Detecting the impacts of humidity, rainfall, temperature, and season on chikungunya, dengue and Zika viruses in Aedes albopictus mosquitoes from selected sites in Cebu city, Philippines.

BACKGROUND: Aedes albopictus is the secondary vector for dengue virus (DENV) in the Philippines, and also harbors chikungunya (CHIKV) and Zika (ZIKV) viruses. This study aimed to determine the minimum infection rates (MIRs) of CHIKV, DENV serotypes, and ZIKV in Ae. albopictus collected from selected two-site categories by altitude (highland [H] and lowland [L] sites) in Cebu city, Philippines during the wet (WS) and dry seasons (DS) of 2021-2022, and to explore the relationships between these arboviral MIRs and the local weather. METHODS: The viral RNA extracts in pooled and reared adult Ae. albopictus collected during the DS and WS from two-site categories were subjected to RT-PCR to amplify and detect gene loci specific for CHIKV, DENV-1 to DENV-4, and ZIKV and analyzed with the weather data. RESULTS: The range of CHIKV MIRs was higher in the WS (13.61-107.38 infected individuals per 1,000 mosquitoes) than in the DS (13.22-44.12), but was similar between the two-site categories. Rainfall (RF) influenced the CHIKV MIR. The MIR ranges of both DENV-2 (WS: H = 0, L = 0; DS: H = 0-5.92; L = 0-2.6) and DENV-4 (WS: H = 0, L = 0-2.90; DS: H = 2.96-6.13, L = 0-15.63) differed by season but not between the two-site categories. Relative humidity (RH), RF, and temperature did not influence DENVs' MIRs. The MIR range of ZIKV was similar in both seasons (WS: 11.36-40.27; DS: 0-46.15) and two-site categories (H = 0-90.91, L = 0-55.56). RH and temperature influenced ZIKV MIR. CONCLUSIONS: RF influenced CHIKV MIR in Ae. albopictus, whereas RH and temperature influenced that of ZIKV. Season influenced the MIRs of CHIKV and DENVs but not in ZIKV. Ae. albopictus were co-infected with CHIKV, DENVs, and ZIKV in both highland and lowland sites in Cebu city. Recommendations include all-year-round implementation of the Philippine Department of Health's  4S enhanced strategy and installation of water pipelines in rural highlands for vector and disease control. Our findings are relevant to protect public health in the tropics in this climate change.

Yearly variations of the genetic structure of Aedes aegypti (Linnaeus) (Diptera: Culicidae) in the Philippines (2017-2019).

Dengue is the fastest emerging arboviral disease in the world, imposing a substantial health and economic burden in the tropics and subtropics. The mosquito, Aedes aegypti, is the primary vector of dengue in the Philippines. We examined the genetic structure of Ae. aegypti populations collected from the Philippine major islands (Luzon, Visayas and Mindanao), each with highland (Baguio city, Cebu city mountains and Maramag, Bukidnon, respectively) and lowland sites (Quezon city; Liloan, Cebu and Cagayan de Oro [CDO] city, respectively) during the wet (2017-2018 and 2018-2019) and dry seasons (2018 and 2019). Mosquitoes (n = 1800) were reared from field-collected eggs and immatures, and were analyzed using 12 microsatellite loci. Generalized linear model analyses revealed yearly variations between highlands and lowlands in the major islands as supported by Bayesian clustering analyses on: 1) stronger selection (inbreeding coefficient, FIS = 0.52) in 2017-2018 than in 2018-2019 (FIS = 0.32) as influenced by rainfall, 2) the number of non-neutral loci indicating selection, and 3) differences of effective population size although at p = 0.05. Across sites except Baguio and CDO cities: 1) FIS varied seasonally as influenced by relative humidity (RH), and 2) the number of non-neutral loci varied as influenced by RH and rainfall indicating selection. Human-mediated activities and not isolation by distance influenced genetic differentiations of mosquito populations within (FST = 0.04) the major islands and across sites (global FST = 0.16). Gene flow (Nm) and potential first generation migrants among populations were observed between lowlands and highlands within and across major islands. Our results suggest that dengue control strategies in the epidemic wet season are to be changed into whole year-round approach, and water pipelines are to be installed in rural mountains to prevent the potential breeding sites of mosquitoes.

