α,ß-trehalose, an intracellular substance in resting cyst of colpodid ciliates as a key to environmental tolerances.

α,α-trehalose is a well-known sugar that plays a key role in establishing tolerance to environmental stresses in many organisms, except unicellular eukaryotes. However, almost nothing is known about α,ß-trehalose, including their synthesis, function, and even presence in living organisms. In this study, we identified α,ß-trehalose in the resting cyst, a dormancy cell form characterized by extreme tolerance to environmental stresses, of the ciliated protist Colpoda cucullus, using high-performance liquid chromatography (HPLC), and a proton nuclear magnetic resonance (1H NMR). Gene expression analysis revealed that the expression of trehalose-6-phosphate synthase (TPS), glycosyltransferase (GT), alpha-amylase (AMY), and trehalose transporter 1 (TRET1), were up-regulated in encystment, while the expression of α-glucosidase 2 (AG2) and trehalase (TREH) was up-regulated in excystment. These results suggest that α,ß-trehalose is synthesized during encystment process, while and contributes to extreme tolerances to environmental stressors, stored carbohydrates, and energy reserve during resting cyst and/or during excystment.

Response of wild aquatic insect communities to thermal variation through comparative landscape transcriptomics.

Fluctuations in temperature are recognized as a potent driver of selection pressure, fostering genomic variations that are crucial for the adaptation and survival of organisms under selection. Notably, water temperature is a pivotal factor influencing aquatic organism persistence. By comprehending how aquatic organisms respond to shifts in water temperature, we can understand their potential physiological adaptations to environmental change in one or multiple species. This, in turn, contributes to the formulation of biologically relevant guidelines for the landscape scale transcriptome profile of organisms in lotic systems. Here, we investigated the distinct responses of seven stream stonefly species, collected from four geographical regions across Japan, to variations in temperature, including atmospheric and water temperatures. We achieved this by assessing the differences in gene expression through RNA-sequencing within individual species and exploring the patterns of community-genes among different species. We identified 735 genes that exhibited differential expressions across the temperature gradient. Remarkably, the community displayed expression levels differences of respiration and metabolic genes. Additionally, the diversity in molecular functions appeared to be linked to spatial variation, with water temperature differences potentially contributing to the overall functional diversity of genes. We found 22 community-genes with consistent expression patterns among species in response to water temperature variations. These genes related to respiration, metabolism and development exhibited a clear gradient providing robust evidence of divergent adaptive responses to water temperature. Our findings underscore the differential adaptation of stonefly species to local environmental conditions, suggesting that shared responses in gene expression may occur across multiple species under similar environmental conditions. This study emphasizes the significance of considering various species when assessing the impacts of environmental changes on aquatic insect communities and understanding potential mechanisms to cope with such changes.

Wastewater-based reproduction numbers and projections of COVID-19 cases in three areas in Japan, November 2021 to December 2022.

BackgroundWastewater surveillance has expanded globally as a means to monitor spread of infectious diseases. An inherent challenge is substantial noise and bias in wastewater data because of the sampling and quantification process, limiting the applicability of wastewater surveillance as a monitoring tool.AimTo present an analytical framework for capturing the growth trend of circulating infections from wastewater data and conducting scenario analyses to guide policy decisions.MethodsWe developed a mathematical model for translating the observed SARS-CoV-2 viral load in wastewater into effective reproduction numbers. We used an extended Kalman filter to infer underlying transmissions by smoothing out observational noise. We also illustrated the impact of different countermeasures such as expanded vaccinations and non-pharmaceutical interventions on the projected number of cases using three study areas in Japan during 2021-22 as an example.ResultsObserved notified cases were matched with the range of cases estimated by our approach with wastewater data only, across different study areas and virus quantification methods, especially when the disease prevalence was high. Estimated reproduction numbers derived from wastewater data were consistent with notification-based reproduction numbers. Our projections showed that a 10-20% increase in vaccination coverage or a 10% reduction in contact rate may suffice to initiate a declining trend in study areas.ConclusionOur study demonstrates how wastewater data can be used to track reproduction numbers and perform scenario modelling to inform policy decisions. The proposed framework complements conventional clinical surveillance, especially when reliable and timely epidemiological data are not available.

