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Because of its importance as a malaria vector, Anopheles coluzzii's Coetzee & Wilkerson olfactory system has been studied extensively. Among this work is a series of studies comparing the expression of chemosensory genes in olfactory organs in females and/or males of these species. These have identified species- and female-biased chemosensory gene expression patterns. However, many questions remain about the role of chemosensation in male anopheline biology. To pave the way for future work we used RNAseq to compare chemosensory gene expression in the male maxillary palps of An. coluzzii and its sibling species An. quadriannulatus Theobald. As expected, the chemosensory gene repertoire is small in the male maxillary palps. Both species express the tuning receptors Or8 and Or28 at relatively high levels. The CO2 receptor genes Gr22-Gr24 are present in both species as well, although at much lower level than in females. Additionally, several chemoreceptors are species-specific. Gr37 and Gr52 are exclusive to An. coluzzii, whereas Or9 and Gr60 were detected only in An. quadriannulatus. Furthermore, several chemosensory genes show differential expression between the two species. Finally, several Irs, Grs, and Obps that show strong differential expression in the female palps, are absent or lowly expressed in the male palps. While many questions remain about the role of chemosensation in anopheline male biology, these results suggest that the male maxillary palps could have both a sex- and species-specific role in the perception of chemical stimuli. This work may guide future studies on the role of the male maxillary palp in these species., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of Entomological Society of America.)

Background: Infectious disease interventions are increasingly tested using cluster-randomized trials (CRTs). These trial settings tend to involve a set of sampling units, such as villages, whose geographic arrangement may present a contamination risk in treatment exposure. The most widely used approach for reducing contamination in these settings is the so-called fried-egg design, which excludes the outer portion of all available clusters from the primary trial analysis. However, the fried-egg design ignores potential intra-cluster spatial heterogeneity and makes the outcome measure inherently less precise. Whereas the fried-egg design may be appropriate in specific settings, alternative methods to optimize the design of CRTs in other settings are lacking., Methods: We present a novel approach for CRT design that either fully includes or fully excludes available clusters in a defined study region, recognizing the potential for intra-cluster spatial heterogeneity. The approach includes an algorithm that allows investigators to identify the maximum number of clusters that could be included for a defined study region and maintain randomness in both the selection of included clusters and the allocation of clusters to either the treatment group or control group. The approach was applied to the design of a CRT testing the effectiveness of malaria vector-control interventions in southern Malawi., Conclusions: Those planning CRTs to evaluate interventions should consider the approach presented here during trial design. The approach provides a novel framework for reducing the risk of contamination among the CRT randomization units in settings where investigators determine the reduction of contamination risk as a high priority and where intra-cluster spatial heterogeneity is likely. By maintaining randomness in the allocation of clusters to either the treatment group or control group, the approach also permits a randomization-valid test of the primary trial hypothesis.

Dosage compensation has evolved in concert with Y-chromosome degeneration in many taxa that exhibit heterogametic sex chromosomes. Dosage compensation overcomes the biological challenge of a "half dose" of X chromosome gene transcripts in the heterogametic sex. The need to equalize gene expression of a hemizygous X with that of autosomes arises from the fact that the X chromosomes retain hundreds of functional genes that are actively transcribed in both sexes and interact with genes expressed on the autosomes. Sex determination and heterogametic sex chromosomes have evolved multiple times in Diptera, and in each case the genetic control of dosage compensation is tightly linked to sex determination. In the Anopheles gambiae species complex (Culicidae), maleness is conferred by the Y-chromosome gene Yob, which despite its conserved role between species is polymorphic in its copy number between them. Previous work demonstrated that male An. gambiae s.s. males exhibit complete dosage compensation in pupal and adult stages. In the present study, we have extended this analysis to three sister species in the An. gambiae complex: An. coluzzii, An. arabiensis, and An. quadriannulatus. In addition, we analyzed dosage compensation in bi-directional F1 hybrids between these species to determine if hybridization results in the mis-regulation and disruption of dosage compensation. Our results confirm that dosage compensation operates in the An. gambiae species complex through the hypertranscription of the male X chromosome. Additionally, dosage compensation in hybrid males does not differ from parental males, indicating that hybridization does not result in the mis-regulation of dosage compensation.