Temperature, season, and latitude influence development-related phenotypes of Philippine Aedes aegypti (Linnaeus): Implications for dengue control amidst global warming.

BACKGROUND: Dengue is endemic in the Philippines. Aedes aegypti is the primary vector. This study aimed to determine the hatching behavior and viability of Ae. aegypti first-generation (F1) eggs when exposed to temperature and photoperiod regimes under laboratory conditions. METHODS: Parental eggs were collected from selected highland and lowland sites in the Philippine big islands (Luzon, Visayas, and Mindanao) during the wet (2017-2018) and dry (2018) seasons. F1 egg cohorts were exposed separately in environmental chambers at 18, 25, and 38 °C with respective photoperiods for 6 weeks. Phenotypes (percent pharate larvae [PPL], hatch rates [HRs], and reproductive outputs [ROs]) were determined. RESULTS: Results of multivariate analyses of variance (MANOVA) between seasons showed significant main effects of temperature, season, and big island on all phenotypes across all sites. Significant interaction effects between seasons on all phenotypes across sites were shown between or among (1) season and big island, (2) season and temperature, (3) big island and temperature, (4) season, big island, and temperature, (5) big island, altitude, and temperature, and (6) season, big island, altitude, and temperature. Factors associated with the big islands might include their ecology, available breeding sites, and day lengths due to latitudinal differences, although they were not measured in the field. MANOVA results within each season on all phenotypes across sites showed (1) significant main effects of big island and temperature, and (2) significant interaction effects between big island and temperature within the wet season and (3) between temperature and photoperiod within the dry season. PPL were highest at 18 °C and were formed even at 38 °C in both seasons. Pharate larvae might play an adaptive role in global warming, expanded distribution to highlands, and preponderance to transmit human diseases. HRs in both seasons were highest at 25 °C and lowest at 38 °C. ROs were highest at 25 °C in the wet season and at 18 °C in the dry season. CONCLUSIONS: Temperature and latitude of Philippine big islands influenced the development-related phenotypes of Ae. aegypti in both seasons. The two seasons influenced the phenotypes and their interaction effects with big island and/or temperature and/or altitude. Recommendations include year-round enhanced 4S control strategies for mosquito vectors and water pipeline installation in rural highlands.

Novel and Broadly Applicable Microsatellite Markers in Identified Chromosomes of the Philippine Dengue Mosquitoes, Aedes aegypti (Diptera: Culicidae).

Dengue is the leading arboviral infection in the Philippines. Its endemicity in the country is due to the presence of its primary mosquito vector, Aedes aegypti (L.). This species has limited microsatellite markers. This study characterized microsatellite markers screened in silico from intergenic regions of the updated reference genome of Ae. aegypti from Liverpool, U.K. Criteria for good markers are: polymorphic, inherited in a Mendelian codominant manner, no null alleles, selectively neutral, randomly associated, and broadly applicable across different regions. Genotypes were scored using ABI Peak Scanner and were screened for the presence of null alleles. Hardy-Weinberg equilibrium, linkage disequilibrium, and robustness of the markers were determined by GENEPOP using Ae. aegypti samples from selected highland and lowland sites (n = 30 each) in the Philippines and outgroups (Thailand and Vietnam). Mendelian codominant inheritance was examined using F1 offspring of Ae. aegypti family (n = 30 each) derived from samples collected from Cebu city highlands and Maramag, Bukidnon. From the 63 randomly selected markers, nine were polymorphic. Two markers (Aaeg1-3D of chromosome 1 and Aaeg3-4C of chromosome 3) satisfied all criteria, hence, are good broadly useful microsatellite markers. Two other markers (Aaeg2-2E of chromosome 2 and Aaeg3-2A of chromosome 3) met all criteria but deviated from Mendelian codominant inheritance. These new markers of the Philippine Ae. aegypti with their chromosomal locations relative to the other published markers are presented, and will ultimately be useful in a variety of population genetic studies of Ae. aegypti to protect the public health.

