High prevalence and lineage diversity of avian malaria in wild populations of great tits (Parus major) and mosquitoes (Culex pipiens).

Avian malaria studies have taken a prominent place in different aspects of evolutionary ecology. Despite a recent interest in the role of vectors within the complex interaction system of the malaria parasite, they have largely been ignored in most epidemiological studies. Epidemiology of the disease is however strongly related to the vector's ecology and behaviour, and there is a need for basic investigations to obtain a better picture of the natural associations between Plasmodium lineages, vector species and bird hosts. The aim of the present study was to identify the mosquito species involved in the transmission of the haemosporidian parasites Plasmodium spp. in two wild populations of breeding great tits (Parus major) in western Switzerland. Additionally, we compared Plasmodium lineages, based on mitochondrial DNA cytochrome b sequences, between the vertebrate and dipteran hosts, and evaluated the prevalence of the parasite in the mosquito populations. Plasmodium spp. were detected in Culex pipiens only, with an overall 6.6% prevalence. Among the six cytochrome b lineages of Plasmodium identified in the mosquitoes, three were also present in great tits. The results provide evidence for the first time that C. pipiens can act as a natural vector of avian malaria in Europe and yield baseline data for future research on the epidemiology of avian malaria in European countries.

Natural malaria infection reduces starvation resistance of nutritionally stressed mosquitoes.

In disease ecology, there is growing evidence that environmental quality interacts with parasite and host to determine host susceptibility to an infection. Most studies of malaria parasites have focused on the infection costs incurred by the hosts, and few have investigated the costs on mosquito vectors. The interplay between the environment, the vector and the parasite has therefore mostly been ignored and often relied on unnatural or allopatric Plasmodium/vector associations. Here, we investigated the effects of natural avian malaria infection on both fecundity and survival of field-caught female Culex pipiens mosquitoes, individually maintained in laboratory conditions. We manipulated environmental quality by providing mosquitoes with different concentrations of glucose-feeding solution prior to submitting them to a starvation challenge. We used molecular-based methods to assess mosquitoes' infection status. We found that mosquitoes infected with Plasmodium had lower starvation resistance than uninfected ones only under low nutritional conditions. The effect of nutritional stress varied with time, with the difference of starvation resistance between optimally and suboptimally fed mosquitoes increasing from spring to summer, as shown by a significant interaction between diet treatment and months of capture. Infected and uninfected mosquitoes had similar clutch size, indicating no effect of infection on fecundity. Overall, this study suggests that avian malaria vectors may suffer Plasmodium infection costs in their natural habitat, under certain environmental conditions. This may have major implications for disease transmission in the wild.

Screening of Bacillus thuringiensis strains effective against mosquitoes

The objective of this work was to evaluate 210 Bacillus thuringiensis strains against Aedes aegypti and Culex quinquefasciatus larvae to select the most effective. These strains were isolated from different regions of Brazil and are stored in a Bacillus spp. collection at Embrapa Recursos Genéticos e Biotecnologia, Brasília, Brazil. The selected strains were characterized by morphological (microscopy), biochemical (SDS-PAGE 10%) and molecular (PCR) methods. Six B. thuringiensis strains were identified as mosquito-toxic after the selective bioassays. None of the strains produced the expected PCR products for detection of cry4, cry11 and cyt1A genes. These results indicate that the activity of mosquitocidal Brazilian strains are not related with Cry4, Cry11 or Cyt proteins, so they could be used as an alternative bioinsecticide against mosquitoes.

