Transinfection of Wolbachia wAlbB into Culex quinquefasciatus mosquitoes does not alter vector competence for Hawaiian avian malaria (Plasmodium relictum GRW4).

Avian malaria is expanding upslope with warmer temperatures and driving multiple species of Hawaiian birds towards extinction. Methods to reduce malaria transmission are urgently needed to prevent further declines. Releasing Wolbachia-infected incompatible male mosquitoes could suppress mosquito populations and releasing Wolbachia-infected female mosquitoes (or both sexes) could reduce pathogen transmission if the Wolbachia strain reduced vector competence. We cleared Culex quinquefasciatus of their natural Wolbachia pipientis wPip infection and transinfected them with Wolbachia wAlbB isolated from Aedes albopictus. We show that wAlbB infection was transmitted transovarially, and demonstrate cytoplasmic incompatibility with wild-type mosquitoes infected with wPip from Oahu and Maui, Hawaii. We measured vector competence for avian malaria, Plasmodium relictum, lineage GRW4, of seven mosquito lines (two with wAlbB; three with natural wPip infection, and two cleared of Wolbachia infection) by allowing them to feed on canaries infected with recently collected field isolates of Hawaiian P. relictum. We tested 73 groups (Ntotal = 1176) of mosquitoes for P. relictum infection in abdomens and thoraxes 6-14 days after feeding on a range of parasitemias from 0.028% to 2.49%, as well as a smaller subset of salivary glands. We found no measurable effect of Wolbachia on any endpoint, but strong effects of parasitemia, days post feeding, and mosquito strain on both abdomen and thorax infection prevalence. These results suggest that releasing male wAlbB-infected C. quinquefasciatus mosquitoes could suppress wPip-infected mosquito populations, but would have little positive or negative impact on mosquito vector competence for P. relictum if wAlbB became established in local mosquito populations. More broadly, the lack of Wolbachia effects on vector competence we observed highlights the variable impacts of both native and transinfected Wolbachia infections in mosquitoes.

Entomological assessment in 'hotspots' of four lymphatic filariasis endemic districts, Central Nepal during post-MDA surveillance.

BACKGROUND OBJECTIVES: Annual mass drug administration (MDA) is the main strategy to interrupt the transmission of lymphatic filariasis (LF) in the community. The main aim of monitoring the MDA program, for its effectiveness and interruption of LF is the post-MDA surveillance using antigen survey in children born after MDA. The latest technique of new research suggests that xenomonitoring is an effective tool for monitoring LF intervention. The objective of this study was to assess the W. bancrofti infection/or infectivity in vector mosquitoes by xenomonitoring during post-MDA surveillance. METHODS: A descriptive cross-sectional study was conducted in the hotspots of selected four districts of Central Nepal. A gravid trap technique was used for sampling mosquitoes. Infection/or infectivity was determined via the dissection of vector mosquitoes. Anopheles, Aedes, Armigerus and Culex species were collected from hotspots of four endemic districts, two from the hilly region (Lalitpur and Dhading) and two from Terai region (Bara and Mahottari) of Central Nepal. RESULTS: A total of 4450 mosquitoes belonging to four genera, Anopheles, Culex, Armigeres, and Aedes were collected from four hotspots. The distribution of Culex quinquefasciatus was found to be the highest, 88.9% (n=3955/4450) followed by Cx. vishnui (4.5%), Armigeres sp (5.8%), An. culicifascies (0.2%), Aedes spp (0.8%). The proportion of female mosquitoes trapped is significantly higher. A total of 3344 parous Cx. quinquefasciatus mosquitoes were dissected for any larval stage of W. bancrofti. We could not find any filarial infection in dissected mosquito samples. INTERPRETATION CONCLUSION: We conclude that the gravid trap is an efficient tool for the collection of gravid Cx. quinquefasciatus mosquitoes for xenomonitoring studies of filariasis endemic regions. Vector composition indicated a maximum number of vector mosquitoes of lymphatic filariasis were trapped compared with the other three species. Distribution and density of Cx. quinquefasciatus was found highest in four hotspots of endemic districts. None of the Cx. quinquefasciatus dissected were found to be infected by larval forms of filaria. Since the low levels of infection persistence in the human population in these hot spots, vector infection and infectivity can't be ignored. Microscopic xenomonitoring at a low level of infection persistent is less likely to be efficient so molecular xenomonitoring along with a large sample should be required in each of the hot spots of the districts. Additionally, area is receptive so further vector control intervention should be required to reduce the risk of resurgence of infection.

