Plasmodium ovale infection in Sri Lanka: distant exposure and incidental detection of hyperparasitemia: a case report.

BACKGROUND: Plasmodium ovale malaria, which was previously endemic to tropical Africa and the Southwest Pacific islands is now being reported from parts of Asia. In Sri Lanka, the indigenous transmission of malaria has not been documented since October 2012. Since then, there have been several imported cases of malaria, including P. ovale, which have been detected sporadically. The reporting case of P. ovale was imported and detected incidentally in 2021, with several atypical presentations. CASE PRESENTATION: A 40-year-old Sri Lankan medical doctor developed continuous fever with chills, rigors, and dysuria a day following removal of a large lipoma at the root of the neck under general anaesthesia. When the fever has been responding to antibiotics, on the 4th postoperative day a mild thrombocytopenia on complete blood count was detected. A blood smear which was done on the 5th postoperative day incidentally found a malaria parasite and confirmed as Plasmodium ovale with a density of 6535 parasites/microliter on the same day. He never had malaria in the past, but he had worked in South Sudan 1 year ago and visited India six months ago. On the 6th postoperative day, he was treated with chloroquine, and hyperparasitemia reduced rapidly by the next day. As the fever recurred with clinical deterioration, he was treated with different antibiotics. During the course of the illness, he did not develop pallor, or icterus except for a palpable soft spleen. The parasite count was zero on the 9th postoperative day and his fever subsided on the next day. Further, he was treated with primaquine to prevent future relapse and transmission. CONCLUSION: A long incubation period, incidental detection of P ovale in a blood smear, and hyperparasitaemia are the atypical presentations of this case. Postoperative bacterial infection and stress may have reactivated the dormant malaria (hyponozoites) in this patient with an unusual picture. Coinfection of malaria with bacterial sepsis is a challenge in the management of the patient. As the Anopheles mosquito vector exists in Sri Lanka, the risk of indigenous transmission is high from such imported cases of P. ovale.

Systematic review of sporozoite infection rate of Anopheles mosquitoes in Ethiopia, 2001-2021.

BACKGROUND: Adult mosquitoes of the genus Anopheles are important vectors of Plasmodium parasites, causative agents of malaria. The aim of this review was to synthesize the overall and species-specific proportion of Anopheles species infected with sporozoites and their geographical distribution in the last 2 decades (2001-2021). METHODS: A comprehensive search was conducted using databases (PubMed, Google Scholar, Science Direct, Scopus, African Journals OnLine) and manual Google search between January 1 and February 15, 2022. Original articles describing work conducted in Ethiopia, published in English and reporting infection status, were included in the review. All the required data were extracted using a standardized data extraction form, imported to SPSS-24, and analyzed accordingly. The quality of each original study was assessed using a quality assessment tool adapted from the Joanna Briggs Institute critical appraisal checklist. This study was registered on PROSPERO (International Prospective Register of Systematic Reviews; registration no. CRD42022299078). RESULTS: A search for published articles produced a total of 3086 articles, of which 34 met the inclusion criteria. Data on mosquito surveillance revealed that a total of 129,410 anophelines comprising 25 species were captured, of which 48,365 comprising 21 species were tested for sporozoites. Anopheles arabiensis was the dominant species followed by An. pharoensis and An. coustani complex. The overall proportion infected with sporozoites over 21 years was 0.87%. Individual proportions included Anopheles arabiensis (1.09), An. pharoensis (0.79), An. coustani complex (0.13), An. funestus (2.71), An. demeilloni (0.31), An. stephensi (0.70), and An. cinereus (0.73). Plasmodium falciparum sporozoites accounted 79.2% of Plasmodium species. Mixed infection of Plasmodium vivax and P. falciparum was only reported from one An. arabiensis sample. CONCLUSIONS: Anopheles arebiensis was the dominant malaria vector over the years, with the highest sporozoite infection proportion of 2.85% and an average of 0.90% over the years. Other species contributing to malaria transmission in the area were An. pharoensis, An. coustani complex, An. funestus, An. stephensi, and An. coustani. The emergence of new vector species, in particular An. stephensi, is particularly concerning and should be investigated further.

