Behavioral and postural analyses establish sleep-like states for mosquitoes that can impact interactions with hosts

Sleep is an evolutionarily conserved process that has been described in different animal systems. For insects, sleep characterization has been primarily achieved using behavioral and electrophysiological correlates in a few systems. Sleep in mosquitoes, which are important vectors of disease-causing pathogens, has not been directly examined. This is surprising as circadian rhythms, which have been well studied in mosquitoes, influence sleep in other systems. In this study, we characterized sleep in mosquitoes using body posture analysis and behavioral correlates, and quantified the effect of sleep deprivation on sleep rebound and host landing. Body and appendage position metrics revealed a clear distinction between the posture of mosquitoes in their putative sleep and awake states for multiple species, which correlates with a reduction in responsiveness to host cues. Sleep assessment informed by these posture analyses indicated significantly more sleep during periods of low activity. Nighttime and daytime sleep deprivation resulting from the delivery of vibration stimuli induced sleep rebound in the subsequent phase in day and night active mosquitoes, respectively. Lastly, sleep deprivation suppressed host landing in both laboratory and field settings when mosquitoes would normally be active. These results suggest that quantifiable sleep states occur in mosquitoes, and highlight the potential epidemiological importance of mosquito sleep.

Long-Range But Not Short-Range Attraction of Male Aedes aegypti (Diptera: Culicidae) Mosquitoes to Humans

Female Aedes aegypti (Linnaeus) mosquitoes integrate multiple sensory cues to locate human hosts for blood meals. Although male Ae. aegypti swarm around and land on humans in nature to mate, direct evidence of attraction to humans is limited. Male mosquito attraction to human host cues is often undetectable in confined laboratory assays, leading to a misconception that male mosquitoes are not attracted to humans. We used semifield experiments to demonstrate robust attraction of male Ae. aegypti to humans. Human-baited traps captured up to 25% of released males within 15 min, whereas control traps without humans as bait failed to capture males. Rapid attraction to humans was further demonstrated through videography. Males swarmed around and landed on human subjects, with no activity recorded in paired unbaited controls. Finally, we confirm the lack of discernible male attraction to humans in small laboratory cages. Our experiments demonstrate that both male and female Ae. aegypti show attraction to humans, but with clear sex-specific behavioral differences at short-range. Male mosquito attraction to humans is likely to be important for mating success in wild populations and its basis should be further explored. Our results highlight the importance of arena size and assay design for mosquito behavioral research. A better understanding of host cues that attract males could help us to improve mosquito surveillance and control.

Changes in antennal gene expression underlying sensory system maturation in Rhodnius prolixus

Background Triatomine bugs are the blood feeding insect vectors transmitting Chagas disease to humans, a neglected tropical disease that affects over 8 million people, mainly in Latin America. The behavioral responses to host cues and bug signals in Rhodnius prolixus are state dependent, i.e., they vary as a function of post-ecdysis age. At the molecular level, these changes in behavior are probably due to a modulation of peripheral and central processes. In the present study, we report a significant modulation of the expression of a large set of sensory-related genes. Results were generated by means of antennal transcriptomes of 5th instar larvae along the first week (days 0, 2, 4, 6 and 8) after ecdysis sequenced using the Illumina platform. Results Age induced significant changes in transcript abundance were established in more than 6,120 genes (54,7 % of 11,186 genes expressed) in the R. prolixus antenna. This was especially true between the first two days after ecdysis when more than 2,500 genes had their expression significantly altered. In contrast, expression profiles were almost identical between day 6 and 8, with only a few genes showing significant modulation of their expression. A total of 86 sensory receptors, odorant carriers and odorant degrading enzymes were significantly modulated across age points and clustered into three distinct expression profiles. Conclusions The set of sensory genes whose expression increased with age (profile 3) may include candidates underlying the increased responsiveness to host cues shown by R. prolixus during the first days after molting. For the first time, we describe the maturation process undergone at the molecular level by the peripheral sensory system is described in an hemimetabolous insect.

