Detection of SARS-CoV-2 and Omicron variant RNA in wastewater samples from Manila, Philippines.

Manila, a highly urbanized city, is listed as one of the top cities with the highest recorded number of coronavirus disease 2019 (COVID-19) cases in the Philippines. This study aimed to detect and quantify the RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the Omicron variant in 51 wastewater samples collected from three locations in Manila, namely Estero de Santa Clara, Estero de Pandacan, which are open drainages, and a sewage treatment plant (STP) at De La Salle University-Manila, between July 2022 and February 2023. Using one-step reverse transcription-quantitative polymerase chain reaction, SARS-CoV-2 and Omicron variant RNA were detected in 78 % (40/51; 4.9 ± 0.5 log10 copies/L) and 60 % (24/40; 4.4 ± 0.3 log10 copies/L) of wastewater samples collected from all sampling sites, respectively. SARS-CoV-2 RNA was detected frequently at Estero de Santa Clara (88 %, 15/17); its highest concentration was at the STP (6.3 log10 copies/L). The Omicron variant RNA was present in the samples collected (4.4 ± 0.3 log10 copies/L) from all sampling sites, with the highest concentration at the STP (4.9 log10 copies/L). Regardless of normalization, using concentrations of pepper mild mottle virus RNA, SARS-CoV-2 RNA concentrations exhibited the highest positive correlation with COVID-19 reported cases in Manila 5 days after the clinical report. These findings revealed that wastewater-based epidemiology may aid in identifying and monitoring of the presence of pathogens in open drainages and STPs in the Philippines. This paper provides the first documentation on SARS-CoV-2 and the Omicron variant in wastewater from Manila.

Geometric Morphometric Wing Analysis of Avian Malaria Vector, Culiseta longiareolata, from Two Locations in Algeria.

The application of geometric morphometry on mosquito wings (Culicidae) is considered a powerful tool for evaluating correlations between the phenotype (e.g., shape) and environmental or genetic variables. However, this has not been used to study the wings of the avian malaria vector, Culiseta longiareolata. Therefore, the goal of this study is to investigate the intra-specific wing variations between male and female Cs. longiareolata populations in different types of larval habitats and climatic conditions in Algeria. A total of 256 Cs. longiareolata mosquito samples were collected from January 2020 to July 2021 in three cities (Annaba, El-Tarf, and Guelma) of northeastern Algeria that have two distinct climatic condition levels (sub-humid and sub-arid) and different types of larval habitats (artificial and natural). Nineteen (19) wing landmarks (LMs) were digitized and analyzed based on geometric morphometry. Our results revealed differences in the wing shape of female and male mosquito populations, indicating sexual dimorphism. Moreover, canonical variance analysis (CVA) showed that factors, such as climatic conditions and type of larval habitats, also affect the wing shape of female and male Cs. longiareolata mosquito populations. Furthermore, the wing shape of male populations was more distinct compared with female populations.

Temporal Correlation Between Urban Microclimate, Vector Mosquito Abundance, and Dengue Cases.

Dengue Hemorrhagic Fever (DHF) is a major mosquito-borne viral disease. Studies have reported a strong correlation between weather, the abundance of Aedes aegypti, the vector of DHF virus, and dengue incidence. However, this conclusion has been based on the general climate pattern of wide regions. In general, however, the human population, level of infrastructure, and land-use change in rural and urban areas often produce localized climate patterns that may influence the interaction between climate, vector abundance, and dengue incidence. Thoroughly understanding this correlation will allow the development of a customized and precise local early warning system. To achieve this purpose, we conducted a cohort study, during January-December 2017, in 16 districts in Bandung, West Java, Indonesia. In the selected areas, local weather stations and modified light mosquito traps were set up to obtain data regarding daily weather and the abundance of adult female Ae. aegypti. A generalized linear model was applied to analyze the effect of local weather and female adult Ae. aegypti on the number of dengue cases. The result showed a significant non-linear correlation among mosquito abundance, maximum temperature, and dengue cases. Using our model, the data showed that the addition of a single adult Ae. aegypti mosquito increased the risk of dengue infection by 1.8%, while increasing the maximum temperature by one degree decreased the risk by 17%. This finding suggests specific actionable insights needed to supplement existing mosquito eradication programs.

An updated checklist of the mosquitoes (Diptera: Culicidae) present in Algeria, with assessments of doubtful records and problematic species.

