Jonathan M. Darbro

Avian Malaria Parasites Share Congeneric Mosquito Vectors

Abstract Assessing parasite specificity to vector is crucial to understanding the emergence of vector-borne diseases and the evolution of parasite diversity. Avian malaria parasites have a cosmopolitan distribution and broad avian host range, which together predict they are vector generalists, but little is known about parasite–vector associations in the wild. We tested this prediction by asking if 5 different mosquito species, known to feed on birds and abundant in the northeastern United States, were naturally infected in the field with identical avian Plasmodium spp. lineages. Mosquitoes were not pooled but rather analyzed individually, and, possibly as a result, lineage diversity was higher than reported in previous avian malaria vector studies. Plasmodium spp. lineages were rare in Aedes canadensis and absent in Aedes aurifer and Culiseta melanura. We sequenced a standard Plasmodium cytochrome b marker from Culex pipiens pipiens, Culex restuans, and Ae. canadensis. Most Plasmodium clades were shared by Cx. pipiens and Cx. restuans. In addition, 4 individual lineages were shared by both mosquito species, including the most common lineage. One Plasmodium clade, however, was only found in Cx. restuans. We therefore found limited support for our prediction that avian Plasmodium spp. vector breadth accompanies host breadth. The association of both Culex species with most Plasmodium clades, and the presence of a single parasite lineage in 3 mosquito species representing 2 genera, suggests that avian Plasmodium species are not tightly coevolved with vector species.

Field evaluation of the establishment potential of wmelpop Wolbachia in Australia and Vietnam for dengue control

BackgroundIntroduced Wolbachia bacteria can influence the susceptibility of Aedes aegypti mosquitoes to arboviral infections as well as having detrimental effects on host fitness. Previous field trials demonstrated that the wMel strain of Wolbachia effectively and durably invades Ae. aegypti populations. Here we report on trials of a second strain, wMelPop-PGYP Wolbachia, in field sites in northern Australia (Machans Beach and Babinda) and central Vietnam (Tri Nguyen, Hon Mieu Island), each with contrasting natural Ae. aegypti densities.MethodsMosquitoes were released at the adult or pupal stages for different lengths of time at the sites depending on changes in Wolbachia frequency as assessed through PCR assays of material collected through Biogents-Sentinel (BG-S) traps and ovitraps. Adult numbers were also monitored through BG-S traps. Changes in Wolbachia frequency were compared across hamlets or house blocks.ResultsReleases of adult wMelPop-Ae. aegypti resulted in the transient invasion of wMelPop in all three field sites. Invasion at the Australian sites was heterogeneous, reflecting a slower rate of invasion in locations where background mosquito numbers were high. In contrast, invasion across Tri Nguyen was relatively uniform. After cessation of releases, the frequency of wMelPop declined in all sites, most rapidly in Babinda and Tri Nguyen. Within Machans Beach the rate of decrease varied among areas, and wMelPop was detected for several months in an area with a relatively low mosquito density.ConclusionsThese findings highlight challenges associated with releasing Wolbachia-Ae. aegypti combinations with low fitness, albeit strong virus interference properties, as a means of sustainable control of dengue virus transmission.

Bird-Baited Traps for Surveillance of West Nile Mosquito Vectors: Effect of Bird Species, Trap Height, and Mosquito Escape Rates

Abstract Host-seeking mosquitoes were sampled in bird-baited traps at four sites in New York state in 2003–2004. Trap placement and efficacy of chickens, Gallus gallus domesticus L., as bait compared with house sparrows, Passer domesticus L., an important reservoir of West Nile virus (family Flaviviridae, genus Flavivirus, WNV), was evaluated. Each site had a chicken-baited trap near ground level (≈1.5 m) and in the tree canopy (≈9 m), and a house sparrow-baited trap at ground level and canopy level. Each trap allowed mosquito access to birds on one end, and an inner mesh screen blocked bird access on the other end. The two most abundant mosquitoes, Culex restuans Theobald and Culex pipiens pipiens L., were differentiated using molecular characters. In 2003, Cx. restuans and Cx. p. pipiens made up 88% of total mosquito catch. In 2004, Cx. restuans comprised 43% of total catch and Cx. p. pipiens comprised 33%. The remaining species representing at least 1% of total catch were Ochlerotatus trivittatus (Coquillett), Coquilletidia perturbans (Walker), and Culiseta morsitans (Theobald). Capture rates were similar for chicken and house sparrow-baited traps; however, significantly more mosquitoes were captured in the canopy for both bird species. Cx. restuans preferred canopy traps, whereas equal numbers of Cx. p. pipiens were captured at ground and canopy levels. Mosquitoes were more likely to escape (74%) when excluded from birds than when allowed free access to birds (54%). Sentinel bird surveillance for WNV can be improved by trapping in the tree canopy in addition to ground level to capture the most important avian vectors.