Evaluation of the protective efficacy of Olyset®Plus ceiling net on reducing malaria prevalence in children in Lake Victoria Basin, Kenya: study protocol for a cluster-randomized controlled trial.

BACKGROUND: In the Lake Victoria Basin of western Kenya, malaria remains highly endemic despite high coverage of interventions such as insecticide-impregnated long-lasting insecticidal nets (LLIN). The malaria-protective effect of LLINs is hampered by insecticide resistance in Anopheles vectors and its repurposing by the community. Ceiling nets and LLIN with synergist piperonyl butoxide (PBO-LLIN) are novel tools that can overcome the problems of behavioral variation of net use and metabolic resistance to insecticide, respectively. The two have been shown to reduce malaria prevalence when used independently. Integration of these two tools (i.e., ceiling nets made with PBO-LLIN or Olyset®Plus ceiling nets) appears promising in further reducing the malaria burden. METHODS: A cluster-randomized controlled trial is designed to assess the effect of Olyset®Plus ceiling nets on reducing malaria prevalence in children on Mfangano Island in Homa Bay County, where malaria transmission is moderate. Olyset®Plus ceiling nets will be installed in 1315 residential structures. Malaria parasitological, entomological, and serological indicators will be measured for 12 months to compare the effectiveness of this new intervention against conventional LLIN in the control arm. DISCUSSION: Wider adoption of Olyset®Plus ceiling nets to complement existing interventions may benefit other malaria-endemic counties and be incorporated as part of Kenya's national malaria elimination strategy. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000045079. Registered on 4 August 2021.

Dried out but alive: how mosquitoes survive 8 months.

How malaria mosquitoes persist during the dry season in the Sahel and rapidly rebound at the onset of rains is unclear. Recently, Faiman and colleagues demonstrated that aestivation, a summer dormancy mechanism, is a major persistence strategy of Anopheles mosquitoes, which could be targeted by vector control.

Composition of mosquito fauna and insecticide resistance status of Anopheles gambiae sensu lato in Itang special district, Gambella, Southwestern Ethiopia.

BACKGROUND: Anopheles arabiensis, member species of the Anopheles gambiae complex, is the primary vector of malaria and is widely distributed in Ethiopia. Anopheles funestus, Anopheles pharoensis and Anopheles nili are secondary vectors occurring with limited distribution in the country. Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) are pillars for the interventions against malaria control and elimination efforts in Ethiopia. However, the emergence and widespread of insecticide resistance in An. gambiae sensu lato (s.l.), might compromise the control efforts of the country. The aim of this study was to investigate composition of mosquito fauna and insecticide resistance status of An. gambiae s.l. in Itang special district ( woreda), Gambella, southwestern Ethiopia. METHODS: Adult mosquitoes were sampled from September 2020 to February 2021 using the CDC light trap and pyrethrum spray catch (PSC). CDC light traps were placed in three selected houses for two consecutive days per month to collect mosquitoes indoor and outdoor from 6:00 P.M. to 06:00 A.M. and PSC was used to collect indoor resting mosquitoes from ten selected houses once in a month from October 2020 to February 2021. Moreover, mosquito larvae were also collected from different breeding sites and reared to adults to assess susceptibility status of populations of An. gambiae s.l. in the study area. Susceptibility tests were conducted on two to three days old non blood fed female An. gambiae s.l. using insecticide impregnated papers with deltamethrin (0.05%), alpha-cypermethrin (0.05%), propoxur (0.1%), pirimiphos-methyl (0.25%) and bendiocarb (0.1%) following World Health Organization (WHO) standard susceptibility test procedure. Molecular diagnostics were done for the identification of member species of An. gambiae s.l. and detection of knockdown resistance (kdr) allele using species specific polymerase chain reaction (PCR) and allele specific PCR. RESULTS: In total, 468 adult mosquitoes were collected from different houses. Culex mosquitoes were the most dominant (80.4%) followed by Anopheles mosquitoes. Three species of Anopheles (Anopheles coustani, An. pharoensis, and An. gambiae s.l.) were identified, of which An. coustani was the dominant (8.1%) species. Higher number of mosquitoes (231) were collected outdoor by CDC light traps. Out of 468 adult mosquitoes, 294 were blood fed, 46 were half-gravid and gravid whereas the remaining 128 were unfed. WHO bioassay tests revealed that the populations of An. gambiae s.l. in the study area are resistant against alpha-cypermethrin and deltamethrin, but susceptible to bendiocarb, pirimiphos-methyl and propoxur. Of the total 86 An. gambiae s.l. specimens assayed, 79 (92%) successfully amplified and identified as An. arabiensis. West African kdr (L1014F) mutation was detected with high kdr allele frequency ranging from 67 to 88%. CONCLUSION: The detection of target site mutation, kdr L1014F allele, coupled with the phenotypic resistance against alpha-cypermethrin and deltamethrin call for continuous resistance monitoring.

