Thermal effect of blood feeding in the telmophagous fly Glossina morsitans morsitans

During feeding on warm-blooded hosts, haematophagous insects are exposed to thermal stress due to the ingestion of a meal which temperature may highly exceed their own body temperature. In order to avoid overheating and its subsequent deleterious effects, these insects respond by setting up molecular protective mechanisms such as heat shock proteins synthesis or by using thermoregulative strategies. Moreover, the duration of contact with the host depends on the way of feeding displayed by the different species (either telmophagous or solenophagous) and thus also impacts their exposure to heat. Solenophagous insects feed directly on blood vessels and are relatively slow feeders while telmophagous insects by lacerating capillaries, facilitate their access to blood and thus feed more quickly. The aim of this work was to investigate to what extent strictly telmophagous insects such as tsetse flies are exposed to thermal stress during feeding and consequently to evaluate the impact of the feeding strategy on the exposition to overheating in haematophagous insects in general. Real time thermographic analysis during feeding revealed that the flies’ body significantly heat up quite homogeneously. At the end of feeding, however, a marked regional heterothermy occurs as a consequence of the alary muscles warm up that precedes take-off. Feeding strategies, either solenophagy or telmophagy, thus appear to have a great impact on both exposition to predation risks and to thermal stress.     

吸血昆虫生殖调控的分子机制

吸血昆虫是可以传播病原微生物的一类节肢动物,包括蚊虫、白蛉、蠓、猎蝽、跳蚤等。由于其特殊的吸血习性,它们成为了疟疾、登革热、丝虫病、锥虫病等急性传染性疾病的媒介载体。虫媒疾病具有传播速度快、扩散面积广和危害重等特点,不仅严重危害人类健康,还容易造成巨大的经济损失。由于针对虫媒传染病的药物匮乏以及虫媒病原对化学药物抗性的不断增加,阻断吸血昆虫的生殖成为控制虫媒疾病传播的有效措施。保幼激素(juvenile hormone, JH)和20-羟基蜕皮激素(20-hydroxyecdysone, 20E)在昆虫生殖过程中扮演着重要的角色。JH与胞内受体复合物Met/Tai结合后调控JH/Met靶基因表达,进而促进卵黄发生过程,为昆虫之后的吸血及产卵提供了必要条件;20E胞内受体为EcR/USP组成的异源二聚体,两者结合后激活下游基因表达,诱导卵黄原蛋白(vitellogenin, Vg)合成,为发育的卵巢提供营养。营养信号通路(胰岛素信号通路以及氨基酸介导的雷帕霉素靶蛋白信号通路)同样可以激活Vg合成,促进昆虫生殖;此外,营养信号通路与JH和20E之间可以相互作用共同调控吸血昆虫发育和繁殖。碳水化合物代谢以及脂代谢等能量代谢过程是昆虫生殖过程中主要能量来源,可以满足吸血昆虫生殖发育不同阶段极高的能量需求。研究表明,JH和20E信号通路在能量代谢过程中起着重要的调控作用;微小RNA在蚊虫这一类吸血昆虫中被证明与肠道微生物稳态、血液消化以及脂代谢等生理学过程密切相关,进一步影响了蚊虫卵巢发育。近年来,随着分子生物学及测序技术的革新,吸血昆虫生殖调控机制的研究不断取得新的进展。本文主要阐述了吸血昆虫生殖调控的分子机制研究进展,以期为通过调控吸血昆虫生殖的方法以阻断病原传播提供重要线索。     

Aedes aegypti ferritin.

Diseases transmitted by hematophagous (blood-feeding) insects are responsible for millions of human deaths worldwide. In hematophagous insects, the blood meal is important for regulating egg maturation. Although a high concentration of iron is toxic for most organisms, hematophagous insects seem unaffected by the iron load in a blood meal. One means by which hematophagous insects handle this iron load is, perhaps, by the expression of iron-binding proteins, specifically the iron storage protein ferritin. In vertebrates, ferritin is an oligomer composed of two types of subunits called heavy and light chains, and is part of the constitutive antioxidant response. Previously, we found that the insect midgut, a main site of iron load, is also a primary site of ferritin expression and that, in the yellow fever mosquito, Aedes aegypti, the expression of the ferritin heavy-chain homologue (HCH) is induced following blood feeding. We now show that the expression of the Aedes ferritin light-chain homologue (LCH) is also induced with blood-feeding, and that the genes of the LCH and HCH are tightly clustered. mRNA levels for both LCH- and HCH-genes increase with iron, H2O2 and hemin treatment, and the temporal expression of the genes is very similar. These results confirm that ferritin could serve as the cytotoxic protector in mosquitoes against the oxidative challenge of the bloodmeal. Finally, although the Aedes LCH has no iron responsive element (IRE) at its 5'-untranslated region (UTR), the 5'-UTR contains several introns that are alternatively spliced, and this alternative splicing event is different from any ferritin message seen to date.     

