Anopheles Coluzzii Mosquitoes Research Articles

Effects of Local and Systemic Immune Challenges on the Expression of Selected Salivary Genes in the Malaria Mosquito Anopheles coluzzii.

Salivary glands play a crucial tripartite role in mosquito physiology. First, they secrete factors that greatly facilitate both sugar and blood meal acquisition. Second, the transmission of pathogens (parasites, bacteria and viruses) to the vertebrate host requires both the recognition and invasion of the salivary glands. Third, they produce immune factors that both protect the organ from invading pathogens and are also able to exert their activity in the crop and the midgut when saliva is re-ingested during feeding. Studies on mosquito sialomes have revealed the presence of several female and/or male salivary gland-specific or enriched genes whose function is completely unknown so far. We focused our attention on these orphan genes, and we selected, according to sequence and structural features, a shortlist of 11 candidates with potential antimicrobial properties. Afterwards, using qPCR, we investigated their expression profile at 5 and 24 h after an infectious sugar meal (local challenge) or thoracic microinjection (systemic challenge) of Gram-negative (Escherichia coli, EC) or Gram-positive (Staphylococcus aureus, SA) bacteria. We observed a general increase in the transcript abundance of our salivary candidates between 5 and 24 h after local challenge. Moreover, transcriptional modulation was determined by the nature of the stimulus, with salivary gland-enriched genes (especially hyp15 upon SA stimulus) upregulated shortly after the local challenge and later after the systemic challenge. Overall, this work provides one of the first contributions to the understanding of the immune role of mosquito salivary glands. Further characterization of salivary candidates whose expression is modulated by immune challenge may help in the identification of possible novel antimicrobial peptides.

Effects of insemination and blood-feeding on locomotor activity of wild-derived females of the malaria mosquito Anopheles coluzzii

BackgroundBehavioural shifts in the canonical location and timing of biting have been reported in natural populations of anopheline malaria vectors following the implementation of insecticide-based indoor vector control interventions. These modifications increase the likelihood of human-vector contact and allow mosquitoes to avoid insecticides, both conditions being favourable to residual transmission of the malarial parasites. The biting behaviour of mosquitoes follows rhythms that are under the control of biological clocks and environmental conditions, modulated by physiological states. In this work we explore modifications of spontaneous locomotor activity expressed by mosquitoes in different physiological states to highlight phenotypic variability associated to circadian control that may contribute to explain residual transmission in the field.MethodsThe F10 generation progeny of field-collected Anopheles coluzzii from southwestern Burkina Faso was tested using an automated recording apparatus (Locomotor Activity Monitor, TriKinetics Inc.) under LD 12:12 or DD light regimens in laboratory-controlled conditions. Activity recordings of each test were carried out for a week with 6-day-old females belonging to four experimental treatments, representing factorial combinations of two physiological variables: insemination status (virgin vs inseminated) and gonotrophic status (glucose fed vs blood fed). Chronobiological features of rhythmicity in locomotor activity were explored using periodograms, diversity indices, and generalized linear mixed modelling.ResultsThe average strength of activity, onset of activity, and acrophase were modulated by both nutritional and insemination status as well as by the light regimen. Inseminated females showed a significant excess of arrhythmic activity under DD. When rhythmicity was observed in DD, females displayed sustained activity also during the subjective day.ConclusionsInsemination and gonotrophic status influence the underlying light and circadian control of chronobiological features of locomotor activity. Overrepresentation of arrhythmic chronotypes as well as the sustained activity of inseminated females during the subjective day under DD conditions suggests potential activity of natural populations of A. coluzzii during daytime under dim conditions, with implications for residual transmission of malarial parasites.Graphical abstract

Anopheles coluzzii stearoyl-CoA desaturase is essential for adult female survival and reproduction upon blood feeding.

