Mosquitoes cool down during blood feeding to avoid overheating

Temperature is one of the most important factors affecting the life of insects [1]. For instance, high temperatures can have deleterious effects on insects' physiology. Therefore, many of them have developed various strategies to avoid the risk of thermal stress [2]. They can seek a fresher environment or adjust their water loss, but hematophagous insects, such as mosquitoes, must confront the issue of thermal stress at each feeding event on a warm-blooded host [3]. To better understand to what extent mosquitoes are exposed to thermal stress while feeding, we conducted a real-time infrared thermographic analysis of mosquitoes' body temperature during feeding on both warm blood and sugar solution. First, our results highlighted differences in temperature between the body parts of the mosquito (i.e., heterothermy) during blood intake, but not during sugar meals. We also found that anopheline mosquitoes can decrease their body temperature during blood feeding thanks to evaporative cooling of fluid droplets, which are excreted and maintained at the end of the abdomen. This mechanism protects the insect itself, probably as well as the sheltered microorganisms, both symbionts and parasites, from thermal stress. These findings constitute the first evidence of thermoregulation among hematophagous insects and explain the paradox of fresh blood excretion during feeding.     

State-dependency of host-seeking in Rhodnius prolixus: The post-ecdysis time

The source of blood of most haematophagous insects plays at the same time the double role of host and potential predator. Feeding behaviour should be triggered only when necessary and should be completed as quickly as possible. From an epidemiological point of view, this modulation has an impact on the feeding frequency of disease vectors and, as a consequence, on the transmission of parasites. At present, not many data are available on the influence of the physiological state on the motivation to feed, and mostly limited to a few mosquito species. We analyzed the host-seeking behaviour of Rhodnius prolixus as a function of the time elapsed since the ecdysis, by testing the response of larvae to a blood source, and long- (CO₂) and short-range (heat) orientation cues associated to their vertebrate hosts. Our experiments demonstrated that during the first days following the ecdysis insects do not respond to any stimuli. The ability to follow chemical and physical cues increases either gradually (heat) or step-wise (CO₂) with post-ecdysis time. A few insects started to feed on day 2, but only at day 7 following the ecdysis 50% of them took a blood meal, to reach the highest motivation to feed on day 10. The reasons for the “maturation period” in feeding behaviour of R. prolixus are discussed.     

Aging affects expression of 70-kDa heat shock proteins in Drosophila

We examined the effect of cellular aging on adult mortality and hsp70 gene expression in Drosophila melanogaster under thermal stress. The results showed that flies exposed to 37 degrees C for various time intervals had reduced survival rate with age. The level of hsp70 mRNA increases in flies up to 23-28 days of age, but then declines as they get older. When flies are shifted to 25 degrees C after 30 min of heat stress, the time-dependent decrease in hsp70 mRNA levels occurs more rapidly in young flies than in old ones. The hsp70 mRNA present during this recovery period is translated into protein, and senescent flies continue to synthesize this protein for up to 5 h after heat shock. The prolonged expression of hsp70 RNA during recovery from heat shock was also observed in young flies fed canavanine, an arginine analogue. These data suggest that in old insects, the accumulation of conformationally altered proteins plays a role in the regulation of hsp70 RNA expression. These results are discussed in relation to the finding that old flies are more sensitive to thermal stress than young ones.     

Modelling the effect of feeding-related mortality on the feeding strategy of tsetse (Diptera: Glossinidae)

Abstract. Free-living haematophagous insects risk death through host grooming responses or through increased susceptibility to predation whenever they take a bloodmeal. In this paper we investigate the effects of these risks on the feeding strategy of tsetse. A model is presented that allows for death of tsetse by starvation if they do not succeed in feeding within a fixed time (set at 6 days in the first instance) and for mortality specifically associated with feeding. In addition there is background mortality that applies to all flies at all times.
The model is used to compute the individual life-time fertility (number of female puparia per female) as a function of the probability of obtaining a meal (indicated by field data to be very high, usually > 0.85 per day) and the day on which flies start to search for a meal. We suggest that the feeding strategy that would be selected for is that which allows the maximum reproductive output. The model shows that this strategy involves making no attempts to feed for 3–4 days after the previous meal and then attempting to feed with the greatest possible probability until a meal is obtained. The predicted feeding interval, obtained independently of any trapping data, agrees closely with all previous estimates from field studies using a variety of methods. Preliminary results from a laboratory experiment reveal an increased risk of predation of recently fed as compared with hungry tsetse. The lower the actual feeding mortality the more frequently will flies be able to feed should conditions so demand. It is adaptive, however, for tsetse to delay attempting to feed for as long as they can, which is made possible by the near certainty of locating and feeding on a host within 1 day, using their sophisticated sensory systems.     