Genomic advances in the study of the mosquito vector during avian malaria infection.

Invertebrate host­parasite associations are one of the keystones in order to understand vector-borne diseases. The study of these specific interactions provides information not only about how the vector is affected by the parasite at the gene-expression level, but might also reveal mosquito strategies for blocking the transmission of the parasites. A very well-known vector for human malaria is Anopheles gambiae. This mosquito species has been the main focus for genomics studies determining essential key genes and pathways over the course of a malaria infection. However, to-date there is an important knowledge gap concerning other non-mammophilic mosquito species, for example some species from the Culex genera which may transmit avian malaria but also zoonotic pathogens such as West Nile virus. From an evolutionary perspective, these 2 mosquito genera diverged 170 million years ago, hence allowing studies in both species determining evolutionary conserved genes essential during malaria infections, which in turn might help to find key genes for blocking malaria cycle inside the mosquito. Here, we extensively review the current knowledge on key genes and pathways expressed in Anopheles over the course of malaria infections and highlight the importance of conducting genomic investigations for detecting pathways in Culex mosquitoes linked to infection of avian malaria. By pooling this information, we underline the need to increase genomic studies in mosquito­parasite associations, such as the one in Culex­Plasmodium, that can provide a better understanding of the infection dynamics in wildlife and reduce the negative impact on ecosystems.

First evidence for development of Plasmodium relictum (Grassi and Feletti, 1891) sporozoites in the salivary glands of Culex modestus Ficalbi, 1889.

The competence of insect vectors to transmit diseases plays a key role in host-parasite interactions and in the dynamics of avian malaria and other haemosporidian infections (Apicomplexa, Haemosporida). However, the presence of parasite DNA in the body of blood-sucking insects does not always constitute evidence for their competence as vectors. In this study, we investigate the susceptibility of wild-caught mosquitoes (Culex spp.) to complete sporogony of Plasmodium relictum (cyt b lineage SGS1) isolated from great tits (Parus major L., 1758). Adult female mosquitoes were collected with a CO2 bait trap overnight. A set of 50 mosquitoes was allowed to feed for 3 h at night on a single great tit infected with P. relictum. This trial was repeated on 6 different birds. The bloodfed mosquitoes that survived (n = 68) were dissected within 1-2 days (for ookinetes, n = 10) and 10-33 days post infection (for oocysts and sporozoites, n = 58) in order to confirm the respective parasite stages in their organs. The experiment confirmed the successful development of P. relictum (cyt b lineage SGS1) to the stage of sporozoites in Culex pipiens L., 1758 (n = 27) and in Culex modestus (n = 2). Our study provides the first evidence that C. modestus is a competent vector of P. relictum isolated from great tits, suggesting that this mosquito species could also play a role in the natural transmission of avian malaria.

Pathology of an iridescent virus in immature Culex pipiens L. (Diptera, Culicidae).

Iridovirus in Culex pipiens was reported for the first time in 2012. Later studies of horizontal transmission were performed, in which an interaction with the parasite Strelkovimermis spiculatus acting as viral vector was recognized. In the present study, we observed aspects of the pathology produced by an invertebrate iridescent virus in laboratory infected immature Cx. pipiens as well as in infected immature Cx. pipiens in the field. In the laboratory infected larvae, the infection and mortality were asynchronous. Signs of infection in larvae exposed to the virus were observed between the second and the fourth days post-exposure in 99% of the cases, while the highest daily record of visible infected larvae (52%) was observed on the third day post exposure. Moreover, 79% of confirmed virus infected larvae died in the first 10 days after exposure. The Median Lethal Time was eight days. Several tissues were found to be infected and the common sites of replication were the fat body, epidermis and epithelial derivatives, such as the imaginal discs and the tracheal epithelium. Moreover, infection in the salivary glands, gastric ceca and posterior gut have not been previously documented on other mosquito iridescent viruses.

