Ovipositional Behavior of Anopheles gambiae Mosquitoes

Mosquito eggs laid within two hours are necessary for transgenic (injection) studies, because mosquito eggs become hard after that period. Thus, in order to have eggs available within this two-hour window, it is important to understand the ovipositional behavior of Anopheles gambiae s.s.. In the present study, the ovipositional behavior of An. gambiae s.s. (Kisumu) was investigated in several different conditions: age of mosquitoes, time post blood meal to access oviposition substrate, and light conditions. Two groups of mosquitoes, 3–5 days old and 9–11 days old were blood-fed. For those mosquito groups, an oviposition dish was set either at 48 hours or 72 hours after the blood meal either in a light condition or in an artificial dark condition. The number of laid eggs was compared among the different conditions. The 3–5 day-old mosquitoes apparently produced a higher number of eggs than 9–11 day-old mosquitoes, while there was no significant difference between the two groups. The number of laid eggs per one surviving blood-fed mosquito in the dark condition was significantly higher than that in the light condition (p = 0.03). Providing an oviposition dish at 72 hours after blood meal resulted in a significantly higher number of laid eggs per one surviving blood-fed mosquito than at 48 hours after blood meal (p = 0.03). In conclusion, the optimal condition to have readily available egg supply for transgenic analysis was as follows: 3–5 day-old mosquitoes with an oviposition dish placed at 72 hours after the blood meal in a dark environment.     

Chikungunya Virus Replication in Salivary Glands of the Mosquito Aedes albopictus

Chikungunya virus (CHIKV) is an emerging arbovirus transmitted to humans by mosquitoes such as Aedes albopictus. To be transmitted, CHIKV must replicate in the mosquito midgut, then disseminate in the hemocele and infect the salivary glands before being released in saliva. We have developed a standardized protocol to visualize viral particles in the mosquito salivary glands using transmission electron microscopy. Here we provide direct evidence for CHIKV replication and storage in Ae. albopictus salivary glands.     

NADP+-dependent farnesol dehydrogenase,a corpora allata enzyme involved in juvenile hormone synthesis

The synthesis of juvenile hormone (JH) is an attractive target for control of insect pests and vectors of disease, but the minute size of the corpora allata (CA), the glands that synthesize JH, has made it difficult to identify important biosynthetic enzymes by classical biochemical approaches. Here, we report identification and characterization of an insect farnesol dehydrogenase (AaSDR-1) that oxidizes farnesol into farnesal, a precursor of JH, in the CA. AaSDR-1 was isolated as an EST in a library of the corpora allata-corpora cardiaca of the mosquito Aedes aegypti. The 245-amino acid protein presents the typical short-chain dehydrogenase (SDR) Rossmann-fold motif for nucleotide binding. This feature, together with other conserved sequence motifs, place AaSDR-1 into the “classical” NADP⁺-dependent cP2 SDR subfamily. The gene is part of a group of highly conserved paralogs that cluster together in the mosquito genome; similar clusters of orthologs were found in other insect species. AaSDR-1 acts as a homodimer and efficiently oxidizes C₁₀ to C₁₅ isoprenoid and aliphatic alcohols, showing the highest affinity for the conversion of farnesol into farnesal. Farnesol dehydrogenase activity was not detected in the CA of newly emerged mosquitoes but significant activity was detected 24 h later. Real time PCR experiments revealed that AaSDR-1 mRNA levels were very low in the inactive CA of the newly emerged female, but increased >30-fold 24 h later during the peak of JH synthesis. These results suggest that oxidation of farnesol might be a rate-limiting step in JH III synthesis in adult mosquitoes.     

Female-specific flightless phenotype for mosquito control

Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated 50–100 million new infections each year. Aedes aegypti is the major vector of dengue viruses in its range and control of this mosquito would reduce significantly human morbidity and mortality. Present mosquito control methods are not sufficiently effective and new approaches are needed urgently. A “sterile-male-release” strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. Transgenic strains of Aedes aegypti were engineered to have a repressible female-specific flightless phenotype using either two separate transgenes or a single transgene, based on the use of a female-specific indirect flight muscle promoter from the Aedes aegypti Actin-4 gene. These strains eliminate the need for sterilization by irradiation, permit male-only release (“genetic sexing”), and enable the release of eggs instead of adults. Furthermore, these strains are expected to facilitate area-wide control or elimination of dengue if adopted as part of an integrated pest management strategy.     

