Effects of a juvenoid, DPE-28, on biology and behaviour of Culex quinquefasciatus, the human filariasis vector

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1 Indian Journal of Experimental Biology Vol. 38, July 2000, pp Effects of a juvenoid, DPE-28, on biology and behaviour of Culex quinquefasciatus, the human filariasis vector J Virnal Raf, Nisha Mathewt & M Kalyanasundararn Vector Control Research Centre (ICMR), Indira Nagar, Pondicherry 5006, India Received I June /999; revised 6 Apri/2000 Studies on the effect of a juvenoid, DPE-28 (2,4-dinitrophenyl-2',6'-di-tertiarybutyl phenyl ether) on biology and behaviour of Cx. quinquefasciatus showed that the developmental duration, sex ratio, mating success and blood feeding were considerably affected by the exposure of larvae and pupae to the compound. Exposure of fourth instar larvae to (E1<}( 1 ) and (EI 50 ) ppm of DPE-28 prolonged the duration of pupation by 58.6 and 52.4 hr and delayed the adult emergence by 35.4 and 17.7 hr in males and 36.8 and 21.1 hr in females respectively. Exposure of freshly ecdysed pupae to I 0 and 5 ppm delayed the adult emergence with respect to the control by 54.3 and 32.4 hr in males and 55.2 and 33.2 hr in females respectively. The sex ratio of the adults emerged from treated larvae and pupae was also affected. The female mosquitoes that survi ved from the exposed fourth instar larvae and pupae exhibited a low blood engorgement ratio. Thi s depression in blood feeding was more pronounced in adults emerged from treated pupae than that of treated fourth instar larvae. A significant proportion of adults emerged from treated larvae and pupae were able to feed only partially. Mating success of the treated popula!ions declined considerabl y when crosses were made between the mal es and females emerged from treated fourth instar larvae and pupae. The adults emerged from treated larvae and pupae showed a significant reduction in the oviposition. The increasing demand for effective insect control agents that do not harm the environment opens avenues for the use of insect growth regulators (IGRs). The IGRs control the insects by interfering with certain aspects of their physiology during their life cycle with minimum effects on non-target organisms 1 The advantages of using IGRs in insect control include their low toxicity to vertebrates and non-target organisms and their biodegradability contributing minimal environmental contamination 2. Compounds belonging to various chemical classes such as amides', diphenyl ethers 4, carbamates 5, benzoyl phenyl ureas 6 7 and diphenyl ureas 8 have been reported for insect growth regulating activity. They are mainly grouped as juvenoids that are not structurally related to the natural juvenile hormones but do showing juvenile hormone activity and chitin synthesis inhibitors, which interfere with chitin synthesis and inhibit cuticle formation. Most IGRs have high potency against mosquitoes and other pest and vector species 9. They exhibit ovicidal and larvicidal effects and disturbances in diapause of insects In addition to the lethal effects, IGRs can also induce other effects such as, *Present address: University of Readi ng, AMS, 7' 11 Floor, White Knights, P.O. Box 228, Reading RG6 6AJ, England. tcorrespondenl author morphogenetic aberrations 13, developmental duration an d sex ratto an d repro d ucttve. f at 1 ures resu I tmg ?J d f rom unsuccess f u I matmg, ovtposttton - an blood feeding. 6 The insect growth regulating activity shown by DPE-28 against the human filariasis vector mosquito Culex quinquefasciatus in earlier studies 4 ' 22 led us to examine the effect of DPE-28 on the biology and behaviour of Cx. quinquefasciatus in detail. Materials and Methods DPE-28 is a substituted diphenyl ether (2,4- dinitrophenyl-2',6'-ditertiary butyl phenyl ether) with a molecular formula C2oH24N20 5, was synthesized in pure fonn and supplied by the Synthetic Chemistry Department of the Product Development Division of Vector Control Research Centre, Pondicherry. One percent solution of DPE-28 was prepared in ethanol from which stock soluti ons of desired lower concentrations were prepared in ethanol. The vector mosquito, Culex quinquefasciatus Say ( 1823) (Diptera: Culicidae) utilized for thi s study was supplied by the cyclic colonies maintained at our Centre. The colony of Cx~ quinquefasciatus was established from field collected females in Pondicherry during They freely mate and fed without forcing. Females fed on chick blood with 12 hr light and 12 hr dark. This colony has been

