Extracellular matrix proteins Pericardin and Lonely heart mediate periostial hemocyte aggregation in the mosquito Anopheles gambiae.

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.

Temperature and age, individually and interactively, shape the size, weight, and body composition of adult female mosquitoes.

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.

The immune deficiency and c-Jun N-terminal kinase pathways drive the functional integration of 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.

Transglutaminase 3 negatively regulates immune responses on the heart of the mosquito, Anopheles gambiae.

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 of TGase2, whereas infection upregulates TGase1, TGase2 and TGase3. RNAi-based knockdown of TGase1 and TGase2 did not alter periostial hemocyte aggregation, but knockdown of TGase3 increased 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 of TGase3 also slightly reduced the number of sessile hemocytes outside of the periostial regions. Taken altogether, these data show that TGase3 negatively regulates periostial hemocyte aggregation, and we hypothesize that this occurs by negatively regulating the immune deficiency pathway and by altering hemocyte adhesion. In conclusion, TGase3 is involved in the functional integration between the immune and circulatory systems of mosquitoes.

Nitric oxide produced by periostial hemocytes modulates the bacterial infection-induced reduction of the mosquito heart rate.

The circulatory and immune systems of mosquitoes are functionally integrated. An infection induces the migration of hemocytes to the dorsal vessel, and specifically, to the regions surrounding the ostia of the heart. These periostial hemocytes phagocytose pathogens in the areas of the hemocoel that experience the highest hemolymph flow. Here, we investigated whether a bacterial infection affects cardiac rhythmicity in the African malaria mosquito, Anopheles gambiae We discovered that infection with Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis, but not Micrococcus luteus, reduces the mosquito heart rate and alters the proportional directionality of heart contractions. Infection does not alter the expression of genes encoding crustacean cardioactive peptide (CCAP), FMRFamide, corazonin, neuropeptide F or short neuropeptide F, indicating that they do not drive the cardiac phenotype. Infection upregulates the transcription of two superoxide dismutase (SOD) genes, catalase and a glutathione peroxidase, but dramatically induces upregulation of nitric oxide synthase (NOS) in both the heart and hemocytes. Within the heart, nitric oxide synthase is produced by periostial hemocytes, and chemically inhibiting the production of nitric oxide using l-NAME reverses the infection-induced cardiac phenotype. Finally, infection induces the upregulation of two lysozyme genes in the heart and other tissues, and treating mosquitoes with lysozyme reduces the heart rate in a manner reminiscent of the infection phenotype. These data demonstrate an exciting new facet of the integration between the immune and circulatory systems of insects, whereby a hemocyte-produced factor with immune activity, namely nitric oxide, modulates heart physiology.

Transstadial immune activation in a mosquito: Adults that emerge from infected larvae have stronger antibacterial activity in their hemocoel yet increased susceptibility to malaria infection.

Larval and adult mosquitoes mount immune responses against pathogens that invade their hemocoel. Although it has been suggested that a correlation exists between immune processes across insect life stages, the influence that an infection in the hemocoel of a larva has on the immune system of the eclosed adult remains unknown. Here, we used Anopheles gambiae to test whether a larval infection influences the adult response to a subsequent bacterial or malaria parasite infection. We found that for both female and male mosquitoes, a larval infection enhances the efficiency of bacterial clearance following a secondary infection in the hemocoel of adults. The adults that emerge from infected larvae have more hemocytes than adults that emerge from naive or injured larvae, and individual hemocytes have greater phagocytic activity. Furthermore, mRNA abundance of immune genes-such as cecropin A, Lysozyme C1, Stat-A, and Tep1-is higher in adults that emerge from infected larvae. A larval infection, however, does not have a meaningful effect on the probability that female adults will survive a systemic bacterial infection, and increases the susceptibility of females to Plasmodium yoelii, as measured by oocyst prevalence and intensity in the midgut. Finally, immune proficiency varies by sex; females exhibit increased bacterial killing, have twice as many hemocytes, and more highly express immune genes. Together, these results show that a larval hemocoelic infection induces transstadial immune activation-possibly via transstadial immune priming-but that it confers both costs and benefits to the emerged adults.

Anopheles gambiae larvae mount stronger immune responses against bacterial infection than adults: evidence of adaptive decoupling in mosquitoes.

BACKGROUND: The 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. RESULTS: We 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. CONCLUSIONS: These 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.

Mosquito aging modulates the heart rate and the proportional directionality of heart contractions.

