QTL Determining Diel Flight Activity in Male Culex pipiens Mosquitoes.

Members of the Culex pipiens complex differ in physiological traits that facilitate their survival in diverse environments. Assortative mating within the complex occurs in some regions where autogenous (the ability to lay a batch of eggs without a blood meal) and anautogenous populations are sympatric, and differences in mating behaviors may be involved. For example, anautogenous populations mate in flight/swarms, while autogenous populations often mate at rest. Here, we characterized flight activity of males and found that anautogenous strain males were crepuscular, while autogenous strain males were crepuscular and nocturnal, with earlier activity onset times. We conducted quantitative trait locus (QTL) mapping to explore the genetic basis of circadian chronotype (crepuscular vs. crepuscular and nocturnal) and time of activity onset. One major-effect QTL was identified for chronotype, while 3 QTLs were identified for activity onset. The highest logarithm of the odds (LOD) score for the chronotype QTL coincides with a chromosome 3 marker that contains a 15-nucleotide indel within the coding region of the canonical clock gene, cryptochrome 2. Sequencing of this locus in 7 different strains showed that the C-terminus of CRY2 in the autogenous forms contain deletions not found in the anautogenous forms. Consequently, we monitored activity in constant darkness and found males from the anautogenous strain exhibited free running periods of ~24 h while those from the autogenous strain were ~22 h. This study provides novel insights into the genetic basis of flight behaviors that likely reflect adaptation to their distinct ecological niches.

First reported enterovirus D68 infection in pediatric patients from the Caribbean region: evidence of spread from the U.S. outbreak.

The 2014 enterovirus D68 (EV-D68) outbreak in the United States raised concerns about the introduction of the virus in the Caribbean region. The objective of this study was to provide rapid evidence of the introduction of EV-D68 strains in the Caribbean region during the 2014 outbreak in the United States, using a relatively simple phylogenetic approach. From October 2014 to May 2015, four EV-D68 cases from two countries (Bermuda and Dominica) were detected at the regional referral laboratory at the Caribbean Public Health Agency (Port of Spain, Trinidad and Tobago) based on molecular testing of respiratory specimens. All cases were children presenting to hospitals with moderate respiratory distress. No cases of acute flaccid paralysis were detected. Phylogenetic analysis of the Caribbean strains showed more than 99% similarity with the 2014 U.S.-outbreak strain, providing evidence of the introduction and circulation of the virus in the region.

Global genetic diversity of Aedes aegypti.

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co-occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub-Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans-Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.

Composite linkage map and enhanced genome map for Culex pipiens complex mosquitoes.

We report here the development of 65 novel microsatellite loci and construction of a composite genetic linkage map for Culex pipiens complex mosquitoes. Microsatellites were identified by in silico screening of the Culex quinquefasciatus genome assembly. Cross-species utility of 73 microsatellites for population studies in C. pipiens sensu stricto and C. quinquefasciatus was evaluated by genotyping a subset of samples collected in Indiana, United States, and Point Fortin, Trinidad. Allele frequencies of 67 microsatellites were within Hardy-Weinberg expectations in both population subsets. A composite linkage map was constructed based on restriction fragment length polymorphism and microsatellite polymorphisms in 12 independent F1 intercross mapping populations. The composite map consists of 61 marker loci totaling 183.9 cM distributed across the 3 linkage groups. These loci cover 29.5, 88.8, and 65.6 cM on chromosomes I-III, respectively, and allow for assignment of 10.4% of the genome assembly and 13.5% of the protein coding genes to chromosome position. Our results suggest that these microsatellites will be useful for mapping and population studies of 2 pervasive species in the C. pipiens complex. Moreover, the composite map presented here will serve as a basis for the construction of high-resolution genetic and physical maps, as well as detection of quantitative trait loci to aid in the investigation of complex genetic traits influencing phenotypes of interest.

Impact of environmental temperatures on resistance to organophosphate insecticides in Aedes aegypti from Trinidad.

