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Changes in the reproductive biology of Biomphalaria glabrata experimentally infected with the nematode Angiostrongylus cantonensis
Journal of Invertebrate Pathology 108 (2011) 220–223
Contents lists available at SciVerse ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Changes in the reproductive biology of Biomphalaria glabrata experimentally infected with the nematode Angiostrongylus cantonensis Vinícius Menezes Tunholi-Alves a, Victor Menezes Tunholi a, Danilo Lustrino a, Ludimila Santos Amaral b, Silvana Carvalho Thiengo c, Jairo Pinheiro a,⇑ a b c
Departmento de Ciências Fisiológicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro (UFRuralRJ), Brazil Departamento de Saneamento e Saúde Ambiental, Escola Nacional de Saúde Pública, Fiocruz, Brazil Laboratório de Referência Nacional em Malacologia Médica do Instituto Oswaldo Cruz—IOC/Fiocruz, Brazil
a r t i c l e
i n f o
Article history: Received 13 May 2011 Accepted 25 August 2011 Available online 10 September 2011 Keywords: Angiostrongylus cantonensis Biomphalaria glabrata Parasitic castration Reproductive biology
a b s t r a c t This study showed for the first time changes in the reproductive biology of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis. The values of all the parameters analyzed (total number of eggs, number of egg masses, number of eggs/mass, number of eggs/snail, percentage of viable eggs and galactogen content in albumen gland) changed with progressive infection. The results indicate the occurrence of partial parasitic castration of B. glabrata by A. cantonensis larvae, probably in response to the depletion of energy reserves, with no injuries to the gonadal tissues. Ó 2011 Elsevier Inc. Open access under the Elsevier OA license.
1. Introduction Angiostrongylus cantonensis is a nematode parasite of rodent lungs and is considered the main agent responsible for human eosinophilic meningoencephalitis. Its life cycle is heteroxenous, with snails as intermediate hosts. This initial phase is essential for the parasite’s development, enabling it to reach the stage where it can infect the definitive host (Stewart et al., 1985). In recent years, much attention has been given to the clinical aspects and the risk of human infection by A. cantonensis in countries of the Americas (Thiengo et al., 2010). In accordance with World Health Organization recommendations (WHO, 1983), the development strategies to control diseases that are transmitted by snail hosts should be based on control of the snail population as well as treatment of infected animals and humans. So, understanding the changes in the reproductive biology of snails infected with A. cantonensis is essential for developing effective methods against the spread of human eosinophilic meningoencephalitis. However, it is surprising that studies of the reproductive activity of A. cantonensis-infected snails have not yet been conducted, since this parasite has great importance to public health and the response to infection is highly variable among snail species infected by different helminths (Tunholi et al., 2011). To shed light on this subject, the present study analyzed for the first time, the changes in the reproductive biology of Biomphalaria glabrata caused by infection ⇑ Corresponding author. Fax: +55 21 26823222. E-mail address: [email protected] (J. Pinheiro). 0022-2011 Ó 2011 Elsevier Inc. Open access under the Elsevier OA license. doi:10.1016/j.jip.2011.08.009
by A. cantonensis during its prepatent period (3 weeks of infection) (Guilhon and Gaalon, 1969), using the parameters total number of eggs, number of egg masses, number of eggs/mass, number of eggs/ snail, percentage of viable eggs, and galactogen content in albumen gland, as well as the histological status of the gonad (ovotestis of infected snails). The different mechanisms possibly related to this phenomenon are also discussed. 2. Material and methods 2.1. Maintenance of the snails and formation of groups The snails were kept in aquariums containing 1500 ml of dechlorinated water, to which 0.5 g of CaCO3 was added. Polystyrene plates measuring ±2 cm2 were placed inside the aquariums to serve as substrate for egg laying. The snails were fed with dehydrated lettuce leaves (Lactuca sativa L.) ad libitum. Six groups were formed: three control groups (uninfected) and three treatment groups (infected). Each aquarium contained 10 snails, reared in the laboratory from hatching to be certain of their age and sexual maturity. The entire experiment was conducted in duplicate, using a total of 120 snails. 2.2. Parasites Third-stage larvae (L3) of A. cantonensis, obtained from specimens of Achatina fulica collected from Olinda, Pernambuco, Brazil