An updated checklist of the mosquitoes (Diptera: Culicidae) of Algeria based on records published from 1903 to 2021 is presented. Fifty-three species belonging to seven genera are known with reasonable confidence to occur in the country, including Aedes (15 species), Anopheles (15), Coquillettidia (2), Culex (14), Culiseta (5), Orthopodomyia (1) and Uranotaenia (1). Two additional species, Culex simpsoni Theobald, 1905 and Uranotaenia balfouri Theobald, 1904, are provisionally regarded as present in Algeria. Published records are listed for each species, with indication of synonymous usage and variant spelling of names where applicable. The type locality of Aedes biskraensis Brunhes, 1999 is restricted to Seriana in Sidi Okba District in Biskra Province. Problematic species and doubtful occurrence records based on apparently misidentified specimens and confused taxonomy are discussed. Notes on taxonomy and medical importance are provided for the more important species.

Dengue disease dynamics are modulated by the combined influences of precipitation and landscape: A machine learning approach.

BACKGROUND: Dengue is an endemic vector-borne disease influenced by environmental factors such as landscape and climate. Previous studies separately assessed the effects of landscape and climate factors on mosquito occurrence and dengue incidence. However, both factors concurrently coexist in time and space and can interact, affecting mosquito development and dengue disease transmission. For example, eggs laid in a suitable environment can hatch after being submerged in rain water. It has been difficult for conventional statistical modeling approaches to demonstrate these combined influences due to mathematical constraints. OBJECTIVES: To investigate the combined influences of landscape and climate factors on mosquito occurrence and dengue incidence. METHODS: Entomological, epidemiological, and landscape data from the rainy season (July-December) were obtained from respective government agencies in Metropolitan Manila, Philippines, from 2012 to 2014. Temperature, precipitation and vegetation data were obtained through remote sensing. A random forest algorithm was used to select the landscape and climate variables. Afterward, using the identified key variables, a model-based (MOB) recursive partitioning was implemented to test the combined influences of landscape and climate factors on ovitrap index (vector mosquito occurrence) and dengue incidence. RESULTS: The MOB recursive partitioning for ovitrap index indicated a high sensitivity of vector mosquito occurrence on environmental conditions generated by a combination of high residential density areas with low precipitation. Moreover, the MOB recursive partitioning indicated high sensitivity of dengue incidence to the effects of precipitation in areas with high proportions of residential density and commercial areas. CONCLUSIONS: Dengue dynamics are not solely influenced by individual effects of either climate or landscape, but rather by their synergistic or combined effects. The presented findings have the potential to target vector surveillance in areas identified as suitable for mosquito occurrence under specific climatic conditions and may be relevant as part of urban planning strategies to control dengue.

The influence of roads on the fine-scale population genetic structure of the dengue vector Aedes aegypti (Linnaeus).

Dengue is endemic in tropical and subtropical countries and is transmitted mainly by Aedes aegypti. Mosquito movement can be affected by human-made structures such as roads that can act as a barrier. Roads can influence the population genetic structure of Ae. aegypti. We investigated the genetic structure and gene flow of Ae. aegypti as influenced by a primary road, España Boulevard (EB) with 2000-meter-long stretch and 24-meters-wide in a very fine spatial scale. We hypothesized that Ae. aegypti populations separated by EB will be different due to the limited gene flow as caused by the barrier effect of the road. A total of 359 adults and 17 larvae Ae. aegypti were collected from June to September 2017 in 13 sites across EB. North (N1-N8) and South (S1-S5) comprised of 211 and 165 individuals, respectively. All mosquitoes were genotyped at 11 microsatellite loci. AMOVA FST indicated significant genetic differentiation across the road. The constructed UPGMA dendrogram found 3 genetic groups revealing the clear separation between North and South sites across the road. On the other hand, Bayesian cluster analysis showed four genetic clusters (K = 4) wherein each individual samples have no distinct genetic cluster thus genetic admixture. Our results suggest that human-made landscape features such as primary roads are potential barriers to mosquito movement thereby limiting its gene flow across the road. This information is valuable in designing an effective mosquito control program in a very fine spatial scale.

A Bioclimate-Based Maximum Entropy Model for Comperiella calauanica Barrion, Almarinez and Amalin (Hymenoptera: Encyrtidae) in the Philippines.