Vitellogenesis and oocyte maturation require anautogenous female Anopheles mosquitoes to obtain a bloodmeal from a vertebrate host. The bloodmeal is rich in proteins that are readily broken down into amino acids in the midgut lumen and absorbed by the midgut epithelial cells where they are converted into lipids and then transported to other tissues including ovaries. The stearoyl-CoA desaturase (SCD) plays a pivotal role in this process by converting saturated (SFAs) to unsaturated (UFAs) fatty acids; the latter being essential for maintaining cell membrane fluidity amongst other housekeeping functions. Here, we report the functional and phenotypic characterization of SCD1 in the malaria vector mosquito Anopheles coluzzii. We show that RNA interference (RNAi) silencing of SCD1 and administration of sterculic acid (SA), a small molecule inhibitor of SCD1, significantly impact on the survival and reproduction of female mosquitoes following blood feeding. Microscopic observations reveal that the mosquito thorax is quickly filled with blood, a phenomenon likely caused by the collapse of midgut epithelial cell membranes, and that epithelial cells are depleted of lipid droplets and oocytes fail to mature. Transcriptional profiling shows that genes involved in protein, lipid and carbohydrate metabolism and immunity-related genes are the most affected by SCD1 knock down (KD) in blood-fed mosquitoes. Metabolic profiling reveals that these mosquitoes exhibit increased amounts of saturated fatty acids and TCA cycle intermediates, highlighting the biochemical framework by which the SCD1 KD phenotype manifests as a result of a detrimental metabolic syndrome. Accumulation of SFAs is also the likely cause of the potent immune response observed in the absence of infection, which resembles an auto-inflammatory condition. These data provide insights into mosquito bloodmeal metabolism and lipid homeostasis and could inform efforts to develop novel interventions against mosquito-borne diseases.

Detection of Plasmodium falciparum in laboratory-reared and naturally infected wild mosquitoes using near-infrared spectroscopy

There is an urgent need for high throughput, affordable methods of detecting pathogens inside insect vectors to facilitate surveillance. Near-infrared spectroscopy (NIRS) has shown promise to detect arbovirus and malaria in the laboratory but has not been evaluated in field conditions. Here we investigate the ability of NIRS to identify Plasmodium falciparum in Anopheles coluzzii mosquitoes. NIRS models trained on laboratory-reared mosquitoes infected with wild malaria parasites can detect the parasite in comparable mosquitoes with moderate accuracy though fails to detect oocysts or sporozoites in naturally infected field caught mosquitoes. Models trained on field mosquitoes were unable to predict the infection status of other field mosquitoes. Restricting analyses to mosquitoes of uninfectious and highly-infectious status did improve predictions suggesting sensitivity and specificity may be better in mosquitoes with higher numbers of parasites. Detection of infection appears restricted to homogenous groups of mosquitoes diminishing NIRS utility for detecting malaria within mosquitoes.

Ammonium transporter AcAmt mutagenesis uncovers reproductive and physiological defects without impacting olfactory responses to ammonia in the malaria vector mosquito Anopheles coluzzii

Anopheline mosquitoes are the sole vectors of malaria and rely on olfactory cues for host seeking in which ammonia derived from human sweat plays an essential role. To investigate the function of the Anopheles coluzzii ammonium transporter (AcAmt) in the mosquito olfactory system, we generated an AcAmt null mutant line using CRISPR/Cas9. AcAmt−/− mutants displayed a series of novel phenotypes compared with wild-type mosquitoes including significantly lower insemination rates during mating and increased mortality during eclosion. Furthermore, AcAmt−/− males showed significantly lower sugar consumption while AcAmt−/− females and pupae displayed significantly higher ammonia levels than their wild-type counterparts. Surprisingly, in contrast to previous studies in Drosophila that revealed that the mutation of the ammonium transporter (DmAmt) induces a dramatic reduction of ammonia responses in antennal coeloconic sensilla, no significant differences were observed across a range of peripheral sensory neuron responses to ammonia and other odorants between wild-type and AcAmt−/− females. These data support the existence in mosquitoes of novel compensatory ammonia-sensing mechanisms that are likely to have evolved as a result of the importance of ammonia in host-seeking and other behaviors.