Phlebotomus papatasi exposure cross-protects mice against Leishmania major co-inoculated with Phlebotomus duboscqi salivary gland homogenate

Leishmania parasites are inoculated into host skin together with sand fly saliva and multiple exposures to uninfected sand fly bites protect mice against Leishmania infection. However, sand fly vectors differ in composition of the saliva and therefore the protection elicited by their salivary proteins was shown to be species-specific. On the other hand, the optimal vaccine based on sand fly salivary proteins should be based on conserved salivary proteins conferring cross-reactivity. In the present study we therefore focused on cross-protective properties of saliva from Phlebotomus papatasi and Phlebotomus duboscqi, the two natural vectors of Leishmania major. Two groups of mice exposed to bites of P. papatasi and two control, non-immunized groups were infected with L. major promastigotes along with either P. papatasi or P. duboscqi salivary gland homogenate. All mice were followed for the development of Leishmania lesions, parasite burdens, specific antibodies, and for production of NO, urea, or cytokines by peritoneal macrophages. Protection against Leishmania infection was observed not only in exposed mice challenged with homologous saliva but also in the group challenged with P. duboscqi saliva. Comparing both exposed groups, no significant differences were observed in parasite load, macrophage activity, or in the levels of anti-L. major and anti-P. papatasi/P. duboscqi antibodies. This is the first study showing cross-protection caused by salivary antigens of two Phlebotomus species. The cross-protective effect suggests that the anti-Leishmania vaccine based on P. papatasi salivary proteins might be applicable also in areas where L. major is transmitted by P. duboscqi.     

Transmission Potential of Two Chimeric Western Equine Encephalitis Vaccine Candidates in Culex tarsalis

Western equine encephalitis virus (WEEV) is a zoonotic alphavirus that circulates in western North America between passerine birds and mosquitoes, primarily Culex tarsalis. Since it was isolated in 1930, WEEV has caused tens of thousands of equine deaths in addition to thousands of human cases. In addition because WEEV is a virus of agricultural importance in addition to a public health threat, we developed two live-attenuated chimeric vaccine candidates that have been shown to be immunogenic and efficacious in mouse models. Vaccine candidate strains were developed by inserting the structural protein genes of WEEV strain McMillan (McM) or CO92-1356 into a Sindbis virus (SINV) strain AR339 backbone. The SIN/McM chimera also derived the N-terminal half of its capsid gene from a North American eastern equine encephalitis virus (EEEV) strain FL39-939 (henceforth referred to as SIN/EEE/McM). Although these vaccines do not generate viremia in mice, we further assessed their safety by exposing Cx. tarsalis to artificial blood meals containing high viral titers of each vaccine candidate. Both viruses exhibited a decreased rate of infection, dissemination, and transmission potential compared with the parental alphaviruses. Specifically, SIN/CO92 infected 37% of mosquitoes and disseminated in 8%, but failed to reach the saliva of the mosquitoes. In contrast, the SIN/EEE/McM virus was unable to infect, disseminate, or be transmitted in the saliva of any mosquitoes. These findings suggest that both vaccine candidates are less competent than the parental strains to be transmitted by the primary mosquito vector, Cx. tarsalis, and are unlikely to be reintroduced into a natural WEEV transmission cycle.     

The Effect of Temperature on Wolbachia-Mediated Dengue Virus Blocking in Aedes aegypti

Dengue fever, caused by dengue virus (DENV), is endemic in more than 100 countries. The lack of effective treatment of patients and the suboptimal efficacies of the tetravalent vaccine in trials highlight the urgent need to develop alternative strategies to lessen the burden of dengue fever. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits the replication of the DENV in the mosquito vector, Aedes aegypti. However, several recent studies have demonstrated the sensitivity of pathogens, vectors, and their symbionts to temperature. To understand how the tripartite interactions between the mosquito, DENV, and Wolbachia may change under different temperature regimes, we assessed the vector competence and transmission potential of DENV-infected mosquitoes reared at a common laboratory setting of a constant 25°C and at two diurnal temperature settings with mean of 25°C and 28°C and a fluctuating range of 8°C (±4°C). Temperature significantly affected DENV infection rate in the mosquitoes. Furthermore, temperature significantly influenced the proportion of mosquitoes that achieved transmission potential as measured by the presence of virus in the saliva. Regardless of the temperature regimes, Wolbachia significantly and efficiently reduced the proportion of mosquitoes achieving infection and transmission potential across all the temperature regimes studied. This work reinforces the robustness of the Wolbachia biocontrol strategy to field conditions in Cairns, Australia, and suggests that similar studies are required for local mosquito genotypes and field relevant temperatures for emerging field release sites globally.     

