Gregor J. Devine

Using Adult Mosquitoes to Transfer Insecticides to Aedes Aegypti Larval Habitats

Vector control is a key means of combating mosquito-borne diseases and the only tool available for tackling the transmission of dengue, a disease for which no vaccine, prophylaxis, or therapeutant currently exists. The most effective mosquito control methods include a variety of insecticidal tools that target adults or juveniles. Their successful implementation depends on impacting the largest proportion of the vector population possible. We demonstrate a control strategy that dramatically improves the efficiency with which high coverage of aquatic mosquito habitats can be achieved. The method exploits adult mosquitoes as vehicles of insecticide transfer by harnessing their fundamental behaviors to disseminate a juvenile hormone analogue (JHA) between resting and oviposition sites. A series of field trials undertaken in an Amazon city (Iquitos, Peru) showed that the placement of JHA dissemination stations in just 3–5% of the available resting area resulted in almost complete coverage of sentinel aquatic habitats. More than control mortality occurred in 95–100% of the larval cohorts of Aedes aegypti developing at those sites. Overall reductions in adult emergence of 42–98% were achieved during the trials. A deterministic simulation model predicts amplifications in coverage consistent with our observations and highlights the importance of the residual activity of the insecticide for this technique.

Potential Use of Pyriproxyfen for Control of Aedes aegypti (Diptera: Culicidae) in Iquitos, Perú

Abstract The effects of pyriproxyfen were tested against a local population of Aedes aegypti (L.) in Iquitos, Perú. Bioassays showed that, when applied to late instars, pyriproxyfen prevented adult emergence at extremely low concentrations (LC50 = 0.012 ppb). There was no adult emergence from water sampled from storage tanks that had been seeded with the equivalent of 50–83 ppb (AI) pyriproxyfen. Five months after treatment, despite constant dilution of these tanks, water sampled from these sources continued to be lethal to larvae and pupae. Additional studies, carried out in the laboratory, showed that groups of five or 20 female blood-fed mosquitoes, exposed to residues of ≈0.003 g (AI) pyriproxyfen/m2, could transfer enough chemical to new oviposition sites to prevent ≈80% of adult emergence from larvae developing in that previously uncontaminated water. Moreover, although the fecundity of the adult females used as the transfer vehicles in these tests was unaffected, the subsequent eclosion of the eggs that these mosquitoes laid was decreased by 70–90%. It also was shown that, at very high concentrations (>30,000 ppb), pyriproxyfen-treated water sources were as likely to be used as oviposition sites as untreated sources. These data suggest that treated sites might act as sinks for mosquito reproduction and moreover that such sites might act as dissemination sources for the horizontal transfer of larvicides to new environments by mature females. We review the literature on the environmental and human health effects of this compound and discuss its potential for use as a mosquito control agent in the field.

Increasing role of Anopheles funestus and Anopheles arabiensis in malaria transmission in the Kilombero Valley, Tanzania

BackgroundIn order to sustain the gains achieved by current malaria control strategies, robust surveillance systems that monitor dynamics of vectors and their roles in malaria transmission over time are essential. This longitudinal study demonstrates the trends in malaria vector dynamics and their relative contribution to malaria transmission in hyperendemic transmission settings in Tanzania.MethodsThe study was conducted in two villages within the Kilombero Valley, in rural Tanzania for five consecutive years (2008–2012). Seventy-two houses were selected per village and each house was sampled for mosquitoes monthly using a CDC light trap. Collected mosquitoes were assessed for species identity and sporozoite infection status using PCR and ELISA, respectively. Anopheles funestus and Anopheles arabiensis susceptibility to insecticides was assessed using WHO guidelines.ResultsA total of 100,810 malaria vectors were collected, of which 76% were Anopheles gambiae s. l. and 24% were An. funestus. Of all An. funestus samples that amplified with PCR (n = 2,737), 97% were An. funestus s.s., 2% were Anopheles rivorulum and 1% Anopheles leesoni. Whereas for An. gambiae s.l. (n = 8,117), 93% were An. arabiensis and 7% were Anopheles gambiae s.s. The proportion of An. gambiae s.s. identified by PCR (2,924) declined from 0.2% in the year 2008 to undetectable levels in 2012. Malaria transmission intensity significantly decreased from an EIR of 78.14 infectious bites/person/year in 2008 to 35 ib/p/yr in 2011 but rebounded to 226 ib/p/yr in 2012 coinciding with an increased role of An. funestus in malaria transmission. Insecticide susceptibility tests indicated high levels of resistance in An. funestus against deltamethrin (87%), permethrin (65%), lambda cyhalothrin (74%), bendiocarb (65%), and DDT (66%). Similarly, An. arabiensis showed insecticide resistance to deltamethrin (64%), permethrin (77%) and lambda cyhalothrin (42%) in 2014.ConclusionThe results indicate the continuing role of An. arabiensis and the increasing importance of An. funestus in malaria transmission, and pyrethroid resistance development in both species. Complementary vector control and surveillance tools are needed that target the ecology, behaviour and insecticide resistance management of these vector species, in order to preserve the efficacy of LLINs.