2 688 INDIAN J EXP BIOL, JULY 2000 periodically out-bred with field collected specimens and maintained at 28 ±2 C and 70-% RH. Bioe.fficacy of DPE-28 against IV instar larvae The Elso and El 90 (indicating the statistical estimate of the dose producing 50 and 90% emergence inhibition calculated from a series of exposure concentrations by comparing the percentage of emergence inhibition in the treated group to control by probit analysis 23 ) values were determined by following standard method 1 The desired concentration of test solution was achieved by adding I ml of an appropriate stock solution to 249 ml of tap water taken in 500ml beaker. Early IV instar larvae (25) of Cx. quinquefasciatus were exposed continuously to various concentrations of DPE-28. A pinch of larval food consisting of yeast powder and dog biscuit (I: I) was provided after 24 hr. Four replicates for each concentration and a simultaneous control (I ml ethanol+ 249 ml tap water) were set up. Larval mortality, pupal mortality and inhibition in adult emergeryce were taken as the criteria of response and observations were recorded daily until all the test larvae either died or emerged. All experiments were conducted at 28 ± 2 C and 70-% RH. Effect on developmental duration and sex ratio of adults-the effect of DPE-28 on the developmental duration and sex ratio was determined for the early IV instar larvae and newly ecdysed pupae of Cx. quinquefasciatus. Early IV instar larvae (25) were continuously exposed to (EI 50 ) and (EI 90 ) ppm concentrations of DPE-28. Similarly, pupae were exposed to I 0 and 5 ppm. Larval food was provided until pupation. The duration taken for pupation and emergence, from the time of DPE-28 treatment was recorded. The sex ratio of emerged adults of each group was also determined. Four replicates with a control (249 ml tap water + I ml ethanol) were set up. The experiment was repeated 8 times. ANOV A analysis was performed to find out the significance of variance between test and control groups 24. Effect on adult blood feeding- Early fourth ins tar larvae (I 000) of Cx. quinquefasciatus were. exposed to and 0.007ppm concentrations of DPE-28 in enamel trays (30 x 25 x 5 em) until pupation. Pupae were collected and separately kept for emergence in clean cages. Males and females (50 each) from the emerged mosquitoes were kept in a separate cage and maintained on I 0% sucrose for 3 days. On the fourth day, a chicken, kept in a restrained cage was offered as blood source. In another experiment 500 freshly molted pupae exposed to I 0 and ) p ;~: 1 1 of DPE-28 in enamel bowls were placed in.;,,m cages. Upon emergence, adults were maintained as in the previous experiment. Females emerged from untreated pupae were maintained as control. The number of females that fully engorged, partially fed and failed to feed were counted and recorded by visual examination 25. Each experiment was repeated 10 times. The blood engorgement ratio was calculated by comparing the percentage of treated females fully engorged with that of controls. Visual observations on the feeding behaviour such as locating, landing, probing, puncturing and penetration were also made. Significance in the proportion of engorged females in the treated group and control was statistically analyzed using ANOV A after normalizing the vanance. Effect on reproduction Mating success: Mating success of the adults was studied by exposing the early IV instar larvae of Cx. quinquefasciatus to and 0.007ppm concentrations. From the surviving adults, 25 males and 25 females of each treated and untreated group were crossed in four combinations: treated male x treated female (T a x T <;> ); treated male x untreated female (T a xu 'i> ); untreated male x treated female (U a x T <;> ); untreated male x untreated female (U a xu ';> -control). A similar experiment was set up for pupae at 1 0 and 5 ppm. The mating success was expressed as insemination rate. Evaluation of mating success was determined by dissection of females to determine the presence of sperm in spermathecae, 72 hr after crossing. The females were collected in a test tube and knocked down by