**3.2 Choriogenesis as an emerging target for safe interventions**

The final checkpoint of oogenesis, before fertilization, is the triggering of the choriogenesis program, in which the multiple layers of the chorion are secreted by the follicle cells that envelop the developing oocytes. Remarkably, while the chorion's primary protective function is conserved in insects, its general composition and structure have evolved in a highly species-specific manner, giving rise to a wide range of morphologies and functional adaptations. The main chorion proteins in insects have been identified in models, such as the silk moth *Bombyx mori*, the fruit fly *D. melanogaster,* and the mosquito *Aedes Aegypti*, and revealed to be broadly unrelated to their counterparts in each of these species [41, 42]. Proteins that are conserved in a wide variety of organisms are not ideal target molecules as vector control agents because of deleterious effects on non-target organisms, such as vertebrates, pollinating agricultural insects, and beneficial predators. As a result, studies on the molecular biology of the chorion biogenesis in insect vectors are biotechnology strategic as they are likely to unravel safe molecular targets that are at the same time essential for reproduction and highly specific to one species.

The *A. aegypti* eggshell is composed of different structural proteins, enzymes, odorant-binding proteins, as well as many uncharacterized proteins of unknown

*Vector Control: Insights Arising from the Post-Genomics Findings on Insects' Reproductive Biology DOI: http://dx.doi.org/10.5772/intechopen.106273*

function. Melanization proteins and enzymes of the eggshell have been identified [43–49], and proteomics studies have been performed [42]. Isoe and colleagues [50] designed *in silico* analyses to identify mosquito-specific genes that are essential for successful embryo development. After systematic RNAi functional screening of over 40 selected genes, the authors identified a chorion-related protein named eggshell organizing factor 1 (EOF1), which is essential for eggshell biogenesis and embryo development. The EOF1 sequence includes an F-box functional motif, which is characterized by the interaction with the SKP1 protein in the SCF E3 ubiquitin ligase complex [51]. Although its exact function in the eggshell has not been elucidated, such findings are very promising in terms of designing safe strategies for vector control.

In *R. prolixus*, some aspects of the chorion ultrastructure and permeability properties were previously explored [52–54] and the identification of the specific chorion proteins Rp30 and Rp45, the latter associated with antifungal activity, was also described [55]. In this model, the cell biology of the follicle cells (FCs), the tissue that synthesizes and secretes the chorion components, has been explored. Early transcriptome analysis showed that the FCs are committed to transcription, translation, and vesicular traffic [56]. Accordingly, electron microscopy evidenced the FC's typical secretory cell morphology with a high content of vesiculated rough endoplasmic reticulum [57, 58]. Systemic RNAi experiments targeting the autophagy-related genes ULK1/ATG1, the autophagy-dedicated E2-conjugating enzyme ATG3 [57, 59], and E1-activating and E2-conjugating ubiquitin enzymes [60] generated particular phenotypes of chorion malformations due to alterations in the general protein homeostasis of the FCs during choriogenesis, resulting in extremely lower rates of embryo viability. Taken together, the data points to a high degree of complexity in the chorion biogenesis program in *R. prolixus*, rendering the process extremely sensitive to changes in proteostasis of the FCs, and, thus, an interesting target for slight but effective interventions.

Resistance to desiccation is another potential intervention target. Although mosquito eggs are laid in water, they are susceptible to dehydration in the first hours of development. Thus, this property directly affects mosquito reproduction. In *A. Aegypti*, the serosal cuticle secretion (an inner layer of the chorion secreted during embryogenesis) coincides with an increase in dry resistance and the presence of chitin as one of the serosal cuticle components has been detected [61–63]. In *R. prolixus*, chitin was detected in the ovaries and the embryonic cuticle [64, 65]. Additionally, exposure to lufenuron (a chitin synthesis inhibitor) and chitin synthase RNAi experiments reduced oviposition and embryo viability [66]. Therefore, the synthesis and deposition of chitin or chitin-like components in the eggshells are also promising targets for reproduction interventions.

Altogether, and combined with the above-mentioned high degree of speciesspecificity of the chorion proteins, choriogenesis has the potential to emerge as the foremost target for the generation of new and environmentally safe strategies to achieve vector control.
