Trypsin and chymotrypsin-like enzymes were detected in the gut of Aedes aegypti in the four larval instar and pupal developmental stages. Although overall the amount of trypsin synthesized in the larval gut was 2-fold higher than chymotrypsin, both enzymes are important in food digestion. Feeding Aea-Trypsin Modulating Oostatic Factor (TMOF) to Ae. aegypti and Culex quinquefasciatus larvae inhibited trypsin biosynthesis in the larval gut, stunted larval growth and development, and caused mortality. Aea-TMOF induced mortality in Ae. aegypti, Cx. quinquefasciatus, Culex nigripalpus, Anopheles quadrimaculatus, and Aedes taeniorhynchus larvae, indicating that many mosquito species have a TMOF-like hormone. The differences in potency of TMOF on different mosquito species suggest that analogues in other species are similar but may differ in amino acid sequence or are transported differently through the gut. Feeding of 29 different Aea-TMOF analogues to mosquito larvae indicated that full biological activity of the hormone is achieved with the tetrapeptide YDPA. Using cytoimmunochemical analysis, intrinsic TMOF was localized to ganglia of the central nervous system in larvae and male and female Ae. aegypti adults. The subesophageal, thoracic, and abdominal ganglia of both larval and adult mosquitoes contained immunoreactive cells. Immunoreactive cells were absent in the corpus cardiacum of newly molted 4th instar larvae but were found in late 4th instar larvae. In both males and females, the intrinsic neurosecretory cells of the corpus cardiacum were filled with densely stained immunoreactive material. These results indicate that TMOF-immunoreactive material is synthesized in sugar-fed male and female adults and larvae by the central nervous system cells.

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Adult cat fleas were exposed to residues of pyriproxyfen and methoprene in glass vials, then fed on a cat 24 h later to investigate the mode of action of juvenoid growth regulators on embryonic development in flea eggs. Eggs laid by pyriproxyfen-treated fleas within 70 h after exposure to this juvenoid were often devoid of yolk and frequently collapsed after oviposition. Minimal amounts of yolk were deposited in eggs laid after 70 h, and no blastoderm was formed. These results are significant because both modes of action were different than those observed earlier by investigators studying ovicidal effects in adult insects treated with juvenile hormone. In contrast to the pyriproxyfen results, eggs laid by methoprene-treated fleas showed no gross morphological effects, and these eggs remained turgid during embryogenesis. However, the eggs either did not hatch or the larvae died within hours after hatching. Histological examination of the eggs revealed that most of the eggs contained segmented embryos which had apparently died during blastokinesis. Although eggs of some insects exposed to juvenile hormone during oogenesis fail to undergo germ band formation, there was no evidence of this effect in methoprene-treated cat fleas.

A second mode of action has been found for the inhibition of chitin synthesis by diflubenzuron. This compound blocks synthesis of the imaginal cuticle by preventing formation of the adult epidermis in the pupal stage of the stable fly (Stomoxys calcitrans L.).

When cat fleas, Ctenocephalides felis (Bouché), were fed concentrations of lufenuron in cattle blood ranging from 0.5 to 4 ppm, adult mortality increased in a dose-dependent manner to a maximum of approximately 24% over a period of 10 d. Fleas treated with 0.5 ppm produced abnormal endocuticle consisting of protein globules embedded in an amorphous chitin matrix. At concentrations of 1.0 ppm or greater, endocuticle formation was inhibited. Ultrastructural studies demonstrated that inhibition of chitin synthesis was associated with degeneration of the epidermal cells. The amount of epidermal cytoplasm decreased and cytoplasmic organelles including mitochondria, ribosomes, and golgi showed lytic changes. At least some mortality of treated fleas was likely the result of a weakened endocuticle and the corresponding decrease in resiliency of the cuticle to expansion during blood-feeding and egg production. An unexpected result of lufenuron treatment was the inhibition of midgut epithelial cell differentiation. At concentrations of 0.5 and 1.0 ppm, partially differentiated epithelial cells were seen in the midgut of bloodfed fleas along with fully differentiated cells.