DrcrMsnn.1986 Coqu t uttt t ottt cn 4ss/pEs rN Melevsle 545 LABORATORY COLONIZATION AND LIFE CYCLE OF COQUILLETTIDIA CRASSIPES IN MALAYSIA G. L. CHIANG, W. H. CHEONG, W, A. SAMAWICKREMA AND K. L. ENG Division of Medical Entomology, Institute for Medical Research, Kuala Lumpur, Malaysia ABSTRACT. Methods are described for the laboratory colonization of Coquillettidia crassipes.The highest rateof inseminationoccurredin60 x 60 x l20cmcageiandbetterinseminationinlaboraioryadaptedFl5 generation. Embryonation and hatchability of eggs ranged from 69.6 to 97 .9% and 63.3 ro 94.3% respectively. Gravid females laid egg rafts on water in 500 ml beakers with small leaves of Salainia for resting. 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. At present 2l generations of Cq. erassipeshave been reared in the laboratory. INTRODUCTION attach themselves to the roots of aquatic plants with consequent difficulties in laboratory colo- uassipes (Van der Wulp) is an Coquillettidia nization. ornithophilic mosquito having a distribution The methods for maintenance of wild from the Indian subcontinent through the caught, blood-fed females till they became Southeast Asian Region to Papua New Guinea gravid, setting up of larval cultures using and North Australia. In Sri Lanka, Niles et al. guinea pig dung and liver-yeast infusions with (1965) Dissanaike and Fernando (1965) and plants and paperr for larval attachment in discovered that Cq. crassipes was the natural indoor and outdoor insectaries, the insectary vector of Cardiofilaria nilesi in poultry. This conditions, frequency of replacement of plants finding was of considerable significance in that and paper, harvesting and maintenance of the prepatient period of the filarioid in poultry pupae were the same as used for Mansonia was as short as days (Niles et al. 1965, Niles 2l (Chiang et al. 1985). and Kulasiri 1970). The host-parasite combina- Unlike the Mansonia species colonized, Cq. tion was described as a promising model for crassipesdoes not mate in 250 ml paper cups or filariasis research. small cages. Mating trials were conducted in The main difficulty experienced in the study cages of three different sizes, 30 cm cube, 60 of C. nilesi was the lack of laboratory-colonized cm cube and 60 x 60 x 120 cm. Males and Cq. crassipes.Attempts to infect chickens with C. females were introduced into the cages in the nilzsifrom Sri Lanka, in London using laboratory- ratio of 2: l. Insemination was compared in the bred Aedes/ogoi (Theobald) (Gooneratne 1969) F1 and the F15 generations. were only partially successful. Later, Niles and Coqu,illettidia crassipes lays boat-shaped egg Kulasiri (1970) used small numbers of laboratory- rafts rather like Culex but broader in the bred and wild caught Cq. uassipes to infect middle. Gravid females were introduced into clean chickens. However, further studies on 500 ml beakers with water and young Salainia Cardiofilaria were abandoned due to the lack of plants and covered with netting. Oviposition colony material of Cq. crassipes. always occurred on the surface of water, not In our studies on Mansonia in relation to the immediately after the females were introduced, transmission of Brugian filariasis in Malaysia, as in Mansonia, but during the night. Samples natural infections of subperiodic Bntgia rnalayi of egg rafts, after the eggs hatched, were and C. nilesi were found in Cq. rassipes (Chiang examined under the stereoscopic microscope et al. 1984, Mak et al. 1984, Chiang et al. 1986). for non-embryonated eggs. Laboratory colonized Cq. crassipesis essential to study its susceptibility to subperiodic B. rnalayi and C. nilesi. Three species of Mansonia, Mansonia unifonnis RESULTS AND DISCUSSION (Theobald), Ma. indiana (Edwards) and Ma. bonneae(Edwards) have recently been colonized Cor-oNrzerroN. Table I shows the results of in the laboratory at the Institute for Medical the mating trials in the paper cups and in the Research, Malaysia (Chiang et al. 1985). The cages of different sizes. Insemination occurred methods have been used with some modifica- in all three cage sizes,the highest rate being in tion to colonize Cq. crassipes. the 60 x 60 x 120 cm cage. Better insemina- MATERIALS AND METHODS I "Keaykolour" ruffia snow white paper (substance The habitats of Cq. crrusipesoverlap with 250 GSM) manufactured