OBJECTIVE: To examine the effects of increasing larval rearing temperatures on the resistance status of Trinidadian populations of Aedes aegypti to organophosphate (OP) insecticides. METHODS: In 2007-2008, bioassays and biochemical assays were conducted on A. aegypti larvae collected in 2006 from eight geographically distinct areas in Trinidad (Trinidad and Tobago). Larval populations were reared at four temperatures (28 ± 2ºC, 32ºC, 34ºC, and 36ºC) prior to bioassays with OP insecticides (fenthion, malathion, and temephos) and biochemical assays for esterase enzymes. RESULTS: Most larval populations reared at 28 ± 2ºC were susceptible to fenthion (>98% mortality) but resistant to malathion and temephos (< 80% mortality). A positive association was found between resistance to OP insecticides and increased activities of α- and ß-esterases in larval populations reared at 28 ± 2ºC. Although larval populations reared at higher temperatures showed variations in resistance to OPs, there was a general increase in susceptibility. However, increases or decreases in activity levels of enzymes did not always correspond with an increase or decrease in the proportion of resistant individuals reared at higher temperatures. CONCLUSIONS: Although global warming may cause an increase in dengue transmission, based on the current results, the use of insecticides for dengue prevention and control may yet be effective if temperatures increase as projected.

Organophosphate resistance in Trinidad and Tobago strains of Aedes aegypti.

Aedes aegypti larvae from 8 sites in Trinidad and 1 in Tobago were assayed against temephos, malathion, and fenthion using the Centers for Disease Control and Prevention time-mortality-based bioassay method. Resistance ratios (RRs) and resistance thresholds (RTs) for each insecticide were calculated in relation to the Caribbean Epidemiology Center reference susceptible strain. Results showed that the Haleland Park and Tobago strains were susceptible to fenthion and malathion, respectively (RRs < 1), while the San Fernando strain had a high RR (33.92) to malathion. All other strains had low-level resistance to fenthion and malathion. Resistance to temephos was more intense with 4 strains showing high-level resistance. The established RT was 60 min for fenthion, 75 min for bendiocarb, and 120 min for temephos and malathion. At the RTs, all Trinidad strains were resistant to temephos (11.50-74.50% mortality), 7 resistant to fenthion (21.25-78.75% mortality), and 5 resistant to malathion (56.25-77.50% mortality). The other strains were incipiently resistant (80-97% mortality). Despite the discrepancies between the RR levels and RT status, it is evident that the organophosphate insecticide resistance is prevalent in Trinidad and Tobago populations of Ae. aegypti. These results suggest that operational failure could soon occur and alternative strategies should be developed and implemented to reduce the probability of further selection pressure on resistant Ae. aegypti populations in Trinidad and Tobago.

Mating Competitiveness of Transgenic Aedes aegypti (Diptera: Culicidae) Males Against Wild-Type Males Reared Under Simulated Field Conditions.

Efforts directed at genetic modification of mosquitoes for population control or replacement are highly dependent on the initial mating success of transgenic male mosquitoes following their release into natural populations. Adult mosquito phenotypes are influenced by the environmental conditions experienced as larvae. Semifield studies conducted to date have not taken that under consideration when testing male mating fitness, and have compared mating success of males reared under identical environmental conditions. We performed pairwise mating challenges between males from a genetically modified laboratory strain (BF2) versus males from a recent Trinidad field isolate of Aedes aegypti (L.), a major vector of multiple arboviruses. We utilized larval density and nutrition to simulate environmental stress experienced by the Trinidad males and females. Our results indicated that environmental stress during larval development negatively influenced the competitiveness and reproductive success of males from the Trinidad population when paired with optimum reared BF2 males. Small (0.027 m3) and large (0.216 m3) trials were conducted wherein stressed or optimum Trinidad males competed with optimum BF2 males for mating with stressed Trinidad females. When competing with stress reared Trinidad males, optimum reared BF2 males were predominant in matings with stress reared Trinidad females, and large proportions of these females mated with males of both strains. When competing with optimum reared Trinidad males, no difference in mating success was observed between them and BF2 males, and frequencies of multiple matings were low. Our results indicate that future mating competition studies should incorporate appropriate environmental conditions when designing mating fitness trials of genetically modified males.

