WHO cone bioassay boards with or without holes: relevance for bioassay outcomes in long-lasting insecticidal net studies.

BACKGROUND: The World Health Organization (WHO) cone bioassay is a key method used to evaluate the bioefficacy of long-lasting insecticidal nets (LLINs) used for malaria control. These tests also play an important role in LLIN product prequalification and longitudinal monitoring. Standardization of these assays is therefore important. While many parameters for WHO cone bioassays are defined in the respective WHO guidelines, others are not. One of these undefined parameters is the exact configuration of the bioassay boards. In cone bioassays, LLIN samples are pinned onto a bioassay board for testing. Anecdotal evidence suggests that bioassay boards with holes behind the LLIN samples lead to greater exposure to insecticide, as the mosquitoes are 'forced to stand on the net material'. This may increase the key assay outcomes of 60 min knockdown (KD60) and 24 h mortality (M24). The present study tested this hypothesis in two facilities using two fully susceptible mosquito colonies. METHODS: WHO cone bioassays were performed using bioassay boards with holes and boards without holes in parallel, following WHO guidelines. Five brands of LLINs with four new and unwashed whole net samples per brand were used (total of n = 20 whole nets). Five pieces per whole net sample were prepared in duplicate resulting in a total of n = 100 pairs. Knock-down (KD) was recorded in 10 min intervals within the first hour after exposure and mortality was recorded at 24 h. Assays with Anopheles farauti were done at the Papua New Guinea Institute of Medical Research (PNGIMR) and assays with Aedes aegypti were done at James Cook University, Australia. RESULTS: Results varied not only with bioassay board configuration but also with mosquito colony. In particular, with An. farauti, a significantly higher M24 was observed when boards with holes were used, while this was not observed with Ae. aegypti. WHO cone bioassay results were systematically biased between the two facilities such that the use of An. farauti at PNGIMR predicted higher KD60 and M24. CONCLUSION: The present study highlights the need for further harmonization of WHO cone bioassay methodology. Parameters such as bioassay board configuration and mosquito species systematically affect the observations, which impedes generalizability of WHO cone bioassay outcomes.

Efficiency of the CO2 -baited omni-directional Fay-Prince trap under semi-field conditions and characterizing response behaviours for the yellow fever mosquito, Aedes aegypti.

Aedes aegypti (L.) (Diptera: Culicidae) is a vector of several serious disease-causing viruses including Dengue, Zika, chikungunya and yellow fever. Effective and efficient trapping methods are essential for meaningful mosquito population and disease-presence surveillance and ultimately, vector control. The Fay-Prince trap (FPT) was developed in the late 1960s as a daytime visual trap for male Ae. aegypti. Since then, its use has been expanded into the trapping of female Ae. aegypti, Aedes albopictus Skuse, other Ae. spp., and Culex spp. The efficiency of the FPT alone and with CO2 was tested under semi-field conditions and the behaviour of responding female Ae. aegypti was characterized. The mean capture efficiency of the FPT with CO2 per 30 min in the greenhouse was 3.07% and the capture rate from the total number of mosquitoes in our semi-field setup was slightly higher at 4.45%. Understanding the behaviours that mosquitoes exhibit during their encounter with particularly a visual trap may recommend trap improvements and contributes to our understanding of host-seeking behaviour and how it might be exploited.

Mosquito Repellent Efficacy of Australian Blue Cypress Callitris intratropica Essential Oil and a Topical Formulation under Laboratory and Field Conditions.

Mosquito repellents are important for personal protection against nuisance and potentially infectious mosquito bites. Repellent activity of Australian blue cypress essential oil (EO) and a commercially formulated skin lotion containing blue cypress EO (topical formulation) were compared with 20% DEET (N, N-diethyl-3 toluamide) against mosquitoes under laboratory and field conditions in North Queensland, Australia. On a volunteer's forearm, 1 mL of candidate material was applied to approximately 600 cm2 of exposed skin. When blue cypress EO was applied at various concentrations (0.5%-10.5%), it did not fully prevent mosquito landing or biting. However, a dose-dependent increase, approaching 80% protection, was observed at high EO concentrations. On the basis of these results, three concentrations (5%, 10%, and 20%) of blue cypress EO were selected for complete protection time (CPT) experiments. Topical formulation (undiluted) was also included in CPT experiments. Although some protection was afforded, mosquito landing/probing were still recorded immediately after application for both blue cypress EO and its topical formulation. Specifically, protection declined for blue cypress EO from 80% to 70% (laboratory) and from 93% to 50% (field) within 1 hour. For topical formulation, protection declined from 85% to 75% in the laboratory and from 63% to 50% in the field. In comparison, DEET maintained a 100% protection throughout the testing period of up to 1 h, and there was no landing/probing observed in volunteers who had applied DEET. To conclude, both blue cypress products provided some protection against mosquito bites, which decreased soon after application.