It has been suggested that Plasmodia manipulate their vertebrate hosts to enhance parasite transmission. Using a dual-choice olfactometer, we investigated the attraction of Anopheles gambiae to 50 Kenyan children (aged 5-12 years) who were naturally infected with Plasmodium falciparum or noninfected controls. Microscopic gametocyte carriers attracted almost 2 times more mosquitoes than children who were parasite free, harbored asexual stages, or had gametocytes at submicroscopic densities. By using highly sensitive stage-specific molecular methods to detect P. falciparum, we show that gametocytes-and not their noninfectious asexual progenitors-induce increased attractiveness of humans to mosquitoes. Our findings therefore support the parasite host manipulation hypothesis., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Several microorganisms have been shown to manipulate their host or vector to enhance their own transmission. Here we examined whether an infection with Borrelia afzelii affects its transmission between its bank vole (Myodes glareolus, Schreber, 1780) host and tick vector. Captive-bred bank voles were inoculated with B. afzelii or control medium, after which host preference of Ixodes ricinus L. nymphs was determined in a Y-tube olfactometer. Thereafter, infected and uninfected bank voles were placed in a semifield arena containing questing larvae to measure larval tick attachment. Engorged larvae were collected from these bank voles, molted into nymphs, weighed, and analyzed for infection by PCR.Nymphs were attracted to the odors of a bank vole compared to ambient air and preferred the odors of an infected bank vole over that of an uninfected bank vole. In the semifield arena, infected male bank voles had greater larval tick burdens then uninfected males, while similar larval tick burdens were observed on females regardless of infection status. Nymphal ticks that acquired a B. afzelii infection had higher body weight than nymphs that did not acquire an infection regardless of the infection status of the vole. These results show that a B. afzelii infection in bank voles increases larval tick burden and that a B. afzelii infection in larvae increases nymphal body weight. This finding provides novel ecological insights into the enzootic cycle of B. afzelii., (© The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The health and demographic surveillance system on Rusinga Island, Western Kenya, was initiated in 2012 to facilitate a malaria intervention trial: the SolarMal project. The project aims to eliminate malaria from Rusinga Island using the nationwide adopted strategy for malaria control (insecticide-treated bed nets and case management) augmented with mass trapping of anopheline mosquitoes. The main purpose of the health and demographic surveillance is to measure the effectiveness of the trial on clinical malaria incidence, and to monitor demographic, environmental and malaria-related data variables. At the end of 2014, the 44 km(2) island had a population of approximately 25 000 individuals living in 8746 residential structures. Three times per year, all individuals are followed up and surveyed for clinical malaria. Following each round of surveillance, a randomly selected cross-section of the population is subject to a rapid diagnostic test to measure malaria. Additionally, extensive monitoring of malaria vectors is performed. Data collection and management are conducted using the OpenHDS platform, with tablet computers and applications with advanced software connected to a centralized database. Besides the general demographic information, other health-related data are collected which can be used to facilitate a range of other studies within and outside the current project. Access to the core dataset can be obtained on request from the authors., (© The Author 2016; all rights reserved. Published by Oxford University Press on behalf of the International Epidemiological Association.)

The effectiveness of CO2-baited and human-baited mosquito traps for the sampling of Anopheles darlingi Root was evaluated and compared with human landing collections in Suriname. Biting preferences of this mosquito on a human host were studied and related to trapping data. Traps used were the Centers for Disease Control and Prevention Miniature Light trap, the BG Sentinel mosquito trap, the Mosquito Magnet Liberty Plus mosquito trap (MM-Plus), and a custom-designed trap. Carbon dioxide and humans protected by a bed net were used as bait in the studies. The number of An. darlingi collected was greater with human landing collections than with all other collection methods. An. darlingi did not show a preference for protected humans over CO2 bait. The BG Sentinel mosquito trap with CO2 or human odor as bait and the MM-Plus proved the best alternative sampling tools for An. darlingi. The BG Sentinel mosquito trap with CO2 or human odor as bait was also very efficient at collecting Culex spp. In a field study on biting preferences of wild An. darlingi, the females showed directional biting behavior (P < 0.001), with a majority of females (93.3%) biting the lower legs and feet when approaching a seated human host. Higher efficiency of the closer-to-the-ground collecting MM-Plus and BG Sentinel mosquito trap when compared with the other trapping methods may be a result of a possible preference of this mosquito species for low-level biting. It is concluded that odor-baited sampling systems can reliably collect An. darlingi, but the odor bait needs to be improved, for instance, by including host-specific volatiles, to match live human baits.