A low-cost repellent for malaria vectors in the Americas: results of two field trials in Guatemala and Peru

BackgroundThe cost of mosquito repellents in Latin America has discouraged their wider use among the poor. To address this problem, a low-cost repellent was developed that reduces the level of expensive repellent actives by combining them with inexpensive fixatives that appear to slow repellent evaporation. The chosen actives were a mixture of para-menthane-diol (PMD) and lemongrass oil (LG).MethodsTo test the efficacy of the repellent, field trials were staged in Guatemala and Peru. Repellent efficacy was determined by human-landing catches on volunteers who wore the experimental repellents, control, or 15% DEET. The studies were conducted using a balanced Latin Square design with volunteers, treatments, and locations rotated each night.ResultsIn Guatemala, collections were performed for two hours, commencing three hours after repellent application. The repellent provided >98% protection for five hours after application, with a biting pressure of >100 landings per person/hour. The 15% DEET control provided lower protection at 92% (p < 0.0001). In Peru, collections were performed for four hours, commencing two hours after repellent application. The PMD/LG repellent provided 95% protection for six hours after application with a biting pressure of >46 landings per person/hour. The 20% DEET control provided significantly lower protection at 64% (p < 0.0001).ConclusionIn both locations, the PMD/LG repellent provided excellent protection up to six hours after application against a wide range of disease vectors including Anopheles darlingi. The addition of fixatives to the repellent extended its longevity while enhancing efficacy and significantly reducing its cost to malaria-endemic communities.

The dynamics of pyrethroid resistance in Anopheles arabiensis from Zanzibar and an assessment of the underlying genetic basis.

BackgroundThe emergence of pyrethroid resistance in the malaria vector, Anopheles arabiensis, threatens to undermine the considerable gains made towards eliminating malaria on Zanzibar. Previously, resistance was restricted to the island of Pemba while mosquitoes from Unguja, the larger of the two islands of Zanzibar, were susceptible. Here, we characterised the mechanism(s) responsible for resistance on Zanzibar using a combination of gene expression and target-site mutation assays.MethodsWHO resistance bioassays were conducted using 1-5d old adult Anopheles gambiae s.l. collected between 2011 and 2013 across the archipelago. Synergist assays with the P450 inhibitor piperonyl-butoxide were performed in 2013. Members of the An. gambiae complex were PCR-identified and screened for target-site mutations (kdr and Ace-1). Gene expression in pyrethroid resistant An. arabiensis from Pemba was analysed using whole-genome microarrays.ResultsPyrethroid resistance is now present across the entire Zanzibar archipelago. Survival to the pyrethroid lambda-cyhalothrin in bioassays conducted in 2013 was 23.5-54.3% on Unguja and 32.9-81.7% on Pemba. We present evidence that resistance is mediated, in part at least, by elevated P450 monoxygenases. Whole-genome microarray scans showed that the most enriched gene terms in resistant An. arabiensis from Pemba were associated with P450 activity and synergist assays with PBO completely restored susceptibility to pyrethroids in both islands. CYP4G16 was the most consistently over-expressed gene in resistant mosquitoes compared with two susceptible strains from Unguja and Dar es Salaam. Expression of this P450 is enriched in the abdomen and it is thought to play a role in hydrocarbon synthesis. Microarray and qPCR detected several additional genes putatively involved in this pathway enriched in the Pemba pyrethroid resistant population and we hypothesise that resistance may be, in part, related to alterations in the structure of the mosquito cuticle. None of the kdr target-site mutations, associated with pyrethroid/DDT resistance in An. gambiae elsewhere in Africa, were found on the islands.ConclusionThe consequences of this resistance phenotype are discussed in relation to future vector control strategies on Zanzibar to support the ongoing malaria elimination efforts on the islands.