Living with predators at the larval stage has differential long-lasting effects on adult life history and physiological traits in two anopheline mosquito species

1.Under predation stress, individuals increase their metabolic rate and adopt different resource allocation strategies to favor their direct survival to the detriment of growth, reproduction and self-maintenance. Species and sexes are both faced with different physiological and life history demands and should adopt different strategies which lead to carry-over effects on adult metabolic reserves and on oxidative status.2.Malaria mosquitoes Anopheles coluzzii and An. gambiae are sibling species. In nature, their larvae are exposed to different levels of predation risk and An. coluzzii larvae are more capable of surviving predation threats than An. gambiae, because of a better ability to gauge the threat level and to produce an adapted behavioral response. Impact of this response on adult physiology is however unknown.3.Here, we investigated the carry-over effects of larval predation stress: first, on life history traits such as development length, size and weight at emergence, fecundity and survival; second, on metabolic resources at emergence such as lipids, proteins, carbohydrates and amino acids; and third, on oxidative status with the total anti-oxidant capacity and the oxidation of proteins in the two species and in both sexes.4.We found that on one hand, it took more time for An. coluzzii to develop in presence of a predator and they emerged with a smaller size but a similar weight as control individuals. On the other hand, An. gambiae exposed to predators developed as fast as controls but with both a smaller size and a smaller weight. Fecundity was negatively impacted in both species following exposure to predation threats. Teneral reserves were affected depending on the species and the sex. The total anti-oxidant capacity of individuals exposed to stress was lower than in the controls but a significant level of damages to proteins was detected in An. gambiae males only.5.Our findings suggest that predation stress induces very different physiological responses even in closely related species and between sexes. Allocation of resources in larvae may differ and depend on the species’ aptitude to develop in presence of predators and on the sex, since males and females might adopt different strategies to fulfill their fitness.

Mutagenesis of the orco odorant receptor co-receptor impairs olfactory function in the malaria vector Anopheles coluzzii

Mosquitoes rely heavily on their olfactory systems for host seeking, selection of oviposition sites, and avoiding predators and other environmental dangers. Of these behaviors, the preferential selection of a human blood-meal host drives the vectorial capacity of anthropophilic female Anopheles coluzzii mosquitoes. Olfactory receptor neurons (ORNs) are dispersed across several appendages on the head and express an obligate odorant receptor co-receptor (Orco) coupled with a “tuning” odorant receptor (OR) to form heteromeric, odor-gated ion channels in the membrane of these neurons. To examine the mechanistic and functional contributions of Orco/OR complexes to the chemosensory processes of An. coluzzii, we utilized CRISPR/Cas9 gene editing to create a line of homozygous, Orco-knockout, mutant mosquitoes. As expected, orco−/− ORNs across both adult and larval stages of An. coluzzii display significantly lower background activity and lack nearly all odor-evoked responses. In addition, blood-meal-seeking, adult female, orco−/− mutant mosquitoes exhibit severely reduced attraction to human- and non-human-derived odors while gravid females are significantly less responsive to established oviposition attractants. These results reinforce observations in other insects that Orco is crucial in maintaining the activity of ORNs. In that light, it significantly influences a range of olfactory-driven behaviors central to the anthropophilic host preference that is critical to the vectorial capacity of An. coluzzii as a primary vector for human malaria.