Identification of Host Blood Meals of Mosquitoes (Diptera: Culicidae) Collected at the Aripo Savannas Scientific Reserve in Trinidad, West Indies.

Surveillance for blood-fed female mosquitoes was performed between August 2015 and February 2016 at sites along the periphery of the Aripo Savannas Environmentally Reserve (ASSR) located in northeastern Trinidad, West Indies. We collected engorged female mosquitoes representing 13 species. DNA extractions from dissected abdomens were subjected to PCR amplification with three primer pairs targeting the mitochondrial cytochrome oxidase I and cytochrome b gene sequences. High-quality sequence information and host identification were obtained for 42 specimens representing eight mosquito species with at least one primer combination. A broad range of vertebrates including humans were identified, but the majority were nonhuman mammals, both domestic and wild. Domestic dogs were the most common host and may represent potential sentinel species for monitoring local enzootic arbovirus activity in Trinidad. Culex declarator Dyer and Knab and Culex nigripalpus Theobald were the most common blood-fed mosquito species comprising 79.1% of the total number identified. These species obtained blood meals from birds, nonhuman mammals, and human hosts, and therefore pose significant risks as potential bridge vectors for epizootic arbovirus transmission in the ASSR area as well as other sylvan areas in Trinidad. These data represent the first such results for Trinidad.

Seasonal profiles of Aedes aegypti (Diptera: Culicidae) larval habitats in an urban area of Costa Rica with a history of mosquito control.

Dengue is the most important arboviral disease worldwide and the principal vector-borne disease in Costa Rica. Control of Aedes aegypti populations through source reduction is still considered the most effective way of prevention and control, although it has proven ineffective or unsustainable in many areas with a history of mosquito control. In this study, seasonal profiles and productivity of Aedes aegypti were analyzed in the city of Puntarenas, Costa Rica, where vector control has been practiced for more than ten years. Households contained more than 80% of larval habitats identified, although presence of habitats was more likely in other locations like lots and streets. In the wet season, habitats in the "other" category, like appliances, small manholes, and miscellaneous containers, were the most frequent habitats observed as well as the most common and productive habitats for Ae. aegypti. In the dry season, domestic animal drinking containers were very common, although concrete washtubs contained 79% of Ae. aegypti pupae collected. Individually, non-disposable habitats were as likely or more likely to contain mosquito larvae, and large containers were more likely to harbor mosquito larvae than the small ones only in the dry season. Considering various variables in the logistic regressions, predictors for Ae. aegypti in a habitat were habitat type (p < 0.001), setting (p = 0.043), and disposability (p = 0.022) in the wet season and habitat capacity in the dry season (p = 0.025). Overall, traditional Ae. aegypti larval indices and pupal indices in Puntarenas were high enough to allow viral transmission during the wet season. In spite of continued vector control, it has not been possible to reduce vector densities below threshold levels in Puntarenas, and the habitat profiles show that non-household locations, as well as non-disposable containers, should be targeted in addition to the standard control activities.

A transcriptomic survey of the impact of environmental stress on response to dengue virus in the mosquito, Aedes aegypti.

Populations of Aedes aegypti naturally exhibit variable susceptibility to dengue viruses. This natural variation can be impacted by nutritional stress resulting from larval-stage crowding, indicating the influence of environment components on the adult mosquito immune response. In particular, larval crowding was previously shown to reduce the susceptibility of adult females of a Trinidad field isolate of A. aegypti to the dengue serotype 2 (JAM1409) virus. Here, we present the first whole transcriptome study to address the impact of environmental stress on A. aegypti response to dengue virus. We examined expression profiles of adult females resulting from crowded and optimum reared larvae from the same Trinidad isolate at two critical early time points-3 and 18 hours post dengue virus infected blood meal. We exposed specimens to either a dengue or naïve blood meal, and then characterized the response in ten gene co-expression modules based on their transcriptional associations with environmental stress and time. We further analyzed the top 30 hub or master regulatory genes in each of the modules, and validated our results via qRT-PCR. These hub genes reveal which functions are critical to the mechanisms that confer dengue virus refractoriness or susceptibility to stress conditioned A. aegypti, as well as the time points at which they are most important.