Transcriptional response of individual Hawaiian Culex quinquefasciatus mosquitoes to the avian malaria parasite Plasmodium relictum.

BACKGROUND: Plasmodium parasites that cause bird malaria occur in all continents except Antarctica and are primarily transmitted by mosquitoes in the genus Culex. Culex quinquefasciatus, the mosquito vector of avian malaria in Hawai'i, became established in the islands in the 1820s. While the deadly effects of malaria on endemic bird species have been documented for many decades, vector-parasite interactions in avian malaria systems are relatively understudied. METHODS: To evaluate the gene expression response of mosquitoes exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i, offspring of wild-collected Hawaiian Cx. quinquefasciatus were fed on a domestic canary infected with a fresh isolate of Plasmodium relictum GRW4 from a wild-caught Hawaiian honeycreeper. Control mosquitoes were fed on an uninfected canary. Transcriptomes of five infected and three uninfected individual mosquitoes were sequenced at each of three stages of the parasite life cycle: 24 h post feeding (hpf) during ookinete invasion; 5 days post feeding (dpf) when oocysts are developing; 10 dpf when sporozoites are released and invade the salivary glands. RESULTS: Differential gene expression analyses showed that during ookinete invasion (24 hpf), genes related to oxidoreductase activity and galactose catabolism had lower expression levels in infected mosquitoes compared to controls. Oocyst development (5 dpf) was associated with reduced expression of a gene with a predicted innate immune function. At 10 dpf, infected mosquitoes had reduced expression levels of a serine protease inhibitor, and further studies should assess its role as a Plasmodium agonist in C. quinquefasciatus. Overall, the differential gene expression response of Hawaiian Culex exposed to a Plasmodium infection intensity known to occur naturally in Hawai'i was low, but more pronounced during ookinete invasion. CONCLUSIONS: This is the first analysis of the transcriptional responses of vectors to malaria parasites in non-mammalian systems. Interestingly, few similarities were found between the response of Culex infected with a bird Plasmodium and those reported in Anopheles infected with human Plasmodium. The relatively small transcriptional changes observed in mosquito genes related to immune response and nutrient metabolism support conclusions of low fitness costs often documented in experimental challenges of Culex with avian Plasmodium.

Gene regulation of the avian malaria parasite Plasmodium relictum, during the different stages within the mosquito vector.

The malaria parasite Plasmodium relictum is one of the most widespread species of avian malaria. As in the case of its human counterparts, bird Plasmodium undergoes a complex life cycle infecting two hosts: the arthropod vector and the vertebrate host. In this study, we examined transcriptomes of P. relictum (SGS1) during crucial timepoints within its vector, Culex pipiens quinquefasciatus. Differential gene-expression analyses identified genes linked to the parasites life-stages at: i) a few minutes after the blood meal is ingested, ii) during peak oocyst production phase, iii) during peak sporozoite phase and iv) during the late-stages of the infection. A large amount of genes coding for functions linked to host-immune invasion and multifunctional genes was active throughout the infection cycle. One gene associated with a conserved Plasmodium membrane protein with unknown function was upregulated throughout the parasite development in the vector, suggesting an important role in the successful completion of the sporogonic cycle. Gene expression analysis further identified genes, with unknown functions to be significantly differentially expressed during the infection in the vector as well as upregulation of reticulocyte-binding proteins, which raises the possibility of the multifunctionality of these RBPs. We establish the existence of highly stage-specific pathways being overexpressed during the infection. This first study of gene-expression of a non-human Plasmodium species in its vector provides a comprehensive insight into the molecular mechanisms of the common avian malaria parasite P. relictum and provides essential information on the evolutionary diversity in gene regulation of the Plasmodium's vector stages.

Vector incrimination and transmission of avian malaria at an aquarium in Japan: mismatch in parasite composition between mosquitoes and penguins.

