186 J. Ar"t. Mosg. Coxrnol Assoc. Vor. l, No.2

LABORATORY COLONIZATION OF UNIFORMIS, MA. INDIANA AND MA. BONNEAE IN

G. L. CHIANG, W. H. CHEONG, K. P. LOONG, K. L. ENG ero W. A. SAMARAWICKREMAT Division of Medical Entomology, Institute for Medical Research, Kuala Lumpur, Malaysia

ABSTRACT. Methods are described for the laboratory colonization of Mansonia unifmmis, Ma. indiana and Ma. bonncae in Malaysia. Gravid females oviposited in 500 ml beakers with a layer of water covered with small leaves of Salainia. Newly hatched larvae were set up in a basal medium of guinea pig dung and water or liver powder, yeast powder and water. Larvae attached to aquatic plants or'Keaykolour' ruffia snow white paper. The cultures with paper gave better yields than those with plants. Production of Ma. unifurmis was higher than the other two species. Twelve generations of Ma. unifmmes and I I generations of Ma. indiana and,Ma. bonneae were monitored in the laboratory.

INTRODUCTION Efforts of Wharron (1962) to colonize Malay- sian Mansonia, using guinea pig dung infusion The study of filarial parasites in wild and and Eichhornia plants yielded small numbers domestic in Malaysia (Poynton and which enabled him to study their susceptibility Hodgkin 1939, Edeson et al. 1955) and in to human and filariae. Recently in Kuala (Buckley et al. 1958) which are mor- Lumpur, efforts were intensified to standardize phologically similar to the human filafiae improved methods and production of Ma. uni- mnlayi, and the necessity to identify these forrnis, Ma. indiana (Edwards) and Ma. btnneae parasites provided an impetus to colonize their (Edwards). Preliminary results were reported vectors, species of Mansonia,. Jayawickreme and by Cheong et al. (1984); details are recorded in Niles (1952), following Bonne-Wepster and this paper. Brug (1939), described a technique for rearing In the meantime Sucharit et al. (1981) in Mansan'ia uniformk (Theobald) and, Ma. an- have reported on yields of adults ob- nulifna (Theobald) in small numbers through tained in their cultures of Ma. uniformis, Ma. six generations utilizing a larval medium of ind;iana, Ma. annulifera and Ma. bonneae. dried powdered guinea pig dung in water. Wharton (1957, 1962) used these methods to MATERIALS AND METHODS rear Ma. uniformis in Malaysia. A successful large scale colonization of Mansoni.a africana MarNrnNeNcE oF ADULTS.Fifty to 100 mos- (Theobald) and Ma. unformis from Africa and quitoes were maintained in 250 ml unwaxed Malaysia was reported from England (Laurence paper cups closed with netting arranged on a and Smith 1958, Laurence et al. 1962). plastic tray. Humidity was maintained at Principal difficulties in the colonizarion of 7V90Vo by placing wet cotton on cups and Marconia have been the provision and mainte- covering this with another ray in a humidified nance of aquatic plants to which the larvae and insectary at 24-26oC. Mating occurred readily pupae attach and the maintenance of an ade- ln paper cups, quate food supply. Early workers used common Adults fed on l0% sugar solution from cot- aquatic plants such as , Salainia and Eiih- ton wool pads placed on the netting. Females hom'ia unlized by the immature stages in the were blood-fed either by exposing the mos- field. Laurence and Smith (1958) and Laurence to a human arm or a restrainedwhite et al. (1962), following Wanson (1944), used lTn:: crepe paper for larval attachment in intusions Mansonia mosquitoes lay eggs on the under of guinea pig dung and animal diet. A piece of surface of leaves of aquatic plants. Following grass sod or turf was placed in the container the method of Laurence and Smith (1958), with the newly hatched larvae. These methods bloodfed females were held in paper cups and were followed by Samarawickrema (1962, 1968) when gravid were introduced into 500 ml beak- to colonize Sri Lankan strains of Ma. unifurrnis ers, closed with netting and containing water and Ma. annulifera in London. Later and young Salainia plants. Most females Samarawickrema (unpublished data) suc- oviposited immediately. cessfully colonized (Walker) in MerNrnNeNcE oF r-lRvAE. Leaves with egg Florida, USA, using white offset board paper masses were allowed to.hatch into a dilute larval (Gestetner 223T), fish meal and liver yeast medium with a few young Salvinia plants in a media. humidified insectary at 24-26"C and photo- period L:D 12:12. The eggs hatched mostly 1W.H.O. Entomologist. overnightbetween 1900 and 0600 hr. The eggs Junr, 1985 J. Av. Mosq. Covrnol Assoc. 