BackgroundThe immune system of adult mosquitoes has received significant attention because of the ability of females to vector disease-causing pathogens while ingesting blood meals. However, few studies have focused on the immune system of larvae, which, we hypothesize, is highly robust due to the high density and diversity of microorganisms that larvae encounter in their aquatic environments and the strong selection pressures at work in the larval stage to ensure survival to reproductive maturity. Here, we surveyed a broad range of cellular and humoral immune parameters in larvae of the malaria mosquito, Anopheles gambiae, and compared their potency to that of newly-emerged adults and older adults.ResultsWe found that larvae kill bacteria in their hemocoel with equal or greater efficiency compared to newly-emerged adults, and that antibacterial ability declines further with adult age, indicative of senescence. This phenotype correlates with more circulating hemocytes and a differing spatial arrangement of sessile hemocytes in larvae relative to adults, as well as with the individual hemocytes of adults carrying a greater phagocytic burden. The hemolymph of larvae also possesses markedly stronger antibacterial lytic and melanization activity than the hemolymph of adults. Finally, infection induces a stronger transcriptional upregulation of immunity genes in larvae than in adults, including differences in the immunity genes that are regulated.ConclusionsThese results demonstrate that immunity is strongest in larvae and declines after metamorphosis and with adult age, and suggest that adaptive decoupling, or the independent evolution of larval and adult traits made possible by metamorphosis, has occurred in the mosquito lineage.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-017-2302-6) contains supplementary material, which is available to authorized users.

Hemolymph circulation in insects is driven primarily by the contractile action of a dorsal vessel, which is divided into an abdominal heart and a thoracic aorta. As holometabolous insects, mosquitoes undergo striking morphological and physiological changes during metamorphosis. This study presents a comprehensive structural and functional analysis of the larval and adult dorsal vessel in the malaria mosquito Anopheles gambiae. Using intravital video imaging we show that, unlike the adult heart, the larval heart contracts exclusively in the anterograde direction and does not undergo heartbeat directional reversals. The larval heart contracts 24% slower than the adult heart, and hemolymph travels across the larval dorsal vessel at a velocity that is 68% slower than what is seen in adults. By fluorescently labeling muscle tissue we show that although the general structure of the heart and its ostia are similar across life stages, the heart-associated alary muscles are significantly less robust in larvae. Furthermore, unlike the adult ostia, which are the entry points for hemolymph into the heart, the larval ostia are almost entirely lacking in incurrent function. Instead, hemolymph enters the larval heart through incurrent openings located at the posterior terminus of the heart. These posterior openings are structurally similar across life stages, but in adults have an opposite, excurrent function. Finally, the larval aorta and heart differ significantly in the arrangement of their cardiomyocytes. In summary, this study provides an in-depth developmental comparison of the circulatory system of larval and adult mosquitoes.

BackgroundAs both larvae and adults, mosquitoes encounter a barrage of immune insults, ranging from microbe-rich 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.ResultsBy 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.ConclusionTogether, these findings show that the functional integration of the circulatory, respiratory, and immune systems of mosquitoes varies drastically across life stages.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0305-y) contains supplementary material, which is available to authorized users.

Summary Aedes aegypti mosquitoes transmit pathogens such as yellow fever, dengue, Zika, and chikungunya viruses to millions of human hosts annually [1]. As such, understanding Ae. aegypti courtship and mating biology could prove crucial to the success of pest and disease control efforts that target reproduction. Potentially to communicate reproductive fitness [2,3], mosquito males and females harmonize their flight tones prior to mating in a behavior known as harmonic convergence (HC) [4]. Furthermore, after mating or treatment with male accessory gland (MAG) extracts, female Ae. aegypti become resistant, or refractory, to re-mating [5]. To test the hypothesis that mating and MAG fluids inhibit a female’s ability to induce HC in males, we recorded audio of pre-copulatory flight interactions between virgin males and either virgin, mated, or MAG extract-injected females and analyzed these recordings for the presence or absence of HC. We further assayed HC with heat-treated MAG- and bovine serum albumin (BSA)-injected females to test if potential MAG effects are due specifically to heat-labile molecules. We found that mating and MAG extract, but not heat-treated MAG or BSA, lower HC occurrence by 53% compared to all other controls. Because MAG-injected females formed only nonproductive pseudocopulas and HC was most often accomplished by male tone modulations, these results suggest that mating may inhibit HC indirectly via the broader range of MAG-induced female refractory mating behaviors. Together, our results demonstrate an important new role for MAG molecules in mediating female post-mating behavior.

BackgroundMutations in spastin are the most common cause of hereditary spastin paraplegia, a neurodegenerative disease. In this study, the role of spastin was examined in Drosophila photoreceptor development.Methodology/Principal FindingsThe spastin mutation in developing pupal eyes causes a mild mislocalization of the apical membrane domain at the distal section, but the apical domain was dramatically reduced at the proximal section of the developing pupal eye. Since the rhabdomeres in developing pupal eyes grow from distal to proximal, this phenotype strongly suggests that spastin is required for apical domain maintenance during rhabdomere elongation. This role of spastin in apical domain modulation was further supported by spastin's gain-of-function phenotype. Spastin overexpression in photoreceptors caused the expansion of the apical membrane domain from apical to basolateral in the developing photoreceptor. Although the localizations of the apical domain and adherens junctions (AJs) were severely expanded, there were no defects in cell polarity.Conclusions/SignificanceThese results strongly suggest that spastin is essential for apical domain biogenesis during rhabdomere elongation in Drosophila photoreceptor morphogenesis.