Tania Y. Estévez-Lao

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 mosquitos 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.

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. aegyptis superior ability to kill bacteria in their hemocoels. Hemocytes from both species swiftly phagocytose bacteria, but phagocytosis does not explain Ae. aegyptis 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. aegyptis increased transcriptional induction of antimicrobial peptides and other humoral immune factors in response to infection.

Cardioacceleratory function of the neurohormone CCAP in the mosquito Anopheles gambiae

SUMMARY 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.

Anopheles gambiae corazonin: gene structure, expression and effect on mosquito heart physiology

Haemolymph flow in mosquitoes is primarily driven by the contraction of a dorsal vessel that is subdivided into an abdominal heart and a thoracic aorta. The factors that regulate mosquito heart contractions are not understood, but in other insects heart physiology is partially controlled by several neurohormones. One of these is corazonin, a neuropeptide initially discovered because of its cardioacceleratory activity in the cockroach Periplaneta americana. In the present study, we describe the corazonin gene and transcript structure in the mosquito Anopheles gambiae, characterize its developmental expression, and test its role in modulating heart physiology. We show that the A. gambiae corazonin gene encodes the most common form of the corazonin peptide ([Arg7]‐corazonin) and that it is alternatively spliced, with the only difference between the transcripts occurring in the 5′ untranslated region. Analysis of the developmental expression of corazonin and the corazonin receptor revealed that transcription of both follows a bimodal distribution, with highest mRNA levels in 2nd instar larvae and during the pupa to adult transition. Finally, experiments where mosquitoes were injected with various doses of corazonin and experiments where the transcription of corazonin and the corazonin receptor were reduced by RNA interference failed to detect a significant role for this neuropeptide in modulating mosquito heart physiology.

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.

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 mosquitos 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.

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.

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.

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.