gently shaking the test tube. After removing the wings and legs, the penultimate segment of the abdomen was pulled apart with two finely pointed needles. The spermathecae were then separated and mounted in normal saline and observed under a compound microscope for motility of the sperm. The experiment was repeated 6 times. ANOVA analysis was performed to determine the significance of variance between treated and untreated groups. Effect on oviposition- The adults of Cx. quinquefasciatus that survived from fourth instar larvae exposed to and 0.007ppm concentrations were separated by sex, mixed at a ratio of I: I for 3 days and females were fed on a restrained chicken. Gravid females (25) were allowed to oviposit in enamel bowl containing water and the experiment

3 YIMAL RAJ et al.: JUVENOID, DPE-28, BIOLOGY & BEHAVIOUR OF CULEX 689 was repeated 6 times. A similar experiment was conducted for adults emerged from pupae exposed to 10 and 5 ppm. The number of ovipositing females was noted. Total number of eggs laid per female was recorded. Simultaneous controls were set up with untreated adults for both experiments. Results Bioefficacy of DPE-28 against IV instar larvae The Elso and El 90 values of DPE-28 against early IV instar larvae of Cx. quinquefasciatus were found to be and ppm with the 95% confidence intervals lying between for El 50 and for El 90 and a ;,l value of Effect on developmental duration and sex ratio of adults-the developmental duration and sex ratio of adults that emerged from treated fourth instar larvae and pupae are given in Tables I and 2 respectively. Exposure of fourth ins tar larvae to and ppm concentrations of DPE-28 significantly (P <O.OI ) prolonged the duration of pupation by 58.6 and 52 hr and delayed the adult emergence by 35.4 and 18 hr in males and 36.8 and hr in females respectively. Exposure of freshly ecdysed pupae to 10 and 5 ppm concentrations of DPE-28 resulted in significant (P < 0.05) delay in adult emergence time of 54.3 and 32.4 hr in males and 55.2 and 33.2 hr in females respectively. The sex ratio of the adults emerged from treated larvae and pupae was also affected significantly (P < 0.05) compared to the control. The proportion of adult males was significantly reduced in the treated populations. Effect on blood feeding- The blood feeding responses of the adult female mosquitoes that emerged from treated fourth instar larvae and pupae are shown in Figs I a and I b respectively. Inhibition in blood feeding was significantly higher (P < 0.05) in mosquitoes treated at El9 0 dose as revealed by the low blood engorgement ratio (0.76) than at the EI 50 dose (0.9). The females that survived from treated pupae showed a similar trend and the blood engorgement Table I - Developmental duration and sex ratio of adult Cx.quinquefasciatus emerged from fourth instar larvae treated with DPE-28 Concent- Pupation Adult emergence Sex ratio ration time (hr time (hr) (mean+ SE) Female: Male (ppm) (mean± SE) Female Male ± ± ±_2.2 I : ± ± ± 1.9 I ; 1.7 Control I: 2.1 ratios being 0.46 and 0.65 respectively for the mosquitoes exposed to I 0 and 5 ppm. In both cases, number of females that failed to feed was found to be significantly (P < 0.05) greater when exposed to higher concentration of DPE-28. Exposure of pupae to 10 and 5 ppm considerably reduced the blood feeding ability of Cx. quinquefasciatus as revealed by the higher proportion of females failing to take the blood meal. At I 0 and 5 ppm, 30.3 and 21 % of female mosquitoes failed to take the blood meal. A significant proportion of the adults that emerged was able to feed only partially. Effect on reproduction Mating success: Mating success of the treated population of Cx. quinquefasciatus significantly (P<0.05) declined when crosses were made between treated males and treated females of the groups exposed to DPE-28 at and ppm as early Table 2 - Developmental duration and sex ratio of adult Cx. quinquefasciatus emerged from pupae treated with DPE-28 Concentration Mean adult emergence time (hr) Sex rati o (ppm) (mean± SE) Female: Male Female Male 10 5 Control 20 a 81.0 ± ± ± ± I: 1.4 1:1.4 1: 2.1 <J) "' c &. "' ~ g> 100 r:-: , '2 1 b <J) u.. '$. 0 Unfed 20 ~ Partially fed ~ Fully fed 10 5 Control Exposure concentration (ppm) Fig. I-Blood feeding response in females emerged fro m treated larvae (a) treated pupae (b).