Mosquito aging impacts a myriad of physiological processes, including digestion, flight, mating, reproductive success, and immunity. In the present study, we conducted intravital video imaging in 1, 3, 5, 10, 15 and 20-day-old Anopheles gambiae female adults to assess whether aging impacts mosquito heart physiology. We found that the heart contraction rate increases over the first 15days of adulthood and then decreases. These changes occur for both contraction directions, although aging results in a relative change in the anterograde versus retrograde contraction rates. That is, whereas for the first 5days of life the anterograde and retrograde contraction rates are similar, from day 10 to day 20 the retrograde contraction rate is higher than the anterograde contraction rate. Aging also biases the proportional directionality of heart contractions, from approximately two thirds of the time being spent contracting in the anterograde direction and two thirds of the contractions propagating anterograde during the first 5days of life to an approximately even split between anterograde and retrograde when the mosquitoes have reached 10 to 20days of age. Transcriptional analyses of crustacean cardioactive peptide (CCAP), FMRFamide, calcium-calmodulin dependent kinase II (CaMKII), pygopus, manganese-iron superoxide dismutase (MnSOD1) and vinculin by quantitative RT-PCR revealed age-associated changes in gene expression, with MnSOD1 and vinculin expression showing a declining trend with age. RNAi-based knockdown of MnSOD1 or vinculin resulted in heart physiology that trended toward the aging phenotype for every parameter that was measured, suggesting that these two genes are involved in cardiac aging.

The neurotransmitters serotonin and glutamate accelerate the heart rate of the mosquito Anopheles gambiae.

Serotonin and glutamate are neurotransmitters that in insects are involved in diverse physiological processes. Both serotonin and glutamate have been shown to modulate the physiology of the dorsal vessel of some insects, yet until the present study, their activity in mosquitoes remained unknown. To test whether serotonin or glutamate regulate dorsal vessel physiology in the African malaria mosquito, Anopheles gambiae, live mosquitoes were restrained, and a video of the contracting heart (the abdominal portion of the dorsal vessel) was acquired. These adult female mosquitoes were then injected with various amounts of serotonin, glutamate, or a control vehicle solution, and additional videos were acquired at 2 and 10 min post-treatment. Comparison of the videos taken before and after treatment revealed that serotonin accelerates the frequency of heart contractions, with the cardioacceleration being significantly more pronounced when the wave-like contractions of cardiac muscle propagate in the anterograde direction (toward the head). Comparison of the videos taken before and after treatment with glutamate revealed that this molecule is also cardioacceleratory. However, unlike serotonin, the activity of glutamate does not depend on whether the contractions propagate in the anterograde or the retrograde (toward the posterior of the abdomen) directions. Serotonin or glutamate induces a minor change or no change in the percentage of contractions and the percentage of the time that the heart contracts in the anterograde or the retrograde directions. In summary, this study shows that the neurotransmitters serotonin and glutamate increase the heart contraction rate of mosquitoes.

Deprivation of both sucrose and water reduces the mosquito heart contraction rate while increasing the expression of nitric oxide synthase.

Adult female mosquitoes rely on carbohydrate-rich plant nectars as their main source of energy. In the present study we tested whether the deprivation of a carbohydrate dietary source or the deprivation of both carbohydrate and water affects mosquito heart physiology. Intravital video imaging of Anopheles gambiae showed that, relative to sucrose fed mosquitoes, the deprivation of both sucrose and water for 24h, but not the deprivation of sucrose alone, reduces the heart contraction rate. Measurement of the protein, carbohydrate and lipid content of mosquitoes in the three treatment groups did not explain this cardiac phenotype. However, while the deprivation of sucrose reduced mosquito weight and abdominal width, the deprivation of both sucrose and water reduced mosquito weight even further without augmenting the change in abdominal width, indirectly suggesting that starvation and dehydration reduces hemolymph pressure. Analysis of the mRNA levels of crustacean cardioactive peptide (CCAP), FMRFamide, corazonin, neuropeptide F and short neuropeptide F then suggested that these neuropeptides do not regulate the cardiac phenotype observed. However, relative to sucrose fed and sucrose deprived mosquitoes, the mRNA level of nitric oxide synthase (NOS) was significantly elevated in mosquitoes that had been deprived of both sucrose and water. Given that nitric oxide suppresses the heart rate of vertebrates and invertebrates, these data suggest a role for this free radical in modulating mosquito heart physiology.

Molecular and functional characterization of Anopheles gambiae inward rectifier potassium (Kir1) channels: a novel role in egg production.