187 of Ma. uniformis, Ma. indiana and Ma. bonneae MerurnreNcE oF pupAE. In the indoor insec- hatched in a mean period of 4, 6 and 5 days, tary, pupation of Ma. uniform,k, Ma. ,na and, respectively, after their deposition. Ma. bonneae occurred in (mean + SD) 26-f 2.3, Larval cultures were set up in transparent 33-+4.6 and 35-17.5 days respectively in guinea plastic tanks 30 x 16 x 20 cm covered with pig dung cultures and in 28+3.2,31-r3.2 and plastic mesh. Media used were: (a) dried pow- 35+3.1 days in liver yeast cultures. In the out- dered guinea pig dung, l0 gm per liter of door insectary pupation in the three species deionized water, allowed to stand for 48 hr and occurred in l8-+1.1, 24-+1.6 and 28-r3.4 days, diluted l:2 when used, (b) an infusion of equal respectively in guinea pig dung cultures and parts of Bacto liver powder and a local brand of l9+1.7, 27-+2.3 and 27-+2.0 days, respectively ground dried yeast, I gm per liter ofdeionized in liver yeast cultures. When pupae appeared water. on the paper, they were gently detached with a The attachment sites for larvae were the soft paint-brush, counted and transferred to a aquatic plants Eichhmnia crassipes and Jussiaea beaker of water. Pupae harvested daily were rebens, the semi-aquatic weed Alternanthera placed inside a cage for adult emergence. 'Keaykolour' ninndra and ruffia snow white paper (substance 250 GSM), manufactured by RESULTS Wiggins Teape Paper Ltd., Birmingham, U.K. Selected plants were individually washed sev- Tables l-3 show the relative successof the eral times and the shoot and root systems cultures of the three species. The best overall cleaned of debris and visible organisms 6efore results were obtained for Ma. unifonnis on placing them in the culture media. Rectangular paper in guinea pig dung infusion at 28-30"C pieces of paper 33 x 16 cm folded into two yielding 53.3% pupation and 37.6% aduh were used, one per culture. emergence (Table l). The next best was 37.37o A typical culture contained 250 newly pupation and 29.6Vo emergence in the liver hatched larvae in 3 liters of medium with paper yeast and paper cultures in the same insectary. or plant substratum. About 250 mg of dried In contrast, the yield of adults of Ma. indi,ana yeast were added to each culture once in 3 days. was very low. Paper and,Jussiaea repens in dung Plants were replaced as they deteriorated in the infusion supported Ma. indiana best, yielding cultures. Papers were replaced with new ones 37Vo mean pupation and,2lVo mean emergence every third day. The pH of the medium in a in the outdoor insectary. Mansoni.a bonneae was sample of cultures in each generation was mea- most often cultured on paper. The mean sured using an Orion research digital pH emergence success of l8.lVo in the indoor in- meter. It changed from 6.8 to 7.2. These sectary was comparable to that of 22.3Vo in the agreed with the values reported by Laurence outdoor insectary (Table 3). and Smith (1958) and Wharton (1962). Results from individual cultures of Ma. uni- Initial cultures were maintained in an indoor formis on paper showed 70.5-70.9Vo of the humidified insectary at 24-26"C. Sub- pupae emerging as adults and, on plants, sequently, most cultures were set up in an out- 70.2-74.1Vo. The percentage of pupae emerg- door unhumidified insectary at 28-30"C. ing as adults of Ma. indi.ana was 47.0-57.2Vo on

Table l. Percentage of pupatidn and emergence from first instar larvae of Mansonia unifurmis set up in two culture media with 250 larvae in each culture.