BACKGROUND: Captive populations of penguins outside of their natural distributions are often maintained in outdoor facilities, such as zoos and aquariums. Consequently, such penguins in captivity are constantly exposed to mosquito vectors and risk of avian malarial infection during their active period from spring to autumn, which can be lethal to these naïve birds. Previous studies have investigated parasite prevalence in mosquitoes or penguins, but simultaneous investigations, which would be crucial to monitor the transmission dynamics and cycle within a facility, have not been done. To identify dominant lineages and trends, multiple-year surveys are recommended. METHODS: Avian malaria parasites (Plasmodium spp.) and related haemosporidia were tested in penguins and mosquitoes at an aquarium in Japan through multiple years from 2011 to 2018. Prevalence and dynamics were confirmed, and molecular analyses targeting the protozoal cytb gene were used to reveal the transmission cycle. Blood meals of mosquitoes were also identified using molecular methods. RESULTS: Parasite detection in penguins tended to fluctuate within an individual. Two Plasmodium lineages were consistently detected in mosquitoes that had fed on penguins and wild birds observed around the aquarium. Plasmodium lineage CXPIP09 was detected from both mosquitoes and penguins, suggesting active transmission at this facility. However, Plasmodium cathemerium PADOM02 was only detected in mosquitoes, which may be due to host, vector or parasite-related factors, or detection methods and their limits. Additionally, Haemoproteus larae SPMAG12 was detected from penguins, suggesting active transmission via biting midges. CONCLUSIONS: The mismatch in parasite composition between penguins and mosquitoes shows that multiple aspects such as captive birds, wild birds and vector insects should be monitored in order to better understand and control avian malarial infection within ex-situ conservation facilities. Furthermore, morphological analyses would be needed to confirm competency and infection dynamics of avian malaria parasites.

Experimental study of newly described avian malaria parasite Plasmodium (Novyella) collidatum n. sp., genetic lineage pFANTAIL01 obtained from South Asian migrant bird.

BACKGROUND: Avian malaria parasites are microorganisms parasitizing erythrocytes and various tissues of the birds; they are common and distributed worldwide. These parasites are known to infect birds of different taxa and be the cause of the deaths of birds in the wild and in captivity. The species of parasites with the ability to colonize new territories and infect local non-migratory birds are of particular interest. This scenario is likely in temperate zones of Europe, because of climate change and its contribution in spreading vectors of southern origin, which can be involved in the transmission of malaria parasites. In the present study, a tropical Plasmodium parasite from a naturally infected long-distance migrant bird was isolated and tested for its ability to develop in common species of mosquitoes and European short-distance migrant birds. METHODS: Plasmodium sp. (pFANTAIL01) was isolated on the Curonian spit of the Baltic sea coast from the naturally infected Common rosefinch, Carpodacus erythrinus in June 2019. The parasite was described based on the morphological features of its blood stages, the partial mitochondrial cytochrome b gene and development after experimental infection of birds and mosquitoes. The parasite was inoculated into Eurasian siskins, Carduelis spinus. Parasitaemia, haematocrit and weight of birds were monitored. At the end of the survey, internal organs were collected to study exoerythrocytic stages of this parasite. Experimental infection of mosquitoes Culex pipiens form molestus and Culex quinquefasciatus was applied to study sporogonic development of the parasite. RESULTS: Based on morphological features, the parasite was described as a new species, Plasmodium collidatum n. sp., and attributed to subgenus Novyella. It was revealed that the obtained pFANTAIL01 lineage is a generalist parasite infecting a wide range of avian hosts and most likely is transmitted in South and Southeast (SE) Asia and Oceania. In Europe, this strain was recorded only in adult migratory birds wintering in South Asia. This parasite developed high parasitaemia in experimentally infected siskins and caused 25 % mortality. Exoerythrocytic stages of pFANTAIL01 were found in the lungs, liver, spleen and kidney of the deceased birds. Sporogonic development did not occur in Cx. pipiens form molestus and Cx. quinquefasciatus mosquitoes. CONCLUSIONS: Plasmodium collidatum is a highly virulent for Eurasian siskin and completes its development in these birds, which can be considered as a potential vertebrate host if the transmission of the infection starts occurring in Europe and temperate zones.

Culex quinquefasciatus (Diptera: Culicidae) survivorship following the ingestion of bird blood infected with Haemoproteus sp. parasites.

