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.

Exploring mosquito abundance and Plasmodium infection through nested-PCR: implications for disease surveillance and control.

The Plasmodium is responsible for malaria which poses a major health threat, globally. This study is based on the estimation of the relative abundance of mosquitoes, and finding out the correlations of meteorological parameters (temperature, humidity and rainfall) with the abundance of mosquitoes. In addition, this study also focused on the use of nested PCR (species-specific nucleotide sequences of 18S rRNA genes) to explore the Plasmodium spp. in female Anopheles. In the current study, the percentage relative abundance of Culex mosquitoes was 57.65% and Anopheles 42.34% among the study areas. In addition, the highest number of mosquitoes was found in March in district Mandi Bahauddin at 21 °C (Tmax = 27, Tmin = 15) average temperature, 69% average relative humidity and 131 mm rainfall, and these climatic factors were found to affect the abundance of the mosquitoes, directly or indirectly. Molecular analysis showed that overall, 41.3% of the female Anopheles pools were positive for genus Plasmodium. Among species, the prevalence of Plasmodium (P.) vivax (78.1%) was significantly higher than P. falciparum (21.9%). This study will be helpful in the estimation of future risk of mosquito-borne diseases along with population dynamic of mosquitoes to enhance the effectiveness of vector surveillance and control programs.

Ecological niche modeling analysis (Cx. pipiens), potential risk and projection of Dirofilaria spp. infection in Greece.

Vector-borne diseases continue to increase worldwide. Dirofilariosis is one of the most common vector-borne zoonotic diseases, mainly caused by Dirofilaria spp. (D. immitis and D. repens) and spread by culicid mosquitoes of different species. Greece is one of the countries in southern Europe where it is traditionally endemic, and its distribution is not homogeneous. The aim of this study was to develop an environmental model for Greece that reflects the suitability of the ecological niche for Dirofilaria spp. infection risk and its projection until 2080. For this purpose, we used the potential distribution of suitable habitats for Culex pipiens calculated using an ecological niche model (ENM) and the potential number of generations of Dirofilaria spp. The ecological niche model of Cx. pipiens in Greece showed good predictive power (AUC=0.897) with the parasite at a resolution of 1 km2. The variables that contributed most to the model were mean annual temperature, rivers and human footprint. The highest risk of infection was found in coastal areas and in riverside areas of the main river basins, as well as in irrigated areas of the mainland and peninsular regions and in the whole territory of island areas, and the lowest risk was found in areas of higher altitude. A positive relationship was found between the risk of dirofilariosis and the location of infected dogs, with 86.65% located in very high and high risk areas. In 2080, the percentage of territory gained by Cx. pipiens will increase by 261.52%. This model provides a high predictive value, predicted presence, and risk of Dirofilaria spp. infection and can serve as a tool for the management and control of this disease.

Avian Plasmodium in invasive and native mosquitoes from southern Spain.

BACKGROUND: The emergence of diseases of public health concern is enhanced by factors associated with global change, such as the introduction of invasive species. The Asian tiger mosquito (Aedes albopictus), considered a competent vector of different viruses and parasites, has been successfully introduced into Europe in recent decades. Molecular screening of parasites in mosquitoes (i.e. molecular xenomonitoring) is essential to understand the potential role of different native and invasive mosquito species in the local circulation of vector-borne parasites affecting both humans and wildlife. METHODS: The presence of avian Plasmodium parasites was molecularly tested in mosquitoes trapped in five localities with different environmental characteristics in southern Spain from May to November 2022. The species analyzed included the native Culex pipiens and Culiseta longiareolata and the invasive Ae. albopictus. RESULTS: Avian Plasmodium DNA was only found in Cx. pipiens with 31 positive out of 165 mosquito pools tested. None of the Ae. albopictus or Cs. longiareolata pools were positive for avian malaria parasites. Overall, eight Plasmodium lineages were identified, including a new lineage described here. No significant differences in parasite prevalence were found between localities or sampling sessions. CONCLUSIONS: Unlike the invasive Ae. albopictus, Cx. pipiens plays a key role in the transmission of avian Plasmodium in southern Spain. However, due to the recent establishment of Ae. albopictus in the area, further research on the role of this species in the local transmission of vector-borne pathogens with different reservoirs is required.

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.

The role of Culex territans mosquitoes in the transmission of Batrachochytrium dendrobatidis to amphibian hosts.

