Reduction in Susceptibility to Brugia Malayi of F2 Progeny of Aedes Togoi Treated with Ethyl Methanesulfonate

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Joumal of the American Mosquito Control Association, 15(4):45g_462, 1999 Copyright @ 1999 by the American Mosquito Control Association, Inc.

REDUCTION IN SUSCEPTIBILITY TO BRUGIA MALAYI OF F2 PROGENY OF AEDES TOGOI TREATED WITH ETHYL METHANESULFONATE

PAUL H. RODRIGUEZ, RICHARD CASTILLON, PATRICK WILSON, DENISE BENOIT rNo DEEMAH NASR-SCHIRF

Division of Life Sciences, University of Texas at San Antonio, San Antonio, TX 78249

ABSTRACT' The susceptibillty to Brugia malayi infection was tested in F, female progeny derived from male and female Aedes togoi treated with ethyl methanesulfonate (EMS). Three-day-old rnales and females were treated with 0.025,0.050, and 0.075,0.10, 0.15, or O.2OVoEMS by allowing them to feed for 5 days on sugau cubes containing EMS and then mated at random. Percentage of susceptibility and mean number of infective larvae (L.) in F, females were analyzed over a 2-wk period. Reductions in susceptibility were significant in the F populations arising from the 3 highest EMS concentrations. F2 infections were reduced by 8O7o, indicating that EMS-induced mutations affect loci associated with filarial nematode susceptibility.

KEY WORDS Aedes togoi, EMS-induced mutations, Brugia malayi

INTRODUCTION (1963) described chemosterilization in Anopheles gambiae and Ae. aegypti with thiotepa. Apholate (L.), ln Aedes aegypti susceptibility to the filar- treatment of Ae. aegypti larvae produced both so- ial nematode Brugia malayi (Brug) is controlled matic and chromosomal damage (Rai 1964), devel- by a sex-linked recessive gene, I (Macdonald opmental abnormalities in adult tissue (Sharma and 1962, Kllama and Craig 1969). The f^ gene (Mac- Rai 1969), and pathological changes in male acces- donald and Ramachandran 1965) also controls the sory glands (Powell and Craig l97O). Ethyl meth- development of the human parasite Wuchereria anesulfonate (EMS) and thiotepa treatment of bancrofti (Cobbold) and the animal filarid Brugia adults induce mutations that also affect reproduc- pahangi (Buckley and Edeson). Research is under tive potential and development in Ae. aegypti (Es- way in our laboratory to extend our information parza and Rodriguez 1975, Thompson and Rodri- on the genetics of filarial nematode susceptibility guez 1979). Specific host-parasite interaction in the laboratory model Ae. aegypti and to deter- experiments in our laboratory showed that treat- mine whether similar or other mechanisms exist in ment with EMS decreased the susceptibility to B. a natural vector (Ramachandran et al. 1963, Mac- pahangi in the F, progeny of a moderately suscep- donald 1971, Mori et al. 1985) such as Aedes togoi tible Ae. aegypti strain (PUGU) (Rodriguez 1985). (Theobald). At present, the genetic mechanisms Other studies (Rodriguez et al. 1992) have pro- involved in the transmission of vector borne dis- duced similar results for the F, progeny of the eases are poorly understood, and the mode of in- BLACK EYE Liverpool strain of Ae. aegypri with heritance in the natural vector Ae. togoi has not B. malayi infection after treatment with EMS. The been adequately determined. Susceptibility to fi- current study determined whether treatment with larial nematode infection could well be polygenic, EMS also altered the susceptibility of Ae. togoi, a with major and minor gene involvement, or may natural B. malayi vector. include cytoplasmic factors, as observed in some studies witb Aedes polynesiensis strains (Tripis at al. 1981). MATERIALS AND METHODS The mutagenic effects of radiation and various alkylating agents in insects have been reported by Mosquitoes originated from the IMR strain of several investigators. Parameters include effects on Ae. togoi obtained from the National University reproduction and development (e.g., Crystal and La of Singapore, Republic of Singapore. The origi- Chance 1962, Thompson and Rodriguez 1979) or nal filarid susceptibility of this strain is as high genetic fitness such as viability, longevity, popula- as 65Vo (Rodriguez, unpublished data), and the F, tion size, or productivity (e.9., Wallace 1958, Cren- progeny, derived from EMS-treated parental shaw 1965, Ayala 1966). However, relatively few males and females, were tested for development studies have focused on the genetic effects of of B. malayi. Clawed Mongolian gerbils were chemical mutagens on the development of a path- used as the vertebrate host of B. malayi (Ash and ogen in a vector-host system (Rodriguez 1985, Riley l97O). Rodriguez, et al. 1992). In mosquitoes, Bertram Mosquitoes were reared and maintained, at 25"C Deceunrn 1999 BRUGIA MAIAYI TN TREATED AEDES I:OGOI 459