Plasmodium evasion of mosquito immunity and global malaria transmission: The lock-and-key theory

Plasmodium falciparum malaria originated in Africa and became global as humans migrated to other continents. During this journey, parasites encountered new mosquito species, some of them evolutionarily distant from African vectors. We have previously shown that the Pfs47 protein allows the parasite to evade the mosquito immune system of Anopheles gambiae mosquitoes. Here, we investigated the role of Pfs47-mediated immune evasion in the adaptation of P. falciparum to evolutionarily distant mosquito species. We found that P. falciparum isolates from Africa, Asia, or the Americas have low compatibility to malaria vectors from a different continent, an effect that is mediated by the mosquito immune system. We identified 42 different haplotypes of Pfs47 that have a strong geographic population structure and much lower haplotype diversity outside Africa. Replacement of the Pfs47 haplotypes in a P. falciparum isolate is sufficient to make it compatible to a different mosquito species. Those parasites that express a Pfs47 haplotype compatible with a given vector evade antiplasmodial immunity and survive. We propose that Pfs47-mediated immune evasion has been critical for the globalization of P. falciparum malaria as parasites adapted to new vector species. Our findings predict that this ongoing selective force by the mosquito immune system could influence the dispersal of Plasmodium genetic traits and point to Pfs47 as a potential target to block malaria transmission. A new model, the “lock-and-key theory” of P. falciparum globalization, is proposed, and its implications are discussed.The most deadly form of malaria in humans is caused by Plasmodium falciparum parasites. Malaria originated in Africa (1, 2) and is transmitted by anopheline mosquitoes. The disease became global as humans migrated to other continents and parasites encountered different mosquito species that were sometimes evolutionarily distant from African vectors (3). For example, anophelines of the subgenus Nyssorhynchus (malaria vectors in Central and South America, such as Anopheles albimanus) diverged from the subgenus Cellia (malaria vectors in Africa, India, and South Asia) about 100 Mya (4). P. falciparum parasites are transmitted by more than 70 different anopheline species worldwide (3), but compatibilities differ between specific vector–parasite combinations (5). For example, P. falciparum NF54 (Pf NF54), of putative African origin, effectively infects Anopheles gambiae, the main malaria vector in sub-Saharan Africa; but A. albimanus is highly refractory to this strain (6–8); whereas Asian P. falciparum isolates infect Anopheles stephensi (Nijmegen strain), a major vector in India, more effectively than A. gambiae (9). Similar differences in compatibility have been reported between Plasmodium vivax and different anopheline species (10, 11). The A. gambiae immune system can mount effective antiplasmodial responses mediated by the complement-like system that limit infection (12). We have previously shown that some P. falciparum lines can avoid detection by the A. gambiae immune system (13) and identified Pfs47 as the gene that mediated immune evasion (14). Here, we present direct evidence of selection of P. falciparum by the mosquito immune system and show that providing P. falciparum with a Pfs47 haplotype compatible for a given anopheline mosquito is sufficient for the parasite to evade mosquito immunity. The implications of P. falciparum selection by mosquitoes for global malaria transmission are discussed.     

Allergie aux piqûres de moustiques

In contrast to hymenoptera stings, anaphylactic reactions due to mosquito bites are very rare. In mosquito-allergic patients, mosquito bites can provoke immediate and/or delayed cutaneous reactions, the former involving IgE and IgG, the latter involving lymphocytes. Such cutaneous reactions may last for several days, and they are often painful and affect the involved person's quality of life. Children, who do not have natural immunity against mosquito salivary allergens, are particularly at risk, especially in heavily infested areas. While immunity may be acquired as a result of repeated bites occurring over a long period of time, specific immunotherapy with whole body extracts has sometimes been proposed as a complement to well-defined preventive measures, but in the absence of standardized extracts this remains questionable. The recent identification of the major allergens in mosquito saliva and the production of recombinant mosquito allergens that cross-react with salivary allergens from various type of mosquitoes should facilitate the diagnosis of mosquito allergy and justify immunotherapy for individuals who have serious allergic reactions following mosquito bites.     