Inward rectifier potassium (Kir) channels play essential roles in regulating diverse physiological processes. Although Kir channels are encoded in mosquito genomes, their functions remain largely unknown. In this study, we identified the members of the Anopheles gambiae Kir gene family and began to investigate their function. Notably, we sequenced the A. gambiae Kir1 (AgKir1) gene and showed that it encodes all the canonical features of a Kir channel: an ion pore that is composed of a pore helix and a selectivity filter, two transmembrane domains that flank the ion pore, and the so-called G-loop. Heterologous expression of AgKir1 in Xenopus oocytes revealed that this gene encodes a functional, barium-sensitive Kir channel. Quantitative RT-PCR experiments then showed that relative AgKir1 mRNA levels are highest in the pupal stage, and that AgKir1 mRNA is enriched in the adult ovaries. Gene silencing of AgKir1 by RNA interference did not affect the survival of female mosquitoes following a blood meal, but decreased their egg output. These data provide evidence for a new role of Kir channels in mosquito fecundity, and further validates them as promising molecular targets for the development of a new class of mosquitocides to be used in vector control.

Myotropic effects of FMRFamide containing peptides on the heart of the mosquito Anopheles gambiae.

FMRFamide-like peptides (FLPs) are produced by invertebrate and vertebrate animals, and regulate diverse physiological processes. In insects, several FLPs modulate heart physiology, with some increasing and others decreasing dorsal vessel contraction dynamics. Here, we describe the FMRFamide gene structure in the mosquito, Anopheles gambiae, quantify the developmental and spatial expression of FMRFamide and its putative receptor (FMRFamideR), and show that the peptides FMRFamide and SALDKNFMRFamide have complex myotropic properties. RACE sequencing showed that the FMRFamide gene encodes eight putative FLPs and is alternatively spliced. Of the eight FLPs, only one is shared by A. gambiae, Aedes aegypti and Culex quinquefasciatus: SALDKNFMRFamide. Quantitative PCR showed that peak expression of FMRFamide and FMRFamideR occurs in second instar larvae and around eclosion. In adults, FMRFamide is primarily transcribed in the head and thorax, and FMRFamideR is primarily transcribed in the thorax. Intravital video imaging of mosquitoes injected FMRFamide and SALDKNFMRFamide revealed that at low doses these peptides increase heart contraction rates. At high doses, however, these peptides decrease heart contraction rates and alter the proportional directionality of heart contractions. Taken altogether, these data describe the FMRFamide gene in A. gambiae, and show that FLPs are complex modulators of mosquito circulatory physiology.

Involvement of the Anopheles gambiae Nimrod gene family in mosquito immune responses.

Insects fight infection using a variety of signaling pathways and immune effector proteins. In Drosophila melanogaster, three members of the Nimrod gene family (draper, nimC1 and eater) bind bacteria, and this binding leads to phagocytosis by hemocytes. The Nimrod gene family has since been identified in other insects, but their function in non-drosophilids remains unknown. The purpose of this study was to identify the members of the Nimrod gene family in the malaria mosquito, Anopheles gambiae, and to assess their role in immunity. We identified and sequenced three members of this gene family, herein named draper, nimrod and eater, which are the orthologs of D. melanogaster draper, nimB2 and eater, respectively. The three genes are preferentially expressed in hemocytes and their peak developmental expression is in pupae and young adults. Infection induces the transcriptional upregulation of all three genes, but the magnitude of this upregulation becomes more attenuated as mosquitoes become older. RNAi-based knockdown of eater, but not draper or nimrod, decreased a mosquito's ability to kill Escherichia coli in the hemocoel. Knockdown of draper, eater, or any combination of Nimrod family genes rendered mosquitoes more likely to die from Staphylococcus epidermidis. Finally, knockdown of Nimrod family genes did not impact mRNA levels of the antimicrobial peptides defensin (def1), cecropin (cecA) or gambicin (gam1), but eater knockdown led to a decrease in mRNA levels of nitric oxide synthase. Together, these data show that members of the A. gambiae Nimrod gene family are positive regulators of the mosquito antibacterial response.

Cardioacceleratory function of the neurohormone CCAP in the mosquito Anopheles gambiae.

Crustacean cardioactive peptide (CCAP) is a highly conserved arthropod neurohormone that is involved in ecdysis, hormone release and the modulation of muscle contractions. Here, we determined the CCAP gene structure in the malaria mosquito Anopheles gambiae, assessed the developmental expression of CCAP and its receptor and determined the role that CCAP plays in regulating mosquito cardiac function. RACE sequencing revealed that the A. gambiae CCAP gene encodes a neuropeptide that shares 100% amino acid identity with all sequenced CCAP peptides, with the exception of Daphnia pulex. Quantitative RT-PCR showed that expression of CCAP and the CCAP receptor displays a bimodal distribution, with peak mRNA levels in second instar larvae and pupae. Injection of CCAP revealed that augmenting hemocoelic CCAP levels in adult mosquitoes increases the anterograde and retrograde heart contraction rates by up to 28%, and increases intracardiac hemolymph flow velocities by up to 33%. Partial CCAP knockdown by RNAi had the opposite effect, decreasing the mosquito heart rate by 6%. Quantitative RT-PCR experiments showed that CCAP mRNA is enriched in the head region, and immunohistochemical experiments in newly eclosed mosquitoes detected CCAP in abdominal neurons and projections, some of which innervated the heart, but failed to detect CCAP in the abdomens of older mosquitoes. Instead, in older mosquitoes CCAP was detected in the pars lateralis, the subesophageal ganglion and the corpora cardiaca. In conclusion, CCAP has a potent effect on mosquito circulatory physiology, and thus heart physiology in this dipteran insect is under partial neuronal control.

