Functional integration of the circulatory, immune, and respiratory systems in mosquito larvae: pathogen killing in the hemocyte-rich tracheal tufts

Background: As both larvae and adults, mosquitoes encounter a barrage of immune insults, ranging from microberich communities in larval habitats to ingested blood-borne pathogens in adult blood meals. Given that mosquito adults have evolved an efficient means of eliminating infections in their hemocoel (body cavity) via the coordinated action of their immune and circulatory systems, the goal of the present study was to determine whether such functional integration is also present in larvae. Results: By fluorescently labeling hemocytes (immune cells), pericardial cells, and the heart, we discovered that fourth instar larvae, unlike adults, contain segmental hemocytes but lack the periostial hemocytes that surround the ostia (heart valves) in abdominal segments 2-7. Instead, larvae contain an abundance of sessile hemocytes at the tracheal tufts, which are respiratory structures that are unique to larvae, are located in the posterior-most abdominal segment, and surround what in larvae are the sole incurrent openings for hemolymph entry into the heart. Injection of fluorescent immune elicitors and bacteria into the larval hemocoel then showed that tracheal tuft hemocytes mount rapid and robust immune responses against foreign insults. Indeed, green fluorescent protein-labeled Escherichia coli flowing with the hemolymph rapidly aggregate exclusively at the tracheal tufts, where they are killed within 24 h post-infection via both phagocytosis and melanization. Conclusion: Together, these findings show that the functional integration of the circulatory, respiratory, and immune systems of mosquitoes varies drastically across life stages.