Natural variation in host-finding behaviour of gastropod parasitic nematodes (Phasmarhabditis spp.) exposed to host-associated cues

The gastropod parasitic nematode Phasmarhabditis hermaphrodita has been formulated into a successful biological control agent (Nemaslug®, strain DMG0001) used to kill slugs on farms and gardens. When applied to soil, P. hermaphrodita uses slug mucus and faeces to find potential hosts. However, there is little information on what cues other species of Phasmarhabditis (P. neopapillosa and P. californica) use to find hosts and whether there is natural variation in their ability to chemotax to host cues. Therefore, using chemotaxis assays, we exposed nine wild isolates of P. hermaphrodita, five isolates of P. neopapillosa and three isolates of P. californica to mucus from the pestiferous slug host Deroceras invadens, as well as 1% and 5% hyaluronic acid – a component of slug mucus that is highly attractive to these nematodes. We found P. hermaphrodita (DMG0010) and P. californica (DMG0018) responded significantly more to D. invadens mucus and 1% hyaluronic acid than other strains. Also, P. hermaphrodita (DMG0007), P. neopapillosa (DMG0015) and P. californica (DMG0017) were superior at locating 5% hyaluronic acid compared to other isolates of the same genera. Ultimately, there is natural variation in chemoattraction in Phasmarhabditis nematodes, with some strains responding significantly better to host cues than others.

Type 1 interferon-dependent repression of NLRC4 and iPLA2 licenses down-regulation ofSalmonellaflagellin inside macrophages

Inflammasomes have been implicated in the detection and clearance of a variety of bacterial pathogens, but little is known about whether this innate sensing mechanism has any regulatory effect on the expression of stimulatory ligands by the pathogen. During infection withSalmonellaand many other pathogens, flagellin is a major activator of NLRC4 inflammasome-mediated macrophage pyroptosis and pathogen eradication.Salmonellaswitches to a flagellin-low phenotype as infection progresses to avoid this mechanism of clearance by the host. However, the host cues thatSalmonellaperceives to undergo this switch remain unclear. Here, we report an unexpected role of the NLRC4 inflammasome in promoting expression of its microbial ligand, flagellin, and identify a role for type 1 IFN signaling in switching ofSalmonellato a flagellin-low phenotype. Early in infection, activation of NLRC4 by flagellin initiates pyroptosis and concomitant release of lysophospholipids which in turn enhance expression of flagellin bySalmonellathereby amplifying its ability to elicit cell death. TRIF-dependent production of type 1 IFN, however, later represses NLRC4 and the lysophospholipid biosynthetic enzyme iPLA2, causing a decline in intracellular lysophospholipids that results in down-regulation of flagellin expression bySalmonella. These findings reveal a previously unrecognized immune-modulating regulatory cross-talk between endosomal TLR signaling and cytosolic NLR activation with significant implications for the establishment of infection withSalmonella.

Lure, retain, and catch malaria mosquitoes. How heat and humidity improve odour-baited trap performance

Background When seeking a human for a blood meal, mosquitoes use several cues to detect and find their hosts. From this knowledge, counter-flow odour-baited traps have been developed that use a combination of CO2, human-mimicking odour, visual cues and circulating airflow to attract and capture mosquitoes. Initially developed for monitoring, these traps are now also being considered as promising vector control tools. The traps are attractive to host-seeking mosquitoes, but their capture efficiency is low. It has been hypothesized that the lack of short-range host cues, such as heat and increased local humidity, often prevent mosquitoes from getting close enough to get caught; this lack might even trigger avoidance manoeuvres near the capture region. Methods This study investigated how close-range host cues affect the flight behaviour of Anopheles female malaria mosquitoes around odour-baited traps, and how this affects trap capture performance. For this, a novel counter-flow odour-baited trap was developed, the M-Tego. In addition to the usual CO2 and odour-blend, this trap can provide the short-range host cues, heat and humidity. Systematically adding or removing these two cues tested how this affected the trap capture percentages and flight behaviour. First, capture percentages of the M-Tego with and without short-range host cues to the BG-Suna trap were compared, in both laboratory and semi-field testing. Then, machine-vision techniques were used to track the three-dimensional flight movements of mosquitoes around the M-Tego. Results With heat and humidity present, the M-Tego captured significantly more mosquitoes as capture percentages almost doubled. Comparing the flight behaviour around the M-Tego with variable close-range host cues showed that when these cues were present, flying mosquitoes were more attracted to the trap and spent more time there. In addition, the M-Tego was found to have a better capture mechanism than the BG-Suna, most likely because it does not elicit previously observed upward avoiding manoeuvres. Conclusions Results suggest that adding heat and humidity to an odour-baited trap lures more mosquitoes close to the trap and retains them there longer, resulting in higher capture performance. These findings support the development of control tools for fighting mosquito-borne diseases such as malaria.

