Jpn. J. Infect. Dis., 55, 78-82, 2002

Original Article

Effect of Anti-Mosquito Hemolymph Antibodies on Fecundity andon the Infectivity of Malarial Parasite Plasmodium vivax toAnopheles stephensi (Diptera:Insecta)
Monika Gulia, Amita Suneja and Surendra K. Gakhar*
Department of Biosciences, Maharshi Dayanand University,
Rohtak, Haryana, India(Received March 6, 2002. Accepted July 18, 2002)
SUMMARY: Rabbit antibodies to hemolymph antigens (102.5, 101, 100, 96, 88, 80, 64, 55, 43, 29, and 23 kDa)of Anopheles stephensi reduced fecundity as well as viability in An. stephensi. However, ingestion of these antibodies
was not associated with a marked effect on the engorgement of mosquitoes but egg laying was significantly
delayed. Antisera raised against hemolymph proteins were also used to identify cross reactive antigens/epitopes
present in other tissues by Western blotting, as well as by in vivo ELISA. In addition, a significant reduction inoocyst development was also observed in An. stephensi mosquitoes that ingested anti-hemolymph antibodies
along with Plasmodium vivax. The results confirmed the feasibility of targeting mosquito antigens as a novelanti-mosquito strategy, as well as confirmed the usefulness of such antigens for the development of a transmissionblocking vaccine.

The hemolymph was collected from 40 adult females after
24 h of blood feeding (semi gravid), as described previously(8); collection proceeded by amputation of the antennae and/or by cutting the wings and/or legs to aspirate the hemolymph
droplets from the wounds, in phosphate buffered saline (PBS)
containing phenylmethylsulfonyl flouride (PMSF). Then, it
was centrifuged at 10,000 g for 15 min at 4°C to remove any
debris. Trichloroacetic acid (TCA)-precipitated hemolymph
proteins (170 μg protein in 0.5 ml 0.1 N NaOH) were injectedsubcutaneously by emulsification with Freund’s complete/
incomplete adjuvant in three groups of rabbits, using apreviously described method (2).
Immunized rabbits boosted with hemolymph protein
antigens from the same group of mosquitoes were used forblood-feeding for periods of up to 6 weeks. Six sets per week(containing about 20 females/set) were generated to observe
the egg-laying pattern in An. stephensi, as described previously(2). All of the data were subjected to Student’s t test.
Seventy mosquitoes were dissected to obtain various
tissues (hemolymph, midgut, ovary, and salivary gland). The
quantity of proteins was estimated by the method of Lowry
et al. (9). Proteins were separated by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) (10) on 10%
polyacrylamide gels under reducing conditions. Gels were
either silver-stained or were used for overnight electrophoretictransfer to 0.45 μm nitrocellulose membranes at 4°C for theWestern blotting (11). Nitrocellulose membranes containing
transferred An. stephensi hemolymph proteins were blockedwith 5% non-fat milk for 1 h. Sheets were incubated withrabbit antisera (1:100) for 1.5 hrs, washed three times with
PBS containing 0.1% Tween-20, and then incubated for 1 h
at room temperature with Alkaline phosphatase conjugatedgoat anti-rabbit IgG (1:5000). Bound antibodies were detectedby using NBT-BCIP substrate.
Antibody titers in the sera of different groups of rabbits
were determined by ELISA using immunizing antigens (10μg/ml) to coat the wells. Bound antigens were incubatedwith dilutions of rabbit antisera followed by addition of

