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.

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.

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.

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.

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.

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.

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.

First reported enterovirus D68 infection in pediatric patients from the Caribbean region: evidence of spread from the U.S. outbreak / Primeros casos notificados en el Caribe de infección por enterovirus D68 en pacientes pediátricos: pruebas de la propagación del brote de los Estados Unidos

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.

El brote de enterovirus D68 (EV-D68) registrado en el 2014 en los Estados Unidos suscitó preocupación acerca de la introducción del virus en el Caribe. El objetivo de este estudio fue aportar pruebas rápidas, mediante la adopción de un enfoque filogénico relativamente sencillo, de que durante ese brote ingresaron en el Caribe cepas del EV-D68. Entre octubre del 2014 y mayo del 2015, el laboratorio regional de referencia ubicado en el Organismo de Salud Pública del Caribe (Puerto España, Trinidad y Tabago) detectó cuatro casos de EV-D68 provenientes de dos países (Bermudas y Dominica) mediante el análisis molecular de muestras respiratorias. Todos los casos correspondían a niños que acudieron al hospital con dificultad respiratoria moderada. No se detectó ningún caso de parálisis flácida aguda. El análisis filogénico de las cepas encontradas en el Caribe demostró una semejanza superior al 99 % con la cepa responsable del brote del 2014 en los Estados Unidos, lo que demuestra la introducción y la circulación del virus en la región.

First reported enterovirus D68 infection in pediatric patients from the Caribbean region: evidence of spread from the U.S. outbreak / Primeros casos notificados en el Caribe de infección por enterovirus D68 en pacientes pediátricos: pruebas de la propagación del brote de los Estados Unidos

ABSTRACT 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.(AU)

RESUMEN El brote de enterovirus D68 (EV-D68) registrado en el 2014 en los Estados Unidos suscitó preocupación acerca de la introducción del virus en el Caribe. El objetivo de este estudio fue aportar pruebas rápidas, mediante la adopción de un enfoque filogénico relativamente sencillo, de que durante ese brote ingresaron en el Caribe cepas del EV-D68. Entre octubre del 2014 y mayo del 2015, el laboratorio regional de referencia ubicado en el Organismo de Salud Pública del Caribe (Puerto España, Trinidad y Tabago) detectó cuatro casos de EV-D68 provenientes de dos países (Bermudas y Dominica) mediante el análisis molecular de muestras respiratorias. Todos los casos correspondían a niños que acudieron al hospital con dificultad respiratoria moderada. No se detectó ningún caso de parálisis flácida aguda. El análisis filogénico de las cepas encontradas en el Caribe demostró una semejanza superior al 99 % con la cepa responsable del brote del 2014 en los Estados Unidos, lo que demuestra la introducción y la circulación del virus en la región.(AU)

The Epidemiology of Fatal road traffic Collisions in Trinidad and Tobago, West Indies (2000-2011).

BACKGROUND: The purpose of the study is to determine the epidemiology of road traffic collisions (RTCs) in Trinidad and Tobago by characterizing RTCs in terms of number of collisions, fatalities, victim profiles, and locations for the purpose of informing accident prevention programs. Previous studies of RTCs in Trinidad and Tobago were primarily concerned with patterns of drivers use of seat belts, road collisions as a cause of mortality in young men, and the economic burden of road collisions. Attempts were made to model road fatalities, but limited epidemiological data meant that it was difficult to determine trends or develop models. METHODS: This study determined the epidemiology of RTCs in Trinidad and Tobago over the period 2000-2011 using data collected by the Trinidad and Tobago Road Traffic Branch of the Police Service and secondary data from the Central Statistical Office. Data were analyzed using Excel, SPSS, and R statistical packages. RESULTS: Fatalities were greater among men (80%) than among women (20%) and were highest on two major freeways in Trinidad [the Churchill-Roosevelt Highway and the Sir Solomon Hochoy Highway]. Most collisions occurred during the night among individuals between the ages of 15 and 44 years. Fatalities among drivers steadily increased over the study period and overtook fatalities among pedestrians, who were the group most affected in 2000. Most fatalities occurred at weekends. CONCLUSIONS: These patterns can inform (i) education programs and (ii) road and traffic control measures.