Remote bednet use monitoring to describe patterns of use and exposure to female Anopheles mosquitoes in an Ugandan cohort.

Background: Long lasting insecticide-treated bednets (LLINs) are the most widely used tool for preventing malaria. There has been a plateau in progress in the highest burden African countries since 2015, leading to questions about the effectiveness of LLINs. In this study, remote LLIN use monitors were deployed in a cohort in Eastern Uganda to explore how LLIN use interacts with mosquito exposure. Methods: The SmartNet study included 20 households from May to October 2019. SmartNet devices recorded, every 15 min, whether an LLIN was unfurled or folded up. Unannounced visits were used to assess SmartNet accuracy. Risk factors associated with poor LLIN use were assessed using generalized linear equations. Female Anopheles exposure was estimated by combining hourly probabilities of exposure from human landing catches and measures of density from biweekly CDC light traps in participants rooms. Mosquito exposure averted by LLINs was quantified using SmartNet measurements and age-related differences were estimated using generalized linear equations, adjusting for relevant covariates and household clustering. Results: 96 individuals contributed 5,640 SmartNet observation nights. In 126 unannounced visits, SmartNet had an area under the curve of 0.869 in classifying whether the LLIN was up or down. The rate of non-use was 13.5% of nights (95% CI: 12.6-14.3%). Compared to children under 5, non-use was 1.8 times higher (95% CI: 1.6-2.1; p < 0.001) in children 5-15 years and 2.6 times higher (95% CI: 2.2-3.1; p < 0.001) in participants aged 15-<30years. There was no difference between children under 5 years and adults > 30 years. LLIN use averted 50.3% of female Anopheles mosquito exposure (95% CI: 40.0-60.0%), with decreasing point estimates of efficacy across age groups: from 61.7% (95% CI: 42.6-80.7%) in children under 5 years to 48.0% (95% CI: 29.1-66.8%) in adults over 30. Conclusions: Objective monitors are accurate and can feasibly be deployed to obtain data about LLIN use. LLINs provided protection from only 50% of female Anopheles mosquito exposure in this cohort and protection was dependent upon age. In assessing the role of LLINs in malaria prevention it is crucial to consider the dynamics between mosquito exposure and LLIN use behaviors.

Functional Constraints on Insect Immune System Components Govern Their Evolutionary Trajectories.

Roles of constraints in shaping evolutionary outcomes are often considered in the contexts of developmental biology and population genetics, in terms of capacities to generate new variants and how selection limits or promotes consequent phenotypic changes. Comparative genomics also recognizes the role of constraints, in terms of shaping evolution of gene and genome architectures, sequence evolutionary rates, and gene gains or losses, as well as on molecular phenotypes. Characterizing patterns of genomic change where putative functions and interactions of system components are relatively well described offers opportunities to explore whether genes with similar roles exhibit similar evolutionary trajectories. Using insect immunity as our test case system, we hypothesize that characterizing gene evolutionary histories can define distinct dynamics associated with different functional roles. We develop metrics that quantify gene evolutionary histories, employ these to characterize evolutionary features of immune gene repertoires, and explore relationships between gene family evolutionary profiles and their roles in immunity to understand how different constraints may relate to distinct dynamics. We identified three main axes of evolutionary trajectories characterized by gene duplication and synteny, maintenance/stability and sequence conservation, and loss and sequence divergence, highlighting similar and contrasting patterns across these axes amongst subsets of immune genes. Our results suggest that where and how genes participate in immune responses limit the range of possible evolutionary scenarios they exhibit. The test case study system of insect immunity highlights the potential of applying comparative genomics approaches to characterize how functional constraints on different components of biological systems govern their evolutionary trajectories.