The role of vectors in the epidemiology of avian malaria

Studies on host-parasite relationships have commonly reported that parasitized hosts undergo changes in their behavioural and life history traits. How do these changes affect the fitness of the hosts? What are the ecological and evolutionary drivers of these changes? These open questions are crucial to predict the parasite spread amongst hosts. Surprisingly, mosquito vectors of diseases to humans and animals have long been seen as passive parasite transporters, being unaffected by the infection though they also function as hosts. Natural parasite-vector interactions are therefore poorly documented in the literature. In this thesis, we seek to address the role of wild vectors in the epidemiology of avian Plasmodium, the etiological agents of malaria in birds. We first conducted avian malaria surveys in field-caught mosquitoes to identify the natural vectors in our temperate study area. We report that ornithophilic Culex pipiens primarily act as a vector for Plasmodium vaughani in spring, this parasite species being progressively replaced by P. relictum along with the season. Season-related factors may thus shape the mosquitoes' vectorial capacity. We then used experimental approaches to determine the effect of avian malaria on wild, naturally infected C. pipiens. We show that infected mosquitoes incur unavoidable physiological costs associated with parasite exploitation, these costs being expressed as a reduced survival under nutritionally stressed conditions only. These results are of significant importance for the epidemiology of avian malaria since seasonal changes in climate may likely influence food quality and quantity available to the mosquitoes. The host-selection preferences of the vectors with respect to the malaria-infection status of their bird hosts largely determine the disease spreading. In a second laboratory experiment, we thus offered wild C. pipiens the opportunity to choose between uninfected and naturally infected great tits, Parus major. We show that host-seeking mosquitoes have innate orientation preferences for uninfected birds. This suggests that avian malaria parasites exert strong selective pressures on their vectors, pushing them to evolve anti-parasite behaviours. We lastly investigated the links between malaria-associated symptoms in birds and resulting attractiveness to the mosquitoes. We show that experimentally malaria-infected canaries, Serinus canaria, suffer severe haematocrit reduction at peak parasitaemia and reduced basal metabolic rate later in the course of the infection. However, no links between infection and bird attractiveness to the mosquitoes were shown in an experiment using canaries as live bait for mosquito trap in the field. These links may have been masked by confounding environmental factors. Using a system where the vectors, parasites and hosts co-occur in sympatry, this thesis illustrates that vectors are not always Plasmodium permissive, which opposes to the traditional view that malaria parasites should have little effect on their vectors. The way that the vectors respond to the parasite threat is largely determined by the environmental conditions. This may have major implications for the epidemiology of avian malaria. - Les études portant sur les relations hôtes-parasites mentionnent souvent que les hôtes parasités subissent des modifications de leurs traits d'histoire de vie ou bien comportementaux. Comment ces changements affectent-ils la valeur sélective des hôtes et celle de leurs parasites ? Quels sont les déterminants de ces modifications ? Ces questions sont d'un grand intérêt en épidémiologie. Pour autant, les moustiques vecteurs de maladies infectieuses ont longtemps été perçus comme de simples transporteurs de parasites, n'étant pas affectés par ces derniers. Cette thèse porte sur le rôle des vecteurs dans l'épidémiologie des Plasmodium aviaires, agents étiologiques de la malaria chez les oiseaux. Dans le but d'identifier les vecteurs naturels de malaria aviaire dans notre zone d'étude, nous avons tout d'abord collecté des moustiques sur le terrain, puis déterminé leur statut infectieux. Nous rapportons que les moustiques Culex pipiens sont principalement impliqués dans la transmission de Plasmodium vaughani au printemps, cette espèce de parasite étant progressivement remplacée par P. relictum au fil de la saison de transmission. Nous avons ensuite conduit une expérience visant à déterminer l'effet de la malaria aviaire sur des C. pipiens sauvages, naturellement infectés. Nous montrons que des coûts sont associés à l'infection pour les moustiques. Ces coûts occasionnent une diminution de la survie des vecteurs seulement lorsque ceux-ci sont privés de ressources nutritionnelles. Des changements saisonniers de climats pourraient affecter la quantité et la qualité des ressources disponibles pour les vecteurs et donc, leur aptitude à transmettre l'infection. Les traits comportementaux des moustiques vecteurs, tels que la recherche et le choix d'un hôte pour se nourrir, sont d'une importance majeure pour la dispersion de la malaria. Pour cela, nous avons offert à des C. pipiens sauvages l'opportunité de choisir simultanément entre une mésange charbonnière (Parus major) saine et une autre naturellement infectée. Nous montrons que les moustiques s'orientent préférentiellement vers des mésanges saines. Les Plasmodium aviaires exerceraient donc de fortes pressions de sélection sur leurs vecteurs, favorisant ainsi l'évolution de comportements d'évitement des parasites. Enfin nous avons cherché à identifier de potentiels liens entre symptômes de l'infection malarique chez les oiseaux et attractivité de ces derniers pour les moustiques. Nous montrons que des canaris (Serinus canaria) expérimentalement infectés sont fortement anémiés au moment du pic infectieux et que leur métabolisme basai diminue plus tard au cours de l'infection. Toutefois, aucun lien entre le statut infectieux et l'attractivité des canaris pour les moustiques n'a pu être montré lors d'une expérience réalisée en nature. Il se peut que ces liens aient été masqués par des facteurs environnementaux confondants. Dans son ensemble, cette thèse illustre que, contrairement aux idées reçues, les vecteurs de malaria aviaire ne sont pas toujours permissifs avec leurs parasites. L'environnement apparaît aussi comme un facteur déterminant dans la réponse des vecteurs face à la menace d'infection malarique. Cela pourrait fortement affecter l'épidémiologie de la malaria aviaire.