4 690 INDIAN J EXP BIOL, JULY 2000 Table 3-Percentage ovi position in adult Cx. quinque.fasciatus emerged from fourth instar larvae and pupae treated with DPE-28 Immature stage Concentration Oviposition (%) (ppm) (mean±se ) Larvae ± ±2.0 Control 95.3± 1.7 Pupae ± ±2.7 Control fourth instar larval stage (Fig. 2a). The reduction in percentage insemination was more in population exposed to hi gher concentration. Adults emerged after exposure of the pupae to DPE-28 at I 0 and 5 ppm showed a reduced mating success (Fig. 2b). The diminished mating success was found to be significantly (P < 0.05) hi gher when exposed to I 0 ppm. The percentage mating differed significantly (P < 0.05) when crosses were made between treated and untreated sexes in various combinations. Percentage. insemination was drastically reduced in the females after crossing of treated males with either untreated or treated females. Effect on oviposttwn: Significant (P < 0.05) reduction of 34.7 and 20.7% in ovipositing females was observed when the fourth instar larvae were exposed to DPE-28 at and ppm respectively (Table 3). When freshly formed pupae were exposed to I 0 ppm, a significant (P < 0.05) reduction of 41.3% egg laying was observed. Similarly pupae exposed to 5 ppm showed a significant (P < 0.05) reduction of 23.3 % egg laying. Discussion The results of the bioefficacy of DPE-28 cornfirmed the earlier reports 4 22 and also revealed that mortality in the larval-pupal intermediate was prominent with this compound. The pupae formed after treatment were pale resulting in hi gh mortality. This study showed that developmental duration and sex ratio were found to be adversely affected by this juvenoid and resulted in a higher proportion of females suggesting that males are more susceptible as reported earlier in the case of substituted geraniols 14 and methoprene 16. The results imply that exposure to DPE-28 affected the blood feeding ability of Cx. quinquefasciatus, especially when freshly formed pupae were exposed to 10 ppm. It was observed that females, which were not successful in taking blood meal, had lost one or more of their legs. Some of the males and females emerged from the treated ones were found with one or c 0,., "' c 0 F (j) VI 100 E 2b 0 ~ 10 5 Exposure concentration (ppm) Fig 2 - Insemination rate in female mosquitoes emerged from treated larvae (a) and treated pupae (b). both the wings unfolded permanently while resting and some mosquitoes were not able to unfold their wings to take off for a flight. Many mosquitoes after landi ng to feed failed to take a blood meal. Few mosquitoes took longer time for blood feeding compared to the control leading to partially fed mosquitoes. In normal feeding, mosquito after alighting on the host applies the tip of the proboscis on the skin of the host with the support of the fore and mid legs. Any structural deformity in the legs or proboscis is known to upset the feeding mechanism and the observed abnormalities in feedi ng response of the adult female mosquitoes may be due to the structural deformities caused by the exposure to DPE-28. Similar results were also reported in mosquitoes treated with hexaflumuron 6 It is apparent that DPE-28 adversely affects the mating success of adults emerged from both treated early fourth instar larvae and pupae. External and internal morphogenetic effects can influence mating and other reproductive functions directly or indirectly. Mating may be affected by the inability of treated males to transfer sperm during copulati on. A reduction in the insemination rate was also observed when untreated males were crossed with treated females, which could be due to the unsuccessful

5 VI MAL RAJ eta/.: JUVENOID, DPE-28, BIOLOGY & BEHAVIOUR OF CULEX 691 mating in females. Normal mating involves both the sexes and any divergence from the normal sequence of process can lead to unsuccessful mating resulting in reduced insemination. Similar results were also obtained in juvenile hormone analogue epoxy famesenic acid treated females of Trypodendron lineatum Olivier (Coleoptera: Scolytidae) 17, females emerged from larvae of Plodia interpunctella Hubner (Lepidoptera: Pyralidae) reared on Cecropia juvenile hormone treated diet 18 and in methoprene treated Aedes aegypti (Diptera: Culicidae) 19. The results obtained in this study agree with the observations made by Fujita and Kurihara 20 on the reduced oviposition of Cx. pipiens Linnaeus (Diptera: Culicidae) exposed to methoprene. The characteristic observation made was that some of the females which alighted on the ovipositing medium (water) failed to extrude their egg