Effects of Beauveria bassiana on Survival, Blood-Feeding Success, and Fecundity of Aedes aegypti in Laboratory and Semi-Field Conditions

The fungus Beauveria bassiana reduces Aedes aegypti longevity in laboratory conditions, but effects on survival, blood-feeding behavior, and fecundity in realistic environmental conditions have not been tested. Adult, female Ae. aegypti infected with B. bassiana (FI-277) were monitored for blood-feeding success and fecundity in the laboratory. Fungal infection reduced mosquito-human contact by 30%. Fecundity was reduced by (mean ± SD) 29.3 ± 8.6 eggs per female per lifetime in the laboratory; egg batch size and viability were unaffected. Mosquito survival, blood-feeding behavior, and fecundity were also tested in 5 meter × 7 meter × 4 meter semi-field cages in northern Queensland, Australia. Fungal infection reduced mosquito survival in semi-field conditions by 59-95% in large cages compared with 61-69% in small cages. One semi-field cage trial demonstrated 80% reduction in blood-feeding; a second trial showed no significant effect. Infection did not affect fecundity in large cages. Beauveria bassiana can kill and may reduce biting of Ae. aegypti in semi-field conditions and in the laboratory. These results further support the use of B. bassiana as a potential biocontrol agent against Ae. aegypti.

Increased container-breeding mosquito risk owing to drought-induced changes in water harvesting and storage in Brisbane, Australia

BACKGROUND Extended drought conditions in south-east Queensland during the early 2000s have resulted in a culture of water harvesting and legislated water restrictions. Aedes notoscriptus is a container-breeding mosquito vector of Ross River and Barmah Forest viruses. METHODS From 2008-2009, the larval habitats and seasonal abundance of domestic container-breeding mosquitoes were recorded from three suburbs of Brisbane. A knowledge, attitudes and practice questionnaire was administered to householders. A low-cost, desktop methodology was used to predict the proportion of shaded premises compared with front-of-property estimates. RESULTS We highlight changes in the frequency of container categories for A. notoscriptus as a response to human behavioural changes to drought. Garden accoutrements, discarded household items and water storage containers accounted for 66.2% (525/793) of positive containers and 77.5% (73 441/94 731) of all immature mosquitoes. Of all household premises surveyed, 52.6% (550/1046) contained rainwater tanks and 29.4% (308/1046) harvested water in other containers, contrasting with a previous 1995 survey where neither category was observed. Both Premise Condition Index and shade directly correlated with positive premises. CONCLUSIONS Human response to drought has resulted in new habitats for domestic container-breeding mosquitoes. This recent trend of prolific water storage is similar to earlier years (1904-1943) in Brisbane when Aedes aegypti was present and dengue epidemics occurred.

Evaluation of entomopathogenic fungi as potential biological control agents of the dengue mosquito, Aedes aegypti (Diptera: Culicidae)

Abstract Dengue is a global health concern. Growing insecticide resistance in the primary mosquito vector, Aedes aegypti, limits the effectiveness of vector control, so alternative tools are urgently needed. One approach is the use of biopesticides comprising entomopathogenic fungi, e.g., Beauveria bassiana and Metarhizium anisopliae. These fungi may decrease disease transmission by reducing mosquito vector longevity and also occur worldwide, although many isolates have not been tested for virulence against mosquitoes. Ninety-three isolates of entomopathogenic fungi representing six species (B. bassiana, M. anisopliae, Isaria fumosorosea, I. farinosa, I. flavovirescens, and Lecanicillium spp.) were screened as potential biological control agents of Aedes aegypti. A hierarchical, multi-criteria experimental design was undertaken to find suitable isolates. Initial screening was performed via in vitro assays measuring radial growth and spore persistence, eliminating isolates with poor growth or viability on nutrient-rich substrate. Subsequent measurements of spore persistence revealed that only nine of 30 strains tested had half-lives exceeding 3 weeks. Ten isolates were chosen for in vivo bioassays against adult Ae. aegypti. From these assays, two Australian isolates of B. bassiana, FI-277 and FI-278, appeared to be most promising. Both isolates were shown to be virulent against Ae. aegypti at 20, 26, and 32°C. Spreading spores manually onto substrate was found to be more efficacious than spraying. Ae. aegypti infected by manually-spread spores on cotton substrate were found to have an LT50 of 3.7±0.3 days. These characteristics suggest that FI-277 has promise as a dengue mosquito biocontrol agent, either alone or combined with conventional chemical insecticides.

