Host Immunity to Mosquitoes: EfiFect of Antimosquito Antibodies on Anopheles tessellatus and Culex quinquefasciatus (Diptera: Culicidae) MANTHRI S. RAMASAMY,1 K. ALAGARATNAM SRIKRISHNARAJ,* SANJEEWA WIJEKOONE, 1 L. S. BRUNO JESUTHASAN,* AND RANJAN RAMASAMY* Institute of Fundamental Studies, Hantana Road, Kandy, Sri Lanka
J. Med. Entomol. 29(6): 934-938 (1992)
ABSTRACT Rabbit antibodies to antigens derived from the tissues of sugar-fed Anopheles tessellatus Theobald were fed to An. tessellatus and Culex quinquefasciatus Say in blood meals. These antibodies tended to reduce the number of eggs produced by An. tessellatus, but had no effect on the fecundity of Cx. quinquefasciatus. Ingestion of the rabbit antibodies did not detectably affect the mortality of An. tessellatus but increased the mortality of Cx. quinquefasciatus. The results provide preliminary evidence for the potential of antimosquito antibodies to disrupt the physiology of mosquito vectors and indicate the need for further studies with high-titer, monospecific antibodies against the relevant target antigens. KEY WORDS host immunity, Anopheles tessellatus, Culex quinquefasciatus
WHEN MOSQUITOES FEED on vertebrate hosts, both antibody and cell-mediated immunological responses are induced by mosquito salivary gland secretions that enter the bloodstream (Wikel 1982, R. Ramasamy & Ramasamy 1990). Mosquito antigens that normally are not introduced into the vertebrate host when a mosquito feeds (e.g., midgut and abdomen components) can induce an artificial immune response (Alger & Cabrera 1972, Sutherland & Ewen 1974, Ramasamy et al. 1988a). Specific IgG antibodies ingested in a blood meal are known to cross the midgut epithelium of Aedes (Ramasamy et al. 1988a), Anopheles and Culex mosquitoes (Vaughan & Azad 1988). Ingested antimosquito antibodies have been shown to increase the mortality of An. stephensi Liston (Alger & Cabrera 1972) and Ae. aegypti (L.) (Hatfield 1988) and also reduce the fecundity of Ae. aegypti (Sutherland & Ewen 1974, Ramasamy et al. 1988a). Antimosquito antibodies ingested with an infected blood meal also interfere with the development of pathogenic organisms in mosquitoes. Antibodies to mosquito midgut-derived antigens reduced the susceptibility of Ae. aegypti to arboviruses (Ramasamy et al. 1990) and of Anopheles farauti Laveran to Plasmodium berghei Vincke (Ramasamy & Ramasamy 1990b). Anopheles tessellatus Theobald and Culex quinquefasciatus Say tissues share many common antigens (Ramasamy et al. 1991). Our investigation determined the effects of antimosquito 1 2
Vector Biology Laboratory. Malaria Laboratory.
antibodies ingested in a blood meal, on the physiology of the malaria vector, An. tessellatus, and the Bancroftian filariasis vector, Cx. quinquefasciatus. Materials and Methods Laboratory colonies of An. tessellatus and Cx. quinquefasciatus were maintained at 28 ± 1°C, 80% RH, and natural day-night photoperiods. An. tessellatus larvae were fed powdered infant cereal and dry yeast, and Cx. quinquefasciatus larvae were fed powdered soya protein. Adult mosquitoes were provided 10% glucose supplemented with multivitamins. An. tessellatus were blood fed on guinea pigs during the day, whereas Cx. quinquefasciatus were blood fed on guinea pigs overnight. Antigen Preparation and Immunization. Sugarfed An. tessellatus (4—7 d old) were held at —20°C until they were dissected. The head and thorax, midgut, and the rest of the abdomen were separated in 0.01 M phosphate-buffered saline (PBS), pH 7.4, and were used as three tissue preparations. Tissue pooled from several batches of sugar-fed An. tessellatus was homogenized in PBS, and suspensions were injected intramuscularly at multiple sites into three groups of four New Zealand white rabbits; the first injection was done in Freund's complete adjuvant (Ramasamy et al. 1991). Four immunizations at 3-4 wk intervals were made before the initial determination of serum antibody titers. Cx. quinquefasciatus (4-6 d old) fed on BALB/C mice were stored at —20°C 30 h after the blood meal.
