Insect Pupae sexUAl Size separatOr
VSI IR-604 Pupae sex separator. A successful application of the sterile insect technique and other strategies designed to eliminate large populations of insects relies on the efficient releases of competitive, sterile males into the natural habitat of the target species. As released sterile females do not contribute to the sterility of population in situ, systems for the separation of male from female individuals are needed. This is especially key for vector-transmitting species like mosquitoes, in which only females bite and transmit diseases.
While several genetic and transgenic approaches have been developed that permit male-female separation for some species, separation based on sexual size dimorphism continue to be a useful technique in the laboratory and in small to medium mass rearing settings for elimination of females. In general, female mosquitoes of many species such as Aedes and Culex but also Anopheles quadrimaculatus and A. albimanus are larger than males in the pupae stage.
Application of the IR-604 device to separate other insect species and development stages (e.g. pupae, larvae) is feasible if the individuals can be i) in an aqueous solution, and ii) hold an explicit size-dimorphism. See below for more information on size dimorphisms in insects and potential rearing effects on its extend.
VSI IR-604 Insect Sex Separator - Specifications
The size-based pupae sex separator system IR-604 is a versatile tool for entomologists working in mass rearing or research facilities. Our model is based on the classical larval-pupal separator (narrowing glass plates, Fay-Morlan glass plate separator) but with a more convenient and efficient entering and washing/collection mechanism and three levels of separation - allowing in particular the more accurate fractionation of populations holding some overlap between the sizes of sexes (e.g. small females and large males). See pdf document below for details/specifications of VSI IR-604 Pupae Sex Separator and a brief manual.
Size Dimorphism in Insects
Size dimorphism in insect larvae and pupae refers to the presence of size differences between males and females of a species. In many insect species, such as A. aegypti (see example Image), females are larger than males, a phenomenon known as "sexual size dimorphism". Here are some key points about size dimorphism in insects:
- Causes: The causes of size dimorphism in insect larvae and pupae are not allways fully understood, but are likely to be related to differences in mating behaviour, reproductive strategy and ecological factors. For example, larger females may have a competitive advantage in mating or may be better equipped to produce larger eggs.
- Developmental differences: Size dimorphism in insect larvae and pupae may be the result of developmental differences between males and females. For example, females may have a longer larval or pupal stage than males, allowing them to grow larger.
- Morphological differences: In some insect species, females have different morphological characteristics than males, which can lead to size dimorphism.
It is important to note that rearing conditions can influence the extent of size dimorphism in insect pupae. Nutrition is one key factor that can influence size dimorphism in insects - rendering a standardized feeding process imperative. Inadequate nutrition can lead to smaller body size, different feed application per tray will likely lead increased size heterogeneity and thus complicate sorting. Other environmental factors that can influence size dimorphism in insect pupae include temperature, humidity and/or photoperiod. For example, low temperatures can slow insect development, while high temperatures can accelerate development. In addition, social factors such as crowding can also influence size dimorphism in insect pupae. Crowding can lead to increased competition for resources, resulting in smaller or more heterogenic body size.
In summary, rearing conditions, including diet, temperature, humidity, photoperiod and social factors, can all influence the extent of size dimorphism in insect pupae. To utilise size dimorphisms to separate insect larvae or pupae by a size separator as the IR-604 (or other methods, as exemplified below), it is thus key to use constant and optimized (nutrition, temperature etc) rearing conditions, e.g. in standardized tray and rack systems, leading to most homogeniously sized male and female larvae or pupae.
Methods to Separate Insect Pupae by Size
There are several methods that can be used to separate insect pupae by size. Here some of the more common methods are given:
- Sieving: Sieving is a simple and effective method of separating pupae by size. Pupae are poured into a sieve with a mesh size appropriate to the size range desired and the sieve is shaken or agitated to separate the larger pupae from the smaller ones. The separated pupae can then be collected and sorted as required. However, the mechanical stress on the pupae by sieving is high, resulting in frequent damage to the pupae.
- Narrow plates: The narrow plate method, also known as Fay–Morlan glass plate separator, as used in the VSI IR-604 sex separator (see above), is another technique that can be used to separate insect pupae by size. Here a narrow plate of plastic, glass or metal forms a (an increasingly narrow) slit or slits of a specified width against another part. The width of the slit/slits can be chosen according to the desired size range of the pupae to be sorted. The mechanical stress on the pupae by narrow plate techniques is generally low, resulting in few damaged pupae.