Analysis of off-target effects in CRISPR-based gene drives in the human malaria mosquito

CRISPR-Cas9 nuclease-based gene drives have been developed toward the aim of control of the human malaria vector Anopheles gambiae. Gene drives are based on an active source of Cas9 nuclease in the germline that promotes super-Mendelian inheritance of the transgene by homology-directed repair (“homing”). Understanding whether CRISPR-induced off-target mutations are generated in Anopheles mosquitoes is an important aspect of risk assessment before any potential field release of this technology. We compared the frequencies and the propensity of off-target events to occur in four different gene-drive strains, including a deliberately promiscuous set-up, using a nongermline restricted promoter for SpCas9 and a guide RNA with many closely related sites (two or more mismatches) across the mosquito genome. Under this scenario we observed off-target mutations at frequencies no greater than 1.42%. We witnessed no evidence that CRISPR-induced off-target mutations were able to accumulate (or drive) in a mosquito population, despite multiple generations’ exposure to the CRISPR-Cas9 nuclease construct. Furthermore, judicious design of the guide RNA used for homing of the CRISPR construct, combined with tight temporal constriction of Cas9 expression to the germline, rendered off-target mutations undetectable. The findings of this study represent an important milestone for the understanding and managing of CRISPR-Cas9 specificity in mosquitoes, and demonstrates that CRISPR off-target editing in the context of a mosquito gene drive can be reduced to minimal levels.     

From the Cover: Transmission potential of Rickettsia felis infection by Anopheles gambiae mosquitoes

A growing number of recent reports have implicated Rickettsia felis as a human pathogen, paralleling the increasing detection of R. felis in arthropod hosts across the globe, primarily in fleas. Here Anopheles gambiae mosquitoes, the primary malarial vectors in sub-Saharan Africa, were fed with either blood meal infected with R. felis or infected cellular media administered in membrane feeding systems. In addition, a group of mosquitoes was fed on R. felis-infected BALB/c mice. The acquisition and persistence of R. felis in mosquitoes was demonstrated by quantitative PCR detection of the bacteria up to day 15 postinfection. R. felis was detected in mosquito feces up to day 14. Furthermore, R. felis was visualized by immunofluorescence in salivary glands, in and around the gut, and in the ovaries, although no vertical transmission was observed. R. felis was also found in the cotton used for sucrose feeding after the mosquitoes were fed infected blood. Natural bites from R. felis-infected An. gambiae were able to cause transient rickettsemias in mice, indicating that this mosquito species has the potential to be a vector of R. felis infection. This is particularly important given the recent report of high prevalence of R. felis infection in patients with “fever of unknown origin” in malaria-endemic areas.In 2002, Rickettsia felis, an obligate intracellular bacterium that belongs to the spotted fever group of Rickettsia, was definitively described (1, 2). Over the past 2 decades, an increasing number of reports have implicated R. felis as a human pathogen, paralleling an increase in reports of the detection of R. felis in arthropod hosts throughout the world (1, 3).By 2011, more than 70 human cases of R. felis had been reported worldwide, including in Central and South America, Asia, northern Africa, and Europe (1). More cases have been published since then, including the first probable human cases in Australia (4). In sub-Saharan Africa, recent studies have challenged the importance of R. felis infection in patients with “fever of unknown origin,” with this bacterium detected in up to 15% of such patients (5–7). In 2011, a potential R. felis primary infection, called “yaaf,” was suspected in the case of an 8-mo-old girl in Senegal with polymorphous skin lesions similar to those seen in patients from Mexico (8). The epidemiologic and clinical picture of this emerging infection in Africa, including its potential vectors, is poorly understood, however.Various arthropods, but primarily fleas, have been associated with R. felis (1, 3). More specifically, the cat flea Ctenocephalides felis is the arthropod in which R. felis has been most frequently detected. To date, it is the sole confirmed biological vector of R. felis, with both horizontal and vertical transmission making this flea a potential reservoir for this bacterium (9–11). However, in some countries where R. felis appears to be highly prevalent, such as Senegal, neither cat fleas nor other arthropods have been implicated in its transmission (12).Mosquitoes are the most important vectors of infectious diseases in humans, with more than one-half of the global population at risk for exposure to mosquito-borne infections (13, 14). Anopheles gambiae is known to be the primary vector of malaria in Africa, whereas Aedes albopictus is a vector of dengue and chikungunya (15, 16). Interestingly, Ae. albopictus and An. gambiae mosquito cells support R. felis growth (1, 17). In 2012, Ae. albopictus from Gabon and An. gambiae molecular form S (the primary African malarial vector) from Ivory Coast tested positive for R. felis by species-specific real-time quantitative PCR (qPCR) (17, 18). More recently, several mosquito species from Senegal were found to harbor R. felis, including Ae. luteocephalus, An. arabiensis, An. ziemanni, An. pharoensis, An. funestus, and Mansonia uniformis (5). These data raise new issues with respect to the epidemiology of R. felis in Africa, including the degree of vector competence of mosquitoes. The objective of this work was to study the acquisition and transmission of R. felis by An. gambiae mosquitoes in an experimental model of infection.     