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.

The Role of Serotype Interactions and Seasonality in Dengue Model Selection and Control: Insights from a Pattern Matching Approach.

The epidemiology of dengue fever is characterized by highly seasonal, multi-annual fluctuations, and the irregular circulation of its four serotypes. It is believed that this behaviour arises from the interplay between environmental drivers and serotype interactions. The exact mechanism, however, is uncertain. Constraining mathematical models to patterns characteristic to dengue epidemiology offers a means for detecting such mechanisms. Here, we used a pattern-oriented modelling (POM) strategy to fit and assess a range of dengue models, driven by combinations of temporary cross protective-immunity, cross-enhancement, and seasonal forcing, on their ability to capture the main characteristics of dengue dynamics. We show that all proposed models reproduce the observed dengue patterns across some part of the parameter space. Which model best supports the dengue dynamics is determined by the level of seasonal forcing. Further, when tertiary and quaternary infections are allowed, the inclusion of temporary cross-immunity alone is strongly supported, but the addition of cross-enhancement markedly reduces the parameter range at which dengue dynamics are produced, irrespective of the strength of seasonal forcing. The implication of these structural uncertainties on predicted vulnerability to control is also discussed. With ever expanding spread of dengue, greater understanding of dengue dynamics and control efforts (e.g. a near-future vaccine introduction) has become critically important. This study highlights the capacity of multi-level pattern-matching modelling approaches to offer an analytic tool for deeper insights into dengue epidemiology and control.

A Novel Xenomonitoring Technique Using Mosquito Excreta/Feces for the Detection of Filarial Parasites and Malaria.

BACKGROUND: Given the continued successes of the world's lymphatic filariasis (LF) elimination programs and the growing successes of many malaria elimination efforts, the necessity of low cost tools and methodologies applicable to long-term disease surveillance is greater than ever before. As many countries reach the end of their LF mass drug administration programs and a growing number of countries realize unprecedented successes in their malaria intervention efforts, the need for practical molecular xenomonitoring (MX), capable of providing surveillance for disease recrudescence in settings of decreased parasite prevalence is increasingly clear. Current protocols, however, require testing of mosquitoes in pools of 25 or fewer, making high-throughput examination a challenge. The new method we present here screens the excreta/feces from hundreds of mosquitoes per pool and provides proof-of-concept for a practical alternative to traditional methodologies resulting in significant cost and labor savings. METHODOLOGY/PRINCIPAL FINDINGS: Excreta/feces of laboratory reared Aedes aegypti or Anopheles stephensi mosquitoes provided with a Brugia malayi microfilaria-positive or Plasmodium vivax-positive blood meal respectively were tested for the presence of parasite DNA using real-time PCR. A titration of samples containing various volumes of B. malayi-negative mosquito feces mixed with positive excreta/feces was also tested to determine sensitivity of detection. Real-time PCR amplification of B. malayi and P. vivax DNA from the excreta/feces of infected mosquitoes was demonstrated, and B. malayi DNA in excreta/feces from one to two mf-positive blood meal-receiving mosquitoes was detected when pooled with volumes of feces from as many as 500 uninfected mosquitoes. CONCLUSIONS/SIGNIFICANCE: While the operationalizing of excreta/feces testing may require the development of new strategies for sample collection, the high-throughput nature of this new methodology has the potential to greatly reduce MX costs. This will prove particularly useful in post-transmission-interruption settings, where this inexpensive approach to long-term surveillance will help to stretch the budgets of LF and malaria elimination programs. Furthermore, as this methodology is adaptable to the detection of both single celled (P. vivax) and multicellular eukaryotic pathogens (B. malayi), exploration of its use for the detection of various other mosquito-borne diseases including viruses should be considered. Additionally, integration strategies utilizing excreta/feces testing for the simultaneous surveillance of multiple diseases should be explored.

Aedes aegypti (L.) in Latin American and Caribbean region: With growing evidence for vector adaptation to climate change?