Host-foraging stable flies,Stomoxys calcitrans,are preferentially attracted to objects with both visual and thermal host-like characteristics

Many haematophagous insects use the heat emitted by warm-blooded animals as a cue for locating suitable hosts. Blood-feeding stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae), are known to respond to visual and olfactory host cues. However, the effects of thermal host cues on the foraging behaviour of these flies remain largely unknown. Here we tested the hypothesis that host-foraging stable flies preferentially land on objects with host-like temperature, and on objects with both visual and thermal host-like cues. In laboratory bioassays, stable flies were offered a choice between paired temperature-controlled copper discs. Flies preferentially landed on the disc with a host-like temperature (40 °C), discriminating against discs that were cooler (26 or 35 °C) or warmer (50 or 60 °C) than vertebrate hosts. Flies that were well fed and thus not in foraging mode, or host-foraging flies that were offered infrared radiation but not the conductive and convective heat of different temperature discs, failed to discriminate between the stimuli. In greenhouse experiments, when flies were offered a choice between paired barrels as surrogate hosts, flies preferentially landed on barrels that were both thermally and visually appealing (38–39 °C, black), discriminating against barrels that were cold (10 °C), white, or both cold and white. Thermal cues augmented the overall landing responses of flies but their initial (mid-range) attraction to barrels was mediated by visual cues. Overall, the data suggest that thermal host cues affect the host-foraging behaviour of stable flies primarily at close range, prompting landing on a host.     

Plastic and evolutionary responses to heat stress in a temperate dung fly: negative correlation between basal and induced heat tolerance?

Extreme weather events such as heat waves are becoming more frequent and intense. Populations can cope with elevated heat stress by evolving higher basal heat tolerance (evolutionary response) and/or stronger induced heat tolerance (plastic response). However, there is ongoing debate about whether basal and induced heat tolerance are negatively correlated and whether adaptive potential in heat tolerance is sufficient under ongoing climate warming. To evaluate the evolutionary potential of basal and induced heat tolerance, we performed experimental evolution on a temperate source population of the dung fly Sepsis punctum. Offspring of flies adapted to three thermal selection regimes (Hot, Cold and Reference) were subjected to acute heat stress after having been exposed to either a hot‐acclimation or non‐acclimation pretreatment. As different traits may respond differently to temperature stress, several physiological and life history traits were assessed. Condition dependence of the response was evaluated by exposing juveniles to different levels of developmental (food restriction/rearing density) stress. Heat knockdown times were highest, whereas acclimation effects were lowest in the Hot selection regime, indicating a negative association between basal and induced heat tolerance. However, survival, adult longevity, fecundity and fertility did not show such a pattern. Acclimation had positive effects in heat‐shocked flies, but in the absence of heat stress hot‐acclimated flies had reduced life spans relative to non‐acclimated ones, thereby revealing a potential cost of acclimation. Moreover, body size positively affected heat tolerance and unstressed individuals were less prone to heat stress than stressed flies, offering support for energetic costs associated with heat tolerance. Overall, our results indicate that heat tolerance of temperate insects can evolve under rising temperatures, but this response could be limited by a negative relationship between basal and induced thermotolerance, and may involve some but not other fitness‐related traits.     

Coevolution of color pattern and thermoregulatory behavior in polymorphic pygmy grasshoppers Tetrix undulata

Ectothermic organisms, such as insects and reptiles, rely on external heat sources to control body temperature and possess physiological and behavioral traits that are temperature dependent. It has therefore been hypothesised that differences in body temperature resulting from phenotypic properties, such as color pattern, may translate into selection against thermally inferior phenotypes. We tested for costs and benefits of pale versus dark coloration by comparing the behaviors (i.e., basking duration and bouts) of pygmy grasshopper (Tetrix undulata) individuals exposed to experimental situations imposing a trade-off between temperature regulation and feeding. We used pairs consisting of two full-siblings of the same sex that represented different (genetically coded) color morphs but had shared identical conditions from the time of fertilization. Our results revealed significant differences in behavioral thermoregulation between dark and pale individuals in females, but not in males. Pale females spent more time feeding than dark females, regardless of whether feeding was associated with a risk of either hypothermia or overheating. In contrast, only minor differences in behavior (if any) were evident between individuals that belonged to the same color morph but had been painted black or gray to increase and decrease their heating rates. This suggests that the behavioral differences between individuals belonging to different color morphs are genetically determined, rather than simply reflecting a response to different heating rates. To test for effects of acclimation on behaviors, we used pairs of individuals that had been reared from hatchlings to adults under controlled conditions in either low or high temperature. The thermal regime experienced during rearing had little effect on behaviors during the experiments reported above, but significantly influenced the body temperatures selected in a laboratory thermal gradient. In females (but not in males) preferred body temperature also varied among individuals born to mothers belonging to different color morphs, suggesting that a genetic correlation exists between color pattern and temperature preferences. Collectively, these findings, at least in females, are consistent with the hypothesis of multiple-trait coevolution and suggest that the different color morphs represent alternative evolutionary strategies.     