Comparison of field-based xenodiagnosis and direct membrane feeding assays for evaluating host infectiousness to malaria vector Anopheles gambiae

Several techniques are currently being used to study host infectiousness to mosquitoes, including the experimental possibility of laboratory reared mosquitoes acquiring infections through membrane feeders or directly on host skin. Here, the relative performance of the laboratory-based membrane feeding method (DMFA) and the field-based xenodiagnosis (XD) of malaria infectious hosts using wild Anopheles mosquitoes were compared. A cross-sectional survey involving a sample of 70 children (aged 3–12 years) living in a malaria endemic area in Western Burkina Faso, was carried out to measure their infectiousness to Anopheles mosquitoes using two approaches. The first approach used the xenodiagnostic procedure in which children were exposed to mosquito bites overnight, being sleeping individually in different sentinel huts from 6 pm to 6 am (4 nights per child). Anopheles sp that had acquired blood-meal on each child were subsequently collected early in the morning, and examined for Plasmodium falciparum oocyst infection on day 7 post-feeding. In the second approach, the infectiousness of the same children was estimated by whole-blood membrane feeding procedure using F0 An. gambiae s.l. that emerged from field-collected larvae cohorts. In the DMFA, 41.4% of the children successfully infected at least one mosquito with the mean oocyst prevalence of only 4.6 ± 1.1% in the 2171 mosquitoes that were examined (mean oocyst intensity: 2.0 ± (std error of mean) 0.3 oocysts per infected midgut). Comparatively 78.6% of children yielded oocysts infection in mosquitoes during the XD approach (Chi square = 20.11, df = 1; p < 0.001), with a mean rate of 19.6 ± 2.0 in the 3752 wild caught mosquitoes (mean intensity: 3.93 ± 0.2 oocysts per infected mosquito). The DMFA failed to reveal a portion (n = 26) of infectious individuals that were sharply evidenced by the XD, particularly at low gametocyte densities or at levels that could not be detected by the classical microscopic examination of blood smears. As opposed to the resource consuming DMFA, which is often mined by technical constraints, using the XD method could be an advantage in experimental investigations of host infectiousness in areas where anopheline species cannot be conveniently reared for the experimental studies. Ethical aspects of this approach, mainly related to exposure of the human subjects to potentially infectious mosquito bites are discussed.     

Visualization of Malaria Parasites in the Skin Using the Luciferase Transgenic Parasite,Plasmodium berghei

We produced a transgenic rodent malaria parasite (Plasmodium berghei) that contained the luciferase gene under a promoter region of elongation factor-1α. These transgenic (TG) parasites expressed luciferase in all stages of their life cycle, as previously reported. However, we were the first to succeed in observing sporozoites as a mass in mouse skin following their deposition by the probing of infective mosquitoes. Our transgenic parasites may have emitted stronger bioluminescence than previous TG parasites. The estimated number of injected sporozoites by mosquitoes was between 34 and 775 (median 80). Since luciferase activity diminished immediately after the death of the parasites, luciferase activity could be an indicator of the existence of live parasites. Our results indicated that sporozoites survived at the probed site for more than 42 hours. We also detected sporozoites in the liver within 15 min of the intravenous injection. Bioluminescence was not observed in the lung, kidney or spleen. We confirmed the observation that the liver was the first organ in which malaria parasites entered and increased in number.     