221
The results were expressed as mean ± standard error and submitted to one-way ANOVA and then the Tukey–Kramer test (P < 0.05%) to compare the means (InStat, GraphPad, v.4.00, Prism, GraphPad, v.3.02, Prism Inc.). 3. Results The infection reduced the number of egg masses/snail of the infected snails (12.18 ± 1.82) in comparison with the control/uninfected animals (23.32 ± 1.37) from the second week of infection. The same variation was observed in relation to the number eggs/ snail, with a gradual decline in the oviposition rate as the infection progressed. Significant declines were observed in the second and
0.55 ± 0.06a 0.38 ± 0.07b 0.39 ± 0.06b
Infected Control
0.58 ± 0.04a 0.59 ± 0.05a 0.54 ± 0.04a 96.93 85.53 84.15 98.95 97.98 98.47 215.00 ± 53.13a 134.36 ± 18.44b 132.73 ± 24.48b 307.67 ± 22.32a 304.58 ± 22.27a 310.87 ± 23.11a 12.68 ± 2.00a 9.73 ± 0.78b 9.60 ± 0.76b 15.23 ± 1.20a 15.19 ± 1.25a 16.86 ± 1.18a 221.80 ± 54.14a 157.09 ± 20.15b 157.73 ± 25.60b
Galactogen X ± SE
Infected Viability (%)
Control Infected
Hatched snails X ± SE
Control Infected
Eggs/egg mass X ± SE
Control
313.20 ± 22.29a 313.12 ± 21.97a 315.29 ± 23.54a 22.80 ± 1.82a 12.18 ± 1.82b 12.60 ± 2.17b
2.7. Statistical analyses
23.06 ± 1.61a 23.32 ± 1.37a 24.00 ± 1.40a
Snails from each period of infection were dissected and transferred to Duboscq-Brasil fixative (Fernandes, 1949). The soft tissues were processed according to routine histological techniques (Humason, 1979). The sections (5 lm) were stained using hematoxylin and eosin and observed under a Zeiss Axioplan light microscope; images were captured with an MRc5 AxioCam digital camera and processed with the Axiovision software.
1 2 3
2.6. Histological analyses
Infected
Each week after infection, ten specimens from each group were randomly chosen, dissected and the albumen gland was collected and maintained at 10 °C. Galactogen was extracted and quantified according to Pinheiro and Gomes (1994), being expressed as mg of galactose/g of tissue, wet weight.
Eggs/snail X ± SE
2.5. Galactogen determination
Control
The polystyrene plates were removed from the aquariums and the numbers of egg masses and eggs laid were counted under a stereoscopic microscope on alternate days until three weeks after infection. After the count, the plates were numbered individually and placed in new aquariums free of snails. Then the egg masses were observed to count the snails hatching. The egg viability, expressed as a percentage, is the number of snails hatched divided by the number of eggs laid in each experimental group, multiplied by 100 (Tunholi et al., 2011).
Infected
2.4. Analysis of the reproductive biology of B. glabrata infected by A. cantonensis
Egg masses/snail X ± SE
The feces of parasitized R. norvergicus were collected to obtain the larvae by the technique of Baermann (Willcox and Coura, 1989). After processing the fecal samples, specimens of B. glabrata (8–12 mm) at 90 days old on average were exposed individually to approximately 1200 L1 larvae (Yousif and Lammler, 1977). After 48 h the snails were transferred to the aquariums.
Control
2.3. Infection of the snails
Weeks
(8°10 000 S/34°510 000 W, altitude 16 m) in 2008, in the area surrounding the home of a human patient diagnosed with eosinophilic meningoencephalitis, were inoculated in Rattus norvegicus in the Laboratório Nacional de Referência em Malacologia Médica and Laboratório de Patologia do Instituto Oswaldo Cruz (Fiocruz, RJ, Brazil), where the cycle is maintained. The first-stage larva (L1) utilized in this study were obtained from this experimental cycle maintained in the mentioned laboratories.
Table 1 Changes on the reproductive biology of Biomphalaria glabrata in response to infection with Angionstrongylus cantonensis in different periods of infection, expressed in weeks (1, 2 and 3 weeks). a,bDifferent letters indicate means differ significantly between control and infected snails in each week of infection (row) (P < 0.05). There were no significant differences between the means of the control groups during the 3 weeks, in any of the parameters analyzed.