0022-2585/92/0934-0938$02.00/0 © 1992 Entomological Society of America
November 1992
RAMASAMY ET AL.: HOST IMMUNITY TO MOSQUITOES
Groups of Cx. quinquefasciatus were dissected, and tissues from several mosquitoes were pooled for antigen preparation. The head—thorax, midgut (containing some undigested mouse blood cells), and abdomen were prepared in a manner similar to that of sugar-fed An. tessellatus tissues, and these preparations (approximately five mosquito equivalents in 0.2 ml PBS) were injected subcutaneously into groups of 4-6-wk-old BALB/C mice. The first immunization was made in Freund's complete adjuvant mixed 1:1 with antigen in PBS. Mice were bled through the tail vein 12 d after the third injection to measure antibody titers. Antibody Titers. Antibody titers and antigens recognized by the rabbit antisera were described previously (Ramasamy et al. 1991). Antibody titers in rabbit and mouse sera were determined by an enzyme-linked immunosorbent assay (ELISA) using the immunizing antigen to coat the wells. Bound antigens were incubated with dilutions of either rabbit or mouse sera, followed by peroxidase-conjugated sheep anti-rabbit IgG or goat antimouse IgG (Silenius, Melbourne, Australia). The immune complex was detected with 2,2'-azinobis (3-ethyl benz-thiazoline-6sulphonic acid)-ABTS (BIORAD, Richmond, CA) and H 2 O 2 (Ramasamy et al. 1991). Mosquito Feeding, Fecundity, and Mortality. Immunized rabbits were boosted with the relevant antigens at 5-9-wk intervals and were used for blood feeding between 8 and 60 d after boosting. An. tessellatus (3-4 d old) were fed to repletion directly on restrained rabbits immunized with antigen derived from sugar-fed An. tessellatus. Because An. tessellatus takes multiple blood meals during each gonotrophic cycle, the same rabbit was presented on successive days to a given group of mosquitoes. On day 3 following the first blood meal, in experiments 1 and 2, fed An. tessellatus were transferred individually into plastic vials (5 by 3.5 cm), the bottoms of which were lined with moist filter paper (as an oviposition site) for up to 14 d. In experiment 3, fed mosquitoes were left in the rearing cages (31 by 31 by 31 cm) with a dish lined with moist filter paper for oviposition. The number of eggs laid and mortality were recorded daily. Mosquitoes were fed 10% glucose supplemented with multivitamins during the course of the experiment. The colony of Cx. quinquefasciatus was adapted to feeding late at night. Therefore, feeding Cx. quinquefasciatus directly on restrained immunized rabbits was impractical. However, these mosquitoes fed readily during the day, when in vitro feeding techniques were used. Cx. quinquefasciatus (4-6 d old) were held in paper cups and fed to engorgement on fresh heparinized rabbit blood from immunized rabbits, either through a water-jacketed membrane feeder at 40°C or on a cotton pledget. Cx. quin-
935
quefasciatus took only one blood meal in each experiment. Cx. quinquefasciatus also were fed directly on a restrained immunized mouse placed inside the cage overnight. Mice that had been immunized up to 5 times were used for feeding 15—50 d after immunization. Engorged females were held in rearing cages with a dish of water for oviposition. Mortality and oviposition (numbers of eggs laid in rafts) by Cx. quinquefasciatus were recorded daily. At the end of the experimental period, all females were dissected, and the number of mature follicles (stages IV—V as described by Clements & Boocock 1984) retained in the ovarioles were counted. The total number of eggs per female (Tables 1 and 2) consisted of all laid eggs plus mature unlaid follicles developed by each blood fed mosquito. Statistical Analysis. The number of eggs produced per female was combined within treatments (experiments 1 and 2 for An. tessellatus (Table 1), experiment 1—4 for Cx. quinquefasciatus (Table 2). Experiment 3 with An. tessellatus (Table 1) were kept separate because mosquitoes were not kept in individual vials. Preliminary analysis of variance (ANOVA) was done by the GLM procedure in the SAS/STAT package (SAS Institute 1985). The number of eggs per female produced by mosquitoes fed on control rabbits combined over experiments then were compared with each of the combined treatments by t tests. Results Immunized rabbits developed high titers of antibodies to the immunizing tissue antigens. As reported previously, after four immunizations, the antibody titers in rabbits measured against the immunizing mosquito tissue ranged from 104 to 109, whereas no antibodies were detected in control rabbits (Ramasamy et al. 1991). Titers of antimosquito antibodies continued to rise after subsequent immunizations and reached levels of >10 8 in all immune rabbits. Titers of serum antibodies to Cx. quinquefasciatus antigen in BALB/C mice ranged from 104 to 106 after three immunizations. Control mice injected with Freund's adjuvant without mosquito antigens followed by PBS without antigen showed a nonspecific reaction with mosquito antigen (titer up to 102), probably because of the stimulation of cross-reactive mouse antibodies by the adjuvant. However, the antibody titers of sera from immunized mice were always >10 4 . An. tessellatus engorged readily on control and immunized rabbits; the corresponding body weights immediately after engorgement were 2.19 ± 0.06 (x ± SE) and 2.09 ± 0.09 mg, respectively, indicating that there was no significant difference between females feeding on the two groups of rabbits.