Comperiella calauanica is a host-specific endoparasitoid and effective biological control agent of the diaspidid Aspidiotus rigidus, whose outbreak from 2010 to 2015 severely threatened the coconut industry in the Philippines. Using the maximum entropy (Maxent) algorithm, we developed a species distribution model (SDM) for C. calauanica based on 19 bioclimatic variables, using occurrence data obtained mostly from field surveys conducted in A. rigidus-infested areas in Luzon Island from 2014 to 2016. The calculated the area under the ROC curve (AUC) values for the model were very high (0.966, standard deviation = 0.005), indicating the model's high predictive power. Precipitation seasonality was found to have the highest relative contribution to model development. Response curves produced by Maxent suggested the positive influence of mean temperature of the driest quarter, and negative influence of precipitation of the driest and coldest quarters on habitat suitability. Given that C. calauanica has been found to always occur with A. rigidus in Luzon Island due to high host-specificity, the SDM for the parasitoid may also be considered and used as a predictive model for its host. This was confirmed through field surveys conducted between late 2016 and early 2018, which found and confirmed the occurrence of A. rigidus in three areas predicted by the SDM to have moderate to high habitat suitability or probability of occurrence of C. calauanica: Zamboanga City in Mindanao; Isabela City in Basilan Island; and Tablas Island in Romblon. This validation in the field demonstrated the utility of the bioclimate-based SDM for C. calauanica in predicting habitat suitability or probability of occurrence of A. rigidus in the Philippines.

Wing geometry and genetic analyses reveal contrasting spatial structures between male and female Aedes aegypti (L.) (Diptera: Culicidae) populations in metropolitan Manila, Philippines.

The population genetic structure of Aedes aegypti (Linnaeus, 1762) has been studied in order to understand its role as an efficient vector. Several studies utilized an integrative approach; to combine genetic and phenotypic data to determine its population structure but these studies have only focused on female populations. To address this particular gap, our study compared the population variability and structuring between its male and female populations using phenotypic and genetic data from a highly-urbanized and dengue-endemic region of the Philippines, Metropolitan Manila. Five mosquito populations comprised of female (n = 137) and male (n = 49) adult mosquitoes were used in this study. All mosquito individuals underwent geometric morphometric (26 landmarks), and genetic (11 microsatellite loci) analyses. Results revealed that FST estimates (genetic) were 0.055 and 0.009 while QST estimates (phenotypic) were 0.318 and 0.309 in in male and female populations, respectively. Wing shape variation plots showed that male populations were distinctly separated from each other while female populations overlapped. Similarly, discriminant analysis of principal components using genetic data revealed that male populations were also distinctly separated from each other while female populations showed near-overlapping populations. Genetic and phenetic dendrograms showed the formation of two groups in male populations but no groups in female populations. Further analysis indicated a significant correlation (r = 0.68, p = 0.02) between the genetic and phenetic distances of male populations. Bayesian analysis using genetic data also detected multiple clusters in male (K = 3) and female (K = 2) populations, while no clusters were detected using the phenotypic data from both sexes. Our results revealed contrasting phenotypic and genetic patterns between male and female Ae. aegypti, indicating that male populations were more spatially structured than female populations.

Surveillance of dengue virus in individual Aedes aegypti mosquitoes collected concurrently with suspected human cases in Tarlac City, Philippines.

BACKGROUND: Vector control measures are critical for the prevention and reduction of dengue virus (DENV) transmission. Effective vector control is reliant not only on knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito-borne infection. Mosquito-based virus surveillance programs typically rely on pool-based mosquito testing, although whether individual-based mosquito testing is a feasible alternative to this has not been widely studied. Applying an individual-based mosquito testing approach, we conducted a 1-month surveillance study of DENV in adult Aedes aegypti mosquitoes in homes of suspected dengue patients during the 2015 peak dengue season in Tarlac City, Philippines to more accurately assess the mosquito infection rate and identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients there. METHODS: We performed a one-step multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection and serotyping of DENV in patients and individual female Ae. aegypti mosquitoes. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENV serotypes in mosquitoes and patients at the genotype level. RESULTS: We collected a total of 583 adult Ae. aegypti mosquitoes, of which we individually tested 359 female mosquitoes for the presence of DENV. Ten (2.8%) of the 359 female mosquitoes were positive for the presence of DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which was consistent with the serotypes concurrently found in infected patients. Sequencing and phylogenetic analyses of the detected DENV serotypes based on the partial sequence of the evelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely DENV-1 genotype IV, DENV-2 Cosmopolitan genotype, and DENV-4 genotype II. CONCLUSIONS: We demonstrated the utility of a one-step multiplex real-time RT-PCR assay for the individual-based DENV surveillance of mosquitoes. Our findings reinforce the importance of detecting and monitoring virus activity in local mosquito populations, which are critical for dengue prevention and control.