rafts completely resulting in a partly oviposited state and subsequently the females succumbed. The reason for the failure or inhibition in oviposition may be due to the interference of the DPE-28 on the hormonal regulation in triggering the ovipositional behaviour, since oviposition and its regulation are under humoral and genetic control 21. While chemical control remains the main thrust of most of the insect control programmes of medical, veterinary and agricultural importance, the deleterious effects of insecticides on the environment and the non-target organisms warrant an alternative method of control. DPE-28 is a promising compound for the control of Cx. quinquefasciatus, the human filariasis vector, with its unique mode of action and control efficacy. Acknowledgement The authors are thankful to Dr. P. K. Das, Director, Vector Control Research Centre, and Dr. A. R. Rajavel, Research Officer for valuable suggestions and Mr. D. Jayakumar and Mr. S. Srinivasan for technical assistance. References I Mimeographed document, Informal consu lt ation on Insect Growth Regul ators, WHO/VBC/ Coats J R, Risks from natural versus synthetic insecticides, Annu Rev Entomo/, 39 ( 1994) Tyagi B K, Kalyanasundaram M, Das P K & Somachary N, Evaluation of a new compound (VCRC/INS/A-23) with juvenile hormone activity against mosquito vectors, Indian J Med Res, 82 ( 1985) 9. 4 Nisha G, Vasuki V & Kalyanasundaram M, Structure-activity relationships in diphenyl ethers for insect growth regulating acti vi ty against mosquitoes, Indian J Med Res, 89 ( 1989) Tyagi, B K, Somachary N, Vasuki V & Das P K, Evaluation of three formulations of a chitin synthesis inhibitor (fenoxycarb) against mosquito vectors, Indian J Med Res, 85 (1987) Vasuki V, The effects of sublethal doses of hexanumuron on the feeding behaviour of mosquitoes (Diptera: Culicidae), Bull Entomol Res, 82 ( 1992) Prasad M P, Nisha G, Vasuki V & Kalyanasundaram M, Synthesis and insect growth regul ating activity of stru cturally modi tied benzoylphenylureas, Indian J Chem, 29 ( 1990) Nisha G & Vasuki V, Quantitative structure activity relationship in diphenyl ureas fo r insect growth regulati ng activity, Indian J Med Res, 93 (199 1) Mulla M S, The future of insect growth regulators in vector control, JAm Mosq Control Assoc, 11 (1995) 269. I 0 Retnakaran A, Granett J & Ennis T, Insect growth regulators, in Comprehensive Insect Physiology Biochemistry and Pharmacology, Vol. 12 edited by G A Kerkurt and L I Gi lbert (Pergamon Press, Oxford), 1985, 529. II Staal G B, Insect growth regulators with juvenile honnone activity, Annu Rev Entomol, 20 ( 1975) Krysan J L, Fenoxycarb and di apause: A possible method of control for pear psyll a (Homoptera: Psyllidae), J Econ Entomo/, 83 ( 1990) Arias J R & Mulla M S, Morphogenetic aberrati ons induced by a juvenile honnone analogue in the mosquito Culex tarsalis (Diptera: Culicidae), J Med Entomol, 12 ( 1975) Pawar P V, Pisale S P & Sharma R N, Effect of some insect growth regul ators on metamorphosis and reproduction of Aedes aegypti, Indian J Med Res, 101 ( 1995) Swaby R, Klowden M J & Sjogren R D, Sublethal effects of larval methoprene exposure on adult mosquito longevity, J Am Mosq Control As soc, 8 ( 1992) Robert L L & Olson J K, Effects of sublethal dosages of insecticides on Culex quinquefasciallls, J Am Mosq Control Assoc, 5 (1989) Fockler C E & Borden J H, Mating activity and ovariole development of Trypodendron linea/urn : effect of a juvenile honnone analogue, Ann Entomol Soc Am, 66 ( 1973) Oberlander H, Sower L & Silhacek D L, Mating behaviour of Plodia interpunctella reared on juvenile hormone-treated diet, J Insect Physiol, 21 ( 1975) ' Donnell P P & Klowden MJ, Methoprene affects the rotation of the male tenninali a of Aedes aegypti, J Am Mosq Control Assoc, 13 ( 1997) I. 20 Fujita K & Kurihara T, Oviposition regul ation by methoprene in Culex pipiens moles/us, Japanese J Sanit Zoo/, 35 (1984) Klowden M J & Blackmer J L, J Insect Physiol, Humoral control of pre oviposition behaviour in the mosquito, Aedes aegypti, 33 ( 1987) Dash A P & Ranjit M R, Comparative efficacy of aphid extracts and some juvenoids against the development of mosquitoes, JAm Mosq Control As soc, 8 ( 1992) Finney D J, Probit Analysis, S. Chand & Company Ltd., New Delhi, 1971, Sakal R R & Rohlf J F, in Biometry Principles and Practice of Statistics in Biological Research, (W. H. Creeman & Co, New York) 198 1, Sella M, Relazione della campagna antianofelica di Fiumiciono ( 1919) con speciale ri guardo all a biologia delgi anofeli ed agli anofeli infetti. Annali di lgiene 30, Supplemento 85. ( 1920).