodidae) of the Oriental Region. Bull. Br. Mw. Nat. Hist. Entomol. 37: 2]7-343. Lewis, D. J. Be R. KilIiek-Kendrick. 1973. Some phlebotomid sandflies and other Diptera of Malaysia and Sri Lanka. TranI. R. Soc. Trop. Med. Hyg. 67: 4-5. Lewis, D. J. Be R. P. Lane. 1976. A taxonomic review of Phlebotolllw (Idiophiebotolllus) (Psychodidae). Syst. Entomot. I: 53-60. Lewis, D. J. Be R. H. Wharton. 1963. Some Malayan sand flies (Diptera: Psychodidae). Proc. R. Entomot. Soc. London Ser. B 32: 117-24. Lindquist, E. E. Be P. H. Vercammen-Grandjean. 1971. Revision of the chigger-like larvae of the genera NeotmmbirliulIl Leonardi and Mopunguis Wharton, with a redefinition of the subfamily Neotrombidiinae Feider in the Trombidiidae (Acarina: Prostigmata). Can. Entomot. 103: 1557-90. Newstead, R. ]914. Notes on Phlebotomll.S with descriptions of new species. Part II. Bull. Entomol. Res. 5: ]79-92. Quate, L. W. 1962. A review of the Indo-Chinese Phlebotominae (Diptera: Psychodidae). Pac. Ins. 4: 251-67.
29]