Asymptomatic humans transmit dengue virus to mosquitoes

Three-quarters of the estimated 390 million dengue virus (DENV) infections each year are clinically inapparent. People with inapparent dengue virus infections are generally considered dead-end hosts for transmission because they do not reach sufficiently high viremia levels to infect mosquitoes. Here, we show that, despite their lower average level of viremia, asymptomatic people can be infectious to mosquitoes. Moreover, at a given level of viremia, DENV-infected people with no detectable symptoms or before the onset of symptoms are significantly more infectious to mosquitoes than people with symptomatic infections. Because DENV viremic people without clinical symptoms may be exposed to more mosquitoes through their undisrupted daily routines than sick people and represent the bulk of DENV infections, our data indicate that they have the potential to contribute significantly more to virus transmission to mosquitoes than previously recognized.With 3.97 billion people living in 128 countries currently at risk for infection, dengue viruses (DENV-1 to -4) cause more human morbidity and mortality worldwide than any other arthropod-borne virus (1, 2). Aedes aegypti mosquitoes are the primary vectors of DENV throughout the tropics (3). Dengue prevention relies on the control of Ae. aegypti populations, which is failing in most parts of the world due to lack of resources, lack of political will, and/or ineffective implementation (4).Virus transmission from infected humans to mosquitoes is a critical step in dengue epidemiology, but due to logistical constraints it has been directly examined only in a handful of studies to date (5). In initial experimental infections of human volunteers during the 1920s (6, 7), the onset of clinical symptoms occurred 4–9 d after virus inoculation by mosquito bite (8). DENV-infected humans were infectious to mosquitoes from 2 d before to 2 d after the onset of symptoms, and Ae. aegypti fed on viremic people were able to transmit virus to another person after at least 11 d of extrinsic incubation (8). Results from later studies indicated that, for naturally infected people with clinically apparent dengue, the duration of detectable viremia was on average 4–5 d after the onset of symptoms, but could range from 2 to 12 d (9, 10). Investigators in Vietnam fed Ae. aegypti directly on 208 symptomatic, hospitalized dengue patients and reported that the probability of successful human-to-mosquito DENV transmission was coincident with the kinetics of viremia (11). Dengue patients were infectious up to 5 d after the onset of symptoms, which generally corresponded with “defervescence” (11).All previous studies on human-to-mosquito DENV transmission were limited to people with overt illness and did not consider subclinical infections. An estimated 300 million of the total 390 million DENV infections per year are clinically inapparent or mildly symptomatic, i.e., no illness that disrupted a person’s daily routine (1). Following Grange et al. (12), we use “inapparent” or “subclinical” interchangeably to denote infections confirmed by virus detection or seroconversion, but with insufficient symptoms to be detected by existing surveillance systems and health care providers. “Asymptomatic” refers to a confirmed DENV infection in the complete absence of reported or detected symptoms. Inapparent human DENV infections are a potentially important component of the overall burden of dengue because they can serve as a previously unrecognized source of mosquito infection (12). Epidemic transmission of DENV associated with low viremia levels and mild illness has been reported (13). It has long been assumed, but not empirically verified, that people with inapparent infections fail to infect mosquitoes because they do not reach sufficiently high viremia levels (5). This assumption is based on the observation that disease severity is positively correlated with the magnitude of DENV viremia (10, 11, 14). To our knowledge, the only study that quantified viral RNA levels in a limited number of asymptomatic DENV infections in humans did not detect a significantly lower viremia (15), but infectiousness to mosquitoes was not evaluated. The aim of the present study was to document variation in DENV infectiousness of naturally infected humans across the spectrum of disease manifestations, including fully asymptomatic infections, and to verify the assumption that people with inapparent infections are not infectious to mosquitoes.     

The suitability of clay pots for indoor sampling of mosquitoes in an arid area in northern Tanzania

Water storage clay pots have been recently explored as method for outdoor mosquito sampling and as novel device for administrating insect-pathogenic fungi to mosquitoes. Their suitability for indoor mosquito sampling in natural conditions is unknown. We tested clay pots as indoor resting sites alongside catches by CDC light trap in an area of low malaria endemicity in northern Tanzania. Mosquitoes were caught by clay pots although the rate of female Anopheles mosquito catches was 22.64 (95% CI 11.26-45.52) times greater for CDC light traps. The proportion of fed female Anophelines was significantly higher for clay pots compared to CDC light trap (p < 0.001), indicating these methods sample different populations of mosquitoes. Although we were able to identify households with a consistently higher exposure to mosquitoes by CDC light trap, there was no apparent heterogeneity in mosquito catches by clay pots. We conclude that clay pots are not a reliable tool to sample mosquitoes in the dry season in an area of low transmission intensity with Anopheles arabiensis as principle vector.     