Cryptic species status of Anopheles (Diptera: Culicidae) mosquitoes in Canada using a multidisciplinary approach

Many species of Anopheles mosquitoes (Diptera: Culicidae) are now recognized as species complexes whose members are often indistinguishable morphologically but identifiable based on ecological, genetic, or behavioural data. Because the members of species complexes often differ in their vector potential, accurate identification of vector species is essential for successful mosquito control. To investigate the cryptic species status of Anopheles mosquitoes in Canada, specimens were collected from across the country and examined using morphological, molecular, and ecological data. Six of the seven traditionally recognised species from Canada were collected from locations in British Columbia, Quebec, Newfoundland and Labrador, and throughout Ontario, including Anopheles barberi, An. earlei, An. freeborni, An. punctipennis, An. quadrimaculatus s.l., and An. walkeri. Variation in polymorphic traits within An. earlei, An. punctipennis, and An. quadrimaculatus s.l. were quantified and egg morphology examined using scanning electron microscopy. Morphological identification of adult and larval specimens suggested that two described cryptic species, An. perplexens and An. smaragdinus, were present in Canada. DNA sequence data were analysed for evidence of cryptic species using three molecular markers: COl, ITS2, and ITS!. Intraspecific COl variation was very low in most species «1 %), except for An. punctipennis with 2% sequence divergence between those from British Columbia (BC) and Ontario (ON), and An. walkeri with 7% sequence divergence between populations from Manitoulin Island (NO) and Long Point Provincial Park (LP). Similar patterns were also seen using ITS2 and ITS 1. Therefore, molecular data revealed the presence of two putative cryptic species within two species examined (i.e., An. walkeri and An. punctipennis), corresponding to collection location (i.e., NO vs. LP and BC vs. ON, respectively). Surprisingly, there was no molecular support for the presence of either An. perplexens or An. smaragdinus in Canada despite the morphological assessments. Ecological data from all collection sites were recorded and are available in an online database designed to manage all collection and identification data. Current bionomic information, including regional abundance, larval habitat, and species associations, was determined for each species. This multidisciplinary study of Anopheles mosquitoes is the first detailed investigation of these potential disease vectors in Canada and demonstrates the importance of an integrated approach to anopheline systematics that includes molecular data.

Influence of dietary nutrients on life history-related traits of black flies and mosquitoes

The sugar-feeding ecology of dipteran vectors has recently been targeted because it presents opportunities to inoculate common food sources for these dipterans with entomopathogenic bacteria as a means of controlling the population of host-seeking adult dipteran vectors. Whereas this approach to vector control holds some promise, differences in the nutrient composition and concentration in sugary food sources can influence the food selection pattern of dipteran vectors and potentially confound the outcomes of field trials on the efficacy of entomopathogenic bacteria as vector control agents. Further, nutrient components of bacteria-inoculated artificial diets may present unintended effects of extending the survivorship or fecundity of the target population and potentially render the whole approach counterproductive. The present study investigated the diet-specific factors that influence the foraging decisions of female Simulium venustum/verecundum (Diptera: Simuliidae) and female Anopheles stephensi (Diptera: Culicidae) on artificial nectar and honeydew. Paired choice experiments showed that the black flies forage more frequently from high calorie diets, which contained melezitose, or those diets that contained amino acids, compared to low calorie melezitose-free diets or amino acid-free diets. The mosquitoes however displayed a more random diet selection pattern. The effects of sugary diets on certain life-history traits considered to be important to the ecological fitness of the black flies and mosquitoes were also investigated. Sugary diets had no significant effect on the survivorship and fecundity of the black flies, but they influenced the resistance of Leucocytozoon-infected flies to the parasite. Amino acid-containing diets appeared to extend the survival of mosquitoes, and also allowed them to take more vertebrate blood when they blood fed.

