Jpn. J. Infect. Dis., 54, 1811183, 2001

Short Communication Anti-MosqultO Ovary Antibodies Reduce the Fecundity ofAnopheles stephensi (Diptera: Insecta)

SI K･ Gakhar*, Anu Jhamb**, Monika Gulia and Rajnikant Dixit Department ofBiosciences, Maharshi Dayanand University, Rohtak-12400l, HaTyana, India

(Received February 26, 2001. Accepted September I 7, 2001)

SUMMARY: Rabbit antibodies to five antigens (AJ** 29, 35, 43, 64, and 80 kDa) derived from the ovaries of Anopheles stephensi tended to reduce the number of eggs produced. Ingestion of anti-mosqultO OVary antibodies did not show a detectable effect on the mortalityof mosquitoes. Antisera raised against An. stephensi ovaries showed cross-reactivlty ln Other tissues and in the ovaries of other Anopheles spp. by Westem blottlng. The results indicate that anti-mosqultO OVary antibodies have the potential to disrupt the reproductive physiology of mosqultOeS, and indicate the need for further studieswith target antlgenS.

MosqultO antlgenS that are not nomally lntrOduced into antlgenS tO COat the wells. Bound antlgenS Were incubated a vertebrate host when a mosquito feeds (i.e., antigens of the with dilutions of rabbit sera, followed by peroxidase conju- midgut, ovary, etc.) can induce an artificial immune response gated sheep anti-rabbit lgG (8). The immune complex was ( 1 -3)･ Specific lgGantibodies also pross the midgut epithelium detected with 2,2′-azinobis, 3-ethyl benz-thiozoline-6- ofAedes aegypti (4). Ingested ant1-mosquito antibodies have sulphonic acid, and H202. been shownto increase mortality of An. stephensi (1) and Ae. 0varian extracts from each species were prepared for aegypti (5) and also reduce fecundity in Ae. aegypti (2) and polyacrylamide electrophoresis under reducing conditions(9). Culex quinquefasciatus (3). However, in most of these earlier The soluble proteins separated by sodium dodecyl su胞te- studies (6) antigensW?re prepared either from the head/thorax Polyacrylamide gel electrophoresis (SDS-PAGE) were either or abdomens or by using the whole body. Efforts need to be silver stained (10) or transferred elecqcally to O･45 LLm directed towards the use of defined antlgenS from various nitrocellulose sheets fわr Western blottlng. Nitrocellulose tissues of mosqultOeS. Therefわre in the present study, it was sheets containlng Separated An. stephensi ovary proteins were demonstrated that mosqultO OVary lmmunOgenS induce the blocked with 1% nonfat milk in PBS, and the sheets were production of anti-mosqultO OVary antibodies that reduce incubated with pre-immune sera in PBS, washed, and then fecundityand also the viability of progeny of An. stephensi incubated in the fbllowlng antisera. Followlng Washing, obtained from the blood meal of immunized rabbit. antibody binding was visualized using a peroxidase substrate The culture of An. stephensi (Delhi strain) was maintained system (ll). in our insectary at 28 ± 2oC and 70 - 80% relative humidity Immunized rabbit boosted with ovary antlgenS Was used

(R月) as described earlier (7). Specially prepared cages (3 feet for blood feeding up to 617 weeks after the last immuniza- by 3 by 3 high) made up ofmuslin cloth were used fわr the tion. Six replicates were made each week to obseⅣe the egg- adult mosqultOeS. laylng Pattem in An. stephensi. Each set contained about 50 Fully engorged female An. stephensi were dissected out females. The females were taken Just after blood feeding. Eggs in no-al saline to collect ovaries because the hemoglobin were laid on wet filter paper. The ovaries of females were content of the blood provides the major protein source for also examined for the presence of any retained eggs after egg development. Ovaries pooled from 50 females were egg laylng. The total number of egg production in the first homogenized in phosphate buffered saline (PBS) containing gonotrophic cycle was then calculated by summlng up phenylmethylsulfonylflouride (PMSF). The homogenate was ovlpOSited and unlaid eggs of immunized as well as control centrifuged at 10,000 g for 15 min. The supematant (0.5 ml, (nomal) blood-fed females･ The number of eggs hatched in 150 LLg proteins) was injected subcutaneously at multiple each set was counted and hatching percentage was also sites in the three rabbit groups a洗er being emulsified with an calculated. Differences in mean egg production, mean number equal volume of Freund's complete adjuvant (8). After a 2 of larvae hatched, reduction percentage in fecundity and week interval, a first booster of protein antlgenS Was Injected mortalitywere also calculated･ All the data was subjected to in the same manner with the exceptlOn Ofincomplete Freund's Student's ∫ test. adjuvant. After I week, a second booster Injection was The antibody titer in rabbit measured agalnSt immunizlng administered as in the first booster dosage. It was glVen in mosquito ovary antigens was very high ( 1 : 1 05- 108). Recently, both sides of the upper am, with 0.5 ml at each site. Almeida and Billingsley (12) have shown the progre苧siop of