Size‐dependent insect flight energetics at different sugar supplies

Under most circumstances, large body size confers a higher fitness and is positively selected, whereas selection against large size is empirically poorly documented. Physiologically, according to the ¾ power law, larger animals have lower relative but higher absolute energy demands, such that large body size may become disadvantageous, particularly under fast locomotion in food‐limited environments. After a period of initial feeding on different sugar concentrations, we investigated size‐dependent energy content (reserves) at baseline and of females unflown (i.e. resting) or flown for 18 h in two (replicate) insect species: the yellow dung fly Scathophaga stercoraria and the yellow fever mosquito Aedes aegypti. Tethered adults of various sizes were tested in a flight mill. In both species, teneral glycogen, sugar, and lipid content increased with sugar availability, and isometrically or even hyper‐allometrically (slope > 1) with body size. Activity treatment also revealed the expected consumption effects. Both species increased their flight distance with sugar supply, although only larger mosquitoes flew longer. Crucially, larger females of both species disproportionately exhausted more glycogen and sugars (but not lipid) during flight. The mosquitoes appeared to adjust their flight more finely to their size‐dependent energy reserves at all sugar availabilities, whereas, in the dung flies, size‐dependent energy demands were detectable only with a low but not with an overly high sugar supply. Although we found a greater absolute and relative locomotory energy demand for the larger flies, which is in agreement with interspecific patterns in insects, this was (more than) compensated by their greater baseline energy reserves, resulting in the greater net flying capacity of larger individuals. Consequently, we found no evidence for energetic mechanisms limiting the performance of large flying insects under food limitation. The differences between the two species presumably relate to mosquitoes inherently being long distance flyers and dung flies being short distance flyers. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ●● , ●●–●●.     

The effects of blood feeding and exogenous supply of tryptophan on the quantities of xanthurenic acid in the salivary glands of Anopheles stephensi (Diptera: Culicidae)

Xanthurenic acid (XA), produced as a byproduct during the biosynthesis of insect eye pigment (ommochromes), is a strong inducer of Plasmodium gametogenesis at very low concentrations. In previous studies, it was shown that XA is present in Anopheles stephensi (Diptera: Culicidae) mosquito salivary glands and that during blood feeding the mosquitoes ingested their own saliva into the midgut. Considering these two facts together, it is therefore likely that XA is discharged with saliva during blood feeding and is swallowed into the midgut where it exerts its effect on Plasmodium gametocytes. However, the quantities of XA in the salivary glands and midgut are unknown. In this study, we used high performance liquid chromatography with electrochemical detection to detect and quantify XA in the salivary glands and midgut. Based on the results of this study, we found 0.28+/-0.05 ng of XA in the salivary glands of the mosquitoes, accounting for 10% of the total XA content in the mosquito whole body. The amounts of XA in the salivary glands reduced to 0.13+/-0.06 ng after mosquitoes ingested a blood meal. Approximately 0.05+/-0.01 ng of XA was detected in the midgut of nonblood fed An. stephensi mosquitoes. By adding synthetic tryptophan as a source of XA into larval rearing water (2 mM) or in sugar meals (10 mM), we evaluated whether XA levels in the mosquito (salivary glands, midgut, and whole body) were boosted and the subsequent effect on infectivity of Plasmodium berghei in the treated mosquito groups. A female specific increase in XA content was observed in the whole body and in the midgut of mosquito groups where tryptophan was added either in the larval water or sugar meals. However, XA in the salivary glands was not affected by tryptophan addition to larval water, and surprisingly it reduced when tryptophan was added to sugar meals. The P. berghei oocyst loads in the mosquito midguts were lower in mosquitoes fed tryptophan treated sugar meals than in mosquitoes reared on tryptophan treated larval water. Our results suggest that mosquito nutrition may have a significant impact on whole body and midgut XA levels in mosquitoes. We discuss the observed parasite infectivity results in relation to XA's relationship with malaria parasite development in mosquitoes.     