- Density separation: This method is separating pupae according to their density. Pupae are placed in a solution of a given density and the solution is then centrifuged or allowed to settle; partially iced water has been used to create density differences in a container. The larger and heavier pupae settle to the bottom, while the smaller and lighter pupae remain suspended in the solution. The separated pupae can then be collected and sorted. There is generally no mechanical stress on the pupae by the technique (if not centrifuged).
- Hand sorting: Hand sorting is a labour intensive but effective method of separating pupae by size. Pupae are visually inspected and sorted by hand into different size categories. This method is often used for small-scale rearing or research projects. Hand sorting is also necessary to select sieve sizes and slit widths prior to, and to check sorting accuracies after, using any of the above methods.
Overall, the choice of method for sizing insect pupae depends on the insect species, in particular size range between large and small fractions, the resources and equipment available, and the amount of pupae to sort. The effectiveness and efficiency of each method should be evaluated and compared to determine the optimal approach for the specific application. Vienna Scientific is willing to contribute to the design of automated, effective sorting systems, please get in contact.
Selected Readings on Mechanical Separation and Size Dimorphism of Insects
- Carvalho, D. O., D. Nimmo, N. Naish, A. R. McKemey, P. Gray, A. Wilke, B. B, M. T. Marrelli, J. F. Virginio, L. Alphey, and M. L. Capurro. 2014. Mass Production of Genetically Modified Aedes aegypti for Field Releases in Brazil. jove e3579.
- Gilles, J. R. L., M. F. Schetelig, F. Scolari, F. Marec, M. L. Capurro, G. Franz, and K. Bourtzis. 2014. Towards mosquito sterile insect technique programmes: Exploring genetic, molecular, mechanical and behavioural methods of sex separation in mosquitoes. Acta Tropica 132, Supplement:S178-S187
- Fay, R., and H. Morlan. 1959. A mechanical device for separating the developmental stages, sexes and species of mosquitoes. Mosquito News 19:144-147.
- Focks, D. A. 1980. An improved separator for the developmental stages, sexes, and species of mosquitoes (Diptera: Culicidae). Journal of Medical Entomology 17:567-568.
- Malfacini, M., Puggioli, A., Balestrino, F., Carrieri, M., Dindo, M. L., & Bellini, R. 2022. Aedes albopictus Sterile Male Production: Influence of Strains, Larval Diet and Mechanical Sexing Tools. Insects, 13(10), 899.
- Mamai, W., Maiga, H., Somda, N. S. B., Wallner, T., Konczal, A., Yamada, H., & Bouyer, J. 2020. Aedes aegypti larval development and pupal production in the FAO/IAEA mass-rearing rack and factors influencing sex sorting efficiency. Parasite, 27.
- Mashatola, T., Ndo, C., Koekemoer, L. L., Dandalo, L. C., Wood, O. R., Malakoane, L., ... & Munhenga, G. 2018. A review on the progress of sex-separation techniques for sterile insect technique applications against Anopheles arabiensis. Parasites & vectors, 11, 127-137.
- Mertins, J. W., and H. C. Coppel. 1972. Seed Dockage Sieves for Sex-Separation of Pine Sawfly Cocoons. Annals of the Entomological Society of America 65:1424-1425.
- Papathanos, P. A., H. C. Bossin, M. Q. Benedict, F. Catteruccia, C. A. Malcolm, L. Alphey, and A. Crisanti. 2009. Sex separation strategies: past experience and new approaches. Malaria journal 8:1.
- Ramakrishnan, S. P., Krishnamurthy, B. S., & Singh, N. N. 1963. A simple technique for rapid separation of mosquito pupae by sudden chilling. Indian Journal of Malariology, 17(2/3), 119-21.
- 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(3), 429.
- Wormington, J. D., and S. A. Juliano. 2014. Sexually dimorphic body size and development time plasticity in Aedes mosquitoes (Diptera: Culicidae). Evolutionary Ecology Research 16:223.
- Zhang, D., Zhang, M., Wu, Y., Gilles, J. R., Yamada, H., Wu, Z., ... & Zheng, X. 2017. Establishment of a medium-scale mosquito facility: optimization of the larval mass-rearing unit for Aedes albopictus (Diptera: Culicidae). Parasites & Vectors, 10(1), 1-9.