Surveillance of dengue fever cases using a novel Aedes aegypti population sampling method in Trinidad, West Indies: the cardinal points approach.

A novel dengue surveillance method is described and used to evaluate 100 suspected dengue fever (DF) cases in county St. Patrick, Trinidad, West Indies. From the 30 confirmed DF cases fully investigated within 48 h of diagnosis, 63% (19/30 houses) of their homes were found harboring Aedes aegypti immature stages. Only houses at the four cardinal points of the index case rather than the entire neighborhood were investigated. The results showed significantly (P<0.001) more Ae. aegypti positive houses were observed to the east (P<0.04) and west (P<0.01) than to the north and south (P>0.9). In addition, from the 150 houses inspected a total of 474 artificial containers were inspected and treated, of which 20.8% (99) were infested with Ae. aegypti immature stages. More than 49% of the containers inspected were small miscellaneous containers, but they only produced 4.0% of the Ae. aegypti immatures, of which only 0.4% were pupae. Water tanks (41.7%), drums (40.4%) and buckets (24.2%) produced over 98% of the pupae. The results of this study imply that dengue vector control programs in Trinidad could increase their efficiency by applying the cardinal points surveillance approach during DF case investigations and concentrating their vector control measures on the most productive containers located at the east and west of the index cases.

Passage of ingested Mansonella ozzardi (Spirurida: Onchocercidae) microfilariae through the midgut of Aedes aegypti (Diptera: Culicidae).

When virus and microfilariae are ingested concurrently by a mosquito, microfilariae (mf) may penetrate the mosquito midgut and introduce virus directly into the mosquito hemocoel, allowing mosquitoes to become infectious much sooner than normal and enhancing transmission of viruses by mosquitoes. Mansonella ozzardi (Manson) is a benign filarial nematode parasite of humans in Latin America and is transmitted by black flies (Diptera: Simuliidae) and biting midges (Diptera: Ceratopogonidae). Because M. ozzardi and dengue are sympatric, we wanted to know whether M. ozzardi mf had the ability to penetrate the midgut of Aedes aegypti (L.) (Diptera: Culicidae) and thus play a potential role in the enhancement of dengue transmission. To test this, the F1 progeny from locally collected Ae. aegypti were fed on M. ozzardi-infected human males in an endemic village in northern Trinidad. Mosquitoes were dissected at various times after feeding and examined for mf in the midguts and thoraces. Microfilariae penetrated the midguts of 43% of 63 mosquitoes that ingested mf. Overall, 11% of mf penetrated the midgut by 17 h after being ingested. The intensity of midgut penetration was positively correlated to the numbers of mf ingested. Because midgut penetration is a key requirement for mf enhancement to occur, the potential exists that M. ozzardi could be involved in the enhancement of dengue virus transmission.

Comparison of mosquito control programs in seven urban sites in Africa, the Middle East, and the Americas.

Mosquito control programs at seven urban sites in Kenya, Egypt, Israel, Costa Rica, and Trinidad are described and compared. Site-specific urban and disease characteristics, organizational diagrams, and strengths, weaknesses, obstacles and threats (SWOT) analysis tools are used to provide a descriptive assessment of each mosquito control program, and provide a comparison of the factors affecting mosquito abatement. The information for SWOT analysis is collected from surveys, focus-group discussions, and personal communication. SWOT analysis identified various issues affecting the efficiency and sustainability of mosquito control operations. The main outcome of our work was the description and comparison of mosquito control operations within the context of each study site's biological, social, political, management, and economic conditions. The issues identified in this study ranged from lack of inter-sector collaboration to operational issues of mosquito control efforts. A lack of sustainable funding for mosquito control was a common problem for most sites. Many unique problems were also identified, which included lack of mosquito surveillance, lack of law enforcement, and negative consequences of human behavior. Identifying common virtues and shortcomings of mosquito control operations is useful in identifying "best practices" for mosquito control operations, thus leading to better control of mosquito biting and mosquito-borne disease transmission.

