The mechanism of cytoplasmic incompatibility is conserved in Wolbachia-infected Aedes aegypti mosquitoes deployed for arbovirus control.

The rising interest and success in deploying inherited microorganisms and cytoplasmic incompatibility (CI) for vector control strategies necessitate an explanation of the CI mechanism. Wolbachia-induced CI manifests in the form of embryonic lethality when sperm from Wolbachia-bearing testes fertilize eggs from uninfected females. Embryos from infected females however survive to sustain the maternally inherited symbiont. Previously in Drosophila melanogaster flies, we demonstrated that CI modifies chromatin integrity in developing sperm to bestow the embryonic lethality. Here, we validate these findings using wMel-transinfected Aedes aegypti mosquitoes released to control vector-borne diseases. Once again, the prophage WO CI proteins, CifA and CifB, target male gametic nuclei to modify chromatin integrity via an aberrant histone-to-protamine transition. Cifs are not detected in the embryo, and thus elicit CI via the nucleoprotein modifications established pre-fertilization. The rescue protein CifA in oogenesis localizes to stem cell, nurse cell, and oocyte nuclei, as well as embryonic DNA during embryogenesis. Discovery of the nuclear targeting Cifs and altered histone-to-protamine transition in both Aedes aegypti mosquitoes and D. melanogaster flies affirm the Host Modification Model of CI is conserved across these host species. The study also newly uncovers the cell biology of Cif proteins in the ovaries, CifA localization in the embryos, and an impaired histone-to-protamine transition during spermiogenesis of any mosquito species. Overall, these sperm modification findings may enable future optimization of CI efficacy in vectors or pests that are refractory to Wolbachia transinfections.

Higher temperature accelerates the aging-dependent weakening of the melanization immune response in 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.

Larvicidal activity of the photosensitive insecticides, methylene blue and rose bengal, in Aedes aegypti and Anopheles gambiae mosquitoes.

BACKGROUND: Insecticides are critical for controlling mosquito populations and mitigating the spread of vector-borne disease, but their overuse has selected for resistant populations. A promising alternative to classical chemical insecticides is photosensitive molecules - here called photosensitive insecticides or PSIs - that when ingested and activated by light, generate broadly toxic reactive oxygen species. This mechanism of indiscriminate oxidative damage decreases the likelihood that target site modification-based resistance evolves. Here, we tested whether the PSIs, methylene blue (MB) and rose bengal (RB), are viable insecticides across the mosquito lineage. RESULTS: MB and RB are phototoxic to both Aedes aegypti and Anopheles gambiae at micromolar concentrations, with greatest toxicity when larvae are incubated in the dark with the PSIs for 2 h prior to photoactivation. MB is ten times more toxic than RB, and microscopy-based imaging suggests that this is because ingested MB escapes the larval gut and disperses throughout the hemocoel whereas RB remains confined to the gut. Adding food to the PSI-containing water has a bidirectional, concentration-dependent effect on PSI toxicity; toxicity increases at high concentrations but decreases at low concentrations. Finally, adding sand to the water increases the phototoxicity of RB to Ae. aegypti. CONCLUSION: MB and RB are larvicidal via a light activated mechanism, and therefore, should be further investigated as an option for mosquito control. © 2023 Society of Chemical Industry.

Mosquito larvae exposed to a sublethal dose of photosensitive insecticides have altered juvenile development but unaffected adult life history traits.

BACKGROUND: Larvicides are critical for the control of mosquito-borne diseases. However, even sublethal exposure to a larvicide can alter development and life history traits, which can then affect population density and disease transmission dynamics. Photosensitive insecticides (PSIs) are a promising class of larvicide that are toxic when ingested and activated by light. We investigated whether the time of day when exposure occurs, or the process of pupation, affects larval susceptibility to PSI phototoxicity in the mosquito Anopheles gambiae, and whether sublethal exposure to PSIs alters life history traits. METHODS: Larvae were treated with lethal concentrations of the PSIs methylene blue (MB) and rose bengal (RB), and larval survival was measured at various times of day. Additionally, larvae were exposed to two concentrations of each PSI that resulted in low and medium mortality, and the life history traits of the surviving larvae were measured. RESULTS: Pupation, which predominantly occurs in the evening, protected larvae from PSI toxicity, but the toxicity of PSIs against larvae that had yet to pupate was unaffected by time of day. Larval exposure to a sublethal concentration of MB, but not RB, shortened the time to pupation. However, larval exposure to a sublethal concentration of RB, but not MB, increased pupal mortality. Neither PSI had a meaningful effect on the time to eclosion, adult longevity, or adult melanization potential. CONCLUSIONS: PSIs are lethal larvicides. Sublethal PSI exposure alters mosquito development, but does not affect adult life history traits.