Spatial and phylogenetic structure of Alpine stonefly assemblages across seven habitats using DNA-species.

Stream ecosystems are spatially heterogeneous, with many different habitat patches distributed within a small area. The influence of this heterogeneity on the biodiversity of benthic insect communities is well documented; however, studies of the role of habitat heterogeneity in species coexistence and assembly remain limited. Here, we investigated how habitat heterogeneity influences spatial structure (beta biodiversity) and phylogenetic structure (evolutionary processes) of benthic stonefly (Plecoptera, Insecta) communities. We sampled 20 sites along two Alpine rivers, including seven habitats in four different reaches (headwaters, meandering, bar-braided floodplain, and lowland spring-fed). We identified 21 morphological species and delineated 52 DNA-species based on sequences from mitochondrial cox1 and nuclear ITS markers. Using DNA-species, we first analysed the patterns of variation in richness, diversity, and assemblage composition by quantifing the contribution of each reach and habitat to the overall DNA-species diversity using an additive partition analysis and distance-based redundancy analysis. Using gene-tree phylogenies, we assessed whether environmental filtering could lead to the co-occurrence of DNA-species using a two-step analysis to detect a phylogenetic signal. All four reaches significantly contributed to DNA-species richness, with the meandering reach having the highest contribution. Habitats had an effect on DNA-species diversity, where glide, riffle and, pool influenced the spatial structure of stonefly assemblage possibly due to the high habitat heterogeneity. Among the habitats, the pool showed significant phylogenetic clustering, suggesting high levels of evolutionary adaptation and strong habitat filtering. This assemblage structure may be caused by long-term stability of the habitat and the similar requirements for co-occurring species. Our study shows the importance of different habitats for the spatial and phylogenetic structure of stonefly assemblage and sheds light on the habitat-specific diversity that may help improve conservation practices.

Sediment-associated microbial community profiling: sample pre-processing through sequential membrane filtration for 16S rRNA amplicon sequencing.

BACKGROUND: Sequential membrane filtration as a pre-processing step for capturing sediment-associated microorganisms could provide good quality and integrity DNA that can be preserved and kept at ambient temperatures before community profiling through culture-independent molecular techniques. However, the effects of sample pre-processing via filtration on DNA-based profiling of sediment-associated microbial community diversity and composition are poorly understood. Specifically, the influences of pre-processing on the quality and quantity of extracted DNA, high-throughput DNA sequencing reads, and detected microbial taxa need further evaluation. RESULTS: We assessed the impact of pre-processing freshwater sediment samples by sequential membrane filtration (from 10, 5 to 0.22 µm pore size) for 16S rRNA-based community profiling of sediment-associated microorganisms. Specifically, we examined if there would be method-driven differences between non- and pre-processed sediment samples regarding the quality and quantity of extracted DNA, PCR amplicon, resulting high-throughput sequencing reads, microbial diversity, and community composition. We found no significant difference in the qualities and quantities of extracted DNA and PCR amplicons, and the read abundance after bioinformatics processing (i.e., denoising and chimeric-read filtering steps) between the two methods. Although the non- and pre-processed sediment samples had more unique than shared amplicon sequence variants (ASVs), we report that their shared ASVs accounted for 74% of both methods' absolute read abundance. More so, at the genus level, the final collection filter identified most of the genera (95% of the reads) captured from the non-processed samples, with a total of 51 false-negative (2%) and 59 false-positive genera (3%). We demonstrate that while there were differences in shared and unique taxa, both methods revealed comparable microbial diversity and community composition. CONCLUSIONS: Our observations highlight the feasibility of pre-processing sediment samples for community analysis and the need to further assess sampling strategies to help conceptualize appropriate study designs for sediment-associated microbial community profiling.

Carotenoid coloration and coloration-linked gene expression in red tilapia (Oreochromis sp.) tissues.