Disease Burden of Dengue in the Philippines: Adjusting for Underreporting by Comparing Active and Passive Dengue Surveillance in Punta Princesa, Cebu City.

AbstractDengue virus (DENV) is a serious threat to public health. Having reliable estimates of the burden of dengue is important to inform policy and research, but surveillance systems are not designed to capture all symptomatic DENV infections. We derived the rate of reporting of dengue by comparing active surveillance of symptomatic DENV infections in a prospective community-based seroepidemiological cohort study (N = 1008) of acute febrile illness in Punta Princesa, Cebu City, Philippines, with passive surveillance data from the Cebu City Health Department. Febrile episodes detected in a weekly follow-up of participants were tested for serotype-specific DENV by hemi-nested reverse transcription-polymerase chain reaction (nested RT-PCR) and acute/convalescent blood samples tested by dengue IgM/IgG enzyme immunoassay. We estimated the burden of dengue in the Philippines in disability-adjusted life years (DALYs), and conducted a probabilistic sensitivity analysis using Monte-Carlo simulations to address uncertainty. The results showed a 21% cumulative reporting rate of symptomatic DENV infections, equivalent to an expansion factor of 4.7 (95% certainty level [CL]: 2.2-15.1). Based on surveillance data in the Philippines for 2010-2014, we estimated 794,255 annual dengue episodes (95% CL: 463,000-2,076,000) and a disease burden of 535 (95% CL: 380-994) DALYs per million population using age weights and time discounting and 997 (95% CL: 681-1,871) DALYs per million population without age and time adjustments. Dengue imposes a substantial burden in the Philippines; almost 10 times higher than estimated for rabies, about twice the burden of intestinal fluke infections, and about 10% of the burden of tuberculosis. Our estimates should inform policy makers and raise awareness among the public.

Natural vertical transmission of dengue viruses in Aedes aegypti in selected sites in Cebu City, Philippines.

We attempted to determine the vertical transmission of dengue virus (DENV) in Aedes aegypti in selected sites in Cebu City, Philippines. Mosquito sub-adults were collected monthly from households and the field during the wet-dry-wet season from November, 2011 to July, 2012 and were laboratory-reared to adults. Viral RNA extracts in mosquitoes were assayed by hemi-nested RT-PCR. Results showed that 62 (36.26%; n=679) out of 171 mosquito pools (n=2,871) were DENV+. The minimum infection rate (MIR) of DENV ranged from 0 in wet months to 48.22/1,000 mosquitoes in April, 2012 (mid-dry). DENVs were detected in larvae, pupae, and male and female adults, with DENV-4, DENV-3, and DENV-1, in that rank of prevalence. DENV-1 co-infected with either DENV-3 or -4 or with both in April, 2012; DENV-3 and -4 were present in both seasons. More DENV+ mosquitoes were collected from households than in field premises (p<0.001) and in the dry than in the wet season (p<0.05), with significant interaction (p<0.05) between sites and premises but no interaction between sites and seasons (p>0.05). By Generalized Linear Mixed models, the type of premises nested in sites and monthly total rainfall were significant predictors of monthly dengue cases (p<0.05) and not MIR, season, temperature, and relative humidity. Surveillance of DENV prevalence in Ae. aegypti and detecting their natural foci in the dry season provide an early warning signal of dengue outbreak.

Economic cost and burden of dengue in the Philippines.

Dengue, the world's most important mosquito-borne viral disease, is endemic in the Philippines. During 2008-2012, the country's Department of Health reported an annual average of 117,065 dengue cases, placing the country fourth in dengue burden in southeast Asia. This study estimates the country's annual number of dengue episodes and their economic cost. Our comparison of cases between active and passive surveillance in Punta Princesa, Cebu City yielded an expansion factor of 7.2, close to the predicted value (7.0) based on the country's health system. We estimated an annual average of 842,867 clinically diagnosed dengue cases, with direct medical costs (in 2012 US dollars) of $345 million ($3.26 per capita). This is 54% higher than an earlier estimate without Philippines-specific costs. Ambulatory settings treated 35% of cases (representing 10% of direct costs), whereas inpatient hospitals served 65% of cases (representing 90% of direct costs). The economic burden of dengue in the Philippines is substantial.