Insect physiology: The mouthparts of moths and butterflies breathe through strategically positioned micropores.

Insects employ a tracheal system to transport oxygen and carbon dioxide to and from the body's cells. A new study discovers a micropore-based mechanism of respiration in the coiling mouthparts of moths and butterflies, which allowed these insects to evolve intricately long mouthparts without also evolving proportionally larger body sizes.

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.

Chemical Control of Mosquitoes and the Pesticide Treadmill: A Case for Photosensitive Insecticides as Larvicides.

Insecticides reduce the spread of mosquito-borne disease. Over the past century, mosquito control has mostly relied on neurotoxic chemicals-such as pyrethroids, neonicotinoids, chlorinated hydrocarbons, carbamates and organophosphates-that target adults. However, their persistent use has selected for insecticide resistance. This has led to the application of progressively higher amounts of insecticides-known as the pesticide treadmill-and negative consequences for ecosystems. Comparatively less attention has been paid to larvae, even though larval death eliminates a mosquito's potential to transmit disease and reproduce. Larvae have been targeted by source reduction, biological control, growth regulators and neurotoxins, but hurdles remain. Here, we review methods of mosquito control and argue that photoactive molecules that target larvae-called photosensitive insecticides or PSIs-are an environmentally friendly addition to our mosquitocidal arsenal. PSIs are ingested by larvae and produce reactive oxygen species (ROS) when activated by light. ROS then damage macromolecules resulting in larval death. PSIs are degraded by light, eliminating environmental accumulation. Moreover, PSIs only harm small translucent organisms, and their broad mechanism of action that relies on oxidative damage means that resistance is less likely to evolve. Therefore, PSIs are a promising alternative for controlling mosquitoes in an environmentally sustainable manner.

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.

Silencing Transglutaminase Genes TGase2 and TGase3 Has Infection-Dependent Effects on the Heart Rate of the Mosquito Anopheles gambiae.

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.

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.

The immune and circulatory systems are functionally integrated across insect evolution.

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.

Adult Mosquitoes Infected with Bacteria Early in Life Have Stronger Antimicrobial Responses and More Hemocytes after Reinfection Later in Life.

The immunological strategies employed by insects to overcome infection vary with the type of infection and may change with experience. We investigated how a bacterial infection in the hemocoel of the African malaria mosquito, Anopheles gambiae, prepares the immune system to face a subsequent bacterial infection. For this, adult female mosquitoes were separated into three groups-unmanipulated, injured, or infected with Escherichia coli-and five days later all the mosquitoes were infected with a different strain of E. coli. We found that an injury or a bacterial infection early in life enhances the ability of mosquitoes to kill bacteria later in life. This protection results in higher mosquito survival and is associated with an increased hemocyte density, altered phagocytic activity by individual hemocytes, and the increased expression of nitric oxide synthase and perhaps prophenoloxidase 6. Protection from a second infection likely occurs because of heightened immune awareness due to an already existing infection instead of memory arising from an earlier, cured infection. This study highlights the dynamic nature of the mosquito immune response and how one infection prepares mosquitoes to survive a subsequent infection.

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.

Parasites and Parasitology in this SARS-CoV-2, COVID-19 World: An American Society of Parasitologists Presidential Address.

The novel coronavirus disease 2019 (COVID-19) is one of the worst global health crises of this generation. The core of this pandemic is the rapid transmissibility of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, its high morbidity and mortality, and the presence of infectious asymptomatic carriers. As a result, COVID-19 has dominated this year's headlines and commanded significant research attention. As we consider SARS-CoV-2 and the COVID-19 pandemic, it is essential that scientists, governments, the media, and the general population also come to grips with the everyday cost of parasitic diseases. Plasmodium (malaria), schistosomes, filarial worms, hookworms, Ascaris, whipworms, and other protozoan and metazoan parasites take a tremendous toll on local communities. Yet, because most of these diseases are no longer endemic to developed countries, their research and intervention are not funded at levels that are proportional to their global morbidity and mortality. The scientific and public health communities must indeed vigorously fight SARS-CoV-2 and COVID-19, but while doing so and beyond, it will be essential to demonstrate steadfast resolve toward understanding and combating the parasitic diseases that for centuries have haunted humankind.

The Insect Circulatory System: Structure, Function, and Evolution.

