Nota científica

 

Efficiency of the separation of Aedes (Stegomyia) albopictus (Diptera: Culicidae) male and female pupae using a sieving device

 

Eficiencia de separación de pupas machos y hembras de Aedes (Stegomyia) albopictus (Diptera: Culicidae) usando un dispositivo de tamizaje

 

Oscar Mikery-Pacheco, Karen Serrano-Domínguez, Pilar Marcelín-Chong & Daniel Sánchez-Guillén

 

El Colegio de la Frontera Sur - Km 2.5 carretera Antiguo Aeropuerto, 30700 Tapachula, Chiapas, México. Correo electrónico: omikery@ecosur.edu.mx

 

Recibido: 14/01/2013;
Aceptado: 11/09/2014.

 

RESUMEN

Después de medir el tamaño del cuerpo, abdomen y cefalotórax de la pupa macho y hembra de Aedes albopictus, decidimos usar el tamaño del cefalotórax de la pupa macho para construir un dispositivo para la separación de individuos por sexo en el estado pupal. De manera interesante, muchas pupas macho no pasaron a través del tamiz y fueron retenidos y sacrificados como pupas hembra; esto puede significar un desperdicio de recursos que no ha sido informado previamente. Por tal motivo se hace énfasis en que la eficiencia de retención de pupas macho debe ser confirmada si se planea su cría masiva.

 

Many experiments with mosquitoes (Diptera, Culicidae) require unmated individuals (Blackmore and Lord 2000, Koenraadt 2008), which are obtained by keeping them separated by sex in non-reproductive stages. Separation of unmated individuals by sex in the adult stage may be difficult because mating can pass unnoticed, forcing the researcher to be aware of the moment they molt into adults to be rapidly removed (Brazil & Brazil 2000). For small experiments this can be a manageable task, by placing larvae or pupae in separate vials (Dame et al. 1974). But if larger numbers are required, as in mark-release-capture experiments or in autocidal control of mosquito populations, the previous approach is merely inappropriate.

Size differences of both male and female mosquito pupae were pointed out by McCray (19(1), Gerberg et al. (1969) and Sharma et al. (1972) who took advantage of this finding to build a sieving-based apparatus for rapid sexing of Aedes (S.) aegypti pupae. However, variables like larval density, temperature, food and others can affect pupae cephalothorax size, making it obligatory to follow strict culture conditions so the device can effectively separate males from females (Papathanos et al. 2009). Actually, the importance of rapid, automatic sexing was evident in a recent study: Bellini et al. (2007) used a sieving device to separate 20,000 male pupae of Ae. albopictus a week with a separation efficiency of 9799%. However, these studies based the designs of their devices on trial and error strategies, instead of using morphological parameters of pupae, which could be a more appropriate approach. Here, we measured three morphological characteristics of female and male Ae. albopictus pupae and show how these characteristics proved to be useful to build a device for rapid sexing of pupae, ideal for research with few requirements of biological material or small laboratories with few resources.

Mosquitoes were collected as larvae in the city of Tapachula, Mexico (N14°53'11.85", W92°17'13.09") and reared following Medici et al. (2011) in laboratories of El Colegio de la Frontera Sur in November 2012; once in the pupal stage, 90 individuals were individually separated in vials and then transferred one at a time to a microscope slide. Cephalothorax, abdomen and body length were measured with a 10 mm scale in a stereoscopic microscope (Fig. 1, A). Next, these pupae were taken back to their respective vials to allow their development into adults to determine sex. Forty pupae corresponded to female and 50 to male pupae. Student's t-tests were applied to data to find differences between male and female pupae characteristics. Statistics were done in R for Mac OS X V2.15. Subsequently with this information we constructed a device with a 1.6 x 1.1 mm mesh size based on the male cephalothorax size.

According to our results, the separation of male and female pupae is a straightforward task, since the measurements of the three morphological characteristics is significantly different between sexes (Figure 1, B). The device designed here, we separated 61 individuals into 30 supposedly male pupae (SMP) and 31 supposedly female pupae (SFP), which were reared to adult to confirm sex. Thirteen percent of SMP were actually female mosquitoes; meanwhile 32% of SFP were confirmed as males.