V.M. Tunholi-Alves et al. / Journal of Invertebrate Pathology 108 (2011) 220–223
222
V.M. Tunholi-Alves et al. / Journal of Invertebrate Pathology 108 (2011) 220–223
Fig. 1. Histological section in the ovotestis region of Biomphalaria glabrata. (a) Section from an uninfected snail showing the ovotestis region functionally active, with the structure of the acinus (a) containing sperm grouped by the anterior end (heads) (sh) and the tail (t) projected to the lumen of the acinus. Scale bar = 50 lm. (b) Histological section of ovotestis region of a snail experimentally infected with Angiostrongylus cantonensis, showing a region of the gonad with an active process of gametogenesis, with the sperm grouped by the head region (sh) and the tail (t) projected to the lumen of the acinus (a) and the complete absence of larval nematodes developing in this region. Scale bar = 50 lm.
third weeks (157.09 ± 20.15 and 157.73 ± 25.6, respectively) in comparison with the control (313.12 ± 21.97 and 315.29 ± 23.54). Also, there was a reduction in the average eggs/egg mass ratio during the infection period. However, only the values referring to the second and third weeks (9.73 ± 0.78 and 9.60 ± 0.76, respectively) differed significantly from the uninfected group (15.19 ± 1.25 and 16.86 ± 1.18, respectively). At the same time, there were differences in relation to the hatching rate, these being significantly lower starting in the second week of infection (134.36 ± 18.44), representing a viability rate of 85.53% in relation to the control group, where the rate was 97.98% (Table 1). The galactogen content also decreased from second week postinfection onward in the infected snails (0.38 ± 0.07) in relation to the uninfected ones (0.59 ± 0.05). A similar profile was observed for the third week after infection (Table 1). The histological analyses did not show significant changes in the gonadal tissues of the infected snails when compared to those from uninfected snails (Fig. 1a and b). In both, the structure of the ovotestis seemed to be preserved, where the process of gametes formation was evident, showing a functional structure of this organ. 4. Discussion Studies focusing on infection with trematodes were the first to report a severe decrease of reproductive activity in the intermediate snail host, an effect named parasitic castration (Koie, 1969). The changes in the reproductive biology are usually described by observing parameters related to ovipository activity and viability of eggs laid. But indications of the mechanism that triggers parasitic castration can be obtained using different investigative tools. Baudoin (1975) stated that parasitic castration may be a direct process, whereby the parasite directly causes damages to gonadal tissues, or an indirect process, in response to withdrawal of nutrients by the parasites. So, to obtain information that may indicate the mechanism involved in the parasitic castration, histological analyses were performed to verify the presence of larvae in gonadal tissues. In addition, the galactogen content in the albumen gland was measured because this is an accessory sexual organ that synthesizes this polymer, which is part of the perivitelinic fluid, the main energy source to the embryos and newly hatched snails (Gomot et al., 1989). Reduction in the galactogen concentration will impair the hatching rate, characterizing the parasitic castration as a nutritional process. In the present study, a continuous reduction of the parameters analyzed regarding the reproductive biology of B. glabrata infected with A. cantonensis was observed. But complete interruption of
reproductive activity did not occur, characterizing a partial parasitic castration phenomenon in this parasite–host system. Harris and Cheng (1975) observed encapsulated nematodes in the mantle and cephalopedal mass of B. glabrata infected with A. cantonensis, but there was no histological damages in reproductive system tissues. In the present study, the histological observation also did not show larvae of A. cantonensis in the gonadal tissues of B. glabrata. Our results evidence a progressive reduction in the galactogen contents, with significantly lower values in the second and third weeks of infection, clearly showing that the larval development of A. cantonensis causes changes in the energetic metabolism of B. glabrata, corroborating the results of Brockelman et al. (1976) and Brockelman and Sithithavorn (1980), which showed a reduction in protein, glycogen and glucose concentrations in A. fulica infected with A. cantonensis. The reproductive parameters analyzed were related with a decrease of galactogen content in the albumen gland in infected snails, which occurred from second week postinfection, compromising the number of eggs laid, hatching rate, number of egg masses and egg viability. So, the castration in this system may be considered an indirect effect. Finally, for the first time the effects of A. cantonensis infection on the reproductive biology of B. glabrata was studied and the parasitic castration phenomenon was reported, being classified as an indirect and partial process. Nevertheless, the lowest values were observed only from the second week post-infection, suggesting that the pathological effects produced by nematode larvae on the reproductive biology occur at this time, and can be explained at least in part by a decrease in the galactogen content in the albumen gland of infected snails. However, further studies must be conducted to clarify the metabolic changes that occur in the snail host in response to larval nematode infection, to gain a better understanding of the mechanisms involved in this process.