The Influence of Male Diet on Life History Traits of Female Mosquitoes

In the field, mosquitoes characteristically feed on sugars soon after emergence and intermittently during their adult lives. Sugar meals are commonly derived from plant nectar and homopteran honeydew, and without them, adults can only survive for a few days on larval reserves. In addition to sugar, females of most species rely on blood for the initiation and maintenance of egg development; thus their reproductive success depends to some extent on the availability of blood hosts. Males, on the other hand, feed exclusively on sugars. Consequently, their sexual maturation and reproductive success is largely dependent upon access to sugar sources. Plant nectar and homopteran honeydew are the two main sugar sources utilized by mosquitoes in the wild. Previous laboratory studies had shown that differences between nectar sources can affect the survivorship and biting frequency of disease vectoring mosquitoes. However, little is known on how sugar composition influence the reproductive processes in male mosquitoes. Male mosquitoes transfer accessory gland proteins and other hormones to their mates along with sperm during mating. In the female, these seminal fluid constituents exert their influence on reproductive genes that control ovulation and vitellogenesis. The present study tests the hypothesis that the mates of males consuming different sugar meals will exhibit varying levels of induction of vitellogenin (a gene which regulates the expression of egg yolk precursor proteins). Real-time quantitative RT-PCR was used to investigate how each sugar meal indirectly influences vitellogenin mRNA abundance in female Anopheles stephensi following mating. Results indicate that mates of nectar-fed males exhibit 2-fold greater change in vitellogenin expression than the mates of honeydew-fed males. However, this response did not occur in non-blood fed controls. These findings suggest that the stimulatory effect of mating on vitellogenesis in blood meal-reliant (i.e. anautogenous) mosquitoes may only be synergistic in nature. The present study also sought to compare the potential fitness costs of mating incurred by females that do not necessarily require a blood meal to initiate a reproductive cycle (i.e., exhibit autogeny). Females of the facultatively autogenous mosquito, Culex molestus were allowed to mate with males sustained on either nectar or honedyew. Mean lifetime fecundity and survivorship of females under the two different mating regimes were then recorded. Additionally, one-dimensional gel electrophoresis was used to verify the transfer of male accessory gland proteins to the sperm storage organs of females during mating.While there was no significant difference in survival between the test treatments, the mates of nectar-fed males produced 11% more eggs on average than mates of honeydew-fed males. However, additional data are needed to justify the extrapolation of these findings to natural settings. These findings prompt further investigation as the differences caused by diet variation in males may be reflected across other life history traits such as mating frequency and insemination capacity.

Distribution of mosquitoes in the South East of Argentina and first report on the analysis based on 18S rDNA and COI sequences

Although Mar del Plata is the most important city on the Atlantic coast of Argentina, mosquitoes inhabiting such area are almost uncharacterized. To increase our knowledge in their distribution, we sampled specimens of natural populations. After the morphological identification based on taxonomic keys, sequences of DNA from small ribosomal subunit (18S rDNA) and cytochrome c oxidase I (COI) genes were obtained from native species and the phylogenetic analysis of these sequences were done. Fourteen species from the genera Uranotaenia, Culex, Ochlerotatus and Psorophora were found and identified. Our 18S rDNA and COI-based analysis indicates the relationships among groups at the supra-species level in concordance with mosquito taxonomy. The introduction and spread of vectors and diseases carried by them are not known in Mar del Plata, but some of the species found in this study were reported as pathogen vectors.

British container breeding mosquitoes: the impact of urbanisation and climate change on community composition and phenology

The proliferation of artificial container habitats in urban areas has benefitted urban adaptable mosquito species globally. In areas where mosquitoes transmit viruses and parasites, it can promote vector population productivity and fuel mosquito-borne disease outbreaks. In Britain, storage of water in garden water butts is increasing, potentially expanding mosquito larval habitats and influencing population dynamics and mosquito-human contact. Here we show that the community composition, abundance and phenology of mosquitoes breeding in experimental water butt containers were influenced by urbanisation. Mosquitoes in urban containers were less species-rich but present in significantly higher densities (100.4±21.3) per container than those in rural containers (77.7±15.1). Urban containers were dominated by Culex pipiens (a potential vector of West Nile Virus [WNV]) and appear to be increasingly exploited by Anopheles plumbeus (a human-biting potential WNV and malaria vector). Culex phenology was influenced by urban land use type, with peaks in larval abundances occurring earlier in urban than rural containers. Among other factors, this was associated with an urban heat island effect which raised urban air and water temperatures by 0.9°C and 1.2°C respectively. Further increases in domestic water storage, particularly in urban areas, in combination with climate changes will likely alter mosquito population dynamics in the UK.