Constitutive and blood meal-induced trypsin genes in Lutzomyia longipalpis

Trypsins constitute some of the most abundant midgut digestive proteases expressed by hematophagous insects upon blood feeding. In addition to their role in the digestion of the blood meal, these proteases also have been implicated in the ability of certain pathogens to infect their natural vector. In sand flies, digestive proteases including trypsins were associated with early killing of Leishmania and are believed to play a role in the species-specificity dictating sand fly vectorial capacity. Our group is involved in studies of midgut digestive proteases in the sand fly Lutzomyia longipalpis, the principal vector of visceral leishmaniasis in Brazil. Here we report on the identification of two cDNAs, Lltryp1 and Lltryp2, which code for putative midgut trypsins in L. longipalpis. Analyses of RNA abundance using semi-quantitative RT-PCR show a different pattern of expression between the two genes. Lltryp1 expression remains undetected until blood feeding and reaches a peak at 12 h post-blood meal (PBM), returning to pre-blood meal levels at 72 h PBM. Additionally, Lltryp1 expression is undetected during larval development. Lltryp2, on the other hand, is constitutively expressed as high levels in the non-blood fed female, but is reduced upon blood feeding. At the end of the digestive cycle, Lltryp2 regains its pre-blood meal levels. This cDNA also is present in all developmental stages and in adult males. This pattern of expression is reminiscent of what is seen in mosquitoes and Old World sand flies, but has characteristics that are unique to L. longipalpis.     

The Plasmodium parasite—a ‘new’ challenge for insect innate immunity

Though lacking adaptive immunity, insects possess a powerful innate immune system, a genome-encoded defence machinery used to confront infections. Studies in the fruit fly Drosophila melanogaster revealed a remarkable capacity of the innate immune system to differentiate between and subsequently respond to different bacteria and fungi. However, hematophagous compared to non-hematophagous insects encounter additional blood-borne infectious agents, such as parasites and viruses, during their lifetime. Anopheles mosquitoes become infected with the malaria parasite Plasmodium during feeding on infected human hosts and may then transmit the parasite to new hosts during subsequent bites. Whether Anopheles has developed mechanisms to confront these infections is the subject of this review. Initially, we review our current understanding of innate immune reactions and give an overview of the Anopheles immune system as revealed through comparative genomic analyses. Then, we examine and discuss the capacity of mosquitoes to recognize and respond to infections, especially to Plasmodium, and finally, we explore approaches to investigate and potentially utilize the vector immune competence to prevent pathogen transmission. Such approaches constitute a new challenge for insect immunity research, a challenge for global health.     

The Plasmodium parasite--a 'new' challenge for insect innate immunity

Though lacking adaptive immunity, insects possess a powerful innate immune system, a genome-encoded defence machinery used to confront infections. Studies in the fruit fly Drosophila melanogaster revealed a remarkable capacity of the innate immune system to differentiate between and subsequently respond to different bacteria and fungi. However, hematophagous compared to non-hematophagous insects encounter additional blood-borne infectious agents, such as parasites and viruses, during their lifetime. Anopheles mosquitoes become infected with the malaria parasite Plasmodium during feeding on infected human hosts and may then transmit the parasite to new hosts during subsequent bites. Whether Anopheles has developed mechanisms to confront these infections is the subject of this review. Initially, we review our current understanding of innate immune reactions and give an overview of the Anopheles immune system as revealed through comparative genomic analyses. Then, we examine and discuss the capacity of mosquitoes to recognize and respond to infections, especially to Plasmodium, and finally, we explore approaches to investigate and potentially utilize the vector immune competence to prevent pathogen transmission. Such approaches constitute a new challenge for insect immunity research, a challenge for global health.     