Acknowledgments This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
References Baudoin, M., 1975. Host castration as a parasitic strategy. Evolution 29, 335–352. Brockelman, C.R., Chusatayanond, W., Baidikul, V., 1976. Growth and localization of Angiostrongylus cantonensis in the molluscan host Achatina fulica Southeast Asian. Journal of Tropical Medicine and Public Health 7, 30–37.
V.M. Tunholi-Alves et al. / Journal of Invertebrate Pathology 108 (2011) 220–223 Brockelman, C.R., Sithithavorn, P., 1980. Carbohydrate reserves and hemolymph sugars of the African giant snail, Achatina fulica in relation to parasitic infection and starvation. Zeitschrift fur Parasitenkunde 62, 285–291. Fernandes, M.C., 1949. Métodos escolhidos de técnicas microscópicas, In: second ed. Imprensa Nacional, Rio de Janeiro, RJ. Gomot, A., Gomot, L., Boukraa, S., Bruckert, S., 1989. Influence of soil on the growth of the land snail Helix aspersa an experimental study of the absorption route for the stimulating factors. J. Mollusc. Studies. 55, 1–8. Guilhon, J., Gaalon, A., 1969. Évolution larvaire d´un nematode parasite de pappariel circulatoire du chien dans l´organisme de mollusques dulçaquicoles. Comp. Rend. Acad. Sci. 268, 612–615. Harris, K.R., Cheng, T.C., 1975. The encapsulation process in Biomphalaria glabrata experimentally infected with metastrongylid Angyonstrongylus cantonensis light microscopy. Int. J. Parasitol. 5, 521–528. Humason, G.L., 1979. Animal Tissue Techniques, 4th Ed. W.H. Freeman, San Francisco, USA. Koie, M., 1969. On the endoparasites of Buccinim undatum L. with special reference to the trematodes. Ophelia 6, 251–279. Pinheiro, J., Gomes, E.M., 1994. A method for glycogen determination in molluscs. Arq. Biol. Technol. 37, 569–576.
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Stewart, G.L., Ubelaker, J.E., Curtis, D., 1985. Pathophysiologic alterations in Biomphalaria glabrata infected with Angiostrongylus costaricensis. J. Invert. Pathol. 45, 152–157. Thiengo, S.C., Maldonado, A., Mota, E.M., Torres, E.J., Caldeira, R., Carvalho, O.S., Oliveira, A.P., Simões, R.O., Fernandez, M.A., Lanfredi, R.M., 2010. The giant African snail Achatina fulica as natural intermediate host of Angiostrongylus cantonensis in Pernanbuco, northeast Brazil. Acta Trop. 115, 194–199. Tunholi, V.M., Lustrino, D., Tunholi-Alves, V.M., Mello-Silva, C.C.C., Maldonado, A., Rodrigues, M.L.A., Pinheiro, J., 2011. Changes in the reproductive biology of Biomphalara glabrata infected with different doses of Echinostoma paraensei miracidia. J. Invert. Pathol. 106, 192–195. Willcox, H.P., Coura, J.R., 1989. Nova concepção para o método de Baermann– Moraes – Coutinho na pesquisa de larvas de nematódeos. Mem. Inst. Oswaldo Cruz 84, 539–565. WHO, 1983. Report of a Scientific Working Group on Plant Molluscicide and Guidelines for Evaluation of Plant Molluscicide. World Health Organization (TDR/SCH-SWE (4)/83.3), Geneva. Yousif, F., Lámmler, G., 1977. The mode of infection with and the distribution of Angionstrongylus cantonensis larvae in the intermediate host Biomphalaria glabrata. Z. Parasitenkd. 53, 247–250.