936
Vol. 29, no. 6
JOURNAL OF MEDICAL ENTOMOLOGY
Table 1. Mortality and fecundity of An. tessellatus fed on rabbits immunized with An. tessellatus antigens Rabbit No. (antigen)"
No. fed females
% Mortality at48h
%Egg development/'
Eggs/9 mean (±SE)
Eggs/9, group mean (±SE)
1 2 1 2 1 2 1 2
R 1(HT) R 2(HT) R 5 (AD) R 6 (AD) R 9(MG) RIO(MG) R13 (Control) R14 (Control) R15 (Control) R 3(HT) R 7 (AD) Rll(MG)
44 27 24 40 44 38 42 27 31 34 44 51
9.4 7.5 10.7 26.3 6.0 8.4 2.1 21.1 25.8 38.2 20.5 33.3
79.5 91.2 75.0 95.0 79.1 92.1 76.2 88.9
55.7 (±3.8) 45.9 (±4.4) 46.8 (±5.1) 46.9 (±3.4) 56.8 (±4.2) 40.0 (±2.7) 65.0 (±4.8) 54.7 (±4.1) 46.8 55.2 58.2 34.9
51.1 (±2.9)
J. Med. Entomol. 29(6): 934-938 (1992)
ABSTRACT Rabbit antibodies to antigens derived from the tissues of sugar-fed Anopheles tessellatus Theobald were fed to An. tessellatus and Culex quinquefasciatus Say in blood meals. These antibodies tended to reduce the number of eggs produced by An. tessellatus, but had no effect on the fecundity of Cx. quinquefasciatus. Ingestion of the rabbit antibodies did not detectably affect the mortality of An. tessellatus but increased the mortality of Cx. quinquefasciatus. The results provide preliminary evidence for the potential of antimosquito antibodies to disrupt the physiology of mosquito vectors and indicate the need for further studies with high-titer, monospecific antibodies against the relevant target antigens. KEY WORDS host immunity, Anopheles tessellatus, Culex quinquefasciatus
WHEN MOSQUITOES FEED on vertebrate hosts, both antibody and cell-mediated immunological responses are induced by mosquito salivary gland secretions that enter the bloodstream (Wikel 1982, R. Ramasamy & Ramasamy 1990). Mosquito antigens that normally are not introduced into the vertebrate host when a mosquito feeds (e.g., midgut and abdomen components) can induce an artificial immune response (Alger & Cabrera 1972, Sutherland & Ewen 1974, Ramasamy et al. 1988a). Specific IgG antibodies ingested in a blood meal are known to cross the midgut epithelium of Aedes (Ramasamy et al. 1988a), Anopheles and Culex mosquitoes (Vaughan & Azad 1988). Ingested antimosquito antibodies have been shown to increase the mortality of An. stephensi Liston (Alger & Cabrera 1972) and Ae. aegypti (L.) (Hatfield 1988) and also reduce the fecundity of Ae. aegypti (Sutherland & Ewen 1974, Ramasamy et al. 1988a). Antimosquito antibodies ingested with an infected blood meal also interfere with the development of pathogenic organisms in mosquitoes. Antibodies to mosquito midgut-derived antigens reduced the susceptibility of Ae. aegypti to arboviruses (Ramasamy et al. 1990) and of Anopheles farauti Laveran to Plasmodium berghei Vincke (Ramasamy & Ramasamy 1990b). Anopheles tessellatus Theobald and Culex quinquefasciatus Say tissues share many common antigens (Ramasamy et al. 1991). Our investigation determined the effects of antimosquito 1 2
Vector Biology Laboratory. Malaria Laboratory.