Blood,sweat, and tears: a review of the hematophagous,sudophagous, and lachryphagous Lepidoptera

Although adult Lepidoptera are not often considered medically relevant, some butterflies and moths are notorious for their consumption of mammalian body fluids. These Lepidoptera can be blood‐feeding (hematophagous), tear‐feeding (lachryphagous), or sweat‐feeding (we use the term “sudophagous”). Blood‐feeding Lepidoptera have been observed piercing the skin of their hosts during feeding, while tear‐feeding Lepidoptera have been observed frequenting the eyes of hosts in order to directly obtain lachrymal fluid. These behaviors have negative human health implications and some potential for disease transmission. In this study, articles concerning feeding behavior of blood, sweat, and tear‐feeding Lepidoptera were reviewed, with emphasis on correlations between morphological characters and feeding behaviors. Harmful effects and vector potential of these Lepidoptera are presented and discussed.     

The impact of mating and sugar feeding on blood-feeding physiology and behavior in the arbovirus vector mosquito Aedes aegypti

BackgroundAedes aegypti mosquitoes are globally distributed vectors of viruses that impact the health of hundreds of millions of people annually. Mating and blood feeding represent fundamental aspects of mosquito life history that carry important implications for vectorial capacity and for control strategies. Females transmit pathogens to vertebrate hosts and obtain essential nutrients for eggs during blood feeding. Further, because host-seeking Ae. aegypti females mate with males swarming near hosts, biological crosstalk between these behaviors could be important. Although mating influences nutritional intake in other insects, prior studies examining mating effects on mosquito blood feeding have yielded conflicting results.Methodology/Principal findingsTo resolve these discrepancies, we examined blood-feeding physiology and behavior in virgin and mated females and in virgins injected with male accessory gland extracts (MAG), which induce post-mating changes in female behavior. We controlled adult nutritional status prior to blood feeding by using water- and sugar-fed controls. Our data show that neither mating nor injection with MAG affect Ae. aegypti blood intake, digestion, or feeding avidity for an initial blood meal. However, sugar feeding, a common supplement in laboratory settings but relatively rare in nature, significantly affected all aspects of feeding and may have contributed to conflicting results among previous studies. Further, mating, MAG injection, and sugar intake induced declines in subsequent feedings after an initial blood meal, correlating with egg production and laying. Taking our evaluation to the field, virgin and mated mosquitoes collected in Colombia were equally likely to contain blood at the time of collection.Conclusions/SignificanceMating, MAG, and sugar feeding impact a mosquito’s estimated ability to transmit pathogens through both direct and indirect effects on multiple aspects of mosquito biology. Our results highlight the need to consider natural mosquito ecology, including diet, when assessing their physiology and behavior in the laboratory.     

Haemolymph monophenol oxidase activity in Armigeres subalbatus infected with Brugia pahangi.

The activity of monophenol oxidase can be elicited in the haemolymph of Armigeres subalbatus by both blood and filaria-infected blood feeding. Haemolymph collected from both blood-fed and filaria-infected mosquitoes was investigated using a quantitative radiometric assay that measured the amount of tritiated water formed during the hydroxylation of L-[3,5-3H]tyrosine to dopa. Enzyme activity in filaria-infected mosquitoes was found to be significantly lower than that found in the blood-fed mosquitoes within 3 days post-ingestion, but still remained measurable 72 h post-ingestion. The decreased enzyme activity coincided in time with the development of capsules around the microfilariae. The consumption of monophenol oxidase by the melanization of migrating microfilariae in the haemocoel of filaria-infected mosquitoes and the effects of excretory and secretory products of developing larvae on monophenol oxidase activity are suggested.     

Dynamics of lipid accumulation by the fat body of Rhodnius prolixus: the involvement of lipophorin binding sites

In insects, lipids are stored in the fat body, mainly as triacylglycerol (TAG). In Rhodnius prolixus, a hematophagous hemipteran, lipids are accumulated after blood meal to be used later on. In adult females, at the second day after feeding, the amount of TAG was 57+/-17 microg/fat body, it increased almost five times and at fourth day it was 244+/-35 microg/fat body. TAG content remained constant until day 13, but it then decreased and, at day 20th it was very low (31+/-4.9 microg/fat body). Radiolabeled free fatty acid was used to follow lipid accumulation by the fat body, as it was previously shown that, in R. prolixus, injected free fatty acids associate with lipophorin, a major hemolymphatic lipoprotein. (3)H-palmitic acid was injected into the hemocoel of R. prolixus females. It disappeared from the hemolymph very rapidly, and radioactivity was incorporated by the fat body. Sixty minutes after injection, radioactivity in the fat body was found mainly in TAGs. The capacity of the fat body to incorporate fatty acids from the hemolymph varied according to the days after blood meal, and it was maximal around the fourth day. Lipophorin binding to specific sites in fat body membrane preparations also showed variation at different days. When membranes obtained from insects at the second, fifth and tenth days were compared, binding was highest at fifth day after feeding.     