Gene editing reveals obligate and modulatory components of the CO2 receptor complex in the malaria vector mosquito, Anopheles coluzzii

The sensitivity to volatile carbon dioxide (CO2) produced by humans and other animals is a critical component in the host preference behaviors of the malaria vector mosquito Anopheles coluzzii. The molecular receptors responsible for the ability to sense CO2 are encoded by three putative gustatory receptor (Gr) genes (Gr22,23,24) which are expressed in a distinctive array of sensory neurons housed in maxillary palp capitate peg sensilla of An. coluzzii. Despite the identification of these components and subsequent studies, there is a paucity of understanding regarding the respective roles of these three GRs in the mosquito's CO2 transduction process. To address this, we have used CRISPR/Cas9-based gene editing technique combined with in vivo electrophysiological recordings to directly examine the role of Gr22,23,24 in detecting CO2 in An. coluzzii. These studies reveal that both Gr23 and Gr24 are absolutely required to maintain in vivo CO2 sensitivity while, in contrast, Gr22 knock out mutants are still able to respond to CO2 stimuli albeit with significantly weaker sensitivity. Our data supports a model in which Gr22 plays a modulatory role to enhance the functionality of Gr23/24 complexes that are responsible for CO2 sensitivity of mosquitoes.

Functional analysis of the three major PGRPLC isoforms in the midgut of the malaria mosquito Anopheles coluzzii

Peptidoglycan recognition proteins (PGRPs) constitute the primary means of bacterial recognition in insects. Recent work in the model organism Drosophila has revealed the mechanisms by which the complement of PGRPs refine the sensitivity of different tissues to bacterial elicitors, permitting the persistence of commensal bacteria in the gut whilst maintaining vigilance against bacterial infection. Here, we use in vivo knockdowns and in vitro pull-down assays to investigate the role of the three major isoforms of the transmembrane receptor of the Imd pathway, PGRPLC, in basal immunity in the Anopheles coluzzii mosquito midgut. Our results indicate that the mosquito midgut is regionalized in its expression of immune effectors and of PGRPLC1. We show that PGRPLC1 and PGRPLC3 are pulled down with polymeric DAP-type peptidoglycan, while PGRPLC2 and PGRPLC3 co-precipitate in the presence of TCT, a peptidoglycan monomer. These data suggest that, as found in Drosophila, discrimination of polymeric and monomeric PGN by Anopheles PGRPLC participates in the regulation of the Imd pathway.

The Influence of Larval Stage and Density on Oviposition Site-Selection Behavior of the Afrotropical Malaria Mosquito Anopheles coluzzii (Diptera: Culicidae).

In the selection of oviposition sites female mosquitoes use various cues to assess site quality to optimize survival of progeny. The presence of conspecific larvae influences this process. Interactive effects of oviposition site selection were studied in the malaria mosquito Anopheles coluzzii Coetzee & Wilkerson in dual- and no-choice assays, by exposing single gravid mosquitoes to oviposition cups containing 1) larvae of different developmental stages, 2) larvae-conditioned water (LCW), and 3) cups where visual cues of conspecific larvae were absent. Early-stage conspecific larvae had a positive effect on the oviposition response. By contrast, late stages of conspecific larvae had a negative effect. Oviposition choice was dependent on larval density. Moreover, in oviposition cups where larvae were hidden from view, late-stage larvae had a significant negative effect on oviposition suggesting the involvement of olfactory cues. LCW had no effect on oviposition response, indicating involvement of chemicals produced by larvae in vivo. It is concluded that the presence of larvae in a breeding site affects the oviposition response depending on the development stage of the larvae. These responses appear to be mediated by olfactory cues emitted by the larval habitat containing live larvae, resulting in the enhanced reproductive fitness of the females.

Commonly Used Insect Repellents Hide Human Odors from Anopheles Mosquitoes.