The key breeding sites by pupal survey for dengue mosquito vectors, Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse), in Guba, Cebu City, Philippines.

We conducted this study to assess how well a pupal survey of dengue mosquito vectors, Aedes aegypti and Aedes albopictus, is able to target the most productive breeding sites. The study was carried out monthly during the rainy season (8 months) in 2008 in Cuba, Cebu City, Philippines. The hypotheses tested were: 1) most pupae of Ae. aegypti or Ae. albopictus were produced in a few types of breeding sites and 2) the most productive types of breeding sites for each species were the most abundant. Approximately 2,500 pupae were collected from 554 breeding sites in 279 houses. Thirty-eight point four percent of ten types of breeding sites were positive for Ae. aegypti, and 11.9% of nine types of sites were positive for Ae. albopictus. Plastic drums (40.2%), metal drums (29.6%), and plastic containers (10.5%) were the key sites for Ae. aegypti pupae, whereas bamboo stumps (28.5%), plastic drums (21.1%), and rubber tires (19.1%) were the key sites for Ae. albopictus. The most productive breeding sites for Ae. aegypti were common but not the most common for Ae. albopictus. These results are relevant for dengue vector control programs.

Effects of latitude and longitude on the population structure of Culex pipiens s.l., vectors of West Nile virus in North America.

We assessed the structure and latitudinal selection that might result in sensitivities to critical day-lengths that trigger diapause between Culex pipiens populations distributed along North-South and East-West axes in eastern North America. Strong population structure between Cx. p. pipiens and Cx. p. quinquefasciatus existed. Among Cx. p. pipiens, a 100-km increase in the latitudinal change resulted in an increased square root of F(ST) by 0.002. A 100-km increase in the longitudinal change caused an increased square root of F(ST) by 0.035. A lack of latitudinal influence on the structure between Cx. p. pipiens populations suggests a uniform signal using the 12 microsatellite markers, which might increase the risk of West Nile virus (WNV) transmission toward northern areas because of longer breeding season, extend host-seeking period, and larger population size. Northern Cx. p. pipiens may have undergone additional generations before diapause is triggered, magnifying population size when WNV amplification is peaking.

A test of the chromosomal theory of ecotypic speciation in Anopheles gambiae.

The role of chromosomal inversions in speciation has long been of interest to evolutionists. Recent quantitative modeling has stimulated reconsideration of previous conceptual models for chromosomal speciation. Anopheles gambiae, the most important vector of human malaria, carries abundant chromosomal inversion polymorphism nonrandomly associated with ecotypes that mate assortatively. Here, we consider the potential role of paracentric inversions in promoting speciation in A. gambiae via "ecotypification," a term that refers to differentiation arising from local adaptation. In particular, we focus on the Bamako form, an ecotype characterized by low inversion polymorphism and fixation of an inversion, 2Rj, that is very rare or absent in all other forms of A. gambiae. The Bamako form has a restricted distribution by the upper Niger River and its tributaries that is associated with a distinctive type of larval habitat, laterite rock pools, hypothesized to be its optimal breeding site. We first present computer simulations to investigate whether the population dynamics of A. gambiae are consistent with chromosomal speciation by ecotypification. The models are parameterized using field observations on the various forms of A. gambiae that exist in Mali, West Africa. We then report on the distribution of larvae of this species collected from rock pools and more characteristic breeding sites nearby. Both the simulations and field observations support the thesis that speciation by ecotypification is occurring, or has occurred, prompting consideration of Bamako as an independent species.

Water quality and immatures of the M and S forms of Anopheles gambiae s.s. and An. arabiensis in a Malian village.

