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

Short Communication

Anti‑MosqultO Ovary Antibodies Reduce the FecundityofAnopheles 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 ofAnopheles 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 ofmosqultOeS, and indicate the need for further studieswith target antlgenS.
MosqultO antlgenS that are not nomally lntrOduced intoantlgenS tO COat the wells. Bound antlgenS Were incubateda vertebrate host when a mosquito feeds (i.e., antigens of thewith 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 havebeen shownto increase mortality of An. stephensi (1) and Ae.

sulphonic acid, and H202.
0varian extracts from each species were prepared foraegypti (5) and also reduce fecundity in Ae. aegypti (2) andpolyacrylamide 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 besilver 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 wassheets containlng Separated An. stephensi ovary proteins weredemonstrated that mosqultO OVary lmmunOgenS induce theblocked with 1% nonfat milk in PBS, and the sheets wereproduction of anti‑mosqultO OVary antibodies that reduceincubated with pre‑immune sera in PBS, washed, and thenfecundityand 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 feetfor blood feeding up to 617 weeks after the last immuniza‑by 3 by 3 high) made up ofmuslin cloth were used fわr thetion. 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 outfemales. 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 werecontent of the blood provides the major protein source foralso examined for the presence of any retained eggs afteregg development. Ovaries pooled from 50 females were
homogenized in phosphate buffered saline (PBS) containing

egg laylng. The total number of egg production in the firstgonotrophic cycle was then calculated by summlng up
ovlpOSited and unlaid eggs of immunized as well as control

phenylmethylsulfonylflouride (PMSF). The homogenate wascentrifuged 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 ancalculated. Differences in mean egg production, mean number

equal volume of Freund's complete adjuvant (8). After a 2of larvae hatched, reduction percentage in fecundity andweek interval, a first booster of protein antlgenS Was Injectedmortalitywere also calculated･ All the data was subjected toin the same manner with the exceptlOn Ofincomplete Freund'sStudent's ∫ test.

adjuvant. After I week, a second booster Injection wasThe antibody titer in rabbit measured agalnSt immunizlng

administered as in the first booster dosage. It was glVen inmosquito 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*Co汀eSpOnding author: Fax: +9 1 ‑ 1 262‑4 1 1 32, E‑mail: gakharsk

by PBS with antigen showed nonspecific reactions (titer upwith Freund's adjuvantwithout mosquito antlgenS followed
to 10 only). This could be due to a cross‑reaction orrabbit@yahoo.co.in

antibodies due to the adjuvant. No antibodies were detected**The antlgenS identified have been named a洗er one of the co‑authors who leftfor heavenly abode.

in the control rabbits. Serum from control rabbits gave noI阜lTable I. Reduction percentage of eggs laid and egg mortahty in Anopheles

preclpitin lines with any antlgen in a double dimlSion test.

stephensi fed on rabbits immunizedwith ovary antlgenS

However, whole ovary antlgenS gave at least two preclpltln
lines with antiserum. The anti‑An. stephensi antiserum wasFeeding a鮎rreacted with ovary extract of An. stephensi recognlZlng uP tO

last booster

five ant.igens: AJ 29, 35･ 43, 64, and 80 kDa (Fig･ 1)･ It wasinterestlng that AJ 29 kDa antlgen Was expressed in all of the(weeks)
1 2 つJ 4 5 ′LU

egg mortality(%)

Reducti onoreggs laid (%)
5 5 4 4 4 tJ 7 つJ ′0 4 5 5con tro 1 experi menta 1
凸7 0 0 上U 7 2738｣｣4four tissues (additional hemolymph, midgut, and salivary′‑U 0 O 7 0 5‑ つJ 2 1 29 4 ′‑U ′hU 7 00 2 0 3 7 つJ

1 つJ 2 I 1gland) examined. However, AJ 43 and 80 kDa were alsopresent in hemolymph and midgut, respectively. Two antlgenS

AJ 35 and 64 kDa were exclusively present in the ovary ofAn. 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･
rabbits suggests that humoral antibodies somehow interfere

jluviatilis only)･ Only one crossIreactive antigen (AJ 64 kDa)

was identified by Westem blottlng (Fig 2).