The mode of action for most mosquito repellents is unknown. This is primarily due to the difficulty in monitoring how the mosquito olfactory system responds to repellent odors. Here, we used the Q-system of binary expression to enable activity-dependent Ca2+ imaging in olfactory neurons of the African malaria mosquito Anopheles coluzzii. This system allows neuronal responses to common insect repellents to be directly visualized in living mosquitoes from all olfactory organs, including the antenna. The synthetic repellents N,N-diethyl-meta-toluamide (DEET) and IR3535 did not activate Anopheles odorant receptor co-receptor (Orco)-expressing olfactory receptor neurons (ORNs) at any concentration, and picaridin weakly activated ORNs only at high concentrations. In contrast, natural repellents (i.e. lemongrass oil and eugenol) strongly activated small numbers of ORNs in the Anopheles mosquito antennae at low concentrations. We determined that DEET, IR3535, and picaridin decrease the response of Orco-expressing ORNs when these repellents are physically mixed with activating human-derived odorants. We present evidence that synthetic repellents may primarily exert their olfactory mode of action by decreasing the amount of volatile odorants reaching ORNs. These results suggest that synthetic repellents disruptively change the chemical profile of host scent signatures on the skin surface, rendering humans invisible to Anopheles mosquitoes.

Current versus future reproductive investment adaptive responses in adult Anopheles coluzzii malaria mosquitoes: hydric-stressed males give it all

BackgroundLife history theory predicts that an individualʼs current reproductive investment should depend on its future reproductive value. A variety of intrinsic biotic and extrinsic factors influence reproductive value, including age, health status and current environmental conditions. Phenotypic plastic reproductive decisions are particularly crucial in species with limited mating and breeding opportunities. In the malaria mosquito Anopheles coluzzii, the combination of male-male competition and female monandry results in male reproductive success being dependent on limited mating opportunities and sperm reserves. Short life spans combined with 3–4 day gonotrophic cycles imply that females can produce only a limited number of egg-batches in their lifetime and rely on a single maleʼs insemination to do so. Here we experimentally tested the effect of hydric stress on male sperm transfer and female sperm maintenance in this important vector species.MethodsVirgin males and females were stressed prior to mating to simulate environmental uncertainty, hence the prospect of a decreased lifespan. They were then paired overnight with non-stressed mates in standardized mating assays. Sperm transfer, uptake and maintenance were quantified using qPCR, and sperm activity determined via video recording.ResultsWhen exposed to hydric stress, males responded by increasing their current reproductive investment and transferred significantly larger amounts of sperm to females. There was no significant increase in the mean number of females inseminated overnight by stressed males. In contrast, females did not significantly change their sperm uptake following stress nor did they alter their sperm maintenance strategy after 7-day post-mating hydric stress as measured through sperm activity level and sperm cells quantification.ConclusionsAs predicted by life-history theory, pre-mating hydric stress was associated with an increase in male current reproductive effort in the form of increased sperm transfer. In contrast, pre and post-mating hydric stress had no impact on sperm uptake and maintenance by females, which is compatible with the prediction that females maximize their reproductive value by withstanding stress periods until a blood meal opportunity and maintain sperm quality towards future egg production.

Evidence of insecticide resistance selection in wild Anopheles coluzzii mosquitoes due to agricultural pesticide use

BackgroundThe wetlands used for some agricultural activities constitute productive breeding sites for many mosquito species. Thus, the agricultural use of insecticide targeting other pests may select for insecticide resistance in malaria mosquitoes. The purpose of this study is to clarify some knowledge gaps on the role of agrochemicals in the development of insecticide resistance in malaria vectors is of utmost importance for vector control.MethodsUsing the CDC bottle test and the log-probit analysis, we investigated for the first time the resistance levels of Anopheles coluzzii mosquitoes to neonicotinoids, insecticides used exclusively for crop protection in Côte d’Ivoire. The study was conducted in two agricultural regions (Tiassale and Gagnoa) and one non-agricultural region (Vitre) between June and August 2017 using clothianidin, acetamiprid and imidacloprid.ResultsMosquito populations from Tiassale and Gagnoa (agricultural settings) were determined to be resistant to acetamiprid with mortality rates being < 85% at 24 h post-exposure. In Vitre (non-agricultural area) however, the mosquito population was susceptible to acetamiprid. In all three localities, mosquito populations were resistant to imidacloprid (mortality rates were 60% in Vitre, 37% in Tiassale, and 13% in Gagnoa) and completely susceptible to clothianidin (100% mortality). An. coluzzii represented 100% of mosquito collected in Gagnoa, 86% in Tiassale and 96% in Vitre.ConclusionsThis study provides strong evidence that agricultural use of insecticides can cause insecticide resistance in malaria vector populations. Insecticide resistance driven by agrochemical usage should be considered when vector control strategies are developed.