BACKGROUND: Mosquitoes are the deadliest organisms in the world, killing an estimated 750,000 people per year due to the pathogens they can transmit. Mosquitoes also pose a major threat to other vertebrate animals. Culex territans is a mosquito species found in temperate zones worldwide that feeds almost exclusively on amphibians and can transmit parasites; however, little is known about its ability to transmit other pathogens, including fungi. Batrachochytrium dendrobatidis (Bd) is a topical pathogenic fungus that spreads through contact. With amphibian populations around the world experiencing mass die-offs and extinctions due to this pathogen, it is critical to study all potential modes of transmission. Because Cx. territans mosquitoes are in contact with their hosts for long periods of time while blood-feeding, we hypothesize that they can transmit and pick up Bd. METHODS: In this study, we first assessed Cx. territans ability to transfer the fungus from an infected surface to a clean one under laboratory conditions. We also conducted a surveillance study of Bd infections in frogs and mosquitoes in the field (Mountain Lake Biological station, VA, USA). In parallel, we determined Cx. territans host preference via blood meal analysis of field caught mosquitoes. RESULTS: We found that this mosquito species can carry the fungus to an uninfected surface, implying that they may have the ability to transmit Bd to their amphibian hosts. We also found that Cx. territans feed primarily on green frogs (Rana clamitans) and bullfrogs (Rana catesbeiana) and that the prevalence of Bd within the frog population at our field site varied between years. CONCLUSIONS: This study provides critical insights into understanding the role of amphibian-biting mosquitoes in transmitting pathogens, which can be applied to disease ecology of susceptible amphibian populations worldwide.

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.

A mass rearing cost calculator for the control of Culex quinquefasciatus in Hawai'i using the incompatible insect technique.

BACKGROUND: Hawai'i's native forest avifauna is experiencing drastic declines due to climate change-induced increases in temperature encroaching on their upper-elevation montane rainforest refugia. Higher temperatures support greater avian malaria infection rates due to greater densities of its primary vector, the southern house mosquito Culex quinquefasciatus, and enhance development of the avian malaria parasite Plasmodium relictum. Here we propose the use of the incompatible insect technique (IIT) or the combined IIT/sterile insect technique (SIT) for the landscape-scale (i.e., area-wide) control of Cx. quinquefasciatus, and have developed a calculator to estimate the costs of IIT and IIT/SIT applications at various sites in Hawai'i. METHODS: The overall cost of the infrastructure, personnel, and space necessary to produce incompatible adult males for release is calculated in a unit of ~ 1 million culicid larvae/week. We assessed the rearing costs and need for effective control at various elevations in Hawai'i using a 10:1 overflooding ratio at each elevation. The calculator uses a rate describing the number of culicids needed to control wild-type mosquitoes at each site/elevation, in relation to the number of larval rearing units. This rate is a constant from which other costs are quantified. With minor modifications, the calculator described here can be applied to other areas, mosquito species, and similar techniques. To test the robustness of our calculator, the Kaua'i-specific culicid IIT/SIT infrastructure costs were also compared to costs from Singapore, Mexico, and China using the yearly cost of control per hectare, and purchasing power parity between sites for the cost of 1000 IIT/SIT males. RESULTS: As a proof of concept, we have used the calculator to estimate rearing infrastructure costs for an application of IIT in the Alaka'i Wilderness Reserve on the island of Kaua'i. Our analysis estimated an initial investment of at least ~ $1.16M with subsequent yearly costs of approximately $376K. Projections of rearing costs for control at lower elevations are ~ 100 times greater than in upper elevation forest bird refugia. These results are relatively comparable to those real-world cost estimates developed for IIT/SIT culicid male production in other countries when inflation and purchasing power parity are considered. We also present supplemental examples of infrastructure costs needed to control Cx. quinquefasciatus in the home range of 'i'iwi Drepanis coccinea, and the yellow fever vector Aedes aegypti. CONCLUSIONS: Our cost calculator can be used to effectively estimate the mass rearing cost of an IIT/SIT program. Therefore, the linear relationship of rearing infrastructure to costs used in this calculator is useful for developing a conservative cost estimate for IIT/SIT culicid mass rearing infrastructure. These mass rearing cost estimates vary based on the density of the targeted organism at the application site.

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.

An update on the mosquito fauna and mosquito-borne diseases distribution in Cameroon.