BACKGROUND: Production, marketability and consumer preference of red tilapia often depends upon the intensity of coloration. Hence, new approaches to develop coloration are now geared to improve market acceptability and profit. This study evaluated the effects of carotenoid-rich diets on the phenotypic coloration, carotenoid level, weight gain and expression of coloration-linked genes in skin, fin and muscle tissues. Carotenoids were extracted from dried Daucus carota peel, Ipomoea aquatica leaves, and Moringa oleifera leaves. Eighty (80) size-14 fish were fed with carotenoid-rich treatments twice a day for 120 days. The phenotypic effect of the carotenoid extracts was measured through a color chart. Skin carotenoid level was measured through UV-vis spectrophotometer. csf1ra, Bcdo2 and StAR expression analysis was done using qRT-PCR. RESULTS: Treatments with carotenoid extracts yielded higher overall scores on phenotypic coloration and tissue carotenoid levels. Differential expression of carotenoid-linked genes such as the elevated expression in csf1ra and lower expression in Bcdo2b following supplementation of the enhanced diet supports the phenotypic redness and increased carotenoid values in red tilapia fed with D. carota peel and I. aquatica leaves. CONCLUSIONS: Overall improvement in the redness of the tilapia was achieved through the supplementation of carotenoid-rich diet derived from readily available plants. Differential expression of coloration-linked genes supports the increase in the intensity of phenotypic coloration and level of carotenoids in the tissues. The study emphasizes the importance of carotenoids in the commercial tilapia industry and highlights the potential of the plant extracts for integration and development of feeds for color enhancement in red tilapia.

Genome-wide signatures of local adaptation among seven stoneflies species along a nationwide latitudinal gradient in Japan.

BACKGROUND: Environmental heterogeneity continuously produces a selective pressure that results in genomic variation among organisms; understanding this relationship remains a challenge in evolutionary biology. Here, we evaluated the degree of genome-environmental association of seven stonefly species across a wide geographic area in Japan and additionally identified putative environmental drivers and their effect on co-existing multiple stonefly species. Double-digest restriction-associated DNA (ddRAD) libraries were independently sequenced for 219 individuals from 23 sites across four geographical regions along a nationwide latitudinal gradient in Japan. RESULTS: A total of 4251 candidate single nucleotide polymorphisms (SNPs) strongly associated with local adaptation were discovered using Latent mixed models; of these, 294 SNPs showed strong correlation with environmental variables, specifically precipitation and altitude, using distance-based redundancy analysis. Genome-genome comparison among the seven species revealed a high sequence similarity of candidate SNPs within a geographical region, suggesting the occurrence of a parallel evolution process. CONCLUSIONS: Our results revealed genomic signatures of local adaptation and their influence on multiple, co-occurring species. These results can be potentially applied for future studies on river management and climatic stressor impacts.

Free-Chlorine Disinfection as a Selection Pressure on Norovirus.

Human noroviruses are excreted in feces from infected individuals and included in wastewater. It is critical to remove/inactivate them in wastewater treatment processes, particularly in the disinfection step, before release to aquatic environments. However, the high mutation rates of human noroviruses raise concerns about the emergence of strains that are less susceptible to disinfectants and can survive even after wastewater treatment. This study aimed to demonstrate the strain-dependent susceptibility of norovirus to free chlorine. A population originated from the murine norovirus strain S7-PP3, a surrogate for human noroviruses in environmental testing, was exposed to free chlorine and then propagated in a host cell. This cycle of free chlorine exposure followed by propagation in cells was repeated 10 times, and populations with lower susceptibility to free chlorine were obtained from two independent trials of chlorine exposure cycles. Open reading frame 2 (ORF2) and ORF3 of the murine norovirus genome were analyzed by next-generation sequencing, and a unique nonsynonymous mutation (corresponding to a change from phenylalanine to serine) at nucleotide (nt) 7280 in ORF3, which encodes the minor capsid protein VP2, was found in chlorine-exposed populations from both trials. It was confirmed that all of the clones from the chlorine-treated population had lower susceptibility to free chlorine than those from the control population. These results indicate that exposure to free chlorine and dilution exert different driving forces to form murine norovirus (MNV) quasispecies, and that there is a selective force to form MNV quasispecies under free chlorine exposure.IMPORTANCE This study showed that free chlorine disinfection exerted a selection pressure for murine norovirus (MNV). The strain-dependent viral susceptibility to the disinfectant elucidated in this study highlights the importance of employing less susceptible strains as representative viruses in disinfection tests, because the disinfection rate values obtained from more susceptible strains would be less useful in predicting the virus inactivation efficiency of circulating strains under practical disinfection conditions.