Contents lists available at SciVerse ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Changes in the reproductive biology of Biomphalaria glabrata experimentally infected with the nematode Angiostrongylus cantonensis Vinícius Menezes Tunholi-Alves a, Victor Menezes Tunholi a, Danilo Lustrino a, Ludimila Santos Amaral b, Silvana Carvalho Thiengo c, Jairo Pinheiro a,⇑ a b c
Departmento de Ciências Fisiológicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro (UFRuralRJ), Brazil Departamento de Saneamento e Saúde Ambiental, Escola Nacional de Saúde Pública, Fiocruz, Brazil Laboratório de Referência Nacional em Malacologia Médica do Instituto Oswaldo Cruz—IOC/Fiocruz, Brazil
a r t i c l e
i n f o
Article history: Received 13 May 2011 Accepted 25 August 2011 Available online 10 September 2011 Keywords: Angiostrongylus cantonensis Biomphalaria glabrata Parasitic castration Reproductive biology
a b s t r a c t This study showed for the first time changes in the reproductive biology of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis. The values of all the parameters analyzed (total number of eggs, number of egg masses, number of eggs/mass, number of eggs/snail, percentage of viable eggs and galactogen content in albumen gland) changed with progressive infection. The results indicate the occurrence of partial parasitic castration of B. glabrata by A. cantonensis larvae, probably in response to the depletion of energy reserves, with no injuries to the gonadal tissues. Ó 2011 Elsevier Inc. Open access under the Elsevier OA license.
1. Introduction Angiostrongylus cantonensis is a nematode parasite of rodent lungs and is considered the main agent responsible for human eosinophilic meningoencephalitis. Its life cycle is heteroxenous, with snails as intermediate hosts. This initial phase is essential for the parasite’s development, enabling it to reach the stage where it can infect the definitive host (Stewart et al., 1985). In recent years, much attention has been given to the clinical aspects and the risk of human infection by A. cantonensis in countries of the Americas (Thiengo et al., 2010). In accordance with World Health Organization recommendations (WHO, 1983), the development strategies to control diseases that are transmitted by snail hosts should be based on control of the snail population as well as treatment of infected animals and humans. So, understanding the changes in the reproductive biology of snails infected with A. cantonensis is essential for developing effective methods against the spread of human eosinophilic meningoencephalitis. However, it is surprising that studies of the reproductive activity of A. cantonensis-infected snails have not yet been conducted, since this parasite has great importance to public health and the response to infection is highly variable among snail species infected by different helminths (Tunholi et al., 2011). To shed light on this subject, the present study analyzed for the first time, the changes in the reproductive biology of Biomphalaria glabrata caused by infection ⇑ Corresponding author. Fax: +55 21 26823222. E-mail address: [email protected] (J. Pinheiro). 0022-2011 Ó 2011 Elsevier Inc. Open access under the Elsevier OA license. doi:10.1016/j.jip.2011.08.009
by A. cantonensis during its prepatent period (3 weeks of infection) (Guilhon and Gaalon, 1969), using the parameters total number of eggs, number of egg masses, number of eggs/mass, number of eggs/ snail, percentage of viable eggs, and galactogen content in albumen gland, as well as the histological status of the gonad (ovotestis of infected snails). The different mechanisms possibly related to this phenomenon are also discussed. 2. Material and methods 2.1. Maintenance of the snails and formation of groups The snails were kept in aquariums containing 1500 ml of dechlorinated water, to which 0.5 g of CaCO3 was added. Polystyrene plates measuring ±2 cm2 were placed inside the aquariums to serve as substrate for egg laying. The snails were fed with dehydrated lettuce leaves (Lactuca sativa L.) ad libitum. Six groups were formed: three control groups (uninfected) and three treatment groups (infected). Each aquarium contained 10 snails, reared in the laboratory from hatching to be certain of their age and sexual maturity. The entire experiment was conducted in duplicate, using a total of 120 snails. 2.2. Parasites Third-stage larvae (L3) of A. cantonensis, obtained from specimens of Achatina fulica collected from Olinda, Pernambuco, Brazil