Microbiota on the human skin plays a major role in body odour production. The human microbial and chemical signature displays a qualitative and quantitative correlation. Genes may influence the chemical signature by shaping the composition of the microbiota. Recent studies on human skin microbiota, using 16S rRNA gene sequencing, found a high inter- and intrapersonal variation in bacterial species on the human skin, which is relatively stable over time. Human body odours mediate the attraction of mosquitoes to their blood hosts. Odours produced by skin microbiota are attractive to mosquitoes as shown by in vitro studies, and variation in bacterial species on the human skin may explain the variation in mosquito attraction between humans. Detailed knowledge of the ecology and genetics of human skin microbiota is needed in order to unravel the evolutionary mechanisms that underlie the interactions between mosquitoes and their hosts., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Females of the African malaria vector, Anopheles gambiae Giles sensu stricto, use human volatiles to find their blood-host. Previous work has shown that ammonia, lactic acid, and aliphatic carboxylic acids significantly affect host orientation and attraction of this species. In the current study, these compounds were tested for their attractiveness relative to human emanations in vivo and in vitro. Emanations from a human hand, incubated sweat, and foot skin residues on a nylon sock were significantly attractive when tested against clean air. In a dual-choice test, foot skin residues were significantly more attractive than emanations from a human hand in vivo. Ammonia alone attracted more mosquitoes than fresh or incubated sweat. However, the odor of a human hand or of foot skin residues were more attractive than ammonia. A known attractive blend of ammonia with lactic acid and carboxylic acids was less effective than natural foot odorants. The results demonstrate that the synthetic blend based on skin odor is attractive for An. gambiae, but that in a choice situation in vitro natural skin odors are still preferred by the mosquito. Differences in volatile organic compound abundances between a worn sock and the synthetic blend may have resulted in stronger attraction to the sock. This suggests that candidate attractants should be evaluated with consideration of the strength of natural odorant sources. The data furthermore suggest that additional unidentified compounds from the human foot are involved in the host-seeking behavior of this mosquito species.

Nymphal Ixodes ricinus ticks (n=180) were collected from three different areas in the Netherlands to investigate the effect of forest composition on tick-associated microbial communities. Sampled habitats differed in thickness of leaf litter and humus layers and vegetation associations and were located near Amsterdam (Beech-Oak), Ede (Birch-Oak) and Veldhoven (Birch-Oak). Analysis of nine 16S rRNA gene clone libraries made from individual ticks showed nearest matches with presumed pathogens Candidatus Neoehrlichia mikurensis and Rickettsia australis and arthropod endosymbionts Wolbachia pipientis and Candidatus Midichloria mitochondrii. Total bacterial species diversity (Shannon index) and Borrelia species infections were determined in I. ricinus by, respectively, PCR-denaturing gradient gel-electrophoresis and PCR-reverse line blot with probes specific for Borrelia burgdorferi sensu stricto, Borrelia afzelii, Borrelia garinii, Borrelia valaisiana, Borrelia lusitaniae and Borrelia ruski. Bacterial diversity differed significantly per area and was lowest in Ede. In contrast, Borrelia species-infected ticks were more abundant in Ede, Candidatus Neoehrlichia mikurensis-infected ticks in Ede and Veldhoven, and R. australis-infected ticks in Amsterdam. Borrelia afzelii was the most common Borrelia species found in all three areas. Bacterial tick diversity was influenced by local differences in forest structure, which is proposed to modulate animal populations that are commonly parasitized by I. ricinus.

Chemical cues play an important role in the host-seeking behavior of blood-feeding mosquitoes (Diptera: Culicidae). A field study was carried out in The Gambia to investigate the effects of human odor or synthetic odor blends on the attraction of mosquitoes. MM-X traps baited with 16 odor blends to which carbon dioxide (CO2) was added were tested in four sets of experiments. In a second series of experiments, MM-X traps with 14 odor blends without CO2 were tested. A blend of ammonia and L-lactic acid with or without CO2 was used as control odor in series 1 and 2, respectively. Centers for Disease Control and Prevention (CDC) traps were placed in a traditional house and an experimental house to monitor mosquito densities during the experiments. The MM-X traps caught a total number of 196,756 mosquitoes, with the most abundant species belonging to the genera Mansonia (70.6%), Anopheles (17.5%), and Culex (11.5%). The most abundant mosquito species caught by the CDC traps (56,290 in total) belonged to the genera Mansonia (59.4%), Anopheles (16.0% An. gambiae s.l. Giles, and 11.3% An. ziemanni Grünberg), and Culex (11.6%). MM-X traps baited with synthetic blends were in many cases more attractive than MM-X traps baited with human odors. Addition of CO2 to synthetic odors substantially increased the catch of all mosquito species in the MM-X traps. A blend of ammonia + L-lactic acid + CO, + 3-methylbutanoic acid was the most attractive odor for most mosquito species. The candidate odor blend shows the potential to enhance trap collections so that traps will provide better surveillance and possible control.