Machine learning methods reveal the temporal pattern of dengue incidence using meteorological factors in metropolitan Manila, Philippines.

BACKGROUND: Several studies have applied ecological factors such as meteorological variables to develop models and accurately predict the temporal pattern of dengue incidence or occurrence. With the vast amount of studies that investigated this premise, the modeling approaches differ from each study and only use a single statistical technique. It raises the question of whether which technique would be robust and reliable. Hence, our study aims to compare the predictive accuracy of the temporal pattern of Dengue incidence in Metropolitan Manila as influenced by meteorological factors from four modeling techniques, (a) General Additive Modeling, (b) Seasonal Autoregressive Integrated Moving Average with exogenous variables (c) Random Forest and (d) Gradient Boosting. METHODS: Dengue incidence and meteorological data (flood, precipitation, temperature, southern oscillation index, relative humidity, wind speed and direction) of Metropolitan Manila from January 1, 2009 - December 31, 2013 were obtained from respective government agencies. Two types of datasets were used in the analysis; observed meteorological factors (MF) and its corresponding delayed or lagged effect (LG). After which, these datasets were subjected to the four modeling techniques. The predictive accuracy and variable importance of each modeling technique were calculated and evaluated. RESULTS: Among the statistical modeling techniques, Random Forest showed the best predictive accuracy. Moreover, the delayed or lag effects of the meteorological variables was shown to be the best dataset to use for such purpose. Thus, the model of Random Forest with delayed meteorological effects (RF-LG) was deemed the best among all assessed models. Relative humidity was shown to be the top-most important meteorological factor in the best model. CONCLUSION: The study exhibited that there are indeed different predictive outcomes generated from each statistical modeling technique and it further revealed that the Random forest model with delayed meteorological effects to be the best in predicting the temporal pattern of Dengue incidence in Metropolitan Manila. It is also noteworthy that the study also identified relative humidity as an important meteorological factor along with rainfall and temperature that can influence this temporal pattern.

Detection and distribution of Wolbachia endobacteria in Culex quinquefasciatus populations (Diptera : Culicidae) from Metropolitan Manila, Philippines.

BACKGROUND & OBJECTIVES: Culex quinquefasciatus is a peridomestic mosquito known for its ability to transmit pathogenic diseases such as filariasis and Japanese encephalitis. The development and use of novel and innovative vector control measures such as the utilization of Wolbachia, along with the existing ones, are necessary to prevent the transmission of these diseases. Studies exploring the diversity of Wolbachia, particularly in Cx. quinquefasciatus are very limited in the Philippines. Thus, the aim of the study was to detect the presence, distribution, and phylogenetic relationship of Wolbachia infections in Cx. quinquefasciatus in Metropolitan Manila, Philippines. METHODS: Adult Cx. quinquefasciatus mosquitoes were collected using a commercially available light-trap from May 2014-January 2015. Based on their sampling grids (n = 51), the adult mosquito abdomens were pooled and subjected to Wolbachia surface protein (wsp) gene amplification assay. Five selected wsp-positive samples were then sequenced and further analyzed to infer their phylogenetic relationship with known Wolbachia strains. RESULTS: A total of 1090 adult Cx. quinquefasciatus mosquitoes were collected. Pooled abdomens (n = 53) were then sorted based on their sampling grids for subsequent screening of wsp gene. Wolbachia infection rate was 59% (31/53). These infections were located at 29 (57%) sampling grids, and were observed to be widely distributed in the study area. Phylogenetic analysis indicated that the sample sequences were Wolbachia pipientis isolated from known hosts, Cx. pipiens and Cx. quinquefasciatus belonging to supergroup B clade. INTERPRETATION & CONCLUSION: The study was able to demonstrate the prevalence and distribution of Wolbachia in Cx. quinquefasciatus in Metropolitan Manila, Philippines. The findings of this study are geared towards proposing a vector control program that utilizes the potential of Wolbachia as a biological control agent in preventing the transmission of Culex-borne diseases.