From the Cover: Increased survival of western corn rootworm on transgenic corn within three generations of on-plant greenhouse selection

To delay evolution of insect resistance to transgenic crops producing Bacillus thuringiensis (Bt) toxins, nearby “refuges” of host plants not producing Bt toxins are required in many regions. Such refuges are expected to be most effective in slowing resistance when the toxin concentration in Bt crops is high enough to kill all or nearly all insects heterozygous for resistance. However, Bt corn, Zea mays, introduced recently does not meet this “high-dose” criterion for control of western corn rootworm (WCR), Diabrotica virgifera virgifera. A greenhouse method of rearing WCR on transgenic corn expressing the Cry3Bb1 protein was used in which approximately 25% of previously unexposed larvae survived relative to isoline survival (compared to 1–4% in the field). After three generations of full larval rearing on Bt corn (Constant-exposure colony), WCR larval survival was equivalent on Bt corn and isoline corn in greenhouse trials, and the LC₅₀ was 22-fold greater for the Constant-exposure colony than for the Control colony in diet bioassays with Cry3Bb1 protein on artificial diet. After six generations of greenhouse selection, the ratio of larval recovery on Bt corn to isoline corn in the field was 11.7-fold greater for the Constant-exposure colony than the Control colony. Removal from selection for six generations did not decrease survival on Bt corn in the greenhouse. The results suggest that rapid response to selection is possible in the absence of mating with unexposed beetles, emphasizing the importance of effective refuges for resistance management.     

Transgenic malaria-resistant mosquitoes have a fitness advantage when feeding on Plasmodium-infected blood

The introduction of genes that impair Plasmodium development into mosquito populations is a strategy being considered for malaria control. The effect of the transgene on mosquito fitness is a crucial parameter influencing the success of this approach. We have previously shown that anopheline mosquitoes expressing the SM1 peptide in the midgut lumen are impaired for transmission of Plasmodium berghei. Moreover, the transgenic mosquitoes had no noticeable fitness load compared with nontransgenic mosquitoes when fed on noninfected mice. Here we show that when fed on mice infected with P. berghei, these transgenic mosquitoes are more fit (higher fecundity and lower mortality) than sibling nontransgenic mosquitoes. In cage experiments, transgenic mosquitoes gradually replaced nontransgenics when mosquitoes were maintained on mice infected with gametocyte-producing parasites (strain ANKA 2.34) but not when maintained on mice infected with gametocyte-deficient parasites (strain ANKA 2.33). These findings suggest that when feeding on Plasmodium-infected blood, transgenic malaria-resistant mosquitoes have a selective advantage over nontransgenic mosquitoes. This fitness advantage has important implications for devising malaria control strategies by means of genetic modification of mosquitoes.     

ANIMAL MODELS FOR THE STUDY OF LEISHMANIASIS IMMUNOLOGY

Leishmaniasis remains a major public health problem worldwide and is classified as Category I by the TDR/WHO, mainly due to the absence of control. Many experimental models like rodents, dogs and monkeys have been developed, each with specific features, in order to characterize the immune response to Leishmania species, but none reproduces the pathology observed in human disease. Conflicting data may arise in part because different parasite strains or species are being examined, different tissue targets (mice footpad, ear, or base of tail) are being infected, and different numbers (“low” 1×10² and “high” 1×10⁶) of metacyclic promastigotes have been inoculated. Recently, new approaches have been proposed to provide more meaningful data regarding the host response and pathogenesis that parallels human disease. The use of sand fly saliva and low numbers of parasites in experimental infections has led to mimic natural transmission and find new molecules and immune mechanisms which should be considered when designing vaccines and control strategies. Moreover, the use of wild rodents as experimental models has been proposed as a good alternative for studying the host-pathogen relationships and for testing candidate vaccines. To date, using natural reservoirs to study Leishmania infection has been challenging because immunologic reagents for use in wild rodents are lacking. This review discusses the principal immunological findings against Leishmania infection in different animal models highlighting the importance of using experimental conditions similar to natural transmission and reservoir species as experimental models to study the immunopathology of the disease.