Attachment host for No. of Mean Vo Mean Va Medium larvae and pupae tests pupation emergence (Range) INDOORS (2.1-26'C) Guinea Pig dung infusion Paper 30 29.3 (0.0-80.0) 20.7 (0.G55.2) Altemanthera 8 l9.l (4.4-33.2) l5.l (3,2-26.4) Liver yeast infusion Paper 25 39.9 (r.2-77.2) 28.9 (0.4-54.0) Eichhomia 5 25.7 (9.L37.2) 10.2 (6.4-20.0) Jussiaea repens ll 29.2 (0.0-74.4) 22.5 (0.L5e.2) Alternanthera 5 7.6 (0.0-18.0) 3.8 (0.G10.4) ouTDooRS (28-30.C) Guinea Pig dung infusion Paper 25 53.3 (0.0-81.2) 37.6 (0.0-65.6) Eichhomi.a 6 27.3 (7.6-54.4) 20.3 (6.4-43.6) Altemanthera 6 l l.5 (4.0-20.4) 8.3 (3.6-le.2) Liver yeast infusion Paper 27 37.3 (0.0-78.8) 29.6 (0.L60.4) Eichhornia I 24*.3 (2.0-*60.8) 18.0 (0.8-41.2) Alternanthera 5 t4.7 (5.2-22.4\ 188 J. At"t. Mosq. CoNrnol Assoc. Vor-.l, No.2

Table 2. Percentage of pupation and emergence from first instar larvae of Marconiaindiana set up in two culture media with 250 larvae in each culture.

Attachment host for No. of Mean Vo Mean Vo Medium larvae and pupae tests pupation (Range) emergence (Range) INDOORS (24-26'C) Guinea Pig dung infusion Paper 26 18.7 (0.0-40.0) 8.8 (0.C27.6) Liver Yeast Infusion Paper 27 21.9 (0.0-66.8) 9.6 (0.L26.4) Jussiaea repens 8 23.2 (r.6-44.0) l2.l (0.0-28.8) ouTDooRS (28-30'C) Guinea Pig dung infusion Paper l9 36.8 (10.0-67.2) 19.5 (2.G34.8) Jussiaea repens 5 34.7 (27.2-46.4) 2l.o (r7.2-24.4) Alternanthera 6 l1.5 (2.4-20.4) 7.r (1.6-14.8) Liver yeast infusion Paper t7 20.0 (4.4-46.4) l1.5 (1.2-28.0) Ei.chhornia 6 rr.7 (2.0-19.2) 6.6 (1.2-l1.2) Jusinza repens 6 l3.l (2.0-26.4) 8.3 (0.G19.6)

paper and 52-60% on plants. In Ma. bonneae parison (Tables l-3), confirming the observa- the emergence was 69.8-74Vo on paper and tions of Laurence and Smith (1958). The pa- 53.1-53.4Vo on plants. The comparable per- pers were uniform, convenient to handle and centages of adult emergence from pupae in the the progress of larval development could be paper and plant cultures of Ma. uniformis and easily assessed by lifting the papers out of the Ma. indinna suggest that the gende dislodging of cultures. In addition. there was less risk of in- the pupae attached to paper with a soft troduction of contamination as in the case of paintbrush did not increase their mortality. plants. Larval counts 1Gl2 days after initiation of The three plants tested gave varying results cultures showed less mortality in Ma. unifurmis for the three species. Although the use of plants than in the other two species(Tables l-3). In all had the convenience of less frequent replace- species, mortality of the larvae in the guinea pig ment of the attachment substrate as compared dung infusion cultures was more than in the with paper, the debris of rotting plants, espe- liver-yeast cultures indoors at 24-26'C, while cially in the case of Eichhornia, probably inter- the reverse was true in the outdoor insectary at fered with larval development. The Eichhmnia 28-30'C. plants deteriorated and withered fast in the or- The numbers of adults produced during the ganically polluted medium under indoor con- colonization of l2 generations of Ma. uniform'is ditions. It proved to be a poor host to Ma. and I I generations each of Ma. ind;iana and Ma. bonneae.Jussiaca repens withstood the conditions bonneaeare given in Table 4. These results show of the culture medium bett€r and its thick, that even though the mean percentage yields hardy stem proved better for larval attachment. were low, especially in Ma. ind,iana and Ma. bon- Predictablv. Altsnnnthcra triandra was the least neae, adequate numbers were produced con- effective. tinuously for experimental studies. The dung medium was used by Wharton (1957) as a finely ground powder. Laurence and Smith (1958) simplified the preparation by DISCUSSION suspending dry dung pellets in tap water and The cultures using paper gave better overall diluting it when setting up the cultures. The yields than those with plants in all but one com- guinea pig dung infusion has the disadvantage