With pupae morphometrics we were able to build a sieving device with good separation efficiency, but far from the one achieved by Bellini et al. (2007). In our study, despite the average larger size of female pupae, the smaller ones could pass trough the sieve. These small females are part of the variation in natural populations of mosquitoes, from which we obtained our biological material. Thus by selecting large females with a lower standard error in their morphometrics this problem could be minimized, a very important aspect not mentioned by Bellini et al. (2007). More important yet is the finding that many males did not pass the sieve, which can be considered a waste of resources. The utility of screening of large numbers of genetically modified or sterile males (Sterile Insect Technique, SIT) is important in biological control by augmentation programs of local populations of mosquitoes (Alphey et al. 2010). However, when considering measures of male standards, the aspect of male mating competitiveness in the field is neglected, where size is a crucial factor in terms of sexual selection in mosquitoes (Sawadogo et al. 2013).

This makes it evident that is not only important to observe the number of females in the SMP, but also the retention of males in the SFP that will be sacrificed for not being separated, which seems to be a larger number than expected. In addition, it should be noted that the selection of a standard size males, likely to result in deficiencies of control programs due to low competitiveness with large males in field.

 

ACKNOWLEDGEMENTS

To Dr. Alfredo Castillo Vera for providing equipment and useful guidance in pupae measurements and to MsC Gloria Elsa Antonio Arreola for her valuable discussion to improve this manuscript.

 

LITERATURE CITED

Alphey L., Benedict, M., Bellini, R., Clark, G. G., Dame, D. A., Service, M. W. & Dobson, S. L. 2010. Sterile-insect methods for control of mosquito-borne diseases: an analysis. Vector Borne Zoonotic Diseases, 10: 295-311.         [ Links ]

Bellini, R., Calvitti, M., Medici, A., Carrieri, M., Celli, G. & Maini, S. 2007. Use of the Sterile Insect Technique against Aedes albopictus in Italy: First Results of a Pilot Trial, pp. 505-515. In: M. J. B. Vreysen, A. S. Robinson & J. Hendrichs (Eds.). Area-Wide Control of Insect Pests. Springer, Netherlands.         [ Links ]

Blackmore, M. S. & Lord, C. C. 2000. The relationship between size and fecundity in Aedes albopictus. Journal of Vector Ecology, 25: 212-217.         [ Links ]

Brazil B. G. & Brazil, R. P. 2000. Sexing sand fly pupae (Diptera: Psychodidae: Phlebotominae). Memorias do Instituto Oswaldo Cruz, 95: 471-472.         [ Links ]

Dame D. A., Lofgren, C. S., Ford, H. R., Boston, M. D., Baldwin, K. F. & Jeffery, G. M. 1974. Release of chemosterilized males for the control of Anopheles albimanus in El Salvador II. Methods of rearing, sterilization, and distribution. The American Journal of Tropical Medicine and Hygiene, 23: 282-287.         [ Links ]

Gerberg E. J., Hopkins, T. M. & Gentry, J. W. 1969. Mass rearing of Culex pipiens L. Mosquito News, 29: 382-385.         [ Links ]

Koenraadt, C. J. M. 2008. Pupal Dimensions as Predictors of Adult Size in Fitness Studies of Aedes aegypti (Diptera: Culicidae). Journal of Medical Entomology, 45: 331-336.         [ Links ]

McCray Jr., E. M. 1961. A mechanical device for the rapid sexing of Aedes aegypti pupae. Journal of Economic Entomology, 54: 819.         [ Links ]

Medici A., Carrieri, M., Scholte, E., Maccagnani, B., Dindo, M. L. & Bellini, R. 2011. Studies on Aedes albopictus Larval Mass-Rearing Optimization. Journal of Economic Entomology, 104: 266-273.         [ Links ]

Papathanos P. A., Bossin, H. C., Benedict, M. Q., Catteruccia, F., Malcolm, C. A., Alphey, L. & Crisanti, A. 2009. Sex separation strategies: past experience and new approaches. Malaria Journal, 8: S5.         [ Links ]

Sawadogo S. P., Diabaté, A., Toé, H. K., Sanon, A., Lefevre, T., Baldet, T., Gilles, J., Simard, F., Gibson, G., Sinkins, S. & Da biré, R. K. 2013. Effects of Age and Size on Anopheles gambiae s.s. Male Mosquito Mating Success. Journal of Medical Entomology, 50: 285-293.         [ Links ]

Sharma V. P., Patterson, R. S. & Ford, H. R. 1972. A device for the rapid separation of male and female mosquito pupae. Bulletin of the World Health Organization, 47: 429-432.         [ Links ]