The Automatic Classification of Pyriproxyfen-Affected Mosquito Ovaries.

Pyriproxyfen (PPF) may become an alternative insecticide for areas where pyrethroid-resistant vectors are prevalent. The efficacy of PPF can be assessed through the dissection and assessment of vector ovaries. However, this reliance on expertise is subject to limitations. We show here that these limitations can be overcome using a convolutional neural network (CNN) to automate the classification of egg development and thus fertility status. Using TensorFlow, a resnet-50 CNN was pretrained with the ImageNet dataset. This CNN architecture was then retrained using a novel dataset of 524 dissected ovary images from An. gambiae s.l. An. gambiae Akron, and An. funestus s.l., whose fertility status and PPF exposure were known. Data augmentation increased the training set to 6973 images. A test set of 157 images was used to measure accuracy. This CNN model achieved an accuracy score of 94%, and application took a mean time of 38.5 s. Such a CNN can achieve an acceptable level of precision in a quick, robust format and can be distributed in a practical, accessible, and free manner. Furthermore, this approach is useful for measuring the efficacy and durability of PPF treated bednets, and it is applicable to any PPF-treated tool or similarly acting insecticide.

A Systematic Review of the Effects of Temperature on Anopheles Mosquito Development and Survival: Implications for Malaria Control in a Future Warmer Climate.

The rearing temperature of the immature stages can have a significant impact on the life-history traits and the ability of adult mosquitoes to transmit diseases. This review assessed published evidence of the effects of temperature on the immature stages, life-history traits, insecticide susceptibility, and expression of enzymes in the adult Anopheles mosquito. Original articles published through 31 March 2021 were systematically retrieved from Scopus, Google Scholar, Science Direct, PubMed, ProQuest, and Web of Science databases. After applying eligibility criteria, 29 studies were included. The review revealed that immature stages of An. arabiensis were more tolerant (in terms of survival) to a higher temperature than An. funestus and An. quadriannulatus. Higher temperatures resulted in smaller larval sizes and decreased hatching and pupation time. The development rate and survival of An. stephensi was significantly reduced at a higher temperature than a lower temperature. Increasing temperatures decreased the longevity, body size, length of the gonotrophic cycle, and fecundity of Anopheles mosquitoes. Higher rearing temperatures increased pyrethroid resistance in adults of the An. arabiensis SENN DDT strain, and increased pyrethroid tolerance in the An. arabiensis SENN strain. Increasing temperature also significantly increased Nitric Oxide Synthase (NOS) expression and decreased insecticide toxicity. Both extreme low and high temperatures affect Anopheles mosquito development and survival. Climate change could have diverse effects on Anopheles mosquitoes. The sensitivities of Anopeheles mosquitoes to temperature differ from species to species, even among the same complex. Notwithstanding, there seem to be limited studies on the effects of temperature on adult life-history traits of Anopheles mosquitoes, and more studies are needed to clarify this relationship.

Forced Egg Laying Method to Establish F1 Progeny from Field Populations and Laboratory Strains of Anopheles Mosquitoes (Diptera: Culicidae) in Thailand.