antibodies ingested in a blood meal, on the physiology of the malaria vector, An. tessellatus, and the Bancroftian filariasis vector, Cx. quinquefasciatus. Materials and Methods Laboratory colonies of An. tessellatus and Cx. quinquefasciatus were maintained at 28 ± 1°C, 80% RH, and natural day-night photoperiods. An. tessellatus larvae were fed powdered infant cereal and dry yeast, and Cx. quinquefasciatus larvae were fed powdered soya protein. Adult mosquitoes were provided 10% glucose supplemented with multivitamins. An. tessellatus were blood fed on guinea pigs during the day, whereas Cx. quinquefasciatus were blood fed on guinea pigs overnight. Antigen Preparation and Immunization. Sugarfed An. tessellatus (4—7 d old) were held at —20°C until they were dissected. The head and thorax, midgut, and the rest of the abdomen were separated in 0.01 M phosphate-buffered saline (PBS), pH 7.4, and were used as three tissue preparations. Tissue pooled from several batches of sugar-fed An. tessellatus was homogenized in PBS, and suspensions were injected intramuscularly at multiple sites into three groups of four New Zealand white rabbits; the first injection was done in Freund's complete adjuvant (Ramasamy et al. 1991). Four immunizations at 3-4 wk intervals were made before the initial determination of serum antibody titers. Cx. quinquefasciatus (4-6 d old) fed on BALB/C mice were stored at —20°C 30 h after the blood meal.
0022-2585/92/0934-0938$02.00/0 © 1992 Entomological Society of America
November 1992
RAMASAMY ET AL.: HOST IMMUNITY TO MOSQUITOES
Groups of Cx. quinquefasciatus were dissected, and tissues from several mosquitoes were pooled for antigen preparation. The head—thorax, midgut (containing some undigested mouse blood cells), and abdomen were prepared in a manner similar to that of sugar-fed An. tessellatus tissues, and these preparations (approximately five mosquito equivalents in 0.2 ml PBS) were injected subcutaneously into groups of 4-6-wk-old BALB/C mice. The first immunization was made in Freund's complete adjuvant mixed 1:1 with antigen in PBS. Mice were bled through the tail vein 12 d after the third injection to measure antibody titers. Antibody Titers. Antibody titers and antigens recognized by the rabbit antisera were described previously (Ramasamy et al. 1991). Antibody titers in rabbit and mouse sera were determined by an enzyme-linked immunosorbent assay (ELISA) using the immunizing antigen to coat the wells. Bound antigens were incubated with dilutions of either rabbit or mouse sera, followed by peroxidase-conjugated sheep anti-rabbit IgG or goat antimouse IgG (Silenius, Melbourne, Australia). The immune complex was detected with 2,2'-azinobis (3-ethyl benz-thiazoline-6sulphonic acid)-ABTS (BIORAD, Richmond, CA) and H 2 O 2 (Ramasamy et al. 1991). Mosquito Feeding, Fecundity, and Mortality. Immunized rabbits were boosted with the relevant antigens at 5-9-wk intervals and were used for blood feeding between 8 and 60 d after boosting. An. tessellatus (3-4 d old) were fed to repletion directly on restrained rabbits immunized with antigen derived from sugar-fed An. tessellatus. Because An. tessellatus takes multiple blood meals during each gonotrophic cycle, the same rabbit was presented on successive days to a given group of mosquitoes. On day 3 following the first blood meal, in experiments 1 and 2, fed An. tessellatus were transferred individually into plastic vials (5 by 3.5 cm), the bottoms of which were lined with moist filter paper (as an oviposition site) for up to 14 d. In experiment 3, fed mosquitoes were left in the rearing cages (31 by 31 by 31 cm) with a dish lined with moist filter paper for oviposition. The number of eggs laid and mortality were recorded daily. Mosquitoes were fed 10% glucose supplemented with multivitamins during the course of the experiment. The colony of Cx. quinquefasciatus was adapted to feeding late at night. Therefore, feeding Cx. quinquefasciatus directly on restrained immunized rabbits was impractical. However, these mosquitoes fed readily during the day, when in vitro feeding techniques were used. Cx. quinquefasciatus (4-6 d old) were held in paper cups and fed to engorgement on fresh heparinized rabbit blood from immunized rabbits, either through a water-jacketed membrane feeder at 40°C or on a cotton pledget. Cx. quin-