An evaluation of some Trinidadian plant extracts against larvae of Aedes aegypti mosquitoes.

In recent times, bioprospecting for plants that show bioactive properties has yielded many chemicals that can be used in controlling mosquitoes. Crude extracts of 4 terrestrial and 3 mangrove plants were assayed against 2-3 larval instars of Aedes aegypti. Among the plants tested, Cordia curassavica showed the highest levels of activity for all the extracts tested. Azadirachta indica showed the least activity, whereas the 2 cultivars of Mangifera indica showed substantial activity for the aqueous extracts. The mangrove species proved to be relatively nontoxic to Ae. aegypti larvae when compared to the terrestrial plants. The results of this study suggest that some common plants in Trinidad may be highly effective in controlling the urban vector of yellow fever and dengue fever, Ae. aegypti.

The influence of house construction on the indoor abundance of mosquitoes.

This study examined the potential effects of different house construction features on the indoor abundance of culicine mosquitoes in Trinidad (TT) and the Dominican Republic (DR) using xenomonitoring surveys. To assess these effects, a survey was taken of different homes in both countries alongside concurrent indoor resting mosquito collections to determine which features may be correlated with a greater abundance. Between June 2002 and April 2003 data were collected from 104 homes in TT and 121 homes in the DR. In TT, 61 (58.65%) of the homes were located in urban areas and 43 (41.35%) were located in rural villages, whereas in the DR 40 (33.06%) were located in rural areas, and 81 (66.94%) in the urban area. Overall, a total of 1,630 mosquitoes were collected in TT, of which 77% were Culex quinquefasciatus, whereas 459 mosquitoes were collected from the DR, of which 46% were Cx. quinquefasciatus. It was found that in TT and the DR the mean number of Cx. quinquefasciatus mosquitoes was greater in cement homes than in either wood or other poorer quality homes (TT cement 17.43, others 14.43; DR cement 4.24, others 3.41). In TT it was found that homes that had painted interiors were significantly more likely to have a high abundance of mosquitoes resting indoors compared to homes without painted interiors (OR 2.90, CI 1.09-8.72). Likewise, having a painted exterior was not significant, but only slightly so, in TT as having a detrimental effect (OR 2.14, CI 0.89-6.67). Similarly, having a painted interior or exterior was also found to be a predictor of a high abundance of indoor resting mosquitoes in the DR (interior OR 3.13, CI 1.41-6.92; exterior OR 1.97, CI .91-4.26). Reduced adult abundance in TT was correlated with homes being built on stilts, with more than four people sleeping in the home, and having a painted interior. In the DR, reductions were correlated with homes where residents slept under a bed net and with people who lived in a rural location. Changes in construction patterns in the Caribbean region could help prevent human-mosquito contact potentially reducing the transmission of certain vector-borne diseases in the population.

Aedes aegypti (L.) mosquitoes in Trinidad, West Indies: longevity case studies.

Few laboratory and field studies have reported long survival periods for Ae. aegypti females and even fewer have designed experiments to characterize this important life history trait. This study was conducted under laboratory conditions to determine the number of blood meals taken by individual females, the number of eggs laid per individual female, the length of the gonotrophic cycle, and the duration of female survival. The results showed individual females oviposited between 670 and 1,500 eggs throughout their lifetimes, females undergoing large numbers of gonotrophic cycles and surviving up to 224 days. These results are discussed in the context of vector competence, unique alternating high and low oviposition patterns observed after week 14, and resource partitioning/allocation by older Ae. aegypti females after blood feeding.