Manipulation of natal host modifies adult reproductive behaviour in the butterfly Heliconius charithonia

Advances in understanding non-genetic inheritance have prompted broader interest in environmental effects. One way in which such effects may influence adaptation is via the transmission of acquired habitat biases. Here I explore how natal experience influences adult host orientation in the oligophagous passion vine butterfly Heliconius charithonia . As an exemplar of the ‘pupal mating' system, this species poses novelty among diurnal Lepidoptera for the extent to which male as well as female reproductive behaviours are guided by olfactory host cues. I sampled wild adult females breeding exclusively upon Passiflora incarnata , assigned their offspring to develop either upon this species or its local alternative Passiflora suberosa , and then assessed the behaviour of F 1 adults in a large rainforest enclosure. Despite the fact that juvenile performance was superior upon P. incarnata , females oviposited preferentially upon their assigned natal species. Mate-seeking males also indicated a bias for the proximity of their natal host, and there was evidence for assortative mating based upon host treatment, although these data are less robust. This study is, to my knowledge, the first to support Hopkins' hostplant principle in butterflies, and points to inducible host preferences capable of reinforcing ecological segregation and ultimately accelerating evolutionary divergence in sympatry.

Does membrane feeding compromise the quality ofAedes aegyptimosquitoes?

ModifiedAedes aegyptimosquitoes are being mass-reared for release in disease control programs around the world. Releases involving female mosquitoes rely on them being able to seek and feed on human hosts. To facilitate the mass-production of mosquitoes for releases, females are often provided blood through artificial membrane feeders. When reared across generations there is a risk that mosquitoes will adapt to feeding on membranes and lose their ability to feed on human hosts. To test adaptation to membrane feeding, we selected replicate populations ofAe. aegyptifor feeding on either human arms or membrane feeders for at least 8 generations. Membrane-selected populations suffered fitness costs, likely due to inbreeding depression arising from bottlenecks. Membrane-selected females had higher feeding rates on membranes than human-selected ones, suggesting adaptation to membrane feeding, but they maintained their attraction to host cues and feeding ability on humans despite a lack of selection for these traits. Host-seeking ability in small laboratory cages did not differ between populations selected on the two blood sources, but membrane-selected females were compromised in a semi-field enclosure where host-seeking was tested over a longer distance. Our findings suggest thatAe. aegyptimay adapt to feeding on blood provided artificially, but this will not substantially compromise field performance or affect experimental assessments of mosquito fitness. However, large population sizes during mass rearing with membrane feeders should be maintained to avoid bottlenecks which lead to inbreeding depression.Author summaryWith modified mosquitoes being mass-reared for release in disease control programs, there is interest in understanding factors that can affect the quality of release stocks. In the laboratory, membrane feeding devices are often used to provide blood to female mosquitoes which they need to lay eggs. Over time, mosquitoes could adapt to membrane feeding and lose their preference for (or ability to feed on) natural hosts. Adaptation could affect the performance of lab-reared mosquitoes when deployed in the field, especially if the success of disease control programs relies on female reproduction such as in gene drive orWolbachia-based approaches. We tested to see ifAedes aegyptimosquitoes, the principal vectors of dengue virus, would adapt to feeding on blood provided via membranes. We found some evidence of adaptation, with membrane-selected mosquitoes having higher feeding rates on membranes, but this didn’t affect their ability to feed on humans or their attraction to host cues. Although membrane feeding alone does not substantially affect mosquito quality, it can lead to inbreeding depression if populations pass through bottlenecks as they adapt to feeding on blood provided artificially. Our results may inform mass-rearing programs involving the release of modified female mosquitoes.