Olfactory receptor neurons (ORNs) in the antenna of insects serve to encode odors in action potential activity conducted to the olfactory lobe of the deuterocerebrum. We performed an analysis of the electrophysiological responses of olfactory neurons in the antennae of the female malaria mosquito Anopheles gambiae s.s. and investigated the effect of blood feeding on responsiveness. Forty-four chemicals that are known to be present in human volatile emanations were used as odor stimuli. We identified 6 functional types of trichoid sensilla and 5 functional types of grooved-peg sensilla (GP) based on a hierarchical cluster analysis. Generalist ORNs, tuned to a broad range of odors, moderate specialist ORNs and 2 ORNs tuned to only one odor were identified in different sensilla types. Neurons in GP were tuned to more polar compounds including the important behavioral attractant ammonia and its synergist L-lactic acid, responses to which were found only in GP. Combinatorial coding is the most plausible principle operating in the olfactory system of this mosquito species. We document for the first time both up- and downregulation of ORN responsiveness after blood feeding. Modulation of host-seeking and oviposition behavior is associated with both qualitative and quantitative changes in the peripheral sensory system.

Host odours play a major role in the orientation and host location of blood-feeding mosquitoes. Anopheles gambiae Giles sensu stricto, which is the most important malaria vector in Africa, is a highly anthropophilic mosquito species, and the host-seeking behaviour of the females of this mosquito is guided by volatiles of human origin. Ammonia, lactic acid and several carboxylic acids are known to be present in the human odour blend. We investigated the effect of these compounds on naive female mosquitoes using a dual-port olfactometer. Ammonia was an attractant on its own, whereas lactic acid was not attractive. Carboxylic acids, offered as a mixture of 12 compounds, were repellent at the concentration tested. The addition of ammonia to the carboxylic acid mixture overruled the repellent effect of the latter. Combining ammonia with either lactic acid or the carboxylic acids did not enhance the attractiveness of ammonia alone. However, a synergistic effect was found when ammonia, lactic acid and the carboxylic acids were applied as a blend. Our findings indicate that An. gambiae s.s. relies on the combination of ammonia, lactic acid and carboxylic acids in its orientation to human hosts. The role of lactic acid in this tripartite synergism differs from that reported for the yellow fever mosquito Aedes aegypti.

In field studies, Anopheles gambiae Giles sensu stricto obtains most blood meals from humans, whereas Anopheles quadriannulatus Theobald bites predominantly bovids. We investigated whether host odors modulate the host preference of these mosquito species. In a dual-choice olfactometer, mosquitoes were given a choice between clean air and putative host-specific odor blends. An. gambiae chose 'human odor' over clean air and clean air over cow odor.' Although Anopheles quadriannulatus did not choose cow odor over clean air, it chose clean air over human odor. Cheese odor, which attracted An. gambiae, did not result in higher trap catch of An. quadriannulatus. We conclude that the degree of anthropophagy of An. gambiae s.l. has an innate olfactory basis.

The effectiveness of traps baited with (5R,6S)-6-acetoxy-5-hexadecanolide (the synthetic oviposition pheromone) and grass infusions in sampling a population of gravid Culex quinquefasciatus Say was conducted in Muheza, Northeast Tanzania. A counterflow geometry (CFG) trap baited with pheromone and set outdoors, adjacent to a pit latrine building, collected more gravid Cx. quinquefasciatus than a CDC trap baited with pheromone and operated without light. Inside pit latrine buildings, significantly more gravid Cx. quinquefasciatus were collected in a CFG trap-baited with pheromone or grass infusion than in traps baited with tap water. CFG traps baited with either grass infusion or pheromone and set outdoors, away from known breeding sites, caught significantly more gravid Cx. quinquefasciatus than traps baited with tap water. CFG traps baited with pheromone + grass infusion caught significantly more gravid Cx. quinquefasciatus than CFG traps baited with either grass infusion or pheromone. In both cases, the proportion of gravid mosquitoes increased as traps were moved away from a natural emergence site. More gravid Cx. quinquefasciatus were collected in a pheromone-baited CFG trap than were egg rafts deposited in a jar with pheromone-treated water. It is concluded that CFG traps baited with oviposition attractants can be used effectively to sample gravid Cx. quinquefasciatus.