Post-feed buzzing in the tsetse, Glossina morsitans morsitans, is an endothermic mechanism

ABSTRACT. Post-feed buzzing in Glossina morsitans morsitans Westw. causes a rise in thoracic temperature relative to the length of the buzz. As lift is proportional to the square of wing-beat frequency, which increases with temperature up to 32°C, buzzing results in an increase in the lift which the fly can produce. Heat generated by buzzing, in combination with the heat received from the host at the time of feeding, may well allow the fly to maximize lift generated in the immediate post-feeding period. Buzzing flies excrete excess water from the meal more rapidly than non-buzzing flies. It is argued that this is due to a rise in abdominal temperature. Maximized lift in the immediate post-feeding period and the rapid elimination of water from the very large blood meals taken by these flies may be expected to have strong selective advantages for the flies.     

Effects of sublethal doses of deltamethrin on the pair Glossina morsitans morsitans-Trypanosoma (Nannomonas) congolense

The presence of Trypanosoma in Glossina morsitans morsitans induced a decrease in the physiological activity which is expressed in particular by a statistically higher mortality rate of infected flies. A similar effect appears with sublethal doses of deltamethrin either in infected or non infected insects. A cumulative activity of both effects was also observed in infected and treated flies. Sublethal doses of the pyrethroid compound showed a delay in feeding, abortions and larval clampings. Moreover, deltamethrin demonstrated an intravectorial effect against Trypanosoma which can reduce the transmission ability of flies.     

Molecular thermal telemetry of free-ranging adult Drosophila melanogaster

The expression of two temperature-sensitive reporter genes, hsp70 and an hsp70-LacZ fusion, in free-ranging adult Drosophila melanogaster indicates that natural thermal stress experienced by such small and mobile insects may be either infrequent or not severe. Levels of the heat-shock protein Hsp70, the major inducible Hsp of Drosophila, were similar in most wild Droso- phila captured after warm days to levels previously reported for unstressed flies in the laboratory. In a transgenic strain transformed with an hsp70-LacZ fusion (i.e., the structural gene encoding bacterial β-galactosidase under control of a heat shock promoter), exposure to temperatures ≥32°C in the laboratory typically resulted in β-galactosidase activities exceeding 140 mOD₄₅₀ h^–1μg^–1 soluble protein. Flies caged in sun frequently had β-galactosidase activities in excess of this level, whereas flies caged in shade and flies released and recaptured on cool days did not. Most flies (>80%) released on warm, sunny days had low β-galactosidase activities upon recapture. Although the balance of recaptured flies had elevated β-galactosidase activities on these days, their β-galactosidase activities were <50% of levels for flies caged in direct sunlight or exposed to laboratory heat shock. These data suggest that even on warm days most flies may avoid thermal stress, presumably through microhabitat selection, but that a minority of adult D. melanogaster undergo mild thermal stress in nature. Both temperature-sensitive reporter genes, however, are limited in their ability to infer thermal stress and demonstrate its absence. Received: 14 July 1999 / Accepted: 21 December 1999     

Thermoregulatory mechanisms in Sarcophaga

Summary The flesh fly, Sarcophaga, is frequently seen feeding on flowers during periods of high radiation when other flies of comparable size avoid exposure because of the dangers of overheating. Sarcophaga is able to maintain its intermittent flower visits due to a cuticle of high thermal reflectance, giving low intrinsic heating rates, and to an ability to shunt blood between thorax and abdomen according to its needs. The fly thus achieves partial thermoregulation and can keep its body temperature within the preferred range for longer periods than its potential entomophilous competitors.     