INTRODUCTION
In spite of numerous studies, anti-arthropod vaccine, analternative to insecticides, remains in its infancy. Limited
success has been achieved to date by vaccination with crude
tissue extracts. A range of mosquito antigen preparations has
been used for immunizing vertebrate hosts in various vaccine
trials, and yet results have rarely been of sufficient efficacy.
In addition, putative antigens have not been identified for
accurate assessment of the feasibility of this approach (1,2).
In our previous reports (2,3), ovary and egg antigens wereidentified, the antibodies of which reduce the fecundityof An. stephensi. Ovarian proteins/insect vitellogens aresynthesized by the female fat body (4) and are transferred in thehemolymph; these vitellogens are then selectively sequestered
to the developing oocyte by receptor-mediated endocytosis(5) and are stored in yolk granules (6). The present study
examines the putative antigenic properties of hemolymph
proteins for the first time and demonstrates strong humoral
reactivity against hemolymph proteins. Here, we identify theputative antigens and describe the mortality and fecundity ofAn. stephensi. Parasite development was simultaneously
blocked in the mosquito that have fed upon them as the next
rational step towards obtaining an anti-mosquito and also atransmission-blocking vaccine.
MATERIALS AND METHODS
The culture of the Delhi strain of An. stephensi (obtained
from Malaria Research Center [MRC], New Delhi, India) was
maintained in our laboratory at 28 ± 2°C and 70-80% relative
humidity (7). Larvae were reared in bowls at a density of 300
larvae/450 ml of water. Specially prepared cages made ofmuslin cloth (1 feet by 1 by 1 high) were used for harboring
adults, as described previously (2).
*Corresponding author: Tel:+91-1262-73147, E-mail: gakharsk@yahoo.co.in78

Alkaline phosphatase conjugated goat anti rabbit IgG(1:20,000). Immune complexes were detected by a p-nitrophenyl
phosphate substrate system.
The cross-reactivity of hemolymph antibodies with othertissues (salivary glands, ovaries, and midgut) from immunized
and control female mosquitoes was observed by in vivo ELISA(12). Twenty mosquitoes were dissected to obtain all four ofthese tissue types after 48 hrs of blood feeding. TCA-precipitated proteins were resuspended in carbonate/bicarbonate
buffer, vortexed thoroughly, and were then used to coat ELISA
plate wells in triplicate. Bound antigens were incubated with
Alkaline phosphatase conjugated goat anti rabbit IgG(1:20,000), as described above.
Sera from immunized rabbits as well as from control rabbits
was ingested together with P. vivax by An. stephensi mosquitoes
using a membrane feeding apparatus to screen out the effects
of antibodies on parasite development in the mosquitoes.
Peak-titer sera from the immunized rabbits was collected,
pooled and stored at –70°C until use. Blood samples infectedwith of P. vivax were obtained from patients visiting the MRC.
Equal volumes of immune and control sera were mixed
with infected blood. Sixty mosquitoes (5-days old) were also
separately membrane-fed on immunized and control sera.
Unfed or partially fed females were removed. The midgut
was dissected in order to count the number of oocysts 8 days
after feeding. Similarly, the salivary glands were excised forobservation of the presence of sporozoites 14 days after feeding.
The percent transmission blockage was determined by themethod described by Ponnundurai et al. (13), namely,

Fig. 1. Antibody titer after last booster measured by antibody capture ELISA.

until the fourth week.
An ELISA-based assay was used to demonstrate the bindingof blood-fed polyclonal antibodies to different tissues (Fig. 5).
Much higher binding was detected as compared to the control
blood-fed. These results demonstrate that the antibodies fed
to the mosquitoes could traverse through the midgut wall andreach their target. The cross-reactivity of these antibodies
was observed with other tissues in findings that were also
confirmed by Western blotting.
In comparison to the control group, a significant reduction

Mean oocyst number in controls–Mean oocyst number in anti-hemolymph—————————————————————————————————— ×100
Mean oocyst number in controls

