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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. 

An infection induces the migration of immune cells called hemocytes to the insect heart, where they aggregate around heart valves called ostia and phagocytose pathogens in areas of high hemolymph flow. Here, we investigated whether the cardiac extracellular matrix proteins, Pericardin (Prc) and Lonely heart (Loh), regulate the infection-induced aggregation of periostial hemocytes in the mosquito, An. gambiae. We discovered that RNAi-based post-transcriptional silencing of Prc or Loh did not affect the resident population of periostial hemocytes in uninfected mosquitoes, but that knocking down these genes decreases the infection-induced migration of hemocytes to the heart. Knocking down Prc or Loh did not affect the proportional distribution of periostial hemocytes along the periostial regions. Moreover, knocking down Prc or Loh did not affect the number of sessile hemocytes outside the periostial regions, suggesting that the role of these proteins is cardiac-specific. Finally, knocking down Prc or Loh did not affect the amount of melanin at the periostial regions, or the intensity of an infection at 24 h after challenge. Overall, we demonstrate that Prc and Loh are positive regulators of the infection-induced migration of hemocytes to the heart of mosquitoes. 

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. 

Most insects are poikilotherms and ectotherms, so their body temperature fluctuates and closely aligns with the temperature of their environment. The rise in global temperatures is affecting the physiology of insects by altering their ability to survive, reproduce, and transmit disease. Aging also impacts insect physiology because the body deteriorates via senescence as the insect ages. Although temperature and age both impact insect biology, these factors have historically been studied in isolation. So, it is unknown whether or how temperature and age interact to shape insect physiology. Here, we investigated the effects of warmer temperature (27 °C, 30 °C and 32 °C), aging (1, 5, 10, and 15 days post-eclosion), and their interaction on the size and body composition of the mosquito, Anopheles gambiae. We found that warmer temperatures result in slightly smaller adult mosquitoes, as measured by abdomen and tibia length. Aging alters both abdominal length and dry weight in a manner that correlates with the increase in energetic resources and tissue remodeling that occurs after metamorphosis and the senescence-based decline that ensues later. Moreover, the carbohydrate and lipid contents of adult mosquitoes are not meaningfully affected by temperature but are altered by aging: carbohydrate content increases with age whereas lipid content increases over the first few days of adulthood and then decreases. Protein content decreases with both rising temperature and aging, and the aging-associated decrease accelerates at warmer temperatures. Altogether, temperature and age, individually and to a lesser extent interactively, shape the size and composition of adult mosquitoes. 

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. 

The immune and circulatory systems of animals are functionally integrated. In mammals, the spleen and lymph nodes filter and destroy microbes circulating in the blood and lymph, respectively. In insects, immune cells that surround the heart valves (ostia), called periostial haemocytes, destroy pathogens in the areas of the body that experience the swiftest haemolymph (blood) flow. An infection recruits additional periostial haemocytes, amplifying heart-associated immune responses. Although the structural mechanics of periostial haemocyte aggregation have been defined, the genetic factors that regulate this process remain less understood. Here, we conducted RNA sequencing in the African malaria mosquito, Anopheles gambiae , and discovered that an infection upregulates multiple components of the immune deficiency (IMD) and c-Jun N-terminal kinase (JNK) pathways in the heart with periostial haemocytes. This upregulation is greater in the heart with periostial haemocytes than in the circulating haemocytes or the entire abdomen. RNA interference-based knockdown then showed that the IMD and JNK pathways drive periostial haemocyte aggregation and alter phagocytosis and melanization on the heart, thereby demonstrating that these pathways regulate the functional integration between the immune and circulatory systems. Understanding how insects fight infection lays the foundation for novel strategies that could protect beneficial insects and harm detrimental ones. 

The immune and circulatory systems of mammals are functionally integrated, as exemplified by the immune function of the spleen and lymph nodes. Similar functional integration exists in the malaria mosquito, Anopheles gambiae , as exemplified by the infection-induced aggregation of hemocytes around the heart valves. Whether this is specific to mosquitoes or a general characteristic of insects remained unknown. We analyzed 68 species from 51 families representing 16 orders and found that infection induces the aggregation of hemocytes and pathogens on the heart of insects from all major branches of the class Insecta. An expanded analysis in the holometabolous mosquito, Aedes aegypti , and the hemimetabolous bed bug, Cimex lectularius , showed that infection induces the aggregation of phagocytic hemocytes on the hearts of distantly related insects, with aggregations mirroring the patterns of hemolymph flow. Therefore, the functional integration of the immune and circulatory systems is conserved across the insect tree of life.