Investigation of the bacterial communities associated with females of Lutzomyia sand fly species from South america

Phlebotomine sand flies are vectors of Leishmania that are acquired by the female sand fly during blood feeding on an infected mammal. Leishmania parasites develop exclusively in the gut lumen during their residence in the insect before transmission to a suitable host during the next blood feed. Female phlebotomine sand flies are blood feeding insects but their life style of visiting plants as well as animals, and the propensity for larvae to feed on detritus including animal faeces means that the insect host and parasite are exposed to a range of microorganisms. Thus, the sand fly microbiota may interact with the developing Leishmania population in the gut. The aim of the study was to investigate and identify the bacterial diversity associated with wild adult female Lutzomyia sand flies from different geographical locations in the New World. The bacterial phylotypes recovered from 16S rRNA gene clone libraries obtained from wild caught adult female Lutzomyia sand flies were estimated from direct band sequencing after denaturing gradient gel electrophoresis of bacterial 16 rRNA gene fragments. These results confirm that the Lutzomyia sand flies contain a limited array of bacterial phylotypes across several divisions. Several potential plant-related bacterial sequences were detected including Erwinia sp. and putative Ralstonia sp. from two sand fly species sampled from 3 geographically separated regions in Brazil. Identification of putative human pathogens also demonstrated the potential for sand flies to act as vectors of bacterial pathogens of medical importance in addition to their role in Leishmania transmission.     

Influence of diet composition on feeding and water excretion by the tsetse fly, Glossina morsitans

Adult Glossina morsitans fed on aqueous salt solutions containing phagostimulant ATP in an in vitro feeding system gave an optimal feeding response only over a narrow pH range equivalent to that of vertebrate blood. There was much less discrimination on the basis of molar concentration.The rate and extent of water excretion by the fly was found to depend on the concentration of Na⁺ ions in the food medium: an active transport mechanism is indicated which enables water to pass from the meal through the anterior midgut wall and into the haemocoele. A favourable osmotic gradient assisted water transport in the presence of Na⁺ ions: the system could not operate efficiently in the presence of Na⁺ ions if the osmotic pressure of the food medium was higher than that of vertebrate blood, nor could it operate efficiently in any solution lacking Na⁺ ions.Normal transfer of a meal from the crop to the anterior midgut occurred only when the food medium was isotonic with vertebrate blood or in the presence of Na⁺ ions if hypotonic. Normal transfer of isotonic solutions was prevented in the presence of excess K⁺ ions, and hypertonic solutions were not transferred normally even in the presence of Na⁺ ions. Thus the rate of water excretion was reduced.Tsetse flies fed on blood in an in vitro feeding system excreted water at a significantly lower rate than flies fed on a living animal. Evidence suggests that this is due to a combined effect of changes in viscosity, effective ionic composition, and osmotic pressure, upon the normal rate and extent of food uptake and manipulation of the meal prior to digestion. The implications of this are discussed in terms of future developments of in vitro feeding techniques for haematophagous insects.     

Repurposing the orphan drug nitisinone to control the transmission of African trypanosomiasis

Tsetse transmit African trypanosomiasis, which is a disease fatal to both humans and animals. A vaccine to protect against this disease does not exist so transmission control relies on eliminating tsetse populations. Although neurotoxic insecticides are the gold standard for insect control, they negatively impact the environment and reduce populations of insect pollinator species. Here we present a promising, environment-friendly alternative to current insecticides that targets the insect tyrosine metabolism pathway. A bloodmeal contains high levels of tyrosine, which is toxic to haematophagous insects if it is not degraded and eliminated. RNA interference (RNAi) of either the first two enzymes in the tyrosine degradation pathway (tyrosine aminotransferase (TAT) and 4-hydroxyphenylpyruvate dioxygenase (HPPD)) was lethal to tsetse. Furthermore, nitisinone (NTBC), an FDA-approved tyrosine catabolism inhibitor, killed tsetse regardless if the drug was orally or topically applied. However, oral administration of NTBC to bumblebees did not affect their survival. Using a novel mathematical model, we show that NTBC could reduce the transmission of African trypanosomiasis in sub-Saharan Africa, thus accelerating current disease elimination programmes.

This study shows that tsetse flies, vectors of African trypanosomiasis, are highly susceptible to killing by nitisinone, a tyrosine catabolism inhibitor currently used to treat human metabolic diseases; this environment-friendly drug could facilitate elimination of African trypanosomiasis and other diseases transmitted by blood feeding insects.     

How do ectoparasitic nycteribiids locate their bat hosts?