Arthropod vectors are frequently exposed to a diverse assemblage of parasites, but the consequence of these infections on their biology and behavior are poorly understood. We experimentally evaluated whether the ingestion of a common protozoan parasite of avian hosts (Haemoproteus spp.; Haemosporida: Haemoproteidae) impacted the survivorship of Culex quinquefasciatus (Say) (Diptera: Culicidae). Blood was collected from wild northern cardinals (Cardinalis cardinalis) in College Station, Texas, and screened for the presence of Haemoproteus spp. parasites using microscopic and molecular methods. Experimental groups of Cx. quinquefasciatus mosquitoes were offered Haemoproteus-positive cardinal blood through an artificial feeding apparatus, while control groups received Haemoproteus-negative cardinal blood or domestic canary (Serinus canaria domestica) blood. Culex quinquefasciatus mosquitoes exposed to Haemoproteus infected cardinal blood survived significantly fewer days than mosquitoes that ingested Haemoproteus-negative cardinal blood. The survival of mosquitoes fed on positive cardinal blood had a median survival time of 18 days post-exposure and the survival of mosquitoes fed on negative cardinal blood exceeded 50% across the 30 day observation period. Additionally, mosquitoes that fed on canary controls survived significantly fewer days than cardinal negative controls, with canary control mosquitoes having a median survival time of 17 days. This study further supports prior observations that Haemoproteus parasites can be pathogenic to bird-biting mosquitoes, and suggests that Haemoproteus parasites may indirectly suppress the transmission of co-circulating vector-borne pathogens by modulating vector survivorship. Our results also suggest that even in the absence of parasite infection, bloodmeals from different bird species can influence mosquito survivorship.

Influence of house characteristics on mosquito distribution and malaria transmission in the city of Yaoundé, Cameroon.

BACKGROUND: Improving house structure is known to limit contact between humans and mosquitoes and reduce malaria transmission risk. In the present study, the influence of house characteristics on mosquito distribution and malaria transmission risk was assessed in the city of Yaoundé. METHODS: The study was conducted from March 2017 to June 2018 in 32 districts of the city of Yaoundé. Mosquito collections were performed indoor in 10 to 15 houses per district using CDC light traps. A total of 467 houses, selected randomly were used. A pretested questionnaire was submitted to participants of the study to collect information on the household: the number of people per house, education level, type of walls, presence of ceilings and eaves, number of windows, usage of long-lasting insecticidal nets (LLINs), number of bedroom and number of window. Mosquitoes collected were identified morphologically. Anophelines were tested by ELISA to detect infection by Plasmodium parasites. General Estimating Equations adjusting for repeated measures in the same house fitting negative binomial analysis were used to assess the influence of house characteristics on mosquito distribution. RESULTS: A total of 168,039 mosquitoes were collected; Culex spp emerged as the predominant species (96.48%), followed by Anopheles gambiae sensu lato (s.l.) (2.49%). Out of the 1033 An. gambiae s.l. identified by PCR, 90.03% were Anopheles coluzzii and the remaining were An. gambiae sensu stricto (s.s.) (9.97%). The high number of people per household, the presence of screens on window and the possession of LLINs were all associated with fewer mosquitoes collected indoors, whilst opened eaves, the high number of windows, the presence of holes in walls and living close to breeding sites were associated with high densities of mosquitoes indoor. Out of 3557 Anophelines tested using ELISA CSP, 80 were found infected by Plasmodium falciparum parasites. The proportion of mosquitoes infected did not vary significantly according to house characteristics. CONCLUSION: The study indicated that several house characteristics such as, the presence of holes on walls, opened eaves, unscreened window and living close to breeding sites, favored mosquito presence in houses. Promoting frequent use of LLINs and house improvement measures, such as the use of screen on windows, closing eaves, cleaning the nearby environment, should be integrated in strategies to improve malaria control in the city of Yaoundé.

Plasmodium transmission differs between mosquito species and parasite lineages.