with the normal process of oogenesis. This lower fecundity

There was no statistically significant difference in therate does not seem to be related to the reduction in ovlpOSition.

corresponding body weight after engorgement in control andThe dissection of a few females from each feeding, 3 daysimmunized rabbits. However, very slgnificant differences inafter they had fed on immunized rabbit blood, showed thatfecundity 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 wastion 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, andabdomen separately.

reduction in fecundity in An. stephensi females that fed on

IgG antibodies have been detected in the hemolymph ofimmunized rabbits compared to those fed on normal or control

Anophelines up to 48 h a洗er blood feeding (1 3)･ Ramasamy

et al. (4) have demonstrated that monoclonal and polyclonalmolecular

antibodies to vitelline alone did not affect the fecundity ofweight

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%, whenAJ 64

mosquitoes fred on antibodies to the head, thorax, and abdomen,respectively. However, Srikrishnaraj et al. ( 14) have shownthat anti一midgut antibodies also reduce vector competence

AJ 43

for the malarial parasite and are also capable of inhibiting theformation of the peritrophic membrane (PM) in the posterior

AJ35

midgut of An. tesselatus (15). Similarly they showed thatmosquitoes fed on rabbits with antibodies to mosquito midgut

AJ 29

also reduced the fecundityby 25%. The high reduction in thenumber of eggs laid as observed during the present investlga‑AB

CD

tions could be attributed to specific anti‑mosqultO antibodies
binding to target antlgenS in the ovary, Interfering with theFig. 1. Westem blot analysis of specific tissue expression of antigenic

nomal process of egg maturation and development.

polypeptides.

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

weightthe homone balance, Or induce the resorptlOn Of mature orimmature oocytes. Altematively, the presence of antibodies(kDa)in Ingested blood may Irritate the gut, reducing the total blood

AJ 80

intake and available nutrients. The amount of blood was notAJ 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･There was no slgnificant mltlgation of the effect of antibodies

AJ 29

by the end of sixth week as compared to control experiment
AB

C(Fig. 3). The number of eggs laid/females increased whensame antisera was fed during subsequent weeks.

Fig･ 2･ Westem blot analysis of spcciesISPeCific expression ofanti‑High titered antibodies agalnSt a specific combination ofantlgenS may be more effective in bringlng about a disruption

genic polypeptides.

A: An. stephensi, B: An. culiclfacies, C: An..Puviatilis.

182

田Control

田Antisera fedof Science and Technology lS also gratefully acknowledged
for its financial assistance.

REFERENCE Sl･ Alger, N･ E･ and Cabrera, E･ J･ (1972):Anincre.ase in deathole∈0‑JOdpEelS6 8‑〇･〇N

rate of Anopheles stephensi fed on rabbits Immunizedwith mosqultO antlgen. ∫. Econ. Entomo1., 65, 165‑168.

2. Sutherland, G. B. and Ewen, A. B. (1974): Fecundity
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3. Ramasamy, M. S., Srikrishnaraj, K. A., Wijekoone, S.,
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on Anopheles tessellatus and Culex quinquefasciatus(Diptera:Culicidae). ∫. Med. Entomo1., 29, 934‑938.

4. Ramasamy, M. S., Ramasamy, R., Kay, B. H. and Kidson,
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2, 87‑93.

5. Hatfield, P.R. (1 988): Anti‑mosquito antibodies and theireffects on feeding, fecundity and mortality ofAedes
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35

6･ Wikel, S. K., Rangappa, N. R. and Bergman, D･ K. (1996):
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Feeding after last booster (Days)

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Ectoparasitic Arthropod Relationships. CAB lntemational,

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8. Ramasamy, 良., Nadesalingam, P. and Ramasamy, M. S.

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the mortality rate in their study †as significant only amoヮg

13. Ⅵlughan, ∫. A. andAzad, A. F. (1988): Passage of hostthose insects that fed on rabbits Immunized with mosqultO

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S･ (1993): Fecundity of Anopheles i.essellatus reduced

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and Ramasamy, R･ ( 1996): Different effect.s of modula‑tion of mosquito (Diptera: Culicidae) trypsln activity on
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ACKNOWLEDGMENTS

lng mOSqultO Cultures for the present work. The Department183