Quantifying the epidemiological impact of vector control on dengue.

[Extract] Dengue virus (DENV) is a self-limiting illness in tropical and subtropical regions around the globe caused by four closely related, but distinct, virus serotypes (DENV-1, -2, -3, and -4) that are transmitted among humans by mosquitoes, primarily Aedes aegypti [1]. Approximately 4 billion people living in more than 128 countries are at risk of infection [2]. Each year there are an estimated 400 million new infections, of which about 100 million manifest as apparent illness [3]. The outcome of human infections ranges from asymptomatic to mild illness to severe, life-threatening disease [4]. DENV not only causes more human morbidity and mortality than any other arthropod-borne virus but it is also a growing public health threat. There has been a dramatic 4-fold increase in dengue cases between 1990–2013 and dengue continues to expand in geographic range [2,3,5,6].

De Novo Generation and Characterization of New Zika Virus Isolate Using Sequence Data from a Microcephaly Case

The major complications of an ongoing Zika virus outbreak in the Americas and Asia are congenital defects caused by the virus’s ability to cross the placenta and infect the fetal brain. The ability to generate molecular tools to analyze viral isolates from the current outbreak is essential for furthering our understanding of how these viruses cause congenital defects. The majority of existing viral isolates and infectious cDNA clones generated from them have undergone various numbers of passages in cell culture and/or suckling mice, which is likely to result in the accumulation of adaptive mutations that may affect viral properties. The approach described herein allows rapid generation of new, fully functional Zika virus isolates directly from deep sequencing data from virus-infected tissues without the need for prior virus passaging and for the generation and propagation of full-length cDNA clones. The approach should be applicable to other medically important flaviviruses and perhaps other positive-strand RNA viruses. ABSTRACT Zika virus (ZIKV) has recently emerged and is the etiological agent of congenital Zika syndrome (CZS), a spectrum of congenital abnormalities arising from neural tissue infections in utero. Herein, we describe the de novo generation of a new ZIKV isolate, ZIKVNatal, using a modified circular polymerase extension reaction protocol and sequence data obtained from a ZIKV-infected fetus with microcephaly. ZIKVNatal thus has no laboratory passage history and is unequivocally associated with CZS. ZIKVNatal could be used to establish a fetal brain infection model in IFNAR−/− mice (including intrauterine growth restriction) without causing symptomatic infections in dams. ZIKVNatal was also able to be transmitted by Aedes aegypti mosquitoes. ZIKVNatal thus retains key aspects of circulating pathogenic ZIKVs and illustrates a novel methodology for obtaining an authentic functional viral isolate by using data from deep sequencing of infected tissues. IMPORTANCE The major complications of an ongoing Zika virus outbreak in the Americas and Asia are congenital defects caused by the virus’s ability to cross the placenta and infect the fetal brain. The ability to generate molecular tools to analyze viral isolates from the current outbreak is essential for furthering our understanding of how these viruses cause congenital defects. The majority of existing viral isolates and infectious cDNA clones generated from them have undergone various numbers of passages in cell culture and/or suckling mice, which is likely to result in the accumulation of adaptive mutations that may affect viral properties. The approach described herein allows rapid generation of new, fully functional Zika virus isolates directly from deep sequencing data from virus-infected tissues without the need for prior virus passaging and for the generation and propagation of full-length cDNA clones. The approach should be applicable to other medically important flaviviruses and perhaps other positive-strand RNA viruses.

Ten years of the Tiger: Aedes albopictus presence in Australia since its discovery in the Torres Strait in 2005