Mosquito and human hepatocyte infections with Plasmodium ovale curtisi and Plasmodium ovale wallikeri.

Human ovale malaria is caused by the two closely related species, Plasmodium ovale curtisi and P. ovale wallikeri. Both species are known to relapse from quiescent hepatic forms months or years after the primary infection occurred. Although some studies have succeeded in establishing mosquito transmission for ovale malaria, none have specifically described transmission and human hepatocyte infection of both sibling species. Here we describe a simplified protocol for successful transmission of both P. ovale curtisi and P. ovale wallikeri to Anopheles coluzzii mosquitoes and streamlined monitoring of infection using sensitive parasite DNA detection, by loop-activated amplification, in blood-fed mosquitoes. In one experimental infection with P. ovale curtisi and one with P. ovale wallikeri, viable sporozoites were isolated from mosquito salivary glands and used to successfully infect cultured human hepatocytes. This protocol provides a method for the utilisation of pretreatment clinical blood samples from ovale malaria patients, collected in EDTA, for mosquito infection studies and generation of the hepatic life cycle stages of P. ovale curtisi and P. ovale wallikeri. We also demonstrate the utility of loop-activated amplification as a rapid and sensitive alternative to dissection for estimating the prevalence of infection in Anopheles mosquitoes fed with Plasmodium-infected blood.

Introgression between Anopheles gambiae and Anopheles coluzzii in Burkina Faso and its associations with kdr resistance and Plasmodium infection

BackgroundInsecticide resistance in Anopheles coluzzii mosquitoes has become widespread throughout West Africa including in Burkina Faso. The insecticide resistance allele (kdr or L1014F) is a prime indicator that is highly correlated with phenotypic resistance in West Africa. Studies from Benin, Ghana and Mali have suggested that the source of the L1014F is introgression of the 2L divergence island via interspecific hybridization with Anopheles gambiae. The goal of this study was to characterize local mosquito populations in the Nouna Department, Burkina Faso with respect to: (i) the extent of introgression between An. coluzzii and An. gambiae, (ii) the frequency of the L1014F mutation and (iii) Plasmodium infection rates.MethodsA total of 95 mosquitoes were collected from ten sites surrounding Nouna town in Kossi Province, Burkina Faso in 2012. The species composition, the extent of introgression in An. coluzzii mosquitoes and their Plasmodium infection rates were identified with a modified version of the “Divergence Island SNP” (DIS) genotyping assay.ResultsThe mosquito collection contained 70.5% An. coluzzii, 89.3% of which carried a 3 Mb genomic region on the 2L chromosome with L1014F insecticide resistance mutation that was introgressed from An. gambiae. In addition, 22.4% in the introgressed An. coluzzii specimens were infected with Plasmodium falciparum, whereas none of the non-introgressed (“pure”) An. coluzzii were infected.ConclusionThis paper is the first report providing divergence island SNP genotypes for natural population of Burkina Faso and corresponding Plasmodium infection rates. These observations warrant further study and could have a major impact on future malaria control strategies in Burkina Faso.