Machine-learning-based detection of adaptive divergence of the stream mayfly Ephemera strigata populations.

Adaptive divergence is a key mechanism shaping the genetic variation of natural populations. A central question linking ecology with evolutionary biology is how spatial environmental heterogeneity can lead to adaptive divergence among local populations within a species. In this study, using a genome scan approach to detect candidate loci under selection, we examined adaptive divergence of the stream mayfly Ephemera strigata in the Natori River Basin in northeastern Japan. We applied a new machine-learning method (i.e., random forest) besides traditional distance-based redundancy analysis (dbRDA) to examine relationships between environmental factors and adaptive divergence at non-neutral loci. Spatial autocorrelation analysis based on neutral loci was employed to examine the dispersal ability of this species. We conclude the following: (a) E. strigata show altitudinal adaptive divergence among the populations in the Natori River Basin; (b) random forest showed higher resolution for detecting adaptive divergence than traditional statistical analysis; and (c) separating all markers into neutral and non-neutral loci could provide full insight into parameters such as genetic diversity, local adaptation, and dispersal ability.

Fine-scale population genetic structure of dengue mosquito vector, Aedes aegypti, in Metropolitan Manila, Philippines.

Dengue is a highly endemic disease in Southeast Asia and is transmitted primarily by the mosquito, Aedes aegypti. The National Capital Region (NCR) of the Philippines, or Metropolitan Manila, is a highly urbanized area that is greatly affected by this arboviral disease. Urbanization has been shown to increase the dispersal of this mosquito vector. For this reason, we conducted a fine-scale population genetic study of Ae. aegypti in this region. We collected adult Ae. aegypti mosquitoes (n = 526 individuals) within the region (n = 21 study areas) and characterized the present population structure and the genetic relatedness among mosquito populations. We genotyped 11 microsatellite loci from all sampled mosquito individuals and analyzed their genetic diversity, differentiation and structure. The results revealed low genetic differentiation across mosquito populations which suggest high gene flow and/or weak genetic drift among mosquito populations. Bayesian analysis indicated multiple genetic structures (K = 3-6), with no clear genetically distinct population structures. This result implies the passive or long-distance dispersal capability nature Ae. aegypti possibly through human-mediated transportation. The constructed dendrogram in this study describes the potential passive dispersal patterns across Metropolitan Manila. Furthermore, spatial autocorrelation analysis showed the limited and active dispersal capability (<1km) of the mosquito vector. Our findings are consistent with previous studies that investigated the genetic structure and dual (active and passive) dispersal capability of Ae. aegypti in a fine-scale highly urbanized area.

Unraveling the Genetic Structure of the Coconut Scale Insect Pest (Aspidiotus rigidus Reyne) Outbreak Populations in the Philippines.

Despite the fact that massive outbreaks of the coconut scale insect pest, Aspidiotus rigidus Reyne (Hemiptera: Diaspididae) are inflicting significant economic losses to the Philippines' coconut industry, little is known about the population and dispersal history of this invasive pest in the country. Here, we examined the genetic diversity, structure and demographic history of A. rigidus sampled from localities with reported outbreaks from 2014 to 2017. We analyzed the genetic structure of the outbreak populations using mitochondrial COI and nuclear EF-1α markers. Both markers and all methods of population genetic structure analyses indicate clear differentiation among the A. rigidus populations separating the north from the southern regions of the Philippines. Very low or no genetic differentiation was observed within and amongst the populations per geographic region indicating two unrelated outbreak events of the pest originating from two genetically uniform populations isolated in each respective region. Historical data supports the resurgence of an established A. rigidus population in the south which could have been driven by sudden climatic changes or human-induced habitat imbalance. Our study provides valuable information on the genetic differentiation of the two A. rigidus groups that would be useful for developing and implementing biological control strategies against this pest in the Philippines.

Detection of Wolbachia in field-collected Aedes aegypti mosquitoes in metropolitan Manila, Philippines.