INTRODUCTION: The associations between the immatures of Anopheles gambiae s.s. (Diptera: Culicidae), its M and S forms, and Anopheles arabiensis among and within larval breeding habitats in Banambani, Mali were investigated under varying conditions of water quality and rainfall. The intent was to elucidate on niche partitioning of these taxa. METHODS: Immatures of An. arabiensis, An. gambiae s.s., and its M and S forms were sampled every alternate day for a month in mid-rainy season from three sampling sites in each of the larval breeding habitats (rock pools, swamp, and puddles). Water quality was characterized by alkalinity, conductivity, dissolved oxygen (D.O.), nitrate, orthophosphate, pH, temperature, total dissolved solids (TDS), and turbidity. A type 3 analysis of the GENMOD model was used to examine the associations between the proportional frequencies of young (first and second instar larvae) and old (third and fourth instar larvae and pupae) or total immatures of species or forms among sampling sites within and among larval breeding habitats during a category of rainfall as influenced by water quality. RESULTS: Of the 4,174 immatures sampled, 1,300 were molecularly identified to species and forms. Significant association between the proportional frequencies of young larvae of An. arabiensis, An. gambiae s.s., its M and S forms was found among sampling sites within habitats but not among larval breeding habitats. The proportional frequencies of young larvae of M and S forms varied daily perhaps due to recruitment, mortality, and dispersal within habitats. Conductivity and TDS had significant effects when the proportional frequencies of young larvae of M and S forms among sampling sites within habitats were significantly associated. Alkalinity, D.O., orthophosphate, pH, nitrate, temperature and turbidity had no effects on niche partitioning of species and forms among sampling sites within habitats. Rainfall did not affect the frequencies of these immatures. CONCLUSION: Conductivity and TDS have significant effects on niche partitioning of young larvae of M and S forms among sampling sites within habitats in Banambani, Mali.

Survivorship and distribution of immature Anopheles gambiae s.l. (Diptera: Culicidae) in Banambani village, Mali.

We observed the survivorship and distribution of larvae and pupae of Anopheles gambiae s.l. Giles immature stages in three habitats (rock pools, swamp, and puddles) in Banambani village. Mali, West Africa, during the mid-rainy season of 2000. Horizontal life tables were constructed for immatures in the laboratory. Times spent in the various immature stages were determined, and laboratory survival was measured. Vertical life tables were obtained from each habitat. We found large day-to-day variation for age class composition within habitats across days. The swamp samples had small but statistically significant different distributions in some instar stages compared with rock pools and puddles as affected by precipitation history. There were obviously unstable age distributions in the swamp and puddles and to some extent in rock pools. There were more individuals in some later age classes than in earlier ones. The daily survival estimates using an exponential decay model were 0.807 in rock pools, 0.899 in the swamp, 0.818 in puddles, and 0.863 in the overall village. Possible reasons for the departure from stable age distribution were cannibalism, predation and other complex interactions, rainfall effects, sampling bias, and differences in physicochemical properties of the water in the habitats.

Spatial and habitat distribution of Anopheles gambiae and Anopheles arabiensis (Diptera: Culicidae) in Banambani village, Mali.

We studied the larval distribution and composition of Anopheles arabiensis Patton, An. gambiae s.s. Giles, and its forms, among local habitats; and their association with the adults between these habitats in Banambani village, Mali during the mid-rainy seasons of 1997-1999. For species and form identification we used polymerase chain reaction (PCR) and PCR-restriction fragment-length polymorphism (RFLP). Differences among species in the distribution of larvae were observed in 1998, but not in 1997 or 1999, although they were on the borderline of statistical significance. Differences among the M and S molecular forms were statistically significant in 1999 when rainfall was high, but not in the two prior, drier sampling periods. Combining all information into the Fisher multiple comparisons test, there were statistically significant differences between species and molecular forms during the 3-yr study period. Hybrid larvae between the M and S forms were observed (0.57%), the first such observation to our knowledge. In spite of differences among larval distribution, no differences of adult species composition were observed among habitats. Factors that influence the distributions of An. gambiae larval populations are discussed.