Table l. Susceptibility to Brugia malayi in female F, Table 2. Variation in susceptibility to Brugia malayi in Aedes togoi (IMR) derived from male and female female Aedes logoi (IMR) derived from male and female parents treated orally with ethyl methanesulfonate parents treated orally with EMS. (EMS). No. L" larvae/female Dose EMS Concen- Females tested for L, larvae (Vo) Week Range Mean + SE tration EMS Vo with L, L, per female 0 I 1-19 2.87 + O.48 (Vo) larvae (n) (Mean + SE) z l-8 t.67 + O.22 o.o25 1 1-10 1.87+ 0.31 0 I 70 (60) 2.87 + O.48 2 l-9 0.70 + 0.09 2 so (60) 1.67+ O.22 I l-5 1.49+ O.2l o.o25 I 60 (60) 1.87+ 0.31 0.050 2 l-3 0.50 + 0.07 2 38 (60) 0.70 + 0.09 0.075 I t2 0.55+ 0.11 0.050 I 60 (55) t.49 + 0.21 z l-8 o.53 + O.O7 2 33 (60) 0.50 + 0.07 0.10 I l-3 0.45 + 0.09 0.075 I 3e (s1) 0.55+ 0.11 z t-J 0.40 + 2 33 (60) o.53 + 0.07 0.05 + 0.15 1 1-6 0.65 + 0.20 0.10 I 35 (60) 0.45 0.09 + (60) + 2 l-4 0.32 O.O4 2 27 0.40 0.05 + (sr) + 0.20 I 7-4 0.58 0.18 0.l5 I 24 0.65 0.20 + 2 20 (60) 0.32 + 0.04 t-4 0.38 0.05 o.20 I 30 (60) 0.58+ 0.18 z 22 (60) 0.38 + 0.05

week were selected, placed in different 3.8-liter mosquito cages, and held in an incubator for 12- and SOVorelative humidity under a 16:8 L:D pho- 14 days. All F, females that remained alive after toperiod in a reach-in environmental chamber (Sci- this holding period were dissected in 2-3 drops of entific Systems, Baton Rouge, LA). Females were Aedes physiological saline (Hayes 1953). Animal infected by techniques similar to those described by use protocols for both mice andjirds were approved Rodriguez (1973). Briefly, males and females were by our institution's Institutional Animal Care and sorted during the pupal stage and kept separated in Use (IACUC) and are in compliance with National 3.8-liter paper carton mosquito cages prior to EMS Institutes of Health regulations. exposure. Four groups of 5O 5-day-old males and Mosquitoes were classified as susceptibleif in- 50 5-day-old females were allowed to feed freely fective L. larvae were present in the proboscis, for 5 days on 4 sugar cubes treated with either head, thorax, or abdomen (Rodriguez and Craig 0.025,0.050, O.U5,0.10, 0.15. or O.2OVoEMS 1973). The numbers of L, larvae per female for (SigmaLaboratories, St. Louis, MO). Each cube each treatment and the 2 separateexperiments were was treated with 250 U.l of the EMS solution for a compared statistically by a X' contingency test, total of I ml per experimental cage (Rodriguez analysis of variance (ANOVA), and Duncan's mul- 1985). A control group of 50 5-day-old males and tiple range test with the GB-Stat statisticalpackage 50 5-day-old females received sugar cubes treated (Dynamic Microsystems 1988). with deionized water (solvent). After exposure to the sugar cubes,2 replicates of RESULTS AND DISCUSSION the 7 groups (1 control and 6 EMS concentrations) of 10 mosquitoes of each sex were transferred to Table I summarizes results from 2 separateex- l.9-liter mosquito cages. F, progeny were derived periments in which F, females were infected with from these F, matings. Mosquitoes were offered the B. malayi to test for changes in fllarial nematode opportunity to bloodfeed twice before mating and susceptibility over the 1- and 2-wk periods. Table at 3-day intervals thereafter on anesthetized mice 2 and Figure 1 show mean L3 variation in sus- (Rodriguez 1985, Rodriguez et al. 1992). Egg col- ceptibility to B. malayi infections inFrAe. togoi. lections were made once during each following Susceptibility in the control population over the week. 2-wk test period was 6OVo.The 0.0257o EMS- F, adult females collected from the l-wk and 2- treated group showed only at LlVo decrease in wk periods were used to test for changes in fllarial susceptibility.At 0.050,0.075, and 0.10% EMS, susceptibility. Briefly, 3-day-old F, females from susceptibility was reduced by as much as 29Vo. each of the 7 groups were starved for 24 h, then In the 0.15 and O.2OVoEMS-treated groups, sus- fed on 4 jirds infected with B. malayi having a mi- ceptibility was decreasedby 38 and34Vo,respec- crofllarial density of 108-240 microfllaria per 2O tively, when compared with the control (1, : mm3 of blood. Four jirds were placed on I cage 22.72,df : 6, P < 0.01; Table l). group, then transferred to each of the other 6 cages. The mean number of L, per F, female varied Overall, l0O engorged F, females per group per among EMS-treated and control populations (F : Jouwer- oF THEAMERICAN Moseurro CoNrrol AssocrerroN Vor. 15,No.4