Anopheles quadriannulatus, the cattle-feeding member of the An. gambiae complex, was fed human blood which contained cultured gametocytes of Plasmodium falciparum, using a membrane feeding system. After 7 days, 33-80% of the mosquitoes that took a blood meal contained developing oocysts. In 7 out of 12 females sporozoites were found in the salivary glands 14 days after the infectious blood meal. Control groups of An. gambiae s.s. and An. stephensi became readily infected with > 90% developing oocysts. All of the An. gambiae dissected 14 days after the infectious blood meal had sporozoites in their salivary glands. The results demonstrate that An. quadriannulatus is susceptible to infections with P. falciparum.

The survival, metabolic reserves, and host-seeking response of unfed Anopheles gambiae Giles sensu stricto females of different size classes were investigated from 1 to 6 d after emergence. In addition, the effect of blood meal size and frequency of feeding on the accumulation of metabolic reserves and reproductive development were investigated during the 1st gonotrophic cycle. Large females survived longer and contained significantly more protein, glycogen, and lipid at emergence than did small females. Over 6 d, the host-seeking response of large mosquitoes was greater than that of small ones. There was no gonotrophic development in unfed small females after emergence, whereas < or = 52% of large females developed oocytes to Christophers stage II. One blood meal, given by enema, resulted in gonotrophic development to stage II in small females, whereas only large females matured oocytes to Christophers stage V. In both large and small females, egg development was related to the size of the blood meal, but large females were better able to utilize the meals. Two successive blood meals enhanced the ability of small females to develop eggs. The pregravid condition commonly observed in An. gambiae appears to be determined by insufficient metabolic reserves at emergence, and the probability for small individuals to be pregravid is greater than for large mosquitoes because of the need to build up sufficient energy reserves before gonotrophic development can occur. The consequence of this behavior in relation to malaria transmission is discussed.

The current geographic range of malaria is much smaller than its potential range. In many regions there exists a phenomena characterized as "Anophelism without malaria." The vectors are present but malaria transmission does not occur. Vectorial capacity often has been used as a parameter to estimate the susceptibility of an area to malaria. Model computations with global climatological data show that a dynamic concept of vectorial capacity can be used as a comparative risk indicator to predict the current extent and distribution of malarious regions in the world. A sensitivity analysis done in 3 distinct geographic areas shows that the areas of largest change of epidemic potential caused by a temperature increase are those where mosquitoes already occur but where development of the parasite is limited by temperature. Computations with the model presented here predict, with different climate scenarios, an increased malaria risk in areas bordering malaria endemic regions and at higher altitudes within malarious regions under a temperature increase of 2-4 degrees C.

Individually occupied tents, modified for sampling mosquitoes, were used to assess the attractiveness of 3 humans for Anopheles gambiae Giles s.l., A. funestus Giles, Culex quinquefasciatus Say and Mansonia spp. in north-east Tanzania. Significant differences (P < 0.025) in attractiveness were observed for the first 3 species, whereas Mansonia did not show any preference. Because of the objective sampling method used, these results provide the first conclusive evidence that isolated people vary in attractiveness to mosquitoes.

Four aspects of olfaction in host location by tsetse flies, Glossina spp., are discussed as follows: (1) host location and its mechanism, (2) factors affecting host location, (3) kairomones and host location, and (4) kairomones and host selection. Flight behavior in the various phases of host location (i.e., ranging, activation, orientation, and landing) in the absence and presence of olfactory cues is summarized. Movement toward an odor source is effected inter alia through optomotor-steered, upwind anemotaxis. It is still unclear how tsetse employ upwind anemotaxis to realize host location, considering the often highly variable wind direction. Olfactorily induced activation is governed by the olfactory cue perceived and threshold levels set by the internal state of the fly. The former depends on the odor source and distance from it; the latter is determined by species, sex, and physiological state. Wind direction and speed, as well as vegetation and the mobility of the host, interfere with successful completion of odor-induced host location. Close-range olfactory cues (including composition and concentration gradients), visual cues, and nutritional state determine whether a fly, having reached the host animal, will land on it. Carbon dioxide is important in host location because it induces landing and long-range attraction. The role of the other kairomones (acetone, 1-octen-3-ol, 4-methyl-phenol, and 3-n-propyl-phenol) is less clear. Apart from the complacency of various host species under tsetse attack, host choice by tsetse is predominantly opportunistic and primarily the result of the frequency of successful tsetse-host encounters. Nevertheless, host selection based on olfactory cues cannot be ruled out.