Streamlined SMFA and mosquito dark-feeding regime significantly improve malaria transmission-blocking assay robustness and sensitivity

BackgroundThe development of malaria transmission-blocking strategies including the generation of malaria refractory mosquitoes to replace the wild populations through means of gene drives hold great promise. The standard membrane feeding assay (SMFA) that involves mosquito feeding on parasitized blood through an artificial membrane system is a vital tool for evaluating the efficacy of transmission-blocking interventions. However, despite the availability of several published protocols, the SMFA remains highly variable and broadly insensitive.MethodsThe SMFA protocol was optimized through coordinated culturing of Anopheles coluzzii mosquitoes and Plasmodium falciparum parasite coupled with placing mosquitoes under a strict dark regime before, during, and after the gametocyte feed.ResultsA detailed description of essential steps is provided toward synchronized generation of highly fit An. coluzzii mosquitoes and P. falciparum gametocytes in preparation for an SMFA. A dark-infection regime that emulates the natural vector-parasite interaction system is described, which results in a significant increase in the infection intensity and prevalence. Using this optimal SMFA pipeline, a series of putative transmission-blocking antimicrobial peptides (AMPs) were screened, confirming that melittin and magainin can interfere with P. falciparum development in the vector.ConclusionA robust SMFA protocol that enhances the evaluation of interventions targeting human malaria transmission in laboratory setting is reported. Melittin and magainin are identified as highly potent antiparasitic AMPs that can be used for the generation of refractory Anopheles gambiae mosquitoes.

Assessment of the developmental success of Anopheles coluzzii larvae under different nutrient regimes: effects of diet quality, food amount and larval density

BackgroundIn a context of increasing resistance of both vectors toward main classes of insecticides used in public health and parasites toward anti-malarial drugs, development of new and complementary molecules or control approaches is fundamental to achieve the objective of controlling or even eliminating malaria. Concerning vector control, the sterile insect technique and other genetic control approaches are among promising complementary tools in an integrated management strategy for malaria control. These approaches rely not only on a good understanding of vector biology (especially during larval stages), but also on the availability of adequate supplies and protocols for efficient mosquito rearing. The aim of this study was to assess the factors impacting the life history of Anopheles coluzzii mosquitoes at the larval stage, in the context of genetic and sterile insect approaches to control malaria vectors.MethodsThe effect of different larval diets and larval rearing densities on the development of An. coluzzii were evaluated in the laboratory. Emergence rate (ER), pre-imaginal developmental time (DT) and adult wing length (WL) were measured under different food regimes. Four diets were tested among which three were provided by the Insect Pest Control Laboratory (IPCL) of the FAO/IAEA Joint division.ResultsData showed significant differences in the quality of the different diets and suggested a negative density dependence in all three life history parameters measured under tested rearing conditions. ER and WL increased with food availability, but decreased with increasing larval density. Conversely DT was shortened with increasing food availability but increased with larval density. These data demonstrates intraspecific larval competition modulated by food amount and space availability. Of the four diets tested, the one made of a mix of tuna meal, bovine liver powder, brewer’s yeast, squid liver powder and vitamin mix (diet 2) yielded the best results as it produced a good balance between ER, DT and WL. Food availability for optimal development (highest survival at shortest time) was in the range of 180–400 µg/larvae/day for the three diets provided by the IPCL.ConclusionThere is an interaction between diet type, diet concentration and larval density. Best results in terms of optimal larvae development parameters happen when moderately high values of those three variables are observed.

Flight behaviour of malaria mosquitoes around odour-baited traps: capture and escape dynamics.

Host-seeking mosquitoes rely on a range of sensory cues to find and approach blood hosts, as well as to avoid host detection. By using odour blends and visual cues that attract anthropophilic mosquitoes, odour-baited traps have been developed to monitor and control human pathogen-transmitting vectors. Although long-range attraction of such traps has already been studied thoroughly, close-range response of mosquitoes to these traps has been largely ignored. Here, we studied the flight behaviour of female malaria mosquitoes (Anopheles coluzzii) in the immediate vicinity of a commercially available odour-baited trap, positioned in a hanging and standing orientation. By analysing more than 2500 three-dimensional flight tracks, we elucidated how mosquitoes reacted to the trap, and how this led to capture. The measured flight dynamics revealed two distinct stereotypical behaviours: (i) mosquitoes that approached a trap tended to simultaneously fly downward towards the ground; (ii) mosquitoes that came close to a trap changed their flight direction by rapidly accelerating upward. The combination of these behaviours led to strikingly different flight patterns and capture dynamics, resulting in contrasting short-range attractiveness and capture mechanism of the oppositely oriented traps. These new insights may help in improving odour-baited traps, and consequently their contribution in global vector control strategies.

