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Transglutaminases are pleiotropic enzymes that in mosquitoes participate in the formation of the mating plug and the wound-induced antimalarial response. Moreover, one transglutaminase, TGase3, negatively regulates the infection-induced aggregation of hemocytes on the heart. Given that TGase3 is an inhibitor of periostial hemocyte aggregation, we used RNAi-based gene silencing followed by intravital video imaging to scrutinize whether any of the three transglutaminases encoded in the genome of the mosquito, Anopheles gambiae, play a role in modulating the heart rate of uninfected and infected mosquitoes. Initially, we confirmed that an infection decreases the heart rate. Then, we uncovered that silencing TGase1 does not impact heart physiology, but silencing TGase2 results in a constant heart rate regardless of infection status, eliminating the infection-induced decrease in the heart rate. Finally, silencing TGase3 decreases the heart rate in uninfected mosquitoes but increases the heart rate in infected mosquitoes. We conclude that TGase2 and TGase3 modulate heart physiology and demonstrate that factors not classically associated with insect circulatory physiology are involved in the functional integration of the immune and circulatory systems of mosquitoes. 

Abstract The immune and circulatory systems of insects are functionally integrated. Following infection, immune cells called hemocytes aggregate around the ostia (valves) of the heart. An earlier RNA sequencing project in the African malaria mosquito,Anopheles gambiae, revealed that the heart-associated hemocytes, called periostial hemocytes, express transglutaminases more highly than hemocytes elsewhere in the body. Here, we further queried the expression of these transglutaminase genes and examined whether they play a role in heart-associated immune responses. We found that, in the whole body, injury upregulates the expression ofTGase2, whereas infection upregulatesTGase1,TGase2andTGase3. RNAi-based knockdown ofTGase1andTGase2did not alter periostial hemocyte aggregation, but knockdown ofTGase3increased the number of periostial hemocytes during the early stages of infection and the sequestration of melanin by periostial hemocytes during the later stages of infection. In uninfected mosquitoes, knockdown ofTGase3also slightly reduced the number of sessile hemocytes outside of the periostial regions. Taken altogether, these data show thatTGase3negatively regulates periostial hemocyte aggregation, and we hypothesize that this occurs by negatively regulating the immune deficiency pathway and by altering hemocyte adhesion. In conclusion,TGase3is involved in the functional integration between the immune and circulatory systems of mosquitoes.