Differences in protein expression among five species of stream stonefly (Plecoptera) along a latitudinal gradient in Japan.

Proteome variation among natural populations along an environmental gradient may provide insights into how the biological functions of species are related to their local adaptation. We investigated protein expression in five stream stonefly species from four geographic regions along a latitudinal gradient in Japan with varying climatic conditions. The extracted proteins were separated by two-dimensional gel electrophoresis and identified by matrix-assisted laser desorption/ionization of time-of-flight (MALDI TOF/TOF), yielding 446 proteins. Low interspecies variation in the proteome profiles was observed among five species within geographical regions, presumably due to the co-occurring species sharing the environments. However, large spatial variations in protein expression were found among four geographic regions, suggesting strong regulation of protein expression in heterogeneous environments, where the spatial variations were positively correlated with water temperature. We identified 21 unique proteins expressed specifically in a geographical region and six common proteins expressed throughout all regions. In warmer regions, metabolic proteins were upregulated, whereas proteins related to cold stress, the photoperiod, and mating were downregulated. Oxygen-related and energy-production proteins were upregulated in colder regions with higher altitudes. Thus, our proteomic approach is useful for identifying and understanding important biological functions related to local adaptations by populations of stoneflies.

Spatial analysis of wing geometry in dengue vector mosquito, Aedes aegypti (L.) (Diptera: Culicidae), populations in Metropolitan Manila, Philippines.

BACKGROUND & OBJECTIVES: Aedes aegypti (L.) is an efficient vector for arboviral diseases such as dengue. The wings of Ae. aegypti has been extensively studied in order to investigate population heterogeneity and structure by utilizing a landmark based geometric morphometrics (GMs) approach. The aim of this study was to examine and assess the wing geometry of Ae. aegypti in Metropolitan Manila. METHODS: In total, 312 Ae. aegypti mosquitoes were collected from 98 sampling points using a mosquito light-trap from May 2014 to January 2015. A complete coverage of the wing was achieved by removing wing scales with chemical and physical treatment, leading to identification of 26 landmarks. Geometric morphometric analyses were employed and the spatial distance pattern was estimated using isolation by distance (IBD) and spatial autocorrelation (SA). RESULTS: The results of the GM analyses revealed population heterogeneity and structuring in Ae. aegypti populations for both sexes using principal component and canonical variate analyses respectively. Moreover, IBD and SA only detected significant spatial structure in male Ae. aegypti populations while female population structures were homogeneous throughout the geographical area. INTERPRETATION & CONCLUSION: The newly modified wing preparation procedure was able to capture a complete coverage of the wings of Ae. aegypti, thus providing a stronger separation power for very close populations in an urban area. It is also noteworthy that the results of IBD and SA supported the findings of GM in the population structuring of male and female Ae. aegypti. The outcome of the study increases our understanding of the vector, which would be useful in developing effective control strategies.

Comparative assessment of primary and secondary infection risks in a norovirus outbreak using a household model simulation.