Successful monitoring of physiological resistance of malaria vectors requires about 150 female mosquitoes for a single set of tests. In some situations, the sampling effort is insufficient due to the low number of field-caught mosquitoes. To address this challenge, we demonstrate the feasibility of using the forced oviposition method for producing F1 from field-caught Anopheles mosquitoes. A total of 430 and 598 gravid Anopheles females from four laboratory strains and five field populations, respectively, were tested. After blood feeding, gravid mosquitoes were individually introduced into transparent plastic vials, containing moistened cotton balls topped with a 4 cm2 piece of filter paper. The number of eggs, hatching larvae, pupation, and adult emergence were recorded daily. The mean number of eggs per female mosquito ranged from 39.3 for Anopheles cracens to 93.6 for Anopheles dirus in the laboratory strains, and from 36.3 for Anopheles harrisoni to 147.6 for Anopheles barbirostris s.l. in the field populations. A relatively high egg hatching rate was found in An. dirus (95.85%), Anopheles minimus (78.22%), and An. cracens (75.59%). Similarly, a relatively high pupation rate was found for almost all test species ranging from 66% for An. minimus to 98.7% for Anopheles maculatus, and lowest for An. harrisoni (43.9%). Highly successful adult emergence rate was observed among 85-100% of pupae that emerged in all tested mosquito populations. The in-tube forced oviposition method is a promising method for the production of sufficient F1 progeny for molecular identification, vector competence, insecticide resistance, and bioassay studies.

Bio-Control of Anopheles Mosquito Larvae Using Invertebrate Predators to Support Human Health Programs in Ethiopia.

Mosquitoes have been a nuisance and health threat to humans for centuries due to their ability to transmit different infectious diseases. Biological control methods have emerged as an alternative or complementary approach to contain vector populations in light of the current spread of insecticide resistance in mosquitoes. Thus, this study aimed to evaluate the predation efficacy of selected potential predators against Anopheles mosquito larvae. Potential invertebrate predators and Anopheles larvae were collected from natural habitats, mainly (temporary) wetlands and ponds in southwest Ethiopia and experiments were conducted under laboratory conditions. Optimal predation conditions with respect to larval instar, water volume and number of predators were determined for each of the seven studied predators. Data analyses were carried out using the Poisson regression model using one way ANOVA at the 5% significant level. The backswimmer (Notonectidae) was the most aggressive predator on Anopheles mosquito larvae with a daily mean predation of 71.5 larvae (95% CI: [65.04;78.59]). Our study shows that larval instar, water volume and number of predators have a significant effect on each predator, except for dragonflies (Libellulidae), with regard to the preference of the larval instar. A selection of mosquito predators has the potential to control Anopheles mosquito larvae, suggesting that they can be used as complementary approach in an integrated malaria vector control strategy.

Spatial and temporal village-level prevalence of Plasmodium infection and associated risk factors in two districts of Meghalaya, India.

BACKGROUND: Despite declining incidence over the past decade, malaria remains an important health burden in India. This study aimed to assess the village-level temporal patterns of Plasmodium infection in two districts of the north-eastern state of Meghalaya and evaluate risk factors that might explain these patterns. METHODS: Primary Health Centre passive malaria case data from 2014 to 2018 were analysed to characterize village-specific annual incidence and temporal trends. Active malaria case detection was undertaken in 2018 and 2019 to detect Plasmodium infections using PCR. A questionnaire collected socio-demographic, environmental, and behavioural data, and households were spatially mapped via GPS. Adult mosquitoes were sampled at a subset of subjects' houses, and Anopheles were identified by PCR and sequencing. Risk factors for Plasmodium infection were evaluated using bivariate and multivariate logistic regression analysis, and spatial cluster analysis was undertaken. RESULTS: The annual malaria incidence from PHC-based passive surveillance datasets in 2014-2018 was heterogenous but declining across villages in both districts. Active surveillance in 2018 enrolled 1468 individuals from 468 households (West Jaintia Hills) and 1274 individuals from 359 households (West Khasi Hills). Plasmodium falciparum prevalence per 100 people varied from 0 to 4.1% in the nine villages of West Jaintia Hills, and from 0 to 10.6% in the 12 villages of West Khasi Hills. Significant clustering of P. falciparum infections [observed = 11, expected = 2.15, Relative Risk (RR) = 12.65; p < 0.001] was observed in West Khasi Hills. A total of 13 Anopheles species were found at 53 houses in five villages, with Anopheles jeyporiensis being the most abundant. Risk of infection increased with presence of mosquitoes and electricity in the households [Odds Ratio (OR) = 1.19 and 1.11], respectively. Households with reported animals had reduced infection risk (OR = 0.91). CONCLUSION: Malaria incidence during 2014-2018 declined in all study villages covered by the passive surveillance data, a period that includes the first widespread insecticide-treated net campaign. The survey data from 2018 revealed a significant association between Plasmodium infection and certain household characteristics. Since species of Plasmodium-competent mosquito vectors continue to be abundant, malaria resurgence remains a threat, and control efforts should continue.