221
The results were expressed as mean ± standard error and submitted to one-way ANOVA and then the Tukey–Kramer test (P < 0.05%) to compare the means (InStat, GraphPad, v.4.00, Prism, GraphPad, v.3.02, Prism Inc.). 3. Results The infection reduced the number of egg masses/snail of the infected snails (12.18 ± 1.82) in comparison with the control/uninfected animals (23.32 ± 1.37) from the second week of infection. The same variation was observed in relation to the number eggs/ snail, with a gradual decline in the oviposition rate as the infection progressed. Significant declines were observed in the second and
0.55 ± 0.06a 0.38 ± 0.07b 0.39 ± 0.06b
Infected Control
0.58 ± 0.04a 0.59 ± 0.05a 0.54 ± 0.04a 96.93 85.53 84.15 98.95 97.98 98.47 215.00 ± 53.13a 134.36 ± 18.44b 132.73 ± 24.48b 307.67 ± 22.32a 304.58 ± 22.27a 310.87 ± 23.11a 12.68 ± 2.00a 9.73 ± 0.78b 9.60 ± 0.76b 15.23 ± 1.20a 15.19 ± 1.25a 16.86 ± 1.18a 221.80 ± 54.14a 157.09 ± 20.15b 157.73 ± 25.60b
Galactogen X ± SE
Infected Viability (%)
Control Infected
Hatched snails X ± SE
Control Infected
Eggs/egg mass X ± SE
Control
313.20 ± 22.29a 313.12 ± 21.97a 315.29 ± 23.54a 22.80 ± 1.82a 12.18 ± 1.82b 12.60 ± 2.17b
2.7. Statistical analyses
23.06 ± 1.61a 23.32 ± 1.37a 24.00 ± 1.40a
Snails from each period of infection were dissected and transferred to Duboscq-Brasil fixative (Fernandes, 1949). The soft tissues were processed according to routine histological techniques (Humason, 1979). The sections (5 lm) were stained using hematoxylin and eosin and observed under a Zeiss Axioplan light microscope; images were captured with an MRc5 AxioCam digital camera and processed with the Axiovision software.
1 2 3
2.6. Histological analyses
Infected
Each week after infection, ten specimens from each group were randomly chosen, dissected and the albumen gland was collected and maintained at 10 °C. Galactogen was extracted and quantified according to Pinheiro and Gomes (1994), being expressed as mg of galactose/g of tissue, wet weight.
Eggs/snail X ± SE
2.5. Galactogen determination
Control
The polystyrene plates were removed from the aquariums and the numbers of egg masses and eggs laid were counted under a stereoscopic microscope on alternate days until three weeks after infection. After the count, the plates were numbered individually and placed in new aquariums free of snails. Then the egg masses were observed to count the snails hatching. The egg viability, expressed as a percentage, is the number of snails hatched divided by the number of eggs laid in each experimental group, multiplied by 100 (Tunholi et al., 2011).
Infected
2.4. Analysis of the reproductive biology of B. glabrata infected by A. cantonensis
Egg masses/snail X ± SE
The feces of parasitized R. norvergicus were collected to obtain the larvae by the technique of Baermann (Willcox and Coura, 1989). After processing the fecal samples, specimens of B. glabrata (8–12 mm) at 90 days old on average were exposed individually to approximately 1200 L1 larvae (Yousif and Lammler, 1977). After 48 h the snails were transferred to the aquariums.
Control
2.3. Infection of the snails
Weeks
(8°10 000 S/34°510 000 W, altitude 16 m) in 2008, in the area surrounding the home of a human patient diagnosed with eosinophilic meningoencephalitis, were inoculated in Rattus norvegicus in the Laboratório Nacional de Referência em Malacologia Médica and Laboratório de Patologia do Instituto Oswaldo Cruz (Fiocruz, RJ, Brazil), where the cycle is maintained. The first-stage larva (L1) utilized in this study were obtained from this experimental cycle maintained in the mentioned laboratories.
Table 1 Changes on the reproductive biology of Biomphalaria glabrata in response to infection with Angionstrongylus cantonensis in different periods of infection, expressed in weeks (1, 2 and 3 weeks). a,bDifferent letters indicate means differ significantly between control and infected snails in each week of infection (row) (P < 0.05). There were no significant differences between the means of the control groups during the 3 weeks, in any of the parameters analyzed.