Antibody titers in rabbit serum were determined by enzyme- irrmune responses in mice during丘ve sequential imnunlZatlOns linked immunosorbent assay (ELISA) using immunizing with An. stephensi mosqultO extracts. Control rabbits Injected with Freund's adjuvantwithout mosquito antlgenS followed

*Co汀eSpOnding author: Fax: +9 1 - 1 262-4 1 1 32, E-mail: gakharsk by PBS with antigen showed nonspecific reactions (titer up @yahoo.co.in to 10 only). This could be due to a cross-reaction orrabbit **The antlgenS identified have been named a洗er one of the co- antibodies due to the adjuvant. No antibodies were detected authors who leftfor heavenly abode. in the control rabbits. Serum from control rabbits gave no

I阜l preclpitin lines with any antlgen in a double dimlSion test. Table I. Reduction percentage of eggs laid and egg mortahty in Anopheles stephensi fed on rabbits immunizedwith ovary antlgenS However, whole ovary antlgenS gave at least two preclpltln lines with antiserum. The anti-An. stephensi antiserum was Feeding a鮎r Reducti on egg mortality(%) reacted with ovary extract of An. stephensi recognlZlng uP tO last booster oreggs laid (%) con tro 1 experi menta 1 five ant.igens: AJ 29, 35･ 43, 64, and 80 kDa (Fig･ 1)･ It was (weeks) ′-U 0 OO 7 0 5 凸7 0 0 上U 7 2 interestlng that AJ 29 kDa antlgen Was expressed in all of the 1 2 つJ 4 5 ′LU 5 5 4 4 4 tJ 7 つJ ′0 4 5 5 7 3 8 ｣ ｣ 4 9 4 ′-U ′hU 7 00 0 2 0 3 7 つJ

four tissues (additional hemolymph, midgut, and salivary - つJ 2 1 2 1 つJ 2 II 1 gland) examined. However, AJ 43 and 80 kDa were also present in hemolymph and midgut, respectively. Two antlgenS AJ 35 and 64 kDa were exclusively present in the ovary of An. stephensi. Antisera raised agalnSt An. stephensi ovaries were also used to identifycross-reactive immunogens in ovary extract for other Anopheles spp. (An. culiclfacies and An･ jluviatilis only)･ Only one crossIreactive antigen (AJ 64 kDa) rabbits suggests that humoral antibodies somehow interfere was identified by Westem blottlng (Fig 2). with the normal process of oogenesis. This lower fecundity There was no statistically significant difference in the rate does not seem to be related to the reduction in ovlpOSition. corresponding body weight after engorgement in control and The dissection of a few females from each feeding, 3 days immunized rabbits. However, very slgnificant differences in after they had fed on immunized rabbit blood, showed that fecundity were observed between mosqultOeS fed on rabbit no egg development had occurred in these female mosqultOeS. blood injected with ovary antlgenS and mosqultOeS fed on Our observations are consistent with the fecundity reduc- control rabbits. The number of eggs laid per female was tion seen in Ae. aegypti ingestlng anti-mosqultO antibodies reduced by about 57% (P < 0.01), when the females were fed (2,4). However these studies used crude preparations of anti- with anti-mosquito ovary antibodies (Table 1 ). The marked gens uslng either the whole body or the head, thorax, and reduction in fecundity in An. stephensi females that fed on abdomen separately. immunized rabbits compared to those fed on normal or control IgG antibodies have been detected in the hemolymph of