935
quefasciatus took only one blood meal in each experiment. Cx. quinquefasciatus also were fed directly on a restrained immunized mouse placed inside the cage overnight. Mice that had been immunized up to 5 times were used for feeding 15—50 d after immunization. Engorged females were held in rearing cages with a dish of water for oviposition. Mortality and oviposition (numbers of eggs laid in rafts) by Cx. quinquefasciatus were recorded daily. At the end of the experimental period, all females were dissected, and the number of mature follicles (stages IV—V as described by Clements & Boocock 1984) retained in the ovarioles were counted. The total number of eggs per female (Tables 1 and 2) consisted of all laid eggs plus mature unlaid follicles developed by each blood fed mosquito. Statistical Analysis. The number of eggs produced per female was combined within treatments (experiments 1 and 2 for An. tessellatus (Table 1), experiment 1—4 for Cx. quinquefasciatus (Table 2). Experiment 3 with An. tessellatus (Table 1) were kept separate because mosquitoes were not kept in individual vials. Preliminary analysis of variance (ANOVA) was done by the GLM procedure in the SAS/STAT package (SAS Institute 1985). The number of eggs per female produced by mosquitoes fed on control rabbits combined over experiments then were compared with each of the combined treatments by t tests. Results Immunized rabbits developed high titers of antibodies to the immunizing tissue antigens. As reported previously, after four immunizations, the antibody titers in rabbits measured against the immunizing mosquito tissue ranged from 104 to 109, whereas no antibodies were detected in control rabbits (Ramasamy et al. 1991). Titers of antimosquito antibodies continued to rise after subsequent immunizations and reached levels of >10 8 in all immune rabbits. Titers of serum antibodies to Cx. quinquefasciatus antigen in BALB/C mice ranged from 104 to 106 after three immunizations. Control mice injected with Freund's adjuvant without mosquito antigens followed by PBS without antigen showed a nonspecific reaction with mosquito antigen (titer up to 102), probably because of the stimulation of cross-reactive mouse antibodies by the adjuvant. However, the antibody titers of sera from immunized mice were always >10 4 . An. tessellatus engorged readily on control and immunized rabbits; the corresponding body weights immediately after engorgement were 2.19 ± 0.06 (x ± SE) and 2.09 ± 0.09 mg, respectively, indicating that there was no significant difference between females feeding on the two groups of rabbits.
936
Vol. 29, no. 6
JOURNAL OF MEDICAL ENTOMOLOGY
Table 1. Mortality and fecundity of An. tessellatus fed on rabbits immunized with An. tessellatus antigens Rabbit No. (antigen)"
No. fed females
% Mortality at48h
%Egg development/'
Eggs/9 mean (±SE)
Eggs/9, group mean (±SE)
1 2 1 2 1 2 1 2
R 1(HT) R 2(HT) R 5 (AD) R 6 (AD) R 9(MG) RIO(MG) R13 (Control) R14 (Control) R15 (Control) R 3(HT) R 7 (AD) Rll(MG)
44 27 24 40 44 38 42 27 31 34 44 51
9.4 7.5 10.7 26.3 6.0 8.4 2.1 21.1 25.8 38.2 20.5 33.3
79.5 91.2 75.0 95.0 79.1 92.1 76.2 88.9
55.7 (±3.8) 45.9 (±4.4) 46.8 (±5.1) 46.9 (±3.4) 56.8 (±4.2) 40.0 (±2.7) 65.0 (±4.8) 54.7 (±4.1) 46.8 55.2 58.2 34.9
51.1 (±2.9)