Table 3. Percentage of pupation and emergence from first instar larvae of Mansonin bonruaz set up in two culture media with 250 larvae in each culture.

Attachment host for No. of Mean Vo Mean Vo Medium larvae and pupae tests pupation (Range) emergence (range) TNDOORS(24-26" C) Guinea Pig dung infusion Paper 22 2l.l (0.0-34.8) t4.7 (0.0-34.8) Liver yeast infusion Paper 5l 2s.3 (0.0-63.2) 18.I (0.0-58.8) Jussiaca repens 6 34.t (13.6-54.0) l9.l (4.8-35.2) ouTDooRs (28-30"c) Guinea Pig dung infusion Paper 23 31.5 (4..+-50.0) 22.3 (4.4-47.2) Liver yeast infusion Paper 20 r5.2 (0.0-46.0) I 1.3 (0.0-31.2) Eichhomia 6 10.7 (0.0-24.8) 3.t (0.0-13.6) Junn, 1985 J. Ar"r.Mosq. Colmnor Assoc. 189

Table 4. Production of adults of Mansonia species in the laboratory through 12, ll and ll generations respectively.

Ma. unifmmis Ma. india:rn Ma. bonwac No. of No. of No. of adults adults adults emerged emerged emerged Generation d I /6emergence 70 emergence 7oemergence FI 550 571 25.5 58 79 4.5 362 351 20.7 F2 439 332 26.I ll7 r24 10.7 226 236 13.2 F3 328 218 9.5 156 166 17.4 294 277 16.9 F4 430 325 t4.4 56 42 9.8 tt7 ll3 18.4 F5 591 602 18.4 202 194 8.8 3ll 313 25.0 F6 673 586 27.4 67 8l 8.2 438 343 14.9 F7 1888 r762 25.0 315 255 7.9 248 166 15.6 F8 482 462 19.5 500 435 13.8 826 736 20.0 F9 1233 l 182 26.7 455 392 10.0 402 387 8.4 Fl0 l09l 999 18.9 273 299 8.9 760 635 15.4 Fll l2l4 l00l 16.4 327 242 13.4 330 250 6.9 Fl2 1558 1633 24.8