RESULTSHigh antibody titer was detected ranging from 1:104 in thefirst week to 1:106 in the third week in immunized rabbits,
whereas control rabbit serum and pre-immune serum showeda negligible amount of antibodies (below threshold), i.e.,
titers up to only 102. The level of antibody titer decreased
during subsequent weeks and showed a minimum value (1:103)
during the sixth week (Fig. 1).
Immunoblotting of anti-hemolymph proteins antibodies
recognized 11 different antigens of molecular weight 102.5,
101, 100, 96, 88, 80, 64, 55, 43, 29, and 23 kDa (Fig. 2). Fiveantigens, 102.5, 88, 64, 55, and 29 kDa were present exclusively in the hemolymph; however, two antigens (43 and 23
kDa) showed cross-reactivity with all of the four tissues. It
was also observed that a 101 kDa antigen was also present in
the midgut and eggs, and 100 and 96 kDa strands were present
in both ovarian tissue as well as in midgut tissue. The 80 kDa
strand cross-reacted only with ovarian tissue.
The maximum reduction in fecundity (47.07%) and increasein mortality (33.3%) was observed during the third and fourthweeks, respectively, after the last booster. Thereafter, the rateof reduction in fecundity (13.3%) and the increase in mortality
declined continuously until the sixth week, when the last
booster was given. (Figs. 3, 4). Total hatchability of mosquito
eggs laid by insects fed on immunized sera was also reducedby 47.5% (P < 0.001). The corresponding body weight after
engorgement decreased by 9.2% during the first week of thestudy (P < 0.05). However, this difference in engorgement
was statistically insignificant during the subsequent weeks(Table 1). It is of note that egg laying was also delayed byabout 12 hrs in the mosquitoes fed upon immunized blood

Fig. 2. Western blot analysis of specific tissue expression of antigenic
polypeptides.
A: Salivary glands, B: Midgut, C: Ovary, D: Hemolymph, E: Eggs.

79

Fig. 3. Effect on fecundity of An. stephensi fed on the blood of rabbits
immunized with hemolymph antigens of An. stephensi.

Fig. 4. Effect on mortality of An. stephensi fed on the blood of rabbits
immunized with hemolymph antigens of An. stephensi.

Table 1. Effect of anti-mosquito hemolymph antibodies on fecundity, hatchability, and viability of An. stephensi
Weeks after
last booster

Reduction in
fecundity(%)

Engorgement(mg)

Hatching/female

C

C

I

I

Reduction inhatchability(%)

Increase inmortality(%)

1

26.37***

0.98
0.89**(±0.059) (±0.028)

39.5(±0.62)

23.16(±3.5)

41.3***

4.76*

2

39.6**

0.93(±0.07)0.85(±0.016)

66.6(±10.0)36.3(±8.05)45.4***

20.0***

3

47.07***

0.97(±1.01)

0.92(±0.37)63.0(±5.9)

31.06(±7.1)

47.5***

25.0***

4

29.9*

0.92
(±0.51)0.91(±1.1)

53.1(±1.2)

33.6(±4.1)

36.7**

33.3***

5

38.7***

0.88(±0.03)0.90(±0.08)88.5(±6.5)

49.8(±2.0)

43.7***

2.4

6

13.3

0.82
(±0.04)0.83(±0.14)84.2(±1.0)

67.4(±7.7)

19.9*—*Significant at P < 0.1.
**Significant at P < 0.05.
***Significant at P < 0.001.

Table 2. Effect of anti-mosquito hemolymph antibodies on malaria parasite development inmosquito An. stephensi
No. of mosquitoes/groupInfected mosquitoes(%)

Control

Anti-HL

Control

Anti-HL

Control

Anti-HL

59

56

73.8

55.8

139.5
±5.1

32.5
±3.9

*Significant at P < 0.05.