The expansion of mosquito-borne diseases such as dengue, yellow fever, and chikungunya in the past 15 years has ignited the need for active surveillance of common and neglected mosquito-borne infectious diseases. The surveillance should be designed to detect diseases and to provide relevant field-based data for developing and implementing effective control measures to prevent outbreaks before significant public health consequences can occur. Mosquitoes are important vectors of human and animal pathogens, and knowledge on their biodiversity and distribution in the Afrotropical region is needed for the development of evidence-based vector control strategies. Following a comprehensive literature search, an inventory of the diversity and distribution of mosquitoes as well as the different mosquito-borne diseases found in Cameroon was made. A total of 290 publications/reports and the mosquito catalogue website were consulted for the review. To date, about 307 species, four subspecies and one putative new species of Culicidae, comprising 60 species and one putative new species of Anopheles, 67 species and two subspecies of Culex, 77 species and one subspecies of Aedes, 31 species and one subspecies of Eretmapodites, two Mansonia, eight Coquillettidia, and 62 species with unknown medical and veterinary importance (Toxorhynchites, Uranotaenia, Mimomyia, Malaya, Hodgesia, Ficalbia, Orthopodomyia, Aedeomyia, and Culiseta and Lutzia) have been collected in Cameroon. Multiple mosquito species implicated in the transmission of pathogens within Anopheles, Culex, Aedes, Eretmapodites, Mansonia, and Coquillettidia have been reported in Cameroon. Furthermore, the presence of 26 human and zoonotic arboviral diseases, one helminthic disease, and two protozoal diseases has been reported. Information on the bionomics, taxonomy, and distribution of mosquito species will be useful for the development of integrated vector management programmes for the surveillance and elimination of mosquito-borne diseases in Cameroon.

Updated distribution maps of predominant Culex mosquitoes across the Americas.

BACKGROUND: Estimates of the geographical distribution of Culex mosquitoes in the Americas have been limited to state and provincial levels in the United States and Canada and based on data from the 1980s. Since these estimates were made, there have been many more documented observations of mosquitoes and new methods have been developed for species distribution modeling. Moreover, mosquito distributions are affected by environmental conditions, which have changed since the 1980s. This calls for updated estimates of these distributions to understand the risk of emerging and re-emerging mosquito-borne diseases. METHODS: We used contemporary mosquito data, environmental drivers, and a machine learning ecological niche model to create updated estimates of the geographical range of seven predominant Culex species across North America and South America: Culex erraticus, Culex nigripalpus, Culex pipiens, Culex quinquefasciatus, Culex restuans, Culex salinarius, and Culex tarsalis. RESULTS: We found that Culex mosquito species differ in their geographical range. Each Culex species is sensitive to both natural and human-influenced environmental factors, especially climate and land cover type. Some prefer urban environments instead of rural ones, and some are limited to tropical or humid areas. Many are found throughout the Central Plains of the USA. CONCLUSIONS: Our updated contemporary Culex distribution maps may be used to assess mosquito-borne disease risk. It is critical to understand the current geographical distributions of these important disease vectors and the key environmental predictors structuring their distributions not only to assess current risk, but also to understand how they will respond to climate change. Since the environmental predictors structuring the geographical distribution of mosquito species varied, we hypothesize that each species may have a different response to climate change.

Dirofilaria immitis and Dirofilaria repens in mosquitoes from Corsica Island, France.

BACKGROUND: Dirofilaria immitis and Dirofilaria repens are the main causative agents of heartworm disease and subcutaneous dirofilariasis in domestic and wild canids, respectively. Both pathogens have zoonotic potential and are transmitted by mosquitoes. The present study aimed to determine the transmission period, prevalence and diversity of Dirofilaria spp. vectors from endemic areas of Corsica (France). METHODS: A monthly point data model based on average temperature recorded by four meteorological stations during 2017 was used to calculate the Dirofilaria transmission period. From June to September 2017, female mosquitoes (n = 1802) were captured using Biogents® Sentinel 2 traps lured with carbon dioxide and BG-Lure™ or octanol. Mosquitoes were identified to species level, pooled accordingly, and screened using multiplex real-time qPCR to detect D. immitis and D. repens. RESULTS: The monthly point data model showed the possible transmission of Dirofilaria spp. from the third week in May to the last week in October in the studied area. Mosquitoes were identified as Ochlerotatus caspius (n = 1432), Aedes albopictus (n = 199), Culex pipiens sensu lato (n = 165) and Aedes vexans (n = 6) and were grouped into 109 pools (from 1 to 27 specimens, mean 11.4 ± 0.7), of which 16 scored positive for Dirofilaria spp. (i.e., n = 13; estimated infection rate [EIR] = 1.1% for D. immitis and n = 3; EIR = 0.2% for D. repens). Specifically, 6 (i.e., EIR = 3.8%) of 15 pools of Ae. albopictus were positive for D. immitis, 2 of 14 of Cx. pipiens s.l. were positive for D. immitis and D. repens, respectively, and 8 of 77 pools of Oc. caspius were positive for D. immitis (i.e., n = 6; EIR = 0.4%) and D. repens (i.e., 2; EIR = 0.1%). The highest mosquito infection rate was recorded in July (EIR = 2.5%), then in June (EIR = 1.3%) and September (EIR = 0.6%). CONCLUSIONS: The data suggest that both Dirofilaria species are endemic and occur possibly in sympatry in the studied area in Corsica, highlighting the need to implement preventive chemoprophylaxis and vector control strategies to reduce the risk of these filarioids in dog and human populations.