of the unpleasant odor and the irritant effects sity of Malaya, Kuala Lumpur, for their crit- on the skin. In London, this medium was aban- icism of the manuscript. doned in favor of an infusion of pellets of bal- The work described in this paper was sup- anced animal diet in tap water (Laurence et al. ported by a grant from the World Health Or- le62). ganization, Western Pacific Regional Office. It Larval media with liver powder and yeast forms a part of the graduate study program of have been widely used in cultures the senior author at the Universiti Sains (Nayar 1967, Nayar and Sauerman 1973). Malaysia,Penang, Malaysia,supported by a Re- Liver-yeast media have also been used to cul- search Training Grant (Grant No. 820515) tlure Maruon'in (Samarawickrema 1968, Nayar et from the UNDP/World BanVWHO Special al. 1973). In our cultures liver yeast infusion Programme for Research and Training in was as effective as the guinea pig dung infusion. Tropical Diseases. Our results for Ma. unifurmi.s are similar to those of Laurence and Smith (1958) and Laur- Rdermces Cited ence et al. (1962). Using a new infusion of liver Bonne-Wepster, and S. L. Brug. 1939. Observa- powder and yeast powder, we have J. successfully tions on the breeding habits of the subgenusMaz- colonized Ma. unifurmis, Ma. indinna and Ma. sonioidts(genus Maruonia, Culicidae). Tijd. Entomol. bonneae, Colonization of four species, Ma. uni,- 82:81-90. fonnis, Ma. anndfera, Ma. indiana and, Ma. bon- Buckley,J.J. C., C. S. Nelsonand R. B. Heisch.1958. neae has been recently reported by Sucharit et On WucherniaPatei n.sp. from the lymphatics of al.-(1981) in Thailand using guinea pig dung catsand dogs and genet catson Pate Island, Kenya. infusion and planrs. No details have been re- J. Helminthol. 30:73-77. ported on the numbers produced and the du- Cheong, W. H., G. L. Chiang, K. P. Loong and W. A. Samarawickrema.1984. Laboratory colonization ration of the colonies. Our results using paper of Mansoniain Malaysia:A preliminary report. Mosq. and liver yeast medium with better yields repre- News44:72-73. sent several improvements on the Thailand Edeson,J. F. B., R. H. Wharton and J. J. C. Buckley. work. Our work aims to standardize liver-veast 1955. Filarial parasites resembling Wu.chereria medium and paper cultures in the colonization malayi in domestic and forest animals in Malaya. of Mansunia. Trans. R. Soc.Trop. Med. Hyg. 49:60.{-605. Jayewickreme,S. H. and W. J. Niles. 1952.A tech- nique for reaing Mansoniaidesmosquitoes ACKNOWLEDGMENTS in the lab oratory. CeylonJ. Sci.,B. 25:l-6. The authors would like to thank the Director. Laurence,B. R. and S. A. Smith. 1958.The breeding Institute for Medical of Taeniorhynchru(subgenus Mansonioides)mos- Research,Kuala Lumpur, quitoesin for the laboratory. Trans. R. Soc.Trop. Med. his supporr and permissionto publish;Dr. Hyg. 52:518-526. Mak Joon Wah, Head of Filariasis and Malaria Laurence, B. R., R. Pageand S. A. Smith. 1962.Labo- Divisions, Institute for Medical Research. and ratory colonization of Mansonia mosquitoes. Bull. Dr. Yong Hoi Sen,Associate Professor, Univer- Entomol.Res. 53:51&-519. 190 J. Ar'r. Mosq. CoNrRoLAssoc. Vor". l, No. 2

Nayar, J. K., 1967. The pupation rhythm in Aedes sonia (Mansonioides)annulifera Theobald from nmiarlrynchus(Diptera: Culicidae) IL Ontogenetic Ceylon. CeylonJ. Med. Sc. 17(2):7-19. timing, rate of development and endogenousdiur- Sucharit, S., V. Kerdibule, C. Apiwathnasorn, C. nal rhythm of pupation. Ann. Entomol. Soc. Am. Harinasutaand R. F. Gass.1981. Studieson the 60:94il971. colonization of Maruonia mosquitoesin Thailand. Nayar,J. K. and D. M. Sauerman.1973. A compara- SoutheastAsian J. Trop. Med. Pub. Hlth. 12:464- tive study of flight performance and fuel utilization 465. as a function of age in females of Florida mos- Wanson, M. 1944. ElevageduTaeniorhynthus (Coquil- quitoes.J. .Physiol. 19:1977-1988. lettidia) metaliczs Theobald. East Afr. Med. J. Poynton,J. O. and E. P. Hodgkin. 1939. Two mi- crofilariae of the Kra monkey (Macacairar). Trans. 2r:269-272. R. Soc. Trop. Med. Hyg. 32:55b-556. Wharton, R. H. 1957. Studieson filariasis in Malaya: Samarawickrema,W. A. 1962. Changes in the ovariole Notes on the breeding of Mawonia (Mansonioi.des) of Marcunia (Mansonfuidzs)mosquitoes in relation to mosquitoes in the laboratory. Ann. Trop. Med. age determination. Ann. Trop. Med. Parasitol. Parasitol.5l:297-300. 56:I l0- 126. Wharton, R. H. 1962. The biology of Mansoniamos- Samarawickrema, W. A. 1968. Laboratory culture quitoes in relation to the transmissionof filariasis in and life cycle of two speciesof mosquitoes,Man- Malaya. Bull. ll, Institute for Medical Research, sonia (Marconioifus) uniformis Theobald and Man- Kuala Lumpur. I 14 p.

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