Diarrheal diseases can be transmitted via both primary infection due to exposures to contaminated materials from the environment and secondary infection due to person-to-person contacts. Usually, the importance of secondary infection is empirically assessed by fitting mathematical models to the epidemic curves. However, these empirical models may not be applicable to other epidemic cases because they are developed only for the target epidemics and they don't consider the detail routes of infection. In our previous study, we developed a theoretical model taking into account the various routes of infection that commonly occur in households (e.g., shaking hands, food handling, and changing diapers). This model was made flexible and applicable to any epidemics by means of adjusting model parameters. In this study, we proposed a new index "Vulnerability indicator to secondary infection (VISI)", which expressed a ratio of secondary infection to primary infection risks and calculated this index in a simulated norovirus (NoV) epidemic that involved 10,000 households. The results demonstrated that households composed of more than three members including infant(s) had much higher levels of VISI (5-45) than two-member-households with VISI (0.1-4). These results concluded that the infants were likely to be a hub of secondary infections in highly dense families and therefore careful handling of diapers was deemed indispensible in such families to effectively control the secondary infections.

Innovative house structures for malaria vector control in Nampula district, Mozambique: assessing mosquito entry prevention, indoor comfort, and community acceptance.

Background: Insecticide-treated mosquito bed nets and indoor residual spraying are widely used for malaria vector control. However, their effectiveness can be affected by household members' habits, requiring alternative approaches toward malaria vector control. Objective: To assess the effectiveness of modified houses in preventing mosquito entry; to assess the impact of house modifications on indoor air conditions and evaluate the acceptability of modified houses in the community where the study was conducted. Methods: Five traditional and five modified houses were constructed in Nampula district, Mozambique and underwent a 90-day overnight indoor mosquito collection using Centers for Disease Control and nitride ultraviolet light traps during the rainy season. Mosquitoes were identified morphologically. Indoor temperature, relative humidity, carbon dioxide levels and wind speed were also collected. The Student's t-test was used to compare the means of the number of mosquitos and environmental factors between both house types. A binomial form of the Generalized Linear Model identified the factors associated with the community volunteer's preference for house type. Results: Modified houses reduced the number of Anopheles by an average of 14.97 mosquitos (95% CI, 11.38-18.56, p < 0.000) and non-Anopheles by 16.66 mosquitoes (95% CI, 8.23-25.09, p < 0.000). Although fewer mosquitoes were trapped in modified houses compared to traditional ones, the modifications were more effective against Anopheles (94% reduction) than for non-Anopheles (71% reduction). The average temperature increased at 0.25°C in modified houses but was not statistically significant (95% CI, -0.62 to 0.12, p = 0.181). Community volunteers preferred modified houses due to reduced mosquito buzzing. The efficacy of modified houses including its acceptability by community, highlight its potential to lower malaria risk. Effective integration of modified houses into the vector control strategy will require raising awareness among communities about malaria risks associated with house structure and training them to modify their houses.

Potential Way to Develop Dengue Virus Detection in Aedes Larvae as an Alternative for Dengue Active Surveillance: A Literature Review.

The burden of dengue has emerged as a serious public health issue due to its impact on morbidity, mortality, and economic burden. Existing surveillance systems are inadequate to provide the necessary data for the prompt and efficient control of dengue. Passive surveillance of dengue cases may lead to underreporting and delayed mitigation responses. Improved dengue control program requires sensitive and proactive methods for early detection of dengue. We collected and reviewed existing research articles worldwide on detecting dengue virus in Aedes species larvae. Searches were conducted in PUBMED and Google Scholar, including all the studies published in English and Bahasa Indonesia. Twenty-nine studies were included in this review in terms of assay used, positivity rate, and dengue serotype detected. The presence of dengue virus in immature mosquitoes was mostly detected using reverse transcription PCR (RT-PCR) in pooled larvae. In one study, dengue virus was detected in larvae from laboratory-infected mosquitoes using enzyme-linked immunosorbent assay (ELISA). The positivity rate of dengue virus detection ranged from 0 to 50% in field-caught larvae. Although various methods can detect the dengue virus, further research encourages the use of low-cost and less laborious methods for active surveillance of dengue in larvae.

Detection and quantification of natural Wolbachia in Aedes aegypti in Metropolitan Manila, Philippines using locally designed primers.