Intravital microscopy and image analysis of Rhodnius prolixus (Hemiptera: Reduviidae) hematophagy: The challenge of blood intake from mouse skin

Hematophagous insects transmit many of the most dangerous parasitic diseases. The transmission usually occurs during hematophagy or just after as this is when the vector and the host are in contact. The contact time is determined by the feeding performance of the insect in each host. In triatomines, feeding performance interferes with both their life cycle and the vectorial competence to transmit the hemoflagellate Trypanosoma cruzi. Triatomine bugs are vessel feeders, obtaining their blood meals directly from the vessels (venules or arterioles) of their vertebrate hosts. The host blood intake rate is not constant during the feeding, and the sucking frequency of triatomines tends to be higher and to contain fewer interruptions in pigeons than in mice. To identify the difficulties encountered by triatomine bugs in obtaining blood meals from mouse skin, we used intravital microscopy techniques associated with electromyograms of the cibarial pump. To monitor the vibration of the cannulated vessels and the blood flow through the head of the insect during the engorgement phase, we introduced a novel method for image analysis. The mean number of vessels used during a Rhodnius prolixus blood meal was 3.4 ± 1.2, and the insects fed more in venules (63%) than in arterioles (37%). An important increase in vascular permeability was observed throughout the feeding. Platelet aggregation, rolling and leukocyte adherence were analyzed on the venular endothelium, showing remarkable increases for some time following the R. prolixus feeding. The reduction in sucking frequency that was observed during insect feeding was likely due to the increased cibarial pump filling time. The monitoring of the vessel wall pulsation also permitted the registration of regurgitation-like movements during blood pumping, with these movements being recorded mostly during the second half of the feeding. The evaluation of blood flow through the head of the insect suggested that the regurgitation-like movements were not true regurgitations and were caused by abrupt difficulties in the function of the cibarial pump. The role of the platelet plugs and the changes in blood viscosity at the R. prolixus feeding site are discussed. The method introduced in the present study to analyze the images brings new insights into the interaction between hematophagous vectors and their hosts, reinforcing the importance of insect saliva throughout the feeding process.     

Malaria Mosquitoes Host-Locate and Feed upon Caterpillars

Adult female mosquitoes need blood to develop their eggs and both sexes use nectar and honeydew as carbohydrate resources for flight, survival and to enhance reproduction. However, there are also a few reports in the literature of mosquitoes feeding on haemolymph of soft-bodied insects such as caterpillars. The frequency and significance of this entomophagous behavior is not well understood, but is thought to be a vestige of ancestral feeding behavior or an opportunistic behavior that has evolved over time. In our current paper we investigated the extent to which the malaria mosquito, Anopheles stephensi, is attracted to, and can successfully feed on, larvae of two common moth species, Manduca sexta and Heliothis subflexa. Using y-tube olfactometer assays we found that female An. stephensi readily flew upwind to and landed on the caterpillars of both moth species. The nature of the volatile cues used in host location remains unclear but respirometer studies suggest a possible role of CO₂. Laboratory cage assays further showed that the female mosquitoes were able to actively feed on moth larvae and gain sufficient nutritional benefit to influence survival. The extent to which such an opportunistic behavior occurs in the field has yet to be explored but our results suggest that this haemolymph feeding behavior could play a role in malaria mosquito life history and could provide a novel mechanism for horizontal transmission of pathogens and other micro-organisms between hosts.     