80

Mean no. of
oocysts/mosquito

Transmission
blocking(%)
76.7*

An. stephensi mosquito extracts. However, monoclonal andpolyclonal antibodies to vitellin alone did not affect the
fecundity of Aedes Aegypti (16), indicating that other target
antigens were also involved.
Fecundity was adversely affected in two ways, namely, by
reduction and delay. A delay of 12 hrs was observed in thegroup for which the blood meals had antibodies against
hemolymph proteins. A similar delay in egg laying was also
observed by Almeida and Billingsley (17) in the case in whichmosquitoes were fed blood with anti-fat body antibodies. The
fat bodies act as site for synthesis of vitellogenins, which are
secreted into the hemolymph in order to transport them to thedeveloping oocytes in the ovary. Therefore, the antibodies
against either of these two may affect egg laying in both ways,i.e., reduction as well as delay. However, the mechanisms
causing these events remain open to speculation.
Western blotting of hemolymph antibodies revealed four
polypeptides, i.e., 80, 64, 43, and 29 kDa, which were also
observed in the case involving anti-mosquito ovary antibodies(2). One polypeptide of 100 kDa was also observed with antimosquito egg antibodies (3). However, Brennan et al. (12)have suggested that this 100 kDa polypeptide is a vitellogenin,
which appears only after a blood meal in An. stephensi.
With this information in mind, we carried out an in vivo
binding assay in order to ascertain the role of anti-hemolymph
protein antibodies as putative candidates for an anti-mosquito
vaccine. We determined by in vivo ELISA that anti-hemolymph
antibodies taken by mosquitoes through a blood meal areindeed capable of binding with hemolymph and, to someextent, to other tissues. Cross-reactivity was extensive andwas attributed to different causes; antigens or epitopes may
be common to other tissues, or non-specific binding may also
occur with low-affinity antibodies.
The present study also demonstrated that antibodies against
hemolymph antigens, when ingested by mosquitoes alongwith infected blood, affected the development of oocysts inthe midgut and/or the migration of sporozoites to the salivary
glands. These results support the findings of Lal et al. (18) and
Brennan et al. (12), who also observed the same phenomena.
Vector control has become increasingly difficult due tothe development of insecticide resistance. Moreover, only
limited funds are available for continuous redistribution forthe purpose of addressing the diversifying health needs ofregions with high malaria endemicity. Therefore, new strategies
of malaria control are currently required. Immune responses
against concealed antigens (in internal organs which were
never exposed to the host during blood feeding) can reduce
the longevity and fecundity of mosquitoes. The anti-mosquito
effects achieved to date are of limited success compared tothose associated with the tick vaccine (19). This difference
can be attributed to differences in the digestive physiology ofthese organisms, as well as to the length of time spent at thehost for blood feeding. A mosquito vaccine approach could
be extremely effective, provided that the vector number werereduced, and parasitic transmission were blocked either
directly (18,20) or else via reducing vector longevity (21).
Although the results of the present study are preliminary, they
do demonstrate that An. stephensi hemolymph proteins can act
as an additional candidate for/putative means of anti-mosquito
activity, as well as acting as a transmission blocking vaccine.

Fig. 5. In vivo ELISA of anti-hemolymph antibodies to various tissuesof An. stephensi.
1: Salivary glands, 2: Midgut, 3: Ovary, 4: Hemolymph.

in parasitic infection was observed in An. stephensi that had
ingested anti-hemolymph antibodies along with P. vivax(Table 2). The infection rate of An. stephensi was reduced
by about 18% when mosquitoes had fed on immune sera.
Moreover, the mean number of oocysts per infected mosquito
was also reduced drastically by about 76.7% (P < 0.05).
However, the infection rate with sporozoites was also reduced
by 23% (data not shown).
DISCUSSION
The efficacy of antibodies raised against the hemolymph
proteins of semi-gravid female mosquitoes was considered
together with a previous study of the development of antibody
responses (2,3). These studies form the foundation for thecharacterization of anti-mosquito antibodies, especially antihemolymph antibodies, which were studied for the first time.
The observed reduction in fecundity as well as viability wasin accordance with the results of previous studies (14-16).
However, prior studies made use of crude antigen preparations, in which either the whole body or the head, thorax, andabdomen were used separately. Almeida and Billingsley (17)have also discussed the effects of induced immunity against
An. stephensi on its survival and fecundity. However, thereduction in fecundity observed in that study was merely 18%,
16%, 21%, and 22% when mosquitoes fed on antibodies tohead, gut, ovaries, and fat bodies, respectively. Recently,
Gakhar et al. (2) also showed ≤ 66% reduction in the fecundityof An. stephensi when the mosquitoes had fed upon antimosquito ovary antibodies. The maximum reduction in
fecundity and viability during the third and fourth weeks,
respectively, after the last booster, coincides with the resultsof ELISA. A high ELISA titer (1:105-106) is also in accordance with the results of a study by Almeida and Billingsley(1), who also demonstrated the progression of immune
responses in mice during five sequential immunizations with

ACKNOWLEDGMENTS
The authors are grateful to the University Grants Commis81

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sion for the financial assistance received for this project. In
addition, Dr. T. Adak, Deputy Director, Malaria ResearchCentre, is to be thanked for providing the mosquito culture.
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