Factors such as the particular combination of parasite-mosquito species, their co-evolutionary history and the host's parasite load greatly affect parasite transmission. However, the importance of these factors in the epidemiology of mosquito-borne parasites, such as avian malaria parasites, is largely unknown. Here, we assessed the competence of two mosquito species [Culex pipiens and Aedes (Ochlerotatus) caspius], for the transmission of four avian Plasmodium lineages (Plasmodium relictum SGS1 and GRW11 and Plasmodium cathemerium-related lineages COLL1 and PADOM01) naturally infecting wild house sparrows. We assessed the effects of parasite identity and parasite load on Plasmodium transmission risk through its effects on the transmission rate and mosquito survival. We found that Cx. pipiens was able to transmit the four Plasmodium lineages, while Ae. caspius was unable to transmit any of them. However, Cx. pipiens mosquitoes fed on birds infected by P. relictum showed a lower survival and transmission rate than those fed on birds infected by parasites related to P. cathemerium. Non-significant associations were found with the host-parasite load. Our results confirm the existence of inter- and intra-specific differences in the ability of Plasmodium lineages to develop in mosquito species and their effects on the survival of mosquitoes that result in important differences in the transmission risk of the different avian malaria parasite lineages studied.

Morphological and molecular characterization of Plasmodium cathemerium (lineage PADOM02) from the sparrow Passer domesticus with complete sporogony in Culex pipiens complex.

Avian malaria is a mosquito-borne disease caused by Plasmodium spp. protozoa. Although these parasites have been extensively studied in North America and Eurasia, knowledge on the diversity of Plasmodium, its vectors and avian hosts in Africa is scarce. In this study, we report on natural malarial infections in free-ranging sparrows (Passer domesticus) sampled at Giza Governorate, Egypt. Parasites were morphologically characterized as Plasmodium cathemerium based on the examination of thin blood smears from the avian host. Sequencing a fragment of the mitochondrial cytochrome b gene showed that the parasite corresponded to lineage PADOM02. Phylogenetic analysis showed that this parasite is closely related to the lineages SERAU01 and PADOM09, both of which are attributed to P. cathemerium. Experimental infection of Culex pipiens complex was successful, with ookinetes first detected at 1-day post infection (dpi), oocysts at 4 dpi and sporozoites at 6 dpi. The massive infection of the salivary glands by sporozoites corroborates that Cx. pipiens complex is a competent vector of PADOM02. Our findings confirm that Plasmodium lineage PADOM02 infects sparrows in urban areas along the Nile River, Egypt, and corroborate that Cx. pipiens complex is a highly competent vector for these parasites. Furthermore, our results demonstrate that this lineage corresponds to the morphospecies P. cathemerium and not P. relictum as previously believed.

Attraction of mosquitoes to primate odours and implications for zoonotic Plasmodium transmission.

Vector-borne diseases often originate from wildlife and can spill over into the human population. One of the most important determinants of vector-borne disease transmission is the host preference of mosquitoes. Mosquitoes with a specialised host preference are guided by body odours to find their hosts in addition to carbon dioxide. Little is known about the role of mosquito host preference in the spillover of pathogenic agents from humans towards animals and vice versa. In the Republic of Congo, the attraction of mosquitoes to primate host odours was determined, as well as their possible role as malaria vectors, using odour-baited traps mimicking the potential hosts of mosquitoes. Most of the mosquito species caught showed a generalistic host preference. Anopheles obscurus was the most abundant Anopheles mosquito, with a generalistic host preference observed from the olfactory response and the detection of various Plasmodium parasites. Interestingly, Culex decens showed a much higher attraction towards chimpanzee odours than to human or cow odours. Human Plasmodium parasites were observed in both human and chimpanzee blood, although not in the Anopheles mosquitoes that were collected. Understanding the role of mosquito host preference for cross-species parasite transmission provides information that will help to determine the risk of spillover of vector-borne diseases.

Development of a molecular xenomonitoring protocol to assess filariasis transmission.

