Getting wild-caught mosquitoes to breed and lay eggs in a lab is often a challenge. A team of researchers at the University of Florida has developed a simple method using small centrifuge tubes to get two important mosquito species to readily lay eggs, providing an easier, cheaper way to establish wild mosquito populations for disease-control studies. (Photo courtesy of Kendra A. Dagg and Edwin R. Burgess IV, Ph.D.)

By Kendra A. Dagg and Edwin R. Burgess IV, Ph.D.

Edwin R. Burgess IV, Ph.D.

Kendra A. Dagg

Mosquito vector research, whether it’s conducting insecticide resistance assays, determining disease vector competency, or product testing, all rely on one key resource: access to large numbers of live mosquitoes. On the whole, there is little worry about when it comes to egg production and numbers of lab-based colonies. This is not the case for wild mosquito populations. Quite often, the most common barriers of wild colony establishment are an insufficient number of field-collected females and poor egg laying success from field blood-fed or gravid females.

In an article published in July 2025 in PLOS Neglected Tropical Diseases, our team at the University of Florida adapted an oviposition encouragement technique for two key mosquito species, Aedes aegypti and Culex quinquefasciatus. Both species are significant vectors to several high-profile human diseases (e.g. West Nile virus, dengue virus, and Zika virus) but more importantly have distinct oviposition preferences and behaviors. Ae. aegypti are “container breeders” with females laying individual eggs on the inner walls of various containers just above the surface level of fresh water. On the other hand, Cx. quinquefasciatus lay their eggs in rafts on the surface of water and prefer water with high organic content.

Oviposition Chamber Setup

Methods for establishing wild Anopheles malaria vector colonies have been developed and tested in multiple countries, but there is a surprising lack of guidance on other mosquito species. The goal of our study was to create an oviposition encouragement protocol that was adaptable, cheap, and easy to do with readily available lab consumables (e.g., centrifuge tubes, filter papers, etc.). Sticking to those criteria, the team developed oviposition chambers with four simple items: cotton, germination/filter paper, a 1.5 milliliter (mL) centrifuge tube, and tap water. A piece of cotton, moistened with tap water, was pushed to the bottom of the tube, a small strip of germination paper was placed over the cotton, and finally a small hole was punched through the top of the tube lid to provide air exchange.

Getting wild-caught mosquitoes to breed and lay eggs in a lab is often a challenge. A team of researchers at the University of Florida has developed a simple method to get two important mosquito species to readily lay eggs. The team developed oviposition chambers with four simple items: cotton, germination/filter paper, a 1.5 milliliter (mL) centrifuge tube, and tap water. A piece of cotton, moistened with tap water, was pushed to the bottom of the tube, a small strip of germination paper was placed over the cotton, and finally a small hole was punched through the top of the tube lid to provide air exchange. (Figure originally published in Dagg et al. 2025, PLOS Neglected Tropical Diseases)

Gravid females were individually transferred into a prepared oviposition tube and left in standard mosquito insectary conditions (27 degrees Celsius, 60% relative humidity, and a light/dark cycle of 12 hours each). The only key difference in preparation between the two species was Cx. quinquefasciatus females preferred a small layer of water remaining in the tube while Ae. aegypti needed all excess water removed.

Female Ae. aegypti took more readily to the technique compared to Cx. quinquefasciatus females, with more than 80% of Ae. aegypti laying eggs within 24 hours, regardless of whether they were from the lab or field. Meanwhile, over 50% of lab or field-caught Cx. quinquefasciatus laid viable egg rafts within 3-7 days.

Does Tube Size Matter?

Interestingly, despite differences in oviposition preference, the team was able to collect large numbers of viable eggs from laboratory and field-caught mosquitoes of both species. But why? One hypothesis is that the success of the method was related to the confined space of the tube, suggesting these mosquitoes had an affinity for tight spaces (i.e., claustrophilia) when laying.

To address this, we tested four different tube sizes (1.5, 5, 15, and 50 mL) and found that in fact, oviposition success did reduce as tube size increased. Both species appeared to display a claustrophilic-like behavior in egg laying—in other words, they had a preference for and higher frequency of egg laying in smaller tubes, with more females laying in 1.5 and 5 mL tubes for Cx. quinquefasciatus and 1.5 mL tubes for Ae. aegypti. Additionally, Ae. aegypti egg counts dropped as tube sized increased. (This was not observed in Cx. quinquefasciatus.)

Mosquito Control Application

As with any research, several limitations and unanswered questions need to be addressed through more rigorous and standardized testing. However, the development of this oviposition encouragement method opens the door to expanding our ability to conduct a range of vector research with multiple key mosquito species.

One of its greatest advantages is ease of use: It requires only a minimal number of females and relies on inexpensive, readily available materials common to most laboratories and mosquito control districts. Additionally, the method proved highly effective in producing large numbers of viable eggs from both long-established lab colonies and newly caught field populations.

Against the ongoing challenge of rearing mosquitoes, this easy-to-use approach proves that sometimes the simplest solutions can have the biggest success.

Kendra A. Dagg is a Ph.D. student in the Entomology and Nematology Department at the University of Florida in Gainesville, Florida, and a medical entomologist with the U.S. Army Medical Department. Email: [email protected] or[email protected]. Edwin R. Burgess IV, Ph.D., is an assistant professor in the Entomology and Nematology Department at the University of Florida in Gainesville, Florida. Email: [email protected].