Although the insect circulatory system is involved in a multitude of vital physiological processes, it has gone grossly understudied. This review highlights this critical physiological system by detailing the structure and function of the circulatory organs, including the dorsal heart and the accessory pulsatile organs that supply hemolymph to the appendages. It also emphasizes how the circulatory system develops and ages and how, by means of reflex bleeding and functional integration with the immune system, it supports mechanisms for defense against predators and microbial invaders, respectively. Beyond that, this review details evolutionary trends and novelties associated with this system, as well as the ways in which this system also plays critical roles in thermoregulation and tracheal ventilation in high-performance fliers. Finally, this review highlights how novel discoveries could be harnessed for the control of vector-borne diseases and for translational medicine, and it details principal knowledge gaps that necessitate further investigation.

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.

Complement-like proteins TEP1, TEP3 and TEP4 are positive regulators of periostial hemocyte aggregation in the mosquito Anopheles gambiae.

The mosquito immune and circulatory systems are functionally integrated. During an infection, hemocytes aggregate around the ostia (valves) of the dorsal vessel - areas of the heart called the periostial regions - where they phagocytose live and melanized pathogens. Although periostial hemocyte aggregation is an immune response that occurs following infection with bacteria and malaria parasites, the molecular basis of this process remains poorly understood. Here, we show that the thioester-containing proteins, TEP1, TEP3 and TEP4 are positive regulators of periostial hemocyte aggregation in the African malaria mosquito, Anopheles gambiae. RNAi-based knockdown of TEP1, TEP3 and TEP4 resulted in fewer periostial hemocytes following Escherichia coli infection, without affecting the adjacent population of non-periostial, sessile hemocytes. Moreover, knockdown of TEP1, TEP3 and TEP4 expression resulted in reduced bacterial accumulation and melanin deposition at the periostial regions. Finally, this study confirmed the role that TEP1 plays in reducing infection intensity in the hemocoel. Overall, this research shows that the complement-like proteins, TEP1, TEP3 and TEP4, are positive regulators of the functional integration between the immune and circulatory systems of mosquitoes.

Insect heart rhythmicity is modulated by evolutionarily conserved neuropeptides and neurotransmitters.

Insects utilize an open circulatory system to transport nutrients, waste, hormones and immune factors throughout the hemocoel. The primary organ that drives hemolymph circulation is the dorsal vessel, which is a muscular tube that traverses the length of the body and is divided into an aorta in the head and thorax, and a heart in the abdomen. The dorsal vessel is myogenic, but its rhythmicity is modulated by neuropeptides and neurotransmitters. This review summarizes how neuropeptides such as crustacean cardioactive peptide (CCAP), FMRFamide-like peptides, proctolin, allatotropin and allatostatin modulate the heart contraction rate and the directionality of heart contractions. Likewise, it discusses how neurotransmitters such as serotonin, octopamine, glutamate and nitric oxide influence the heart rate, and how transcriptomic and proteomic approaches are advancing our understanding of insect circulatory physiology. Finally, this review argues that the immune system may modulate heart rhythmicity, and discusses how the myotropic activity of cardioactive factors extends to the accessory pulsatile organs, such as the auxiliary hearts of the antennae.

Mosquito Hemocytes Associate With Circulatory Structures That Support Intracardiac Retrograde Hemolymph Flow.

A powerful immune system protects mosquitoes from pathogens and influences their ability to transmit disease. The mosquito's immune and circulatory systems are functionally integrated, whereby intense immune processes occur in areas of high hemolymph flow. The primary circulatory organ of mosquitoes is the dorsal vessel, which consists of a thoracic aorta and an abdominal heart. In adults of the African malaria mosquito, Anopheles gambiae, the heart periodically alternates contraction direction, resulting in intracardiac hemolymph flowing toward the head (anterograde) and toward the posterior of the abdomen (retrograde). During anterograde contractions, hemolymph enters the dorsal vessel through ostia located in abdominal segments 2-7, and exits through an excurrent opening located in the head. During retrograde contractions, hemolymph enters the dorsal vessel through ostia located at the thoraco-abdominal junction, and exits through posterior excurrent openings located in the eighth abdominal segment. The ostia in abdominal segments 2 to 7-which function in anterograde intracardiac flow-are sites of intense immune activity, as a subset of hemocytes, called periostial hemocytes, respond to infection by aggregating, phagocytosing, and killing pathogens. Here, we assessed whether hemocytes are present and active at two sites important for retrograde intracardiac hemolymph flow: the thoraco-abdominal ostia and the posterior excurrent openings of the heart. We detected sessile hemocytes around both of these structures, and these hemocytes readily engage in phagocytosis. However, they are few in number and a bacterial infection does not induce the aggregation of additional hemocytes at these locations. Finally, we describe the process of hemocyte attachment and detachment to regions of the dorsal vessel involved in intracardiac retrograde flow.