Escaping blood-fed malaria mosquitoes minimize tactile detection without compromising on take-off speed.

To escape after taking a blood meal, a mosquito must exert forces sufficiently high to take off when carrying a load roughly equal to its body weight, while simultaneously avoiding detection by minimizing tactile signals exerted on the host's skin. We studied this trade-off between escape speed and stealth in the malaria mosquito Anopheles coluzzii using 3D motion analysis of high-speed stereoscopic videos of mosquito take-offs and aerodynamic modeling. We found that during the push-off phase, mosquitoes enhanced take-off speed using aerodynamic forces generated by the beating wings in addition to leg-based push-off forces, whereby wing forces contributed 61% of the total push-off force. Exchanging leg-derived push-off forces for wing-derived aerodynamic forces allows the animal to reduce peak force production on the host's skin. By slowly extending their long legs throughout the push-off, mosquitoes spread push-off forces over a longer time window than insects with short legs, thereby further reducing peak leg forces. Using this specialized take-off behavior, mosquitoes are capable of reaching take-off speeds comparable to those of similarly sized fruit flies, but with weight-normalized peak leg forces that were only 27% of those of the fruit flies. By limiting peak leg forces, mosquitoes possibly reduce the chance of being detected by the host. The resulting combination of high take-off speed and low tactile signals on the host might help increase the mosquito's success in escaping from blood-hosts, which consequently also increases the chance of transmitting vector-borne diseases, such as malaria, to future hosts.

The use of sequential mark-release-recapture experiments to estimate population size, survival and dispersal of male\xa0mosquitoes of the\xa0 Anopheles gambiae complex in Bana, a west African humid savannah village

BackgroundVector control is a major component of the malaria control strategy. The increasing spread of insecticide resistance has encouraged the development of new tools such as genetic control which use releases of modified male mosquitoes. The use of male mosquitoes as part of a control strategy requires an improved understanding of male mosquito biology, including the factors influencing their survival and dispersal, as well as the ability to accurately estimate the size of a target mosquito population. This study was designed to determine the seasonal variation in population size via repeated mark-release-recapture experiments and to estimate the survival and dispersal of male mosquitoes of the Anopheles gambiae complex in a small west African village.MethodsMark-release-recapture experiments were carried out in Bana Village over two consecutive years, during the wet and the dry seasons. For each experiment, around 5000 (3407–5273) adult male Anopheles coluzzii mosquitoes were marked using three different colour dye powders (red, blue and green) and released in three different locations in the village (centre, edge and outside). Mosquitoes were recaptured at sites spread over the village for seven consecutive days following the releases. Three different capture methods were used: clay pots, pyrethroid spray catches and swarm sampling.ResultsSwarm sampling was the most productive method for recapturing male mosquitoes in the field. Population size and survival were estimated by Bayesian analyses of the Fisher-Ford model, revealing an about 10-fold increase in population size estimates between the end of dry season (10,000–50,000) to the wet season (100,000–500,000). There were no detectable seasonal effects on mosquito survival, suggesting that factors other than weather may play an important role. Mosquito dispersal ranged from 40 to 549 m over the seven days of each study and was not influenced by the season, but mainly by the release location, which explained more than 44% of the variance in net dispersal distance.ConclusionThis study clearly shows that male-based MRR experiments can be used to estimate some parameters of wild male populations such as population size, survival, and dispersal and to estimate the spatial patterns of movement in a given locality.