by Wiggins Teape Paper, those of Mansonia.The larvae of both genera Ltd., Birmingham,U.K. 546 JounNer-oF THEArurnrceN Mosqurro coNrnol Assocrerrox vor. 2. No. 4 Table l. Results of natural mating of Coquitlettidia crassipesin different sizes of containers. Ratio No. of female mosquitoes Generation 9:d Size of of the usedper No. of Insemination Container container mosquito contarnel experiments Dissected rate (%) A Papercup (250 ml) F1 6:12 2 12 0 Frs 6:72 l7 0 B Cage30 x 30 x 30 cm F1 15:30 I t4 91 4 Frs 15:30 9 28 25.0 C Cage60 x x 60 60 cm Fr 30:60 2 5+ 27.8 Frs 30:60 2 57 54.4 D Cage120 x 60 x 60 cm Fl 45:90 2 83 74.7 Fts 45:90 2 87 93.l * 9 9 and d d were allowed to mate in the different conrainers for 5 days before g g were dissected and examined for insemination. tion was observed in the laboratory adapted F15 (Niles and Kulasiri 1970). Eichhornia proved, generation in all three cage sizes. g1s1i-t1!le, the highest mean emergence at Embryonation and hatchability of eggs of 28-30"C being 7.1/o. Alternanthera gave a females mated in the 60 x 60 x 120 cm cage consistent emergence ranging from 10.2 to were monitored through 9 generations. Sam- ll.87o. ples of 22, 16,22, 18, 17, 16,12, 7 and 9 egg Results from individual cultures showed rafts were examined during the successive roughly similar proporrions of pupae emerg- generations. The mean egg counts ranged ing as adults from the two types of attachment from 102.1 to 196.2. Embryonation and harch- sites, 50.9-58.9% from papei and 48.b41.9Vo ability showed a range of 69.6-97.9Vo and from plants. The comparable percentages of 63.3-9 4.3 Vo,respectively. adult emergence from pupae with the paper Table 2 gives the relative success of the and plant cukures suggest that in Cq. crassipes, cultures in the two insectaries. The best results as in,Mansonia,gentle dislodging of the pupae were obtained with paper using guinea pig attached to paper with a small soft paintbrush dung infusion at 28-3b"b with yiEld"sof aS.by'o did not increase their mortality. pupation and 25.5% adult emergence. As in , The percentage pupation, emergence and the case of Mansonia, the cultures with paper the number of adults emerged in eich of rhe gave better overall yields. Only two types of first 9 generations and in ii-re generations 16 plants were tested, Eichhornia which is the and l7 are given in Table 3. Pupation and common aquatic plant in Malaysia associated emergence were better in the later generations. with Mansmia and Coquillzttidia, and, Alternanthzra, While the dung medium provided the best a weed associated with Cq. crassipesin Sri Lanka yields with paper ar 28-30"C, there was no Table 2. Percentage of pupation and emergence and number of pupae and adults from first instar larvae of Coquillettidia crassipesset up in different culture media wlin ZSO larvae in each culture. No. of Vo Mean Va Attachment host tests punation (Range) emergence (Range) Indoor Insectary (24-26"C) Guinea pig dung Paper 29 26.6 (0.8-68.4) t4.5 (0.0-34.4) Eichhornin 5 10.8 (0.0-24.7) 5.1 (0.0-14.1) Alternanthera 5 I9.r (0.0-37.2) 10.2 (0.0-26.4) Liver-yeastinfusion Paper 16 26.r (l .6-50.8) l9 A (1.2-28.4) Eichhornia 6 r1.5 (0.0-r8.5) 6.6 (0.0-12.1) Alternanthera 9 19.2 (0.0-49.2) I 1.8 (0.0-34.8) Outdoor Insectary (28-30'C) Guinea pig dung Paper 34 43.5 (0.0-86.8) 25.5 (0.0-54.8) Eichhornia l l.9 (1.5-20.4) a1 (0.0-14.8) Liver-yeast infusion Paper l9 23.8 (0.0-53.2) 12.r (0.0-50.8) Eichhornin 5 7.9 (0.0-18.5) 3.8 (0.0-10.6) Alternanthera l0 20.2 (0.0-46.4) I 1.6 (0.0-23.2) Dncrunnn, Coqut ur,rt n u, cRlsslpls tN MnleYsre 547 Table 3. Production of adults of Coquillettidia crassipesin the laboratory. No. of adults emerged Generation 7o pupation Males Females % emergence F, 30.7 t,467 1,439 t6.5 F2 3r.2 207 90ri 18.3 F3 57.8 9(\9 183 33.2 F4 22.7 126 109 I 1.3 F5 39.2 248 237 23.9 F6 32.r J5I 310 20.9 F- 24.0 244 189 15.5 F8 22.0 223 168 t2.4 Fe 30.6 133 l3l 20.5 Fro 62.0 640 712 38.6 Frz 68.8 506 485 36.0 difference between the emergences from the The work described in this paper was other cultures with dung and liver yeast media. supported by a grant from the World Health The results suggest that the standardized liver Organization, Western Pacific Regional Office. yeast medium and paper can be used to It formed a part of the graduate study colonize Cq.