ty, and population size (Wallace 1958, Crenshaw 1965, Ayala 1966, Esparza and Rodriguez 1975). A mutagen study with the flour beetle-cestode E system also reduced productivity a or genetic fitness ! upon mutagenic exposures (Rodriguez 1970). Our f own studies with the mosquito-filarial nematode o E system have shown significant differences in firar- a o ial larval development between susceptible and re- I fractory strains 2-3 days postinfection (Rodriguez E et al. 1984). Related studies also indicated high z titers of acid hydrolase enzyme activities in refractory strains of Ae. aegypti and an EMS-induced partially refractory F, strain of Ae. togoi 0.000 0.025 0.0y) 0.075 0.100 0.125 0.150 0.175 02m challenged with B. malayi infections (Schirf and oy'oEthyl methanesulf onate Rodriguez, unpublished). Experiments with B. malayi infections and the Fig. l. Variation in mean L. larvae (+SEM) to Brugia BLACK EYE malayi infections in female F, Aedes rogoi (IMR) over a Liverpool strain of Ae. aegypri have 2-wk period derived from male and female parents ini- given similar results in F, and Fo progeny. These tially treated orally with ethyl methanesulfonate (EMS). progeny also were derived from parental males and females exposed to EMS (Rodriguez et al. 1992'). Susceptibility in O.IOVo EMS-selected Frs, for example, was reduced by 25Vo, whereas mean L3 18.71, df : 6,810, P < 0.0001; Table 1 and Fig. numbers decreased by 7 l%o. Fo-selected populations l). The control population had an average of 2.27 derived from the 0.1070 EMS treatments were 43Vo L. per infected female after 2 wk. The 0.025, 0.050, less susceptible when compared with the controls, and O.O75VoEMS F, females had a 44-76Vo de- and mean L.s were reduced by 77Vo. crease in mean L, numbers. At 0.10, 0.15, and Currently, studies are being pursued to deter- O.2O7oEMS, mean L3 numbers were reduced by 81, mine specific genetic mechanisms for filarial sus- 79, and 79Vo, respectively (Thble 1). Differences ceptibility in Ae. togoi. Partially refractory lines between weeks 1 and 2 occurred only in the con- were established, and biochemical procedures are trols and the O.O25Vo and. O.O5O7oEMS groups. being developed to compare variation in acid hy- Multiple range tests (Sokal and Rohlf 1981) also drolase enzyme titers (Rodriguez et al. 1998) and indicated that most significant decreases in mean electrophoretic variants in different filarial wonn- L.s per female between control and EMS-treated susceptible and partially refractory genotypes of populations occurred in both weeks 1 and 2 for the Ae. togoi. Experiments with another mosquito, Ae- highest groups, O.Oi54.2OVo. des albopictus, indicate that EMS and low levels Relatively few investigators have reported the of thiotepa induce dominant lethal mutations and effects of mutagens on susceptibility and devel- reduce reproductive potential in this species. Re- opment of a pathogen in the mosquito vector duced levels of enzyme activity or titers of acid (Rodriguez 1985). Genetic (Macdonald 1962) and hydrolases like acid phosphatase, a-glucosidase, environmental factors, including radiation (Richey and B-glucuronidase also demonstrated strong cor- and Rodriguez 1976) and temperature (Rodriguez relation with decreases in reproductive potential 1975), can affect filarial susceptibility and devel- (Gonzalez, Brown and Rodriguez, unpublished). opment. Filarial susceptibility in progeny of Ae. Perhaps exposure to EMS or other mutagens af- aegypti also was altered with EMS (Rodriguez fects genes as well associated with these acid hy- 198s). drolases. Acid hydrolases have been proposed as The present study indicates that EMS-induced humoral or cell-mediated defenses to immunologic mutations affect filarial susceptibility in F, prog- challenge in the snail host (Cheng et. al 1977, eny of Ae. togoi. The phenotypic expression itself Cheng and Garrabrant 1977, Cheng and Dough- was manifest by both a reduced susceptibility and erty 1989) as well as the mosquito vector and may number of infective larvae in F, Ae. togoi derived play an important role in genetic mechanisms re- from adult parents exposed to EMS. Susceptibility lated to filarial nematode susceptibility (Soderhall to filarial nematode infection could well be mul- and Smith 1986, Stoffolano 1986, Rodriguez et al. tigenic, and EMS exposure could have affected 1998). various genes, including viability, longevity, or other genetic fitness alleles. One group of inves- ACKNOWLEDGMENTS tigators, for example, provided evidence for a complex mode of inheritance of Plasmodiunt sus- This study was supported in part by Coordinating ceptibility inAe. aegyptt (Thathy et al. 1994). Pre- Board Grant ATP01O115-273, and NIH grant GM- vious studies with other insect systems produced 08194, National Institute of General Medical Sci- mutagenic changes affecting viability, productivi- ences, with co-funding through the National Insti- DecrNlsen 1999 BRUGIA MAI-AW IN TREATED AEDES TOGOI 461 tute of Allergy and Infectious Diseases. The clawed Macdonald, W. W and C. P Ramachandran. 1965. 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