[Surveillance of malaria vectors in Anhui Province from 2016 to 2018].

OBJECTIVE: To understand the population distribution, density, seasonal fluctuation and nocturnal activity of malaria vectors in Anhui Province from 2016 to 2018, so as to provide a data support for formulating the control strategy for imported malaria during the malaria post-elimination stage. METHODS: The malaria vectors were monitored in 105 counties (cities or districts) of Anhui Province from 2016 to 2018, and the population density, seasonal fluctuation and nocturnal activity of the mosquitoes were observed using the lamp trapping and human bait trapping methods. The density of Anopheles mosquitoes was compared among different years, regions and mosquito-capturing sites. RESULTS: Anopheles mosquitoes were captured in 103 counties (cities or districts) of Anhui Province during the period from 2016 to 2018, and a total of 32 494 mosquitoes were captured using the lamp trapping method and 36 228 captured using the human bait trapping method. All captured mosquitoes were morphologically identified as Anopheles sinensis, and no An. anthropophagus was found. The density of An. sinensis peaked from June to August, and the peak nocturnal activity was found during the period between 19∶00 and 23∶00. Among all mosquito-capturing sites, the highest mosquito density was seen in the livestock and poultry sheds (H = 18.835, P < 0.05). The density of An. sinensis varied significantly in regions in 2016 and 2017 (H = 16.655 and 11.566, P < 0.01), and a low density was found in north of the Huai River. CONCLUSIONS: An. sinensis is widely distributed in Anhui Province, which is the currently predominant malaria vector in the province. During the malaria post-elimination stage, the malaria vector monitoring should be intensified and vector control interventions should be timely adopted in epidemic foci of Anhui Province to prevent the local re-transmission of overseas imported malaria.

Targeting Plasmodium Plasmepsin V: Hitting Two Birds with One Stone.

A recent report by Jennison et al. reveals an important role for plasmepsin V (PMV), an aspartyl protease, in the development of malaria transmission stages. The authors showed that PMV activity is critical for protein export in these stages and that specific PMV inhibitors block parasite transmission to mosquitoes.

Surveillance of malaria vectors in Anhui Province from 2016 to 2018 / 中国血吸虫病防治杂志

Objective To understand the population distribution, density, seasonal fluctuation and nocturnal activity of malaria vectors in Anhui Province from 2016 to 2018, so as to provide a data support for formulating the control strategy for imported malaria during the malaria post-elimination stage. Methods The malaria vectors were monitored in 105 counties (cities or districts) of Anhui Province from 2016 to 2018, and the population density, seasonal fluctuation and nocturnal activity of the mosquitoes were observed using the lamp trapping and human bait trapping methods. The density of Anopheles mosquitoes was compared among different years, regions and mosquito-capturing sites. Results Anopheles mosquitoes were captured in 103 counties (cities or districts) of Anhui Province during the period from 2016 to 2018, and a total of 32 494 mosquitoes were captured using the lamp trapping method and 36 228 captured using the human bait trapping method. All captured mosquitoes were morphologically identified as Anopheles sinensis, and no An. anthropophagus was found. The density of An. sinensis peaked from June to August, and the peak nocturnal activity was found during the period between 19∶00 and 23∶00. Among all mosquito-capturing sites, the highest mosquito density was seen in the livestock and poultry sheds (H = 18.835, P < 0.05). The density of An. sinensis varied significantly in regions in 2016 and 2017 (H = 16.655 and 11.566, P < 0.01), and a low density was found in north of the Huai River. Conclusions An. sinensis is widely distributed in Anhui Province, which is the currently predominant malaria vector in the province. During the malaria post-elimination stage, the malaria vector monitoring should be intensified and vector control interventions should be timely adopted in epidemic foci of Anhui Province to prevent the local re-transmission of overseas imported malaria.

