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The immune deficiency pathway (IMD) is an important component of the antibacterial, antimalarial and antiviral response in mosquitoes. The IMD pathway also drives the infection induced migration of hemocytes to the heart. During an infection, periostial hemocytes kill pathogens in areas of high hemolymph flow and produce nitric oxide that reduces the heart rate. Here, we investigated the consequences of repressing the IMD pathway by silencing the transcription factor, rel2, or activating the pathway by silencing the negative regulator, caspar, in Anopheles gambiae. In uninfected mosquitoes, repression of the IMD pathway does not affect the circulatory system. However, activating the IMD pathway decreases the heart rate, and this correlates with increased transcription and activity of nitric oxide synthase (NOS), but not increased transcription of the lysozymes, LysC1 or LysC2. In infected mosquitoes, however, activation of the IMD pathway does not affect the heart rate but repression of the pathway decreases the heart rate. This latter phenotype correlates with increased transcription and activity of nitric oxide synthase, which is likely due to an increase in infection intensity. In conclusion, we demonstrate that a major immune signaling pathway that regulates periostial hemocyte aggregation, the IMD pathway, reduces the heart rate via a nitric oxide-based mechanism. 

Mosquitoes cannot use metabolism to regulate their body temperature and therefore climate warming is altering their physiology. Mosquitoes also experience a physiological decline with aging, a phenomenon called senescence. Because both high temperature and aging are detrimental to mosquitoes, we hypothesized that high temperatures accelerate senescence. Here, we investigated how temperature and aging, independently and interactively, shape the antimicrobial immune response of the mosquito Anopheles gambiae. Using a zone-of-inhibition assay that measures the antimicrobial activity of hemolymph, we found that antimicrobial activity increases following infection. Moreover, in infected mosquitoes, antimicrobial activity weakens as the temperature rises to 32°C, and antimicrobial activity increases from 1 to 5 days of age and stabilizes with further aging. Importantly, in E. coli-infected mosquitoes, higher temperature causes an aging-dependent decline in antimicrobial activity. Altogether, this study demonstrates that higher temperature can accelerate immune senescence in infected mosquitoes, thereby interactively shaping their ability to fight an infection. 

The body temperature of mosquitoes, like most insects, is dictated by the environmental temperature. Climate change is increasing the body temperature of insects and thereby altering physiological processes such as immune proficiency. Aging also alters insect physiology, resulting in the weakening of the immune system in a process called senescence. Although both temperature and aging independently affect the immune system, it is unknown whether temperature alters the rate of immune senescence. Here, we evaluated the independent and combined effects of temperature (27°C, 30°C and 32°C) and aging (1, 5, 10 and 15 days old) on the melanization immune response of the adult female mosquito, Anopheles gambiae. Using a spectrophotometric assay that measures phenoloxidase activity (a rate limiting enzyme) in hemolymph, and therefore, the melanization potential of the mosquito, we discovered that the strength of melanization decreases with higher temperature, aging, and infection. Moreover, when the temperature is higher, the aging-dependent decline in melanization begins at a younger age. Using an optical assay that measures melanin deposition on the abdominal wall and in the periostial regions of the heart, we found that melanin is deposited after infection, that this deposition decreases with aging, and that this aging-dependent decline is accelerated by higher temperature. This study demonstrates that higher temperature accelerates immune senescence in mosquitoes, with higher temperature uncoupling physiological age from chronological age. These findings highlight the importance of investigating the consequences of climate change on how disease transmission by mosquitoes is affected by aging.