Background: The Philippines bears health and economic burden caused by high dengue cases annually. Presently, the Philippines still lack an effective and sustainable vector management. The use of Wolbachia, a maternally transmitted bacterium, that mitigate arbovirus transmission has been recommended. Cytoplasmic incompatibility and viral blocking, two characteristics that make Wolbachia suitable for vector control, depend on infection prevalence and density. There are no current Wolbachia release programs in the Philippines, and studies regarding the safety of this intervention. Here, we screened for Wolbachia in Aedes aegypti collected from Metropolitan Manila, Philippines. We designed location-specific primers for qPCR to test whether this improved Wolbachia detection in Ae. aegypti. We explored if host sex and Wolbachia strain could be potential factors affecting Wolbachia density. Methods: Ae. aegypti mosquitoes (n=429) were screened for natural Wolbachia by taqman qPCR using location-specific Wolbachia surface protein primers (wspAAML) and known 16S rRNA primers. Samples positive for wspAAML (n=267) were processed for Sanger sequencing. We constructed a phylogenetic tree using IQ-TREE 2 to further characterize Wolbachia present in the Philippine Ae. aegypti. We then compared Wolbachia densities between Wolbachia groups and host sex. Statistical analyses were done using GraphPad Prism 9.0. Results: Wolbachia prevalence for 16S rRNA (40%) and wspAAML (62%) markers were high. Wolbachia relative densities for 16S rRNA ranged from -3.84 to 2.71 and wspAAML from -4.02 to 1.81. Densities were higher in male than female mosquitoes. Wolbachia strains detected in Ae. aegypti clustered into supergroup B. Some 54% (123/226) of these sequences clustered under a group referred to here as "wAegML," that belongs to the supergroup B, which had a significantly lower density than wAegB/wAlbB, and wAlbA strains. Conclusion: Location-specific primers improved detection of natural Wolbachia in Ae. aegypti and allowed for relative quantification. Wolbachia density is relatively low, and differed between host sexes and Wolbachia strains. An economical way of confirming sporadic or transient Wolbachia in Ae. aegypti is necessary while considering host sex and bacterial strain.

In vitro characterization of cell-fusing agent virus DNA forms in Aedes aegypti mosquitoes.

How non-retroviral endogenous viral elements (EVEs) are established is a long-standing question. Viral DNA (vDNA) forms of RNA viruses are likely to be EVE precursors. Cell-fusing agent virus (CFAV) is a major insect-specific virus (ISV) in the Aedes aegypti mosquitoes and one of the few existing non-retroviral RNA viruses found as EVEs. We characterized CFAV-derived vDNA in the cell line to understand the mechanism of why current viruses are rarely endogenized. vDNA production was affected by cell culture media independent of CFAV replication. vDNAs that correspond to different regions covering the entire viral genome were detected, implying multiple initiation sites exist. A considerable proportion of vDNAs corresponded to ssDNA. Higher vDNA copies were detected in the cytoplasm than the nucleus. Our findings provide valuable insights into the intracellular characteristics of ISV-derived vDNAs, which will aid in understanding the underlying mechanisms of non-retroviral EVE formation.

Fine-scale adaptive divergence and population genetic structure of Aedes aegypti in Metropolitan Manila, Philippines.