A Gut Symbiotic Bacterium Serratia marcescens Renders Mosquito Resistance to Plasmodium Infection Through Activation of Mosquito Immune Responses.

The malaria development in the mosquito midgut is a complex process that results in considerable parasite losses. The mosquito gut microbiota influences the outcome of pathogen infection in mosquitoes, but the underlying mechanisms through which gut symbiotic bacteria affect vector competence remain elusive. Here, we identified two Serratia strains (Y1 and J1) isolated from field-caught female Anopheles sinensis from China and assessed their effect on Plasmodium development in An. stephensi. Colonization of An. stephensi midgut by Serratia Y1 significantly renders the mosquito resistant to Plasmodium berghei infection, while Serratia J1 has no impact on parasite development. Parasite inhibition by Serratia Y1 is induced by the activation of the mosquito immune system. Genome-wide transcriptomic analysis by RNA-seq shows a similar pattern of midgut gene expression in response to Serratia Y1 and J1 in sugar-fed mosquitoes. However, 24 h after blood ingestion, Serratia Y1 modulates more midgut genes than Serratia J1 including the c-type lectins (CTLs), CLIP serine proteases and other immune effectors. Furthermore, silencing of several Serratia Y1-induced anti-Plasmodium factors like the thioester-containing protein 1 (TEP1), fibrinogen immunolectin 9 (FBN9) or leucine-rich repeat protein LRRD7 can rescue parasite oocyst development in the presence of Serratia Y1, suggesting that these factors modulate the Serratia Y1-mediated anti-Plasmodium effect. This study enhances our understanding of how gut bacteria influence mosquito-Plasmodium interactions.

A chromosome-scale assembly of the major African malaria vector Anopheles funestus.

BACKGROUND: Anopheles funestus is one of the 3 most consequential and widespread vectors of human malaria in tropical Africa. However, the lack of a high-quality reference genome has hindered the association of phenotypic traits with their genetic basis in this important mosquito. FINDINGS: Here we present a new high-quality A. funestus reference genome (AfunF3) assembled using 240× coverage of long-read single-molecule sequencing for contigging, combined with 100× coverage of short-read Hi-C data for chromosome scaffolding. The assembled contigs total 446 Mbp of sequence and contain substantial duplication due to alternative alleles present in the sequenced pool of mosquitos from the FUMOZ colony. Using alignment and depth-of-coverage information, these contigs were deduplicated to a 211 Mbp primary assembly, which is closer to the expected haploid genome size of 250 Mbp. This primary assembly consists of 1,053 contigs organized into 3 chromosome-scale scaffolds with an N50 contig size of 632 kbp and an N50 scaffold size of 93.811 Mbp, representing a 100-fold improvement in continuity versus the current reference assembly, AfunF1. CONCLUSION: This highly contiguous and complete A. funestus reference genome assembly will serve as an improved basis for future studies of genomic variation and organization in this important disease vector.

Screening of Plasmodium parasite in vectors and humans in three villages in Aswan Governorate, Egypt.