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.

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.

Multiple cytochrome P450 genes: conferring high levels of permethrin resistance in mosquitoes, Culex quinquefasciatus.

Insecticides, especially pyrethroids, are the most important in the insect pest control and preventing insect vector-borne human diseases. However, insect pests, including mosquitoes, have developed resistance in the insecticides that used against them. Cytochrome P450s are associated with insecticide resistance through overexpression and detoxification mechanisms in insect species. In this study, we utilized a powerful tool, the RNAi technique, to determine the roles of key P450 genes overexpressed in permethrin resistant mosquitoes that confer insecticide resistance to unravel the molecular basis of resistance mechanisms in the mosquito Culex quinquefasciatus. The results showed that knockdown of 8 key P450 genes using RNAi techniques significantly decreased resistance to permethrin in resistant mosquitoes. In silico modeling and docking analysis further revealed the potential metabolic function of overexpressed P450 genes in the development of insecticide resistance in mosquitoes. These findings not only highlighted the functional importance of these P450 genes in insecticide resistance, but also revealed that overexpression of multiple P450 genes was responsible for the high levels of insecticide resistance in a mosquito population of Culex quinquefasciatus.

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.

Screening for Viruses and Lemur-Associated Filara in Wild-Caught Mosquitoes From Madagascar.

Madagascar is a hotspot of biodiversity, but poverty and population growth provoke a high risk of conflict between food security and biodiversity conservation in this tropical country. Numerous vector-borne diseases, including viral infections, affect public health in Madagascar and a continuous expansion of anthropogenically used areas intensifies contact on the human-wildlife interface. However, data on human and animal pathogens in potential insect vectors is limited. Therefore, we conducted a parasitological and virological survey of 785 adult female mosquitoes between March and May 2016 at the Ankarafantsika National Park in northwestern Madagascar. Screening included Alpha-, Phlebo-, and Flaviviridae and the recently described filarial nematode species, Lemurfilaria lemuris. The predominant mosquito genus was Culex (91%), followed by Mansonia (4.1%), Anopheles (3.4%), and Aedes (0.9%). Viral screening revealed no arboviruses, but an insect-specific flavivirus in two Culex sitiens pools. No pools screened positive for the lemur-specific filarial nematode L. lemuris.

Lymphatic filariasis, infection status in Culex quinquefasciatus and Anopheles species after six rounds of mass drug administration in Masasi District, Tanzania.

BACKGROUND: Lymphatic filariasis (LF) elimination program in Tanzania started in 2000 in response to the Global program for the elimination of LF by 2020. Evidence shows a persistent LF transmission despite more than a decade of mass drug administration (MDA). It is advocated that, regular monitoring should be conducted in endemic areas to evaluate the progress towards elimination and detect resurgence of the disease timely. This study was therefore designed to assess the status of Wuchereria bancrofti infection in Culex quinqefasciatus and Anopheles species after six rounds of MDA in Masasi District, South Eastern Tanzania. METHODS: Mosquitoes were collected between June and July 2019 using Center for Diseases Control (CDC) light traps and gravid traps for indoor and outdoor respectively. The collected mosquitoes were morphologically identified into respective species. Dissections and PCR were carried out to detect W. bancrofti infection. Questionnaire survey and checklist were used to assess vector control interventions and household environment respectively. A Poisson regression model was run to determine the effects of household environment on filarial vector density. RESULTS: Overall, 12 452 mosquitoes were collected of which 10 545 (84.7%) were filarial vectors. Of these, Anopheles gambiae complex, An. funestus group and Cx. quinquefasciatus accounted for 0.1%, 0.7% and 99.2% respectively. A total of 365 pools of Cx. quinquefasciatus (each with 20 mosquitoes) and 46 individual samples of Anopheles species were analyzed by PCR. For Cx. quinquefasciatus pools, 33 were positive for W. bancrofti, giving an infection rate of 0.5%, while the 46 samples of Anopheles species were all negative. All 1859 dissected mosquitoes analyzed by microscopy were also negative. Households with modern latrines had less mosquitoes than those with pit latrines [odds ratio (OR) = 0.407, P < 0.05]. Houses with unscreened windows had more mosquitoes as compared to those with screened windows (OR = 2.125, P < 0.05). More than 80% of the participants own bednets while 16.5% had no protection. CONCLUSIONS: LF low transmission is still ongoing in Masasi District after six rounds of MDA and vector control interventions. The findings also suggest that molecular tools may be essential for xenomonitoring LF transmission during elimination phase.

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.