The “Asian tiger mosquito”, Aedes albopictus, is highly invasive, an aggressive biter and a major arbovirus vector. It is not currently present on mainland Australia despite being intercepted on numerous occasions at international ports and infesting the Torres Strait of Australia since at least 2004. In the current paper, we describe the invasion and current status of Ae. albopictus in the Torres Strait, as well as research conducted to assess the threat of this species becoming established in arbovirus transmission cycles on the Australian mainland. Genetic analysis of the invading population demonstrated that the Indonesian region was the likely origin of the invasion and not Papua New Guinea (PNG) as initially suspected. There was also intermixing between Torres Strait, PNG and Indonesian populations, indicating that the species could be re-introduced into the Torres Strait compromising any successful eradication programme. Vector competence experiments with endemic and exotic viruses revealed that Ae. albopictus from the Torres Strait are efficient alphavirus vectors, but less efficient flavivirus vectors. Ae.albopictus obtains blood meals from a range of vertebrate hosts (including humans), indicating that it could play a role in both zoonotic and human-mosquito arbovirus transmission cycles in Australia. Predictive models coupled with climate tolerance experiments suggest that a Torres Strait strain of Ae. albopictus could colonise southern Australia by overwintering in the egg stage before proliferating in the warmer months. Cohabitation experiments demonstrated that the presence of Aedes notoscriptus larvae in containers would not prevent the establishment of Ae. albopictus. Evidence from these studies, coupled with global experience suggests that we need to be prepared for the imminent invasion of Australia by Ae. albopictus by thoroughly understanding its biology and being willing to embrace emerging control technologies.

Holding back the tiger: Successful control program protects Australia from Aedes albopictus expansion.

Background The Asian tiger mosquito, Aedes albopictus, is an important vector of dengue, chikungunya and Zika viruses and is a highly invasive and aggressive biter. Established populations of this species were first recognised in Australia in 2005 when they were discovered on islands in the Torres Strait, between mainland Australia and Papua New Guinea. A control program was implemented with the original goal of eliminating Ae. albopictus from the Torres Strait. We describe the evolution of management strategies that provide a template for Ae. albopictus control that can be adopted elsewhere. Methodology / Principal findings The control strategy implemented between 2005 and 2008 targeted larval habitats using source reduction, insect-growth regulator and pyrethroid insecticide to control larvae and adults in the containers. However, the infrequency of insecticide reapplication, the continual accumulation and replacement of containers, and imminent re-introduction of mosquitoes through people’s movement from elsewhere compromised the program. Consequently, in 2009 the objective of the program changed from elimination to quarantine, with the goal of preventing Ae albopictus from infesting Thursday and Horn islands, which are the transport hubs connecting the Torres Strait to mainland Australia. However, larval control strategies did not prevent the species establishing on these islands in 2010. Thereafter, an additional strategy adopted by the quarantine program in early 2011 was harborage spraying, whereby the vegetated, well shaded resting sites of adult Ae. albopictus were treated with a residual pyrethroid insecticide. Inclusion of this additional measure led to a 97% decline in Ae. albopictus numbers within two years. In addition, the frequency of container treatment was increased to five weeks between treatments, compared to an average of 8 weeks that occurred in the earlier iterations of the program. By 2015 and 2016, Ae. albopictus populations on the two islands were undetectable in 70–90% of surveys conducted. Importantly, a comprehensive surveillance network in selected strategic areas has not identified established populations of this species on the Australian mainland. Conclusions / Significance The program has successfully reduced Ae. albopictus populations on Thursday Island and Horn Island to levels where it is undetectable in up to 90% of surveys, and has largely removed the risk of mainland establishment via that route. The vector management strategies adopted in the later years of the program have been demonstrably successful and provide a practical management framework for dengue, chikungunya or Zika virus outbreaks vectored by Ae. albopictus. As of June 2016, Ae. albopictus had not established on the Australian mainland and this program has likely contributed significantly to this outcome.

Heat Sensitivity of wMel Wolbachia during Aedes aegypti Development

The wMel strain of Wolbachia bacteria is known to prevent dengue and Zika virus transmission in the mosquito vector Aedes aegypti. Accordingly, the release of wMel-infected A. aegypti in endemic regions has been recommended by the World Health Organization as a potential strategy for controlling dengue and Zika outbreaks. However, the utility of this approach could be limited if high temperatures in the aquatic habitats where A. aegypti develop are detrimental to Wolbachia. We exposed wMel-infected A. aegypti eggs and larvae to fluctuating daily temperatures of 30–40°C for three, five, or seven days during their development. We found that Wolbachia levels in females emerging from heat treatments were significantly lower than in the controls that had developed at 20–30°C. Notably, seven days of high temperatures starting at the egg stage reduced Wolbachia levels in emerging females to less than 0.1% of the wMel control levels. However, after adult females returned to 20–30°C for 4–7 days, they experienced differing degrees of Wolbachia recovery. Our findings suggest that the spread of Wolbachia in wild A. aegypti populations and any consequent protection from dengue and Zika viruses might be limited in ecosystems that experience periods of extreme heat, but Wolbachia levels recover partially after temperatures return to normal.