Egypt has successfully eliminated malaria during 2010-2013, however, between May to mid-June 2014, an outbreak was reported in Al-Adwa village, Aswan Governorate indicating that malaria may be potentially re-emerging in the country. The aim of this work was to reevaluate malaria in Al-Adwa and surrounding villages, 1 year after malaria cases have been reported through entomological and human screening in collaboration with the Ministry of Health and Population. Four trips were conducted during the period from March 2015 to March 2016. Mosquitoes were collected, sorted and identified. Anopheles species were processed for detection of Plasmodium by polymerase chain reaction (PCR), and engorged abdomens in blood-fed mosquitoes were analyzed for host preference using multiplex PCR. Thick and thin blood films were prepared from all apparently healthy children (n = 188) attending El-Sheikh Mostafa preparatory school. Results showed that Anopheles genus existed only in East Al-Adwa village. A total of 38 Anopheles mosquitoes were collected and identified as Anopheles multicolor 70% (A. multicolor), A. sergenti 20% and A. pharoensis 10%. The latter showed 100% human blood preference compared to A. sergenti (20%) and An. multicolor (0%). All female Anopheles were 100% negative for Plasmodium DNA, and all blood films showed no detectable parasite. The absence of Plasmodium in the area under study does not rule out the risk of future infections as the vector is still present together with the imported cases and carriers. Regular screening for the presence of Plasmodium in the area is absolutely paramount for early warning.

Guy1, a Y-linked embryonic signal, regulates dosage compensation in Anopheles stephensi by increasing X gene expression.

We previously showed that Guy1, a primary signal expressed from the Y chromosome, is a strong candidate for a male-determining factor that confers female-specific lethality in Anopheles stephensi (Criscione et al., 2016). Here, we present evidence that Guy1 increases X gene expression in Guy1-transgenic females from two independent lines, providing a mechanism underlying the Guy1-conferred female lethality. The median level gene expression (MGE) of X-linked genes is significantly higher than autosomal genes in Guy1-transgenic females while there is no significant difference in MGE between X and autosomal genes in wild-type females. Furthermore, Guy1 significantly upregulates at least 40% of the 996 genes across the X chromosome in transgenic females. Guy1-conferred female-specific lethality is remarkably stable and completely penetrant. These findings indicate that Guy1 regulates dosage compensation in An. stephensi and components of dosage compensation may be explored to develop novel strategies to control mosquito-borne diseases.

Application of matrix-assisted laser desorption/ionization mass spectrometry to identify species of Neotropical Anopheles vectors of malaria.

BACKGROUND: Malaria control in Panama is problematic due to the high diversity of morphologically similar Anopheles mosquito species, which makes identification of vectors of human Plasmodium challenging. Strategies by Panamanian health authorities to bring malaria under control targeting Anopheles vectors could be ineffective if they tackle a misidentified species. METHODS: A rapid mass spectrometry identification procedure was developed to accurately and timely sort out field-collected Neotropical Anopheles mosquitoes into vector and non-vector species. Matrix-assisted laser desorption/ionization (MALDI) mass spectra of highly-abundant proteins were generated from laboratory-reared mosquitoes using different extraction protocols, body parts, and sexes to minimize the amount of material from specimen vouchers needed and optimize the protocol for taxonomic identification. Subsequently, the mass spectra of field-collected Neotropical Anopheles mosquito species were classified using a combination of custom-made unsupervised (i.e., Principal component analysis-PCA) and supervised (i.e., Linear discriminant analysis-LDA) classification algorithms. RESULTS: Regardless of the protocol used or the mosquito species and sex, the legs contained the least intra-specific variability with enough well-preserved proteins to differentiate among distinct biological species, consistent with previous literature. After minimizing the amount of material needed from the voucher, one leg was enough to produce reliable spectra between specimens. Further, both PCA and LDA were able to classify up to 12 mosquito species, from different subgenera and seven geographically spread localities across Panama using mass spectra from one leg pair. LDA demonstrated high discriminatory power and consistency, with validation and cross-validation positive identification rates above 93% at the species level. CONCLUSION: The selected sample processing procedure can be used to identify field-collected Anopheles species, including vectors of Plasmodium, in a short period of time, with a minimal amount of tissue and without the need of an expert mosquito taxonomist. This strategy to analyse protein spectra overcomes the drawbacks of working without a reference library to classify unknown samples. Finally, this MALDI approach can aid ongoing malaria eradication efforts in Panama and other countries with large number of mosquito's species by improving vector surveillance in epidemic-prone sites such as indigenous Comarcas.