BACKGROUND: The adaptive divergence of Aedes aegypti populations to heterogeneous environments can be a driving force behind the recent expansion of their habitat distribution and outbreaks of dengue disease in urbanized areas. In this study, we investigated the population genomics of Ae. aegypti at a regional scale in Metropolitan Manila, Philippines. METHODS: We used the Pool-Seq double digestion restriction-site association DNA sequencing (ddRAD-Seq) approach to generate a high number of single nucleotide polymorphisms (SNPs), with the aim to determine local adaptation and compare the population structure with 11 microsatellite markers. A total of 217 Ae. aegypti individuals from seven female and seven male populations collected from Metropolitan Manila were used in the assays. RESULTS: We detected 65,473 SNPs across the populations, of which 76 were non-neutral SNPs. Of these non-neutral SNPs, the multivariate regression test associated 50 with eight landscape variables (e.g. open space, forest, etc.) and 29 with five climate variables (e.g. air temperature, humidity, etc.) (P-value range 0.005-0.045) in female and male populations separately. Male and female populations exhibited contrasting spatial divergence, with males exhibiting greater divergence than females, most likely reflecting the different dispersal abilities of male and female mosquitoes. In the comparative analysis of the same Ae. aegypti individuals, the pairwise FST values of 11 microsatellite markers were lower than those of the neutral SNPs, indicating that the neutral SNPs generated via pool ddRAD-Seq were more sensitive in terms of detecting genetic differences between populations at fine-spatial scales. CONCLUSIONS: Overall, our study demonstrates the utility of pool ddRAD-Seq for examining genetic differences in Ae. aegypti populations in areas at fine-spatial scales that could inform vector control programs such as Wolbachia-infected mosquito mass-release programs. This in turn would provide information on mosquito population dispersal patterns and the potential barriers to mosquito movement within and around the release area. In addition, the potential of environmental adaptability observed in Ae. aegypti could help population control efforts.

Genetic structure and Rickettsia infection rates in Ixodes ovatus and Haemaphysalis flava ticks across different altitudes.

Ixodid ticks, such as Ixodes ovatus and Haemaphysalis flava, are important vectors of tick-borne diseases in Japan, such as Japanese spotted fever caused by Rickettsia japonica. This study describes the Rickettsia infection rates influenced by the population genetic structure of I.ovatus and H. flava along an altitudinal gradient. A total of 346 adult I. ovatus and 243 H. flava were analyzed for the presence of Rickettsia by nested PCR targeting the 17kDA, gltA, rOmpA, and rOmpB genes. The population genetic structure was analyzed utilizing the mitochondrial cytochrome oxidase 1 (cox1) marker. The Rickettsia infection rates were 13.26% in I. ovatus and 6.17% in H. flava. For I. ovatus, the global FST value revealed significant genetic differentiation among the different populations, whereas H. flava showed non-significant genetic differentiation. The cox1 I. ovatus cluster dendrogram showed two cluster groups, while the haplotype network and phylogenetic tree showed three genetic groups. A significant difference was observed in Rickettsia infection rates and mean altitude per group between the two cluster groups and the three genetic groups identified within I. ovatus. No significant differences were found in the mean altitude or Rickettsia infection rates of H. flava. Our results suggest a potential correlation between the low gene flow in I. ovatus populations and the spatially heterogeneous Rickettsia infection rates observed along the altitudinal gradient. This information can be used in understanding the relationship between the tick vector, its pathogen, and environmental factors, such as altitude, and for the control of tick-borne diseases in Japan.

No detectable fitness cost of infection by cell-fusing agent virus in Aedes aegypti mosquitoes.

Aedes mosquitoes are well-known vectors of arthropod-borne viruses (arboviruses). Mosquitoes are more frequently infected with insect-specific viruses (ISVs) that cannot infect vertebrates. Some ISVs interfere with arbovirus replication in mosquito vectors, which has gained attention for potential use against arbovirus transmission. Cell-fusing agent virus (CFAV), a widespread ISV, can reduce arbovirus dissemination in Ae. aegypti. However, vectorial capacity is largely governed by other parameters than pathogen load, including mosquito survival and biting behaviour. Understanding how ISVs impact these mosquito fitness-related traits is critical to assess the potential risk of using ISVs as biological agents. Here, we examined the effects of CFAV infection on Ae. aegypti mosquito fitness. We found no significant reduction in mosquito survival, blood-feeding behaviour and reproduction, suggesting that Ae. aegypti is tolerant to CFAV. The only detectable effect was a slight increase in human attraction of CFAV-infected females in one out of eight trials. Viral tolerance is beneficial for introducing CFAV into natural mosquito populations, whereas the potential increase in biting activity must be further investigated. Our results provide the first insight into the link between ISVs and Aedes mosquito fitness and highlight the importance of considering all aspects of vectorial capacity for arbovirus control using ISVs.