Increased survivorship following bacterial infection by the mosquito Aedes aegypti as compared to Anopheles gambiae correlates with increased transcriptional induction of antimicrobial peptides.

Mosquitoes defend themselves from pathogens by mounting cellular and humoral innate immune responses. Bioinformatic analyses have revealed considerable divergence in immune gene repertoires between mosquito species, but interspecies empirical comparisons of immune responses are lacking. Here, we present a comparative analysis of the antimicrobial responses of two distantly related disease vectors: Aedes aegypti and Anopheles gambiae. Survival studies showed that Ae. aegypti are more proficient in surviving a bacterial infection than An. gambiae, and this correlates with Ae. aegypti's superior ability to kill bacteria in their hemocoels. Hemocytes from both species swiftly phagocytose bacteria, but phagocytosis does not explain Ae. aegypti's increased robustness: An. gambiae contain more circulating hemocytes and display a higher phagocytic index, but the phagocytic capacity of individual hemocytes is greater in Ae. aegypti. Then, profiling of 19 immunity genes revealed that transcriptional induction following infection is significantly elevated in Ae. aegypti when compared to An. gambiae, with the largest change seen in the transcription of cecropin and defensin. These data show that Ae. aegypti is better equipped to survive a bacterial infection than An. gambiae, and this correlates with Ae. aegypti's increased transcriptional induction of antimicrobial peptides and other humoral immune factors in response to infection.

Bursicon-expressing neurons undergo apoptosis after adult ecdysis in the mosquito Anopheles gambiae.

Neuropeptides are important regulators of diverse processes during development. The insect neuropeptide bursicon, a 30 kDa heterodimer, controls the hardening of the new cuticle after the shedding of the old one (ecdysis) and the inflation and maturation of adult wings. Given this specific functional role, its expression should only be required transiently because adult insects no longer undergo ecdysis. Here we report the transient expression of bursicon in the mosquito, Anopheles gambiae. Quantitative RT-PCR revealed that transcription of the bursicon monomers, burs and pburs, steadily increases through the larval stages, peaks in the black pupa stage, and decreases to below detectable levels by 8 h after adult ecdysis (eclosion). Immunohistochemistry on the adult nervous system showed that bursicon is co-expressed with crustacean cardioactive peptide (CCAP) in specific neurons of the abdominal ganglia, but that labeling intensity wanes by 14 h post-eclosion. Finally, detection of disintegrating DNA by TUNEL labeling demonstrated that the bursicon expressing neurons successively undergo apoptosis following eclosion. Taken altogether, these data describe A. gambiae as another holometabolous insect in which bursicon ceases to be produced in adults, and in which the bursicon expressing neurons are removed from the ventral nerve cord.

Nitric oxide is an essential component of the hemocyte-mediated mosquito immune response against bacteria.

Nitric oxide is a signaling and immune effector molecule synthesized by the enzyme nitric oxide synthase. In mosquitoes, nitric oxide functions as a parasite antagonist in the midgut but little is known about its function in the hemocoel. Here, we characterized the temporal and spatial expression of the Anopheles gambiae nitric oxide synthase gene and explored the role nitric oxide plays in the antibacterial response in the mosquito hemocoel. Quantitative PCR and Western blot analyses showed that nitric oxide synthase is expressed in hemocytes and fat body, and is upregulated in response to systemic infection with Escherichia coli and Micrococcus luteus. Diaphorase staining and immunofluorescence showed that nitric oxide synthase is abundant in the granulocyte subpopulation of hemocytes, and both the staining intensity and the percentage of cells that stain for nitric oxide synthase significantly increase after a bacterial challenge. When nitric oxide production was inhibited, the mosquito's ability to kill E. coli was significantly reduced. Accordingly, inhibiting nitric oxide production increased the mortality rate of mosquitoes with systemic E. coli infections. Taken altogether, these data show that nitric oxide is a crucial player in the antibacterial immune response in the mosquito hemocoel.