Julian F. Hillyer

The cardiac extracellular matrix provides structural support to the primary circulatory organ of an insect: a dorsal vessel that is divided into an aorta in the thorax and a heart in the abdomen. In the fruit fly, Drosophila melanogaster , the extracellular matrix components, Pericardin and Lonely heart, are needed for proper heart contractility, as they mediate adhesion between cardiomyocytes, pericardial cells, hemocytes, and alary muscles. In the mosquito, Anopheles gambiae , Pericardin and Lonely heart are necessary for the infection‐induced migration of hemocytes to the heart. In these infected mosquitoes, hemocytes decrease the heart rate by releasing nitric oxide. Here, we demonstrate in mosquitoes that Pericardin is transcribed throughout the abdomen, but that Lonely heart is transcribed mainly in the heart. Moreover, we demonstrate that Pericardin and Lonely heart influence the infection‐induced disproportional decrease in the anterograde and retrograde heart rates. We also demonstrate that, in infected mosquitoes, Pericardin and Lonely heart alter the directionality of heart contractions, shifting to more contractions propagating anterograde. These findings further explain how the immune and circulatory systems of insects are functionally integrated by demonstrating that the cardiac extracellular matrix, in addition to driving hemocyte organization on the heart, modulates heart rhythmicity in infected mosquitoes.

Background: Global environmental temperatures are rising, which is increasing the body temperature of mosquitoes. This increase in body temperature is accelerating senescence, thereby weakening immune responses and reproductive processes, and shortening lifespan. To determine how warmer temperature and aging, individually and interactively, shape the transcriptome of the African malaria mosquito, Anopheles gambiae , we conducted RNA-sequencing and network-analysis in naïve and immune-induced mosquitoes that had been reared at 27 °C, 30 °C or 32 °C and were 1, 5, 10 or 15 days into adulthood. Results: We demonstrate that immune induction, warmer temperature, and aging alter the transcriptome. Notably, the transcriptome of 1-day-old mosquitoes is pronouncedly different from older mosquitoes, and importantly, warmer temperature modifies the aging-dependent changes to accelerate senescence. For example, warmer temperature amplifies the aging-dependent decrease in immune gene expression but dampens both the aging-dependent decrease in metabolic gene expression and the aging-dependent increase in DNA repair gene expression. Conclusions: Altogether, warmer temperature accelerates senescence, shaping the transcriptome in ways that alter the mosquito’s ability to fight infection and survive in a warming environment.

Background: Global environmental temperatures are rising, which is increasing the body temperature of mosquitoes. This increase in body temperature is accelerating senescence, thereby weakening immune responses and reproductive processes, and shortening lifespan. To determine how warmer temperature and aging, individually and interactively, shape the transcriptome of the African malaria mosquito, Anopheles gambiae , we conducted RNA-sequencing and network-analysis in naive and immune-induced mosquitoes that had been reared at 27C, 30C or 32C and were 1, 5, 10 or 15 days into adulthood. Results: We demonstrate that immune induction, warmer temperature, and aging alter the transcriptome. Notably, the transcriptome of 1-day-old mosquitoes is pronouncedly different from older mosquitoes, and importantly, warmer temperature modifies the aging-dependent changes to accelerate senescence. For example, warmer temperature amplifies the aging-dependent decrease in immune gene expression but dampens both the aging-dependent decrease in metabolic gene expression and the aging-dependent increase in DNA repair gene expression. Conclusions: Altogether, warmer temperature accelerates senescence, shaping the transcriptome in ways that alter the mosquitos ability to fight infection and survive in a warming environment.

Infection induces the aggregation of hemocytes on the dorsal vessel of mosquitoes. These hemocytes, called periostial hemocytes, phagocytose pathogens and produce immune factors on the abdominal portion of the dorsal vessel, called the heart. One of these immune factors, nitric oxide, is a pleiotropic free radical that is an antimicrobial and a heartbeat reducer. But nitric oxide is not just produced by hemocytes. It is also synthesized by pericardial cells that flank the heart, and other tissues. To determine whether it is the periostial hemocytes or the pericardial cells that modulate the heart following infection, we chemically ablated the hemocytes using clodronate liposomes and measured immune responses and heart physiology. We demonstrate that clodronate liposomes ablate the sessile hemocytes, including the periostial hemocytes, while leaving the pericardial cells and heart integrity unaffected. Moreover, ablating hemocytes abolishes the phagocytosis of bacteria, alters the deposition of melanized bacteria, and decreases nitric oxide synthase activity on the heart. Importantly, hemocyte ablation eliminates the infection induced reduction of the heart rate, mainly by modifying the anterograde heart rate. Therefore, periostial hemocytes drive immune responses on the heart and infection-induced changes to circulatory physiology.

In most mosquito species, reproduction requires mating between the female and the male, followed by the female blood-feeding, completing oogenesis, and laying eggs. Warmer environmental temperature and aging both reduce mosquito fecundity and fertility, and warmer temperature accelerates the aging-dependent decline in reproduction such that reproductive impairment manifests earlier in life. To shed light on how this warming-based acceleration of reproductive senescence occurs, we investigated how temperature (27 °C, 30 °C, and 32 °C) and aging interactively shape female and male reproductive tissue size in the African malaria mosquito, Anopheles gambiae. In blood-fed females, we discovered that warmer temperature accelerates the aging-dependent decrease in the size of the ovaries but not the spermatheca. In males, we discovered that warmer temperature lessens and delays the aging-dependent increase in the size of the male accessory glands but not the testes. Next, we measured the expression of reproductive genes in females and males. In female reproductive tissues, warmer temperature accelerates the aging-dependent decrease in the expression of vitellogenin and the aging-dependent increase in the expression of MISO and HPX15. In male reproductive tissues, warmer temperature accelerates an aging-dependent decrease in the expression of Plugin, TGase3, phLP, and CYP315A1. Altogether, these data shed light on how physical and transcriptional changes underpin the warming-based acceleration of an aging-dependent decline in mosquito fecundity and fertility.

To reproduce, most female mosquitoes must ingest blood to obtain nutrients for viable eggs. Global warming is increasing mosquito body temperature and hampering their reproduction. Moreover, because it takes several days to produce eggs and mosquitoes are short-lived, the age of blood feeding determines whether reproduction is feasible. Given that warmer temperature and aging both impair reproduction, we scrutinized whether temperature modifies the aging-based decline in fecundity and fertility. By rearing the African malaria mosquito, Anopheles gambiae, at three temperatures and offering them a blood meal at four ages, we demonstrate that warmer temperature reduces blood feeding propensity and blood meal mass. Warmer temperature and aging decrease survival, delay oviposition, and reduce oviposition success, fecundity, and fertility. Importantly, warmer temperature quickens the onset of the aging-dependent decline in fecundity and fertility, and at the warmest temperature of 32°C, mosquitoes are infertile. Warmer temperature accelerates reproductive senescence, which has implications for disease transmission in this warming world.