According to the World Health Organization, lymphatic filariasis (LF), a mosquito-borne neglected tropical disease (NTD), should be eliminated as a public health concern by the end of 2020. To this end, the goals of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) include interrupting transmission through mass drug administration (MDA). After two decades, several countries have implemented MDA and are now ready to confirm whether transmission has been interrupted. The method for detecting the parasites in mosquito vectors known as xenomonitoring is a non-invasive tool for assessing the current transmission status of the filarial nematode Wuchereria bancrofti (which is responsible for 90% of cases) by their vectors. There are several methods available for detection of the worm in mosquito samples, such as dissection or polymerase chain reaction (PCR). However, most of these techniques still produce a considerable number of false-negative results. The present study describes a new duplex PCR protocol, which is an improvement on the traditional PCR methodology, enhanced by introducing the actin gene as an endogenous control gene. After adjusting the mosquito pool size, DNA extraction, and WbCx PCR duplex design, we achieved a reliable and sensitive molecular xenomonitoring protocol. This assay was able to eliminate 5% of false negative samples and detected less than one Wb larvae. This high sensitivity is particularly valuable after MDA, when prevalence declines. This new method could reduce the number of false-negative samples, which will enable us to improve our ability to generate accurate results and aid the monitoring strategies used by LF elimination programmes.

Avian malaria alters the dynamics of blood feeding in Culex pipiens mosquitoes.

BACKGROUND: Some Plasmodium species have the ability to modify the behaviour of their mosquito vectors. This is thought to be an adaptive strategy that maximizes the parasite's transmission. METHODS: The effect of Plasmodium relictum infections on the blood feeding behaviour of Culex pipiens quinquefasciatus mosquitoes was monitored. RESULTS: Plasmodium infections did not alter the proportion of blood fed mosquitoes but they did affect the dynamics and the size of the blood meal. Sporozoite-infected mosquitoes completed their blood meal 1.3 times later than uninfected mosquitoes and ended up with smaller blood meals. CONCLUSION: The potential adaptive nature of this manipulation of mosquito behaviour is discussed in the light of previous studies on other malaria models.

Isolation and growth inhibition potential of entomopathogenic nematodes against three public health important mosquito vectors.

The prevalence of mosquito vector borne diseases and the resistance of mosquitoes to conventional pesticides have been of important public concern to the mosquito endemic countries. Present study was conducted to identify the native bio-larvicidal potential of the entomopathogenic nematodes; Steinernema siamkayai (KPR-4) Heterohabditis indica (KPR-8), Steinernema glaseri and Steinernema abbasi. The isolated nematodes were subsequently cultured and evaluated their larvicidal potential against the larvae of Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus. Among the tested four different nematode species, the S. abassi exerted the highest mortality against A. aegypti (97.33%), the H. indica (KPR-8) against A. stephensi (97.33%) and the S. siamkayai (KPR-4) against C. quinquefasciatus (98.67%). The maximal mosquito-larvicidal property of EPNs was found with the LC50 and LC90 values (IJs/larvae): S. abbasi = 12.47 & 54.35 on A. aegypti; H. indica KPR-8 = 19.88 & 66.81 on A. stephensi and S. siamkayai KPR-4 = 16.69 & 58.97 on C. quinquefasciatus, respectively. The presently generated data on the molecular and larvicidal characteristics of the entomopathogenic nematodes form an important baseline data that upon further research would lead to the development of eco-friendly mosquito-control agent.

Behavior manipulation of mosquitoes by a mermithid nematode.

We examined manipulation of mosquito behavior by the parasitic mermithid nematode, Strelkovimermis spiculatus. This nematode species typically infects early instar host larvae and emerges after parasitic development to kill last-instar larvae. Parasitized adults, however, have occasionally been reported from field collections. We obtained low rates (1.7-11.5%) of parasitized adults in laboratory exposures only when Culex pipiens pipiens fourth-instar larvae nearing pupation were exposed to infective nematodes. This did not allow an adequate interval for parasitic development in immature host stages. Parasitized adult females in a multiple-choice assay were three times more likely to seek water than a blood source (63.1 vs. 20.5%), whereas uninfected females were twice as likely to seek blood than water (64%3.9 vs. 32.6%). This altered host behavior benefits the parasite by providing the only mechanism for dispersal and colonization of new host habitats while concurrently avoiding risks from the defensive behaviors associated with blood-feeding. Behavioral alternation in Cx. p. pipiens larval hosts was also examined using larvae infected as second instars to allow for a normal duration of parasitic development. As larvae neared pupation and parasite emergence, parasitized larvae became more spatially aggregated than unparasitized larvae. This altered host behavior benefits the parasite by providing a corresponding increase in post-parasite aggregation, which facilitates formation of large mating clusters and concomitantly reproductive success. Parasites derive fitness gains by overriding host autonomy, whereas hosts have zero fitness once parasitism is established, suggesting a coevolutionary response is inoperative and that the behavioral modifications may be adaptive.