Morphological and fecundity traits of Culex mosquitoes caught in gravid traps in urban and rural Berkshire, UK

Culex pipiens s.l. is one of the primary vectors of West Nile Virus in the USA and Continental Europe. The seasonal abundance and eco-behavioural characteristics of the typical form, Cx. pipiens pipiens, make it a key putative vector in Britain. Surveillance of Culex larvae and adults is essential to detect any changes to spatial and seasonal activity or morphological traits that may increase the risk of disease transmission. Here we report the use of the modified Reiter gravid box trap, which is commonly used in the USA but scarcely used in the UK, to assess its suitability as a tool for British female Culex mosquito surveillance. Trapping was carried out at 110 sites in urban and rural gardens in Berkshire in May, July and September 2013. We tested if reproductively active adult female Culex are more abundant in urban than rural gardens and if wing characteristic traits and egg raft size are influenced by location and seasonal variations. Gravid traps were highly selective for Culex mosquitoes, on average catching significantly more per trap in urban gardens (32.4 ± 6.2) than rural gardens (19.3 ± 4.0) and more in July than in May or September. The majority of females were caught alive in a good condition. Wing lengths were measured as an indicator of size. Females flying in September were significantly smaller than females in May or July. Further non-significant differences in morphology and fecundity between urban and rural populations were found that should be explored further across the seasons.

Quinine, mosquitoes and empire: reassembling malaria in British India, 1890-1910

The drug quinine figured as an object of enforced consumption in British India between the late 1890s and the 1910s, when the corresponding diagnostic category malaria itself was redefined as a mosquito-borne fever disease. This article details an overlapping milieu in which quinine, mosquitoes and malaria emerged as intrinsic components of shared and symbiotic histories. It combines insights from new imperial histories, constructivism in the histories of medicine and literature about non-humans in science studies to examine the ways in which histories of insects, drugs, disease and empire interacted and shaped one another. Firstly, it locates the production of historical intimacies between quinine, malaria and mosquitoes within the exigencies and apparatuses of imperial rule. In so doing, it explores the intersections between the worlds of colonial governance, medical knowledge, vernacular markets and pharmaceutical business. Secondly, it outlines ways to narrate characteristics and enabling properties of non-humans (such as quinines and mosquitoes) while retaining a constructivist critique of scientism and empire. Thirdly, it shows how empire itself was reshaped and reinforced while occasioning the proliferation of categories and entities like malaria, quinine and mosquitoes.

Forest-obligate Sabethes mosquitoes suggest palaeoecological perturbations

The origin of tropical forest diversity has been hotly debated for decades. Although specific mechanisms vary, many such explanations propose some vicariance in the distribution of species during glacial cycles and several have been supported by genetic evidence in Neotropical taxa. However, no consensus exists with regard to the extent or time frame of the vicariance events. Here, we analyse the cytochrome oxidase II mitochondrial gene of 250 Sabethes albiprivus B mosquitoes sampled from western Sao Paulo in Brazil. There was very low population structuring among collection sites (Phi(ST) = 0.03, P = 0.04). Historic demographic analyses and the contemporary geographic distribution of genetic diversity suggest that the populations sampled are not at demographic equilibrium. Three distinct mitochondrial clades were observed in the samples, one of which differed significantly in its geographic distribution relative to the other two within a small sampling area (similar to 70 x 35 km). This fact, supported by the inability of maximum likelihood analyses to achieve adequate fits to simple models for the population demography of the species, suggests a more complex history, possibly involving disjunct forest refugia. This hypothesis is supported by a genetic signal of recent population growth, which is expected if population sizes of this forest-obligate insect increased during the forest expansions that followed glacial periods. Although a time frame cannot be reliably inferred for the vicariance event leading to the three genetic clades, molecular clock estimates place this at similar to 1 Myr before present.