Anophelines up to 48 h a洗er blood feeding (1 3)･ Ramasamy et al. (4) have demonstrated that monoclonal and polyclonal molecular antibodies to vitelline alone did not affect the fecundity of weight Ae. Aegypti, indicatlng that other target antlgenS are also (kDa) involved. These polyclonal antibodies produced from crude preparations were not very effective in reducing fecundityin An･ tefsellatus･ Ramasamy et al･ (3) had reported that the - AJ80 reductlOn Of fecundity was just 15%, 23%, and 20%, when AJ 64 mosquitoes fred on antibodies to the head, thorax, and abdomen, respectively. However, Srikrishnaraj et al. ( 14) have shown

that anti一midgut antibodies also reduce vector competence AJ 43 for the malarial parasite and are also capable of inhibiting the AJ35 formation of the peritrophic membrane (PM) in the posterior midgut of An. tesselatus (15). Similarly they showed that AJ 29 mosquitoes fed on rabbits with antibodies to mosquito midgut also reduced the fecundityby 25%. The high reduction in the number of eggs laid as observed during the present investlga- A B C D tions could be attributed to specific anti-mosqultO antibodies

Fig. 1. Westem blot analysis of specific tissue expression of antigenic binding to target antlgenS in the ovary, Interfering with the polypeptides. nomal process of egg maturation and development. A: Ovary, B: Hemolymph, C: Salivary glands, D: Midgut. The antibodies'mode of action is also unknown but could involve or a combination ofseveral factors, i.e., inhibition of the metabolism or transport of vitellogenic proteins in insect molecular hemolymph or in their uptake by developlng OOCyStS, upset weight the homone balance, Or induce the resorptlOn Of mature or (kDa) immature oocytes. Altematively, the presence of antibodies in Ingested blood may Irritate the gut, reducing the total blood AJ 80 intake and available nutrients. The amount of blood was not AJ 64 assessed in the present study, but the females that fed appeared to engorgefully. SerologlCal tests indicated a strong response to Injected antlgenS. AJ 43 The activities of these antibodies in terms of their effects

on egg laylng pattem Was also observed f♭r about 6 weeks･ AJ 29 There was no slgnificant mltlgation of the effect of antibodies by the end of sixth week as compared to control experiment A B C (Fig. 3). The number of eggs laid/females increased when same antisera was fed during subsequent weeks. Fig･ 2･ Westem blot analysis of spcciesISPeCific expression ofanti- High titered antibodies agalnSt a specific combination of genic polypeptides. A: An. stephensi, B: An. culiclfacies, C: An..Puviatilis. antlgenS may be more effective in bringlng about a disruption

182 田Control 田Antisera fed of Science and Technology lS also gratefully acknowledged for its financial assistance.

REFERENCE S

ole∈0-JOdpEelS668-〇･〇N l･ Alger, N･ E･ and Cabrera, E･ J･ (1972):Anincre.ase in death rate of Anopheles stephensi fed on rabbits Immunized

with mosqultO antlgen. ∫. Econ. Entomo1., 65, 165-168. 2. Sutherland, G. B. and Ewen, A. B. (1974): Fecundity decreases in mosqultOeS lngeStlng bloodfrom specifically sensitized mammals. J. Insect. PhyslO1., 20, 655-660. 3. Ramasamy, M. S., Srikrishnaraj, K. A., Wijekoone, S.,

Jesuthasan, L S. and Ramasamy, 良. (1992): Host immu- nlty tO mOSqultOeS: effect of anti-mosquito antibodies on Anopheles tessellatus and Culex quinquefasciatus