Survey of Muscalure [(Z)-9-tricosene] on House Flies (Diptera: Muscidae) from Field Populations in California

Abstract The sex pheromone component muscalure [(Z)-9-tricosene] on the cuticles of female house flies (Musca domestica L.) was quantified using gas chromatography. Laboratory and wild-strain house fly colonies were derived from poultry and dairy farms and a horse-riding stable. Muscalure levels from strain MB females were highest after 13 and 23 generations in culture, but 30–70% of females from early generations (F8–F12) had no detectable levels of muscalure. Flies <2 d old from the MB laboratory colony (F23) had less muscalure than did older flies, and levels tended to increase as flies aged from 3 to 8 d. Of 10 wild house fly populations sampled at 3 d of age (72–96 h old) between F1 and F7 in culture, 1 had no detectable muscalure, and 8 of 10 averaged ≤218 ng of muscalure per female, comprising ≤1.6% of total cuticular hydrocarbons. In 6 of 10 field populations, ≥67% of females had no detectable muscalure. In contrast, two field strains had average levels of 559 and 1,113 ng muscalure/female (comprising 3.2 and 5.0% of total hydrocarbons), which was comparable to long-established laboratory strains such as the UCR strain (751 ng muscalure/female, comprising 3.0% of total hydrocarbons). More than 70% of females from those two field strains had muscalure. Flies from the Cooper laboratory strain also lacked detectable muscalure. Muscalure amounts were highly variable; it was rare or lacking in many field populations and thus does not seem to be essential for house fly reproduction.

Mapping the virome in wild-caught Aedes aegypti from Cairns and Bangkok

Medically important arboviruses such as dengue, Zika, and chikungunya viruses are primarily transmitted by the globally distributed mosquito Aedes aegypti. Increasing evidence suggests that transmission can be influenced by mosquito viromes. Herein RNA-Seq was used to characterize RNA metaviromes of wild-caught Ae. aegypti from Bangkok (Thailand) and from Cairns (Australia). The two mosquito populations showed a high degree of similarity in their viromes. BLAST searches of assembled contigs suggest up to 27 insect-specific viruses may infect Ae. aegypti, with up to 23 of these currently uncharacterized and up to 16 infecting mosquitoes from both Cairns and Bangkok. Three characterized viruses dominated, Phasi Charoen-like virus, Humaita-Tubiacanga virus and Cell fusing agent virus, and comparisons with other available RNA-Seq datasets suggested infection levels with these viruses may vary in laboratory-reared mosquitoes. As expected, mosquitoes from Bangkok showed higher mitochondrial diversity and carried alleles associated with knock-down resistance to pyrethroids. Blood meal reads primarily mapped to human genes, with a small number also showing homology with rat/mouse and dog genes. These results highlight the wide spectrum of data that can be obtained from such RNA-Seq analyses, and suggests differing viromes may need to be considered in arbovirus vector competence studies.

Laboratory colonization of the European invasive mosquito Aedes ( Finlaya ) koreicus

BackgroundAedes (Finlaya) koreicus (Edwards) is a mosquito that has recently entered Europe from Asia. This species is considered a potential threat to newly colonized territories, but little is known about its capacity to transmit pathogens or ability to compete with native mosquito species. The establishment of a laboratory colony is a necessary first step for further laboratory studies on the biology, ecology and vector competence of Ae. koreicus.ResultsA self-mating colony was established at QIMR Berghofer Medical Research Institute (Brisbane, Australia) from eggs of the F1 progeny of individuals collected as free-living larvae in northeastern Italy (Belluno province). Mosquitoes are currently maintained on both defibrinated sheep blood provided via an artificial membrane system and human blood from volunteers. Larvae are maintained in rain water and fed with Tetramin® fish food (©2015 Spectrum Brands - Pet, Home and Garden Division, Tetra-Fish). Morphometric measurements related to body size were taken and a fecundity index, based on wing length, was calculated. An in vivo technique for differentiating male and female pupae has been optimized. Our findings provide the basis for further studies on the ecology and physiology of Ae. koreicus.ConclusionWe describe the establishment of an Ae. koreicus colony in the laboratory and identify critical requirements for the maintenance of this mosquito species under artificial conditions. The laboratory colony will facilitate studies investigating the vector potential of this species for human pathogens.