# Claustrophilic oviposition: Oviposition performance depends on container size in a novel forced oviposition method for Culex quinquefasciatus and Aedes aegypti

## TL;DR

A new method for forcing mosquitoes to lay eggs in small containers is effective for two species, improving lab research and colony establishment.

## Contribution

A modified forced oviposition method is introduced and tested for Aedes aegypti and Culex quinquefasciatus, showing species-specific efficacy and claustrophilic oviposition behavior.

## Key findings

- Aedes aegypti showed higher oviposition success (>80%) compared to Culex quinquefasciatus (50–60%).
- Both species preferred smaller oviposition tubes (1.5 and 5 mL) for laying eggs.
- The method successfully produced F1 progeny from both lab and wild-caught mosquitoes.

## Abstract

Mosquito vector control research relies heavily on the use of live organisms, with wild populations playing a critical role in surveillance, field product validation, and strengthening the overall efficacy and accuracy of studies. However, too often wild mosquito rearing and colony establishment in laboratory settings is consistently challenging. Here we present a modified version of a forced oviposition method for use against two different mosquito species, Aedes aegypti and Culex quinquefasciatus. The efficacy of the technique was tested with both laboratory and wild-caught strains placed in oviposition tubes consisting of a 1.5 mL tube containing moistened cotton and a strip of germination paper. To determine if size had an impact on oviposition rate, an additional test was conducted using four different size oviposition tubes; 1.5, 5, 15, and 50 mL. Overall, the forced oviposition method was highly effective, successfully generating eggs from lab reared and field collected strains. The method was more effective with Ae. aegypti (>80% females oviposited) compared to Cx. quinquefasciatus (50–60%) in both strains. Culex quinquefasciatus holding time was longer (3–7 days) compared to Ae. aegypti females, which oviposited within 24 hrs of being transferred into tubes. Intact versus broken egg rafts affected egg hatch rate in laboratory Cx. quinquefasciatus strains but had less impact on the wild strain. Additionally, both Ae. aegypti and Cx. quinquefasciatus displayed a claustrophilic oviposition behavior with a higher percentage of females ovipositing in the 1.5 and 5 mL tubes. This study demonstrates that the forced oviposition method can be easily adapted to other mosquito vector species and effective in producing F1 progeny needed for critical vector research.

Mosquito vector control programs rely heavily on data and information generated from research and phenotypic insecticide resistance monitoring, which require the use of live mosquitoes, both from laboratory colonies and field caught populations. Here we tested a modified version of a force oviposition technique originally designed for establishing wild Anopheles spp. colonies, on two key mosquito vectors, Culex quinqufasciatus and Aedes aegypti. We find that, despite their distinct oviposition preferences and behaviors, both species of mosquitoes generated adequate numbers of F1 eggs needed to start either laboratory or wild colonies. A key advantage to this method is its ease of use, only requiring minimal numbers of female mosquitoes and inexpensive, easily accessible materials commonly found in most laboratories and mosquito control districts. Further application of the forced oviposition method has important implications for enhancing our capacity to conduct a range of vector research with various key mosquito species. This could include key vector research projects such as, determining single female sex ratios, producing genetic isolines by controlling specific genetic backgrounds, and conducting heritability studies focused on selective mating.