Protozoan parasites in Culex pipiens mosquitoes in Vienna.

Avian malaria (Plasmodium spp.) and kinetoplastid (Trypanosoma spp.) parasites are common vector-borne pathogens in birds worldwide; however, knowledge about vector competence of different mosquito species is currently lacking. For a pilot project examining vector competence of mosquitoes of the Culex pipiens complex and Culex torrentium for protozoan parasites in the city of Vienna, 316 individual mosquitoes were sampled in the months June-August 2017 around the campus of the Veterinary University of Vienna. Since vector competence for avian Plasmodium can only be ascertained by finding infectious sporozoites in mosquito salivary glands, special emphasis was on examining these, or at least insect thoraxes, which contain the salivary glands. After species identification, the mosquitoes were processed in three different ways to determine the best method of visually detecting protozoan parasites in salivary glands: (1) microscopic examination of individual, fixed and Giemsa-stained salivary glands, (2) microscopic examination of stained sections of individually fixed and embedded mosquito thoraxes and (3) stained sections of individual whole insects. Material from all three groups was also subjected to PCR to detect avian haemosporidian and trypanosomatid parasite DNA. PCR was performed on all 316 collected mosquitoes, with 37 pools (n = 2-10) of 263 individuals and 53 single individuals in all together 90 PCR reactions. Avian Plasmodium was found in 18 (20%) and trypanosomatid parasites were found in 10 (11.1%) of the examined samples and pools yielded a higher proportion of positives than did individual samples. Six different species of protozoan parasites were identified, namely Plasmodium vaughani SYAT05 which was the most common, P. elongatum GRW6, P. relictum SGS1, Trypanosoma avium, T. culicavium and Crithidia dedva. Seventy-seven mosquito salivary glands were dissected and stained with Giemsa solution. Of these, one (1.3%) featured sporozoites and one (1.3%) trypanosomatid parasites. While the trypanosomes were identified as T. avium, the avian Plasmodium species were present in a mixed infection with P. vaughani SYAT05 as the dominant species. In conclusion, mosquitoes of the Culex pipiens complex are very likely vectors of different avian Plasmodium and Trypanosoma species and PCR was the most successful and reliable method for parasite detection in mosquito samples, delivering higher rates and more accurate results. The visual detection of parasite stages in the salivary glands was more difficult and only a few specimens were detected using Giemsa stain and chromogenic in situ hybridization. For further studies on vector competence of different protozoan parasites in mosquitoes, the use of PCR-based methods would be preferable.

Molecular detection of vector-borne pathogens from mosquitoes collected in two zoological gardens in Germany.

In Germany, knowledge of disease agents transmitted by arthropods in zoological gardens is scarce. In the framework of ecological studies, mosquitoes were therefore collected in German zoological gardens and examined for mosquito-borne pathogen DNA and RNA. In total, 3840 mosquitoes were screened for filarial nematodes and three groups of viruses (orthobunyaviruses, flaviviruses, alphaviruses) while 405 mosquitoes were tested for avian malaria parasites. In addition to the filarial nematode species Dirofilaria repens (n = 1) and Setaria tundra (n = 8), Sindbis virus (n = 1) and the haemosporidian genera Haemoproteus (n = 8), Leucocytozoon (n = 10) and Plasmodium (n = 1) were demonstrated. Identified pathogens have the potential to cause disease in zoo and wild animals, but some of them also in humans. Positive mosquitoes were collected most often in July, indicating the highest infection risk during this month. Most of the pathogens were found in mosquito specimens of the Culex pipiens complex, suggesting that its members possibly act as the most important vectors in the surveyed zoos, although the mere demonstration of pathogen DNA/RNA in a homogenised complete mosquito is not finally indicative for a vector role. Outcomes of the study are not only significant for arthropod management in zoological gardens, but also for the general understanding of the occurrence and spread of mosquito-borne disease agents.