Discovery of Mosquito Saliva MicroRNAs during CHIKV Infection

Mosquito borne pathogens are transmitted to humans via saliva during blood feeding. Mosquito saliva is a complex concoction of many secretory factors that modulate the feeding foci to enhance pathogen infection and establishment. Multiple salivary proteins/factors have been identified/characterized that enhance pathogen infection. Here, we describe, for the first time, the identification of exogenous microRNAs from mosquito saliva. MicroRNAs are short, 18–24 nucleotide, non-coding RNAs that regulate gene expression, and are generally intracellular. However, circulating miRNAs have been described from serum and saliva of humans. Exogenous miRNAs have not been reported from hematophagous arthropod saliva. We sought to identify miRNAs in the mosquito saliva and their role in Chikungunya virus (CHIKV) infection. Next generation sequencing was utilized to identify 103 exogenous miRNAs in mosquito saliva of which 31 miRNAs were previously unidentified and were designated novel. Several miRNAs that we have identified are expressed only in the CHIKV infected mosquitoes. Five of the saliva miRNAs were tested for their potential to regulated CHIKV infection, and our results demonstrate their functional role in the transmission and establishment of infection during blood feeding on the host.     

Is the expression of autogeny by Culex molestus Forskal (Diptera: Culicidae) influenced by larval nutrition or by adult mating,sugar feeding,or blood feeding?

Culex molestus Forskal is suspected to have been introduced into southern Australia during the 1940s. Investigations to determine factors influencing the expression of autogeny, the response of this mosquito to potential blood meals, and the subsequent influence on oviposition were undertaken. Immature mosquitoes raised at five feeding regimes had mortality rates, development rates, wing length, and autogenous egg raft size measured. All surviving female mosquitoes laid autogenous eggs but there was a significant difference between the mean number of eggs per raft. For mosquitoes raised at each of the feeding regimes, there was a significant linear relationship between the number of eggs per autogenous egg raft and wing length. Newly emerged mosquitoes were offered a blood meal (i.e., rodent) daily but no blood feeding occurred until the autogenous egg raft was laid. There was no statistical difference in the rate of autogenous oviposition or post-oviposition blood feeding between control or treatment groups. The results of this study indicate that Cx. molestus is perfectly adapted to subterranean habitats in close association with human habitation, but their preference to delay blood feeding until up to day 8 following emergence may reduce their relative importance as a vector of arboviruses.     

Interactions of dietary protein and carbohydrate determine blood sugar level and regulate nutrient selection in the insect Manduca sexta L

The non-homeostatic regulation of blood sugar concentration in the insect Manduca sexta L. was affected by nutritional status. Larvae maintained on diets lacking sucrose displayed low concentrations of trehalose, the blood sugar of insects, which varied from 5 to 15 mM with increasing dietary casein level between 12.5 and 75 g/l. These insects were glucogenic, as demonstrated by the selective 13C enrichment of trehalose synthesized from [3-13C]alanine, and de novo synthesis was the sole source of blood sugar. The distribution of 13C in glutamine established that following transamination of the 13C substituted substrate, [3-13C]pyruvate carboxylation rather than decarboxylation was the principal pathway of Pyr metabolism. The mean blood trehalose level was higher in insects maintained on diets with sucrose. At the lowest dietary casein level blood trehalose was approximately 50 mM, and declined to 20 mM at the highest casein level. Gluconeogenesis was detected in insects maintained on sucrose-free diets at the higher protein levels examined, but [3-13C]pyruvate decarboxylation and TCA cycle metabolism was the principal fate of [3-13C]alanine following transamination, and dietary carbohydrate was the principal source of glucose for trehalose synthesis. Feeding studies established a relationship between nutritional status, blood sugar level and dietary self-selection. Insects preconditioned by feeding on diets without sucrose had low blood sugar levels regardless of dietary casein level, and when subsequently given a choice between a sucrose diet or a casein diet, selected the former. Larvae preconditioned on a diet containing sucrose and the lowest level of casein had high blood sugar levels and subsequently selected the casein diet. Larvae maintained on the sucrose diet with the highest casein level had low blood sugar and self-selected the sucrose diet. When preconditioned on diets with sucrose and intermediate levels of casein, insects selected more equally between the sucrose and the casein diets. It is concluded that blood sugar level may be intimately involved in dietary self-selection by M. sexta larvae, and that in the absence of dietary carbohydrate, gluconeogenesis provides sufficient blood sugar to ensure that larvae choose a diet or diets that produce an optimal intake of dietary protein and carbohydrate.