Nycteribiids (Diptera: Nycteribiidae) are specific haematophagous ectoparasites of bats, which spend nearly all their adult lives on hosts. However, females have to leave bats to deposit their larva on the walls of the roosts, where they later emerge as adult flies. Nycteribiids had thus to evolve efficient sensorial mechanisms to locate hosts from a distance. We studied the sensory cues involved in this process, experimentally testing the role of specific host odours, and general cues such as carbon dioxide, body heat, and vibrations. As models we used two nycteribiids (Penicillidia conspicua and Penicillidia dufourii) and their primary bat hosts (Miniopterus schreibersii and Myotis myotis, respectively). Carbon dioxide was the most effective cue activating and orientating the responses of nycteribiids, followed by body heat and body odours. They also responded to vibration, but did not orientate to its source. In addition, sensory cues combined (carbon dioxide and body heat) were more effective in orientating nycteribiids than either cue delivered alone. Results suggest that nycteribids have some capacity to distinguish specific hosts from a distance, probably through their specific body odours. However, the strong reliance of nycteribiids on cues combined indicates that they follow these to orientate to nearby multispecies bat clusters, where the chances of finding their primary hosts are high. The combination of sensory cues seems therefore an effective strategy used by nycteribiids to locate bat hosts at a distance.     

Thermal constraints and optimization of winter feeding and habitat choice in white-tailed deer

I examined the activity and habitat choice of white-tailed deer in winter to determine how deer balance feeding in habitats which may expose them to cold with resting in sheltered habitats. Average heat gain or loss of deer for feeding, resting and walking in four habitats in each of 24 h were estimated for two winter periods (early-mid and late winter). These values were used in a dynamic optimization model to predict the hourly behavior-habitat choices which enable deer to maintain thermal homeostasis, minimize the risk of exceeding lethal body temperatures and satisfy daily energy requirements. The behavior and habitat choices observed under natural conditions were consistent with the model predictions. Deer foraged for the maximum time that was thermally possible in both sampling periods and they used exposed habitats without incurring high heat losses. Deer appeared to avoid overheating in sheltered habitats during daylight and avoid hypothermia in exposed habitats at night.     

Cloning and expression of the yolk protein of the tsetse fly Glossina morsitans morsitans

Two major families of nutritional proteins exist in insects, namely the vitellogenins and the yolk proteins. While in other insects only vitellogenins are found, cyclorraphan flies only contain yolk proteins. Possible sites of yolk protein synthesis are the fat body and the follicle cells surrounding the oocyte. We report the cloning of the yolk protein of the tsetse fly Glossina morsitans morsitans, a species with adenotrophic viviparity. The tsetse fly yolk protein could be aligned with other dipteran yolk proteins and with some vertebrate lipases. In contrast to the situation in most fly species, only a single yolk protein gene was found in the tsetse fly. Northern blot analysis showed that only the ovarian follicle cells, and not the fat body represents the site of yolk protein synthesis.     

Validity of thermal ramping assays used to assess thermal tolerance in arthropods

Proper assessment of environmental resistance of animals is critical for the ability of researchers to understand how variation in environmental conditions influence population and species abundance. This is also the case for studies of upper thermal limits in insects, where researchers studying animals under laboratory conditions must select appropriate methodology on which conclusions can be drawn. Ideally these methods should precisely estimate the trait of interest and also be biological meaningful. In an attempt to develop such tests it has been proposed that thermal ramping assays are useful assays for small insects because they incorporate an ecologically relevant gradual temperature change. However, recent model-based papers have suggested that estimates of thermal resistance may be strongly confounded by simultaneous starvation and dehydration stress. In the present study we empirically test these model predictions using two sets of independent experiments. We clearly demonstrate that results from ramping assays of small insects (Drosophila melanogaster) are not compromised by starvation- or dehydration-stress. Firstly we show that the mild disturbance of water and energy balance of D. melanogaster experienced during the ramping tests does not confound heat tolerance estimates. Secondly we show that flies pre-exposed to starvation and dehydration have "normal" heat tolerance and that resistance to heat stress is independent of the energetic and water status of the flies. On the basis of our results we discuss the assumptions used in recent model papers and present arguments as to why the ramping assay is both a valid and ecologically relevant way to measure thermal resistance in insects.     

Effects of stable flies (Diptera: Muscidae) and heat stress on weight gain and feed efficiency of feeder cattle.

Cattle respond to the feeding of stable flies, Stomoxys calcitrans (L.), by bunching to protect their front legs. This bunching can increase heat stress which indirectly accounts for much of the reduction in cattle weight gains. We used fly-screened, self-contained feedlot pens which allowed regulation of fly populations feeding on cattle. The indirect fly effects (bunching and heat stress) accounted for 71.5% of the reduced weight gain. The direct effect of the biting flies and energy loss involved in fighting flies accounted for 28.5% of the reduced weight gain.