(Diptera:Culicidae). ∫. Med. Entomo1., 29, 934-938. 4. Ramasamy, M. S., Ramasamy, R., Kay, B. H. and Kidson, C. ( 1 988): Anti-mosquito antibodies decrease the repro- ductive capaclty OfAedes aegypti. Med. Vet. Entomo1., 2, 87-93. 5. Hatfield, P.R. (1 988): Anti-mosquito antibodies and their effects on feeding, fecundity and mortality ofAedes aegwti. Med. Vet. Entomol･, 2, 331-338. 0 7 14 21 28 35 6･ Wikel, S. K., Rangappa, N. R. and Bergman, D･ K. (1996): Arthropod modulation of host immune responses. p. 1 07- Feeding after last booster (Days) 130. In Wikel, S.K. (ed.), The lmmunology of Host- Fig. 3. FecundityofAnopheles stephensi fed on the blood of rabbits Ectoparasitic Arthropod Relationships. CAB lntemational, immunized with ovary antigens of An. stephensi. Oxon. 7･ Gakhar, SI K･, Singh, S･ and Shandilya, H･ (1997): Changes in soluble proteins during the development of malarla of mosquito reproductive physiology･ This possibilityrequires vector Anopheles stephensi (Diptera:Insecta). Proc. Natl. further investlgation uslng a combination of monospecific Acad. S°i., B63, 289-298. polyclonal and monoclonal antibodies produced agalnSt 8. Ramasamy, 良., Nadesalingam, P. and Ramasamy, M. S. purified or synthetic antlgen. The antibodies did not appear ( 1 99 1 ): Antigenic similarity between the mosquito vectors to be present in rabbit in effective concentrationsat 5-6 Weeks ofmalaria and filariasis. J. Med. Entomo1., 28, 7601762. after the last antlgen Injection. When the same rabbit was 9. Laemmli, U. K. (1970): Cleavage orstmctural proteins used 6 weeks later to infect An. stephensi, the increase in the during the assembly of the head of bacteriophage T4. number of eggs was attributed to a decline in antibody titer Nature, 227, 680-585. below an effective threshold. 10. Hames, B. D. andRickwood, D. (1990): ln Gel electro- The percentage of larva hatched from eggs laid by females phoresis in practical approach. Oxford UniversityPress, fed on imnunized rabbit showed that the difference in mor- Oxfbrd. tality rates was greater during first 3 weeks. However, this ll. Leary, ∫. ∫., Brigati, D. ∫. andWard, D. C. (1983): Rapid difference was reduced to an almost inslgniBcant level, i.e., the and sensitive colorimetric method for visualizing biotin- mortalityrate was almost same in the control and experimental labeled DNA probes hybridized to DNA or RNA irrmum0- (imunized) mosquitoes dWing the subsequent 3 weeks. This bilized on nitrocellulose: Bio-blots. Proc. Natl. Acad. S°i. indicated that the effect of immunization had declined by the USA, 80, 4045-4049. end of the sixth week. 12. Almeida, A. P.and Billingsley, P. F. (1999): Induced immu- We also did not observe increased mortality among female nityagainst the mosquito Anopheles stephensi: reactivity mosqultOeS that had fed on immunized rabbits, in contrast to characteristics of immune sera. Med. Vet. Entomo1., 1 3, the findings ofAlger and Cabrera (1). lt must be noted that 53-64. the mortality rate in their study †as significant only amoヮg 13. Ⅵlughan, ∫. A. andAzad, A. F. (1988): Passage of host those insects that fed on rabbits Immunized with mosqultO immunoglobulin G from blood meal into hemolymph of midgut. Our experiments showed that the next generation was selected mosquito species (Diptera:Culicidae). ∫. Med. not affected by their parents having fed on irrmunized rabbits. Entomo1., 25, 472-474. Several hundreds of eggs produced by all feedings were reared 14. Srikrishnaraj, K. A., Ramasamy, R. and Ramasamy, M. through next generation and the percentages or egg laylng S･ (1993): Fecundity of Anopheles i.essellatus reduced and hatching were calculated. These values did not differ by the ingestion ofmurine anti-mosqultO antibodies. Med. slgnificantly between control and experimental mosqultOeS･ Vet. Entomol., 7, 66-68. 15･ Ramasamy, M. S., Kulasekera, R., Srikrishnaraj, K. A. and Ramasamy, R･ ( 1996): Different effect.s of modula- ACKNOWLEDGMENTS tion of mosquito (Diptera: Culicidae) trypsln activity on The authors are grateful to Dr. S. K. Subbarao and Dr. T. the infectivity of two human malaria (Hemosporidia:

Adak, Malaria Research Center (MRC), New Delhi for provid- Plasmodidae) parasites. ∫. Med. Entomo1., 33, 777-782. lng mOSqultO Cultures for the present work. The Department

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