Within Latin America and the Caribbean region the impact of climate change has been associated with the effects of rainfall and temperature on seasonal outbreaks of dengue but few studies have been conducted on the impacts of climate on the behaviour and ecology of Aedes aegypti mosquitoes.This study was conducted to examine the adaptive behaviours currently being employed by A. aegypti mosquitoes exposed to the force of climate change in LAC countries. The literature on the association between climate and dengue incidence is small and sometimes speculative. Few laboratory and field studies have identified research gaps. Laboratory and field experiments were designed and conducted to better understand the container preferences, climate-associated-adaptive behaviour, ecology and the effects of different temperatures and light regimens on the life history of A. aegypti mosquitoes. A. aegypti adaptive behaviours and changes in container preferences demonstrate how complex dengue transmission dynamics is, in different ecosystems. The use of underground drains and septic tanks represents a major behaviour change identified and compounds an already difficult task to control A. aegypti populations. A business as usual approach will exacerbate the problem and lead to more frequent outbreaks of dengue and chikungunya in LAC countries unless both area-wide and targeted vector control approaches are adopted. The current evidence and the results from proposed transdisciplinary research on dengue within different ecosystems will help guide the development of new vector control strategies and foster a better understanding of climate change impacts on vector-borne disease transmission.

The Anopheles arabiensis genetic sexing strain ANO IPCL1 and its application potential for the sterile insect technique in integrated vector management programmes.

The Anopheles arabiensis genetic sexing strain ANO IPCL1 was developed based on a dieldrin resistant mutation. The strain has been shown to be practical and reliable in terms of female elimination by dieldrin treatments at larval stages, but has provided some difficulties when treatments were applied at the egg stage. The high natural sterility of this strain has advantages and disadvantages in both mass rearing and the sterilization process. In addition, its recombination rate, although relatively low, poses a threat of strain deterioration if left unchecked in a mass-rearing setting. The males of the ANO IPCL1 have been shown to be equally competitive as lab-reared males of the wild-type Dongola strain, but competitiveness decreased by half when irradiated with 75 Gy­a dose conferring >98% sterility. More controversial issues surround the use of dieldrin­a highly persistent organochlorine that is known to bioaccumulate in the food chain. The prospective use of large volumes of dieldrin in a mass-rearing facility and the retention of its residues by the male mosquitoes makes the use of the strain in the context of the sterile insect technique against this vector highly questionable, and therefore its implementation at a large scale cannot be recommended.

Exploratory space-time analysis of dengue incidence in Trinidad: a retrospective study using travel hubs as dispersal points, 1998-2004.

BACKGROUND: Dengue is an acute arboviral disease responsible for most of the illness and death in tropical and subtropical regions. Over the last 25 years there has been increase epidemic activity of the disease in the Caribbean, with the co-circulation of multiple serotypes. An understanding of the space and time dynamics of dengue could provide health agencies with important clues for reducing its impact. METHODS: Dengue Haemorrhagic Fever (DHF) cases observed for the period 1998-2004 were georeferenced using Geographic Information System software. Spatial clustering was calculated for individual years and for the entire study period using the Nearest Neighbor Index. Space and time interaction between DHF cases was determined using the Knox Test while the Nearest Neighbor Hierarchical method was used to extract DHF hot spots. All space and time distances calculated were validated using the Pearson r significance test. RESULTS: Results shows that (1) a decrease in mean distance between DHF cases correlates with activity leading up to an outbreak, (2) a decrease in temporal distance between DHF cases leads to increased geographic spread of the disease, with an outbreak occurrence about every 2 years, and (3) a general pattern in the movement of dengue incidents from more rural to urban settings leading up to an outbreak with hotspot areas associated with transportation hubs in Trinidad. CONCLUSION: Considering only the spatial dimension of the disease, results suggest that DHF cases become more concentrated leading up to an outbreak. However, with the additional consideration of time, results suggest that when an outbreak occurs incidents occur more rapidly in time leading to a parallel increase in the rate of distribution of the disease across space. The results of this study can be used by public health officers to help visualize and understand the spatial and temporal patterns of dengue, and to prepare warnings for the public. Dengue space-time patterns and hotspot detection will provide useful information to support public health officers in their efforts to control and predict dengue spread over critical hotspots allowing better allocation of resources.