V.M. Tunholi-Alves et al. / Journal of Invertebrate Pathology 108 (2011) 220–223
222
V.M. Tunholi-Alves et al. / Journal of Invertebrate Pathology 108 (2011) 220–223
Fig. 1. Histological section in the ovotestis region of Biomphalaria glabrata. (a) Section from an uninfected snail showing the ovotestis region functionally active, with the structure of the acinus (a) containing sperm grouped by the anterior end (heads) (sh) and the tail (t) projected to the lumen of the acinus. Scale bar = 50 lm. (b) Histological section of ovotestis region of a snail experimentally infected with Angiostrongylus cantonensis, showing a region of the gonad with an active process of gametogenesis, with the sperm grouped by the head region (sh) and the tail (t) projected to the lumen of the acinus (a) and the complete absence of larval nematodes developing in this region. Scale bar = 50 lm.
third weeks (157.09 ± 20.15 and 157.73 ± 25.6, respectively) in comparison with the control (313.12 ± 21.97 and 315.29 ± 23.54). Also, there was a reduction in the average eggs/egg mass ratio during the infection period. However, only the values referring to the second and third weeks (9.73 ± 0.78 and 9.60 ± 0.76, respectively) differed significantly from the uninfected group (15.19 ± 1.25 and 16.86 ± 1.18, respectively). At the same time, there were differences in relation to the hatching rate, these being significantly lower starting in the second week of infection (134.36 ± 18.44), representing a viability rate of 85.53% in relation to the control group, where the rate was 97.98% (Table 1). The galactogen content also decreased from second week postinfection onward in the infected snails (0.38 ± 0.07) in relation to the uninfected ones (0.59 ± 0.05). A similar profile was observed for the third week after infection (Table 1). The histological analyses did not show significant changes in the gonadal tissues of the infected snails when compared to those from uninfected snails (Fig. 1a and b). In both, the structure of the ovotestis seemed to be preserved, where the process of gametes formation was evident, showing a functional structure of this organ. 4. Discussion Studies focusing on infection with trematodes were the first to report a severe decrease of reproductive activity in the intermediate snail host, an effect named parasitic castration (Koie, 1969). The changes in the reproductive biology are usually described by observing parameters related to ovipository activity and viability of eggs laid. But indications of the mechanism that triggers parasitic castration can be obtained using different investigative tools. Baudoin (1975) stated that parasitic castration may be a direct process, whereby the parasite directly causes damages to gonadal tissues, or an indirect process, in response to withdrawal of nutrients by the parasites. So, to obtain information that may indicate the mechanism involved in the parasitic castration, histological analyses were performed to verify the presence of larvae in gonadal tissues. In addition, the galactogen content in the albumen gland was measured because this is an accessory sexual organ that synthesizes this polymer, which is part of the perivitelinic fluid, the main energy source to the embryos and newly hatched snails (Gomot et al., 1989). Reduction in the galactogen concentration will impair the hatching rate, characterizing the parasitic castration as a nutritional process. In the present study, a continuous reduction of the parameters analyzed regarding the reproductive biology of B. glabrata infected with A. cantonensis was observed. But complete interruption of
reproductive activity did not occur, characterizing a partial parasitic castration phenomenon in this parasite–host system. Harris and Cheng (1975) observed encapsulated nematodes in the mantle and cephalopedal mass of B. glabrata infected with A. cantonensis, but there was no histological damages in reproductive system tissues. In the present study, the histological observation also did not show larvae of A. cantonensis in the gonadal tissues of B. glabrata. Our results evidence a progressive reduction in the galactogen contents, with significantly lower values in the second and third weeks of infection, clearly showing that the larval development of A. cantonensis causes changes in the energetic metabolism of B. glabrata, corroborating the results of Brockelman et al. (1976) and Brockelman and Sithithavorn (1980), which showed a reduction in protein, glycogen and glucose concentrations in A. fulica infected with A. cantonensis. The reproductive parameters analyzed were related with a decrease of galactogen content in the albumen gland in infected snails, which occurred from second week postinfection, compromising the number of eggs laid, hatching rate, number of egg masses and egg viability. So, the castration in this system may be considered an indirect effect. Finally, for the first time the effects of A. cantonensis infection on the reproductive biology of B. glabrata was studied and the parasitic castration phenomenon was reported, being classified as an indirect and partial process. Nevertheless, the lowest values were observed only from the second week post-infection, suggesting that the pathological effects produced by nematode larvae on the reproductive biology occur at this time, and can be explained at least in part by a decrease in the galactogen content in the albumen gland of infected snails. However, further studies must be conducted to clarify the metabolic changes that occur in the snail host in response to larval nematode infection, to gain a better understanding of the mechanisms involved in this process.
Acknowledgments This study was supported in part by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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