BACKGROUND: Recent reports reveal the presence of Wolbachia in Ae. aegypti. Our study presents additional support for Wolbachia infection in Ae. aegypti by screening field-collected adult mosquitoes using two Wolbachia-specific molecular makers. METHODS: A total of 672 Ae. aegypti adult mosquitoes were collected from May 2014 to January 2015 in Metropolitan Manila. Each individual sample was processed and screened for the presence of Wolbachia by selected markers, Wolbachia-specific 16S rDNA and its surface protein (wsp), under optimized PCR conditions and sequenced. RESULTS: Totals of 113 (16.8%) and 89 (13.2%) individual mosquito samples were determined to be infected with Wolbachia using the wsp and 16S rDNA markers, respectively. The Ae. aegpyti wsp sample sequences were similar or identical to five known Wolbachia strains belonging to supergroups A and B while the majority of 16S rDNA sample sequences were similar to strains belonging to supergroup B. Overall, 80 (11.90%) individual mosquito samples showed positive amplifications in both markers and 69% showed congruence in supergroup identification (supergroup B). CONCLUSIONS: By utilizing two Wolbachia-specific molecular makers, our study demonstrated the presence of Wolbachia from individual Ae. aegypti samples. Our results showed a low Wolbachia infection rate and inferred the detected strains belong to either supergroups A and B.

Using Google Trends to Examine the Spatio-Temporal Incidence and Behavioral Patterns of Dengue Disease: A Case Study in Metropolitan Manila, Philippines.

Dengue is a major public health concern and an economic burden in the Philippines. Despite the country's improved dengue surveillance, it still suffers from various setbacks and needs to be complemented with alternative approaches. Previous studies have demonstrated the potential of Internet-based surveillance such as Google Dengue Trends (GDT) in supplementing current epidemiological methods for predicting future dengue outbreaks and patterns. With this, our study has two objectives: (1) assess the temporal relationship of weekly GDT and dengue incidence in Metropolitan Manila from 2009⁻2014; and (2) examine the health-seeking behavior based on dengue-related search queries of the population. The study collated the population statistics and reported dengue cases in Metropolitan Manila from respective government agencies to calculate the dengue incidence (DI) on a weekly basis for the entire region and annually per city. Data processing of GDT and dengue incidence was performed by conducting an 'adjustment' and scaling procedures, respectively, and further analyzed for correlation and cross-correlation analyses using Pearson's correlation. The relative search volume of the term 'dengue' and top dengue-related search queries in Metropolitan Manila were obtained and organized from the Google Trends platform. Afterwards, a thematic analysis was employed, and word clouds were generated to examine the health behavior of the population. Results showed that weekly temporal GDT pattern are closely similar to the weekly DI pattern in Metropolitan Manila. Further analysis showed that GDT has a moderate and positive association with DI when adjusted or scaled, respectively. Cross-correlation analysis revealed a delayed effect where GDT leads DI by 1⁻2 weeks. Thematic analysis of dengue-related search queries indicated 5 categories namely; (a) dengue, (b) sign and symptoms of dengue, (c) treatment and prevention, (d) mosquito, and (e) other diseases. The majority of the search queries were classified in 'signs and symptoms' which indicate the health-seeking behavior of the population towards the disease. Therefore, GDT can be utilized to complement traditional disease surveillance methods combined with other factors that could potentially identify dengue hotspots and help in public health decisions.

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.

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.

Ecdysis period and rate deviations of dengue mosquito vector, Aedes aegypti reared in different artificial water-holding containers.

BACKGROUND & OBJECTIVES: Artificial water-holding containers (AWHCs) have been well-documented in many Aedes aegypti studies for dengue surveillance and developmental research. Hence, we investigated the role of different AHWCs on the development and ecdysis period of Ae. aegypti dengue vector, a container breeding mosquito. METHODS: Nine types of AWHCs, namely glass, polystyrene foam, rubber, steel, porcelain, plastic, aluminum, clay and concrete, were chosen for the study. All AWHCs were subjected to the developmental assay for an observation period of 10 days. Regression and hazard analyses were employed to the developmental stages and the characteristics of the AWHCs. RESULTS: The observations revealed that Ae. aegypti development is fastest in glass and polystyrene containers while slowest in concrete containers. Moreover, pupal ecdysis appears to be the most affected by the characteristics of the AWHCs based on regression and hazard analyses. INTERPRETATION & CONCLUSION: Characteristics of the container that can regulate water temperature seem to be the driving force with regards to the slow or fast development of Ae. aegypti, more notably in pupal ecdysis. The results of the study further strengthen our understanding on the dynamics of Ae. aegypti's developmental biology to different characteristics of artificial water containers. This, in turn, would aid in devising vector control strategies against dengue especially in endemic areas.