Description of Brachymeria philornisae sp. n. (Hymenoptera: Chalcididae), a parasitoid of the bird parasite Philornis trinitensis (Diptera: Muscidae) in Tobago, with a review of the sibling species.

In this paper, we describe and illustrate a new species of parasitoid wasp as Brachymeria philornisae Delvare, sp. nov. The new species was reared from Philornis trinitensis Dodge & Aitken (Diptera: Muscidae) puparia that were found in the nests of the bird species Mimus gilvus (Vieillot) (Mimidae) and Tiaris bicolor (L.) (Thraupidae) in Tobago. The new species is of particular interest as it may be considered a potential biological control agent in locations where Philornis species are invasive, such as the Galapagos Islands. Closely related Brachymeria species had taxonomically ambiguous relationship in the past and are compared and reviewed. The species have been classified in the subgenus Pseudobrachymeria, but are here treated within the newly defined subconica species-group of Brachymeria as part of a sibling species complex designated as the subrugosa complex. Species assigned to the subconica species-group are listed and five, one unnamed, are assigned to the subrugosa complex and their females keyed. Species are separated by qualitative characters and morphometry using distance measurements. Trigonura annulipes Costa Lima is renamed as Brachymeria costalimai Delvare nom. nov. because of secondary homonymy.

Larval stress alters dengue virus susceptibility in Aedes aegypti (L.) adult females.

In addition to genetic history, environmental conditions during larval stages are critical to the development, success and phenotypic fate of the Aedes aegypti mosquito. In particular, previous studies have shown a strong genotype-by-environment component to adult mosquito body size in response to optimal vs stressed larval conditions. Here, we expand upon those results by investigating the effects of larval-stage crowding and nutritional limitation on the susceptibility of a recent field isolate of Aedes aegypti to dengue virus serotype-2. Interestingly, female mosquitoes from larvae subjected to a stressed regime exhibited significantly reduced susceptibility to disseminated dengue infection 14days post infection compared to those subjected to optimal regimes. Short term survivorship post-infected blood feeding was not significantly different. As with body size, dengue virus susceptibility of a mosquito population is determined by a combination of genetic and environmental factors and is likely maintained by balancing selection. Here, we provide evidence that under different environmental conditions, the innate immune response of field-reared mosquitoes exhibits a large range of phenotypic variability with regard to dengue virus susceptibility. Further, as with body size, our results suggest that mosquitoes reared under optimal laboratory conditions, as employed in all mosquito-pathogen studies to date, may not always be realistic proxies for natural populations.

Population dynamics of an endogenous meiotic drive system in Aedes aegypti in Trinidad.

An endogenous meiotic drive system was previously reported to be segregating in the yellow fever mosquito Aedes aegypti L. (Diptera: Culicidae) population in Trinidad. The meiotic driver (M(D)) is tightly linked to the male determining locus and selectively targets sensitive responders linked to the female determining allele, causing fragmentation of female gametes. This results in highly male-biased progeny. The M(D) system was initially studied as a genetic tool for population control with limited success, but recently interest has focused on its potential for population replacement. This study examines the distribution and dynamics of the M(D) system in Trinidad natural populations. We obtained ovitrap samples from seven geographically distinct regions and determined the allele frequencies of the driver (M(D)) and sensitive (m(s)) versus insensitive (m(i)) responders, respectively. Frequencies of the M(D) allele ranged from 0.1 to 0.5 and were low at the two major port cities, Port of Spain and San Fernando, suggesting the effects of frequent immigration by non-driving genotypes. Frequencies of the m(i) allele ranged from 0.4 to 0.7, suggesting the effects of strong selection by the driver. In addition, our results show that the driver and sensitivity of responders in the Trinidad populations are highly polymorphic. Continued studies of the dynamics of the M(D) system in natural populations are critical to considerations of its use in population replacement.