Within the genus Aedes, the reproductive biology of two species has been most thoroughly studied: Aedes aegypti and Aedes albopictus. In these species, females tend to copulate with one or more males once sexually mature. Within a few hours after an initial insemination, most females become refractory to insemination for the rest of their lives. Aedes females store sperm in three sclerotized spherical structures called spermathecae, where they can remain viable for >3 mo after copulation. This protocol outlines a quick (∼3–5 min per female once you are practiced in dissection) and effective method for dissecting the female spermathecae from Ae. aegypti and Ae. albopictus mosquitoes and scoring them for the presence or absence of sperm in cases in which the researcher does not need the female for further studies.

Aedes mosquitoes are the vectors of several arboviruses that cause human disease. A better understanding of their reproduction helps to improve their management and contributes insights into the fundamental biology of mosquitoes. During mating, inseminated mosquito females receive seminal fluids and sperm from males that they then store in the spermathecae. In Aedes aegypti and Aedes albopictus, most mated females become resistant to further insemination within 2 h of initial insemination. Although the male seminal fluids are known to be involved in initiating the resistance of inseminated females to further insemination, the mechanism underlying this resistance is not well-understood. The determination of insemination status is a key step in investigating the behavioral and molecular interactions between males and females and for exploring the proximate influences and evolutionary implications of interspecific copulations. Several methods exist for determining insemination status, as discussed here. The choice of method depends on the research question and the availability of resources.

Acute injury to the adult zebrafish retina triggers the release of pro-inflammatory cytokines and growth factors that stimulate multiple gene regulatory networks, which ultimately stimulate Müller glia to proliferate and regenerate neurons. In contrast, zebrafish carrying mutations in cep290 or bbs2 undergo progressive loss of cone photoreceptors and exhibit signs of microglia activation and inflammation, but the mutants fail to stimulate a regeneration response. To identify transcriptional changes that occur in zebrafish mutants undergoing progressive photoreceptor degeneration, RNA-seq transcriptional profiling was performed on cep290−/− and bbs2−/− retinas. The PANTHER Classification System was used to identify biological processes and signaling pathways that were differentially expressed between mutants and wild-type siblings during degeneration. As expected, genes associated with phototransduction were downregulated in cep290 and bbs2 mutants compared to wild-type siblings. Although both cep290 and bbs2 mutants undergo proliferation of rod precursors in response to retinal degeneration, the process of negatively regulating proliferation is enriched for upregulated genes, and this negative regulation may restrict proliferation of Müller glia and inhibit regeneration. A total of 815 differentially expressed genes (DEGs) were shared by cep290 and bbs2 retinas. Genes in pathways associated with inflammation, apoptosis, stress response, and PDGF signaling were overrepresented. Identifying the genes and biological pathways that are common in zebrafish models of inherited retinal degeneration provides a foundation for future studies on the mechanisms that regulate cell death as well as processes that prohibit Müller cell reprogramming or proliferation in a model capable of retinal regeneration. The pathways will provide targets for future interventions that may promote successful regeneration of lost photoreceptors.