Verifying the efficiency of the Biogents Sentinel trap in the field and investigating microclimatic influences on responding Aedes aegypti behavior.

Successful surveillance and control of mosquito arbovirus vectors requires effective and sensitive trapping methods for adult insects. The Biogents Sentinel (BGS) trap is widely used for mosquito trapping but has low capture efficiency for both female and male Aedes aegypti under semi-field conditions. Efficiency refers to the proportion of mosquitoes that are trapped of those encountering the trap. We verified the efficiency of the BGS under field conditions in suburban Riverside, California, U.S.A., following our previous work determining the efficiency under semi-field conditions in Cairns, Northern Australia. The efficiency of the BGS with CO2 and a human skin odor mimic (BG-Lure) for both Ae. aegypti sexes in the field was 9%. This closely aligns with the results of our previous study, the efficiency for females being 5% and males being 9%. In the present study microclimatic conditions were monitored and capture occurred during periods of significantly lower mean temperature. There were no discernible changes in wind directionality or strength in the 60 s leading up to mosquito capture by the BGS. Our results support our previous findings that capture efficiency of the BGS for Ae. aegypti is low.

Long-Range But Not Short-Range Attraction of Male Aedes aegypti (Diptera: Culicidae) Mosquitoes to Humans.

Female Aedes aegypti (Linnaeus) mosquitoes integrate multiple sensory cues to locate human hosts for blood meals. Although male Ae. aegypti swarm around and land on humans in nature to mate, direct evidence of attraction to humans is limited. Male mosquito attraction to human host cues is often undetectable in confined laboratory assays, leading to a misconception that male mosquitoes are not attracted to humans. We used semifield experiments to demonstrate robust attraction of male Ae. aegypti to humans. Human-baited traps captured up to 25% of released males within 15 min, whereas control traps without humans as bait failed to capture males. Rapid attraction to humans was further demonstrated through videography. Males swarmed around and landed on human subjects, with no activity recorded in paired unbaited controls. Finally, we confirm the lack of discernible male attraction to humans in small laboratory cages. Our experiments demonstrate that both male and female Ae. aegypti show attraction to humans, but with clear sex-specific behavioral differences at short-range. Male mosquito attraction to humans is likely to be important for mating success in wild populations and its basis should be further explored. Our results highlight the importance of arena size and assay design for mosquito behavioral research. A better understanding of host cues that attract males could help us to improve mosquito surveillance and control.

Attraction Versus Capture II: Efficiency of the BG-Sentinel Trap Under Semifield Conditions and Characterizing Response Behaviors of Male Aedes aegypti (Diptera: Culicidae).

Aedes aegypti (L.) is an important vector of viruses causing dengue, Zika, chikungunya, and yellow fever and as such presents a serious threat to public health in tropical regions. Control programs involving 'rear and release' of modified male Ae. aegypti are underway and require effective trapping methods for surveillance of both the released insects and the impacted wild mosquito population. The BG-Sentinel trap (BGS) is widely used in Ae. aegypti surveillance but its level of efficiency, that is, what proportion of the mosquitoes encountering the trap are captured, is unknown. This is especially true for male mosquitoes, the behavior of which is incompletely understood. We tested the efficiency of two versions of the BGS for capturing male Ae. aegypti under semifield conditions with and without CO2 and a human skin odor mimic lure and with these baits combined. A navy-blue BGS trap emitting CO2 and a human skin odor mimic captured 18% of the released male Ae. aegypti, with a capture efficiency of 9 % (of the total encounters with the trap). Male Ae. aegypti had multiple encounters with the BGS that did not result in capture; they crossed over the trap entrance without being captured or landed on the sides of the trap. Swarming behavior around the BGS was also recorded, even when only a visual cue was present. Understanding male Ae. aegypti behaviors during an encounter with the BGS can inform improvement of trap design and therefore capture efficiency for surveillance in control programs.

Attraction Versus Capture: Efficiency of BG-Sentinel Trap Under Semi-Field Conditions and Characterizing Response Behaviors for Female Aedes aegypti (Diptera: Culicidae).

Aedes aegypti (L.) is an important vector of viruses causing dengue, Zika, chikungunya, and yellow fever and as such is a threat to public health worldwide. Effective trapping methods are essential for surveillance of both the mosquito species and disease presence. The BG-Sentinel (BGS) is a widely used to trap Ae. aegypti but little is known of its efficiency, i.e., what proportion of the mosquitoes encountering the trap are captured. The first version of the BGS trap was predominantly white, and the current version is mostly navy blue. While this trap is often deployed without any olfactory lure, it can also be deployed with CO2 and/or a human skin odor mimic lure to increase capture rates. We tested the efficiency of capturing Ae. aegypti under semi-field conditions for the original white version without lures as well the blue version with and without various lure combinations. None of the configurations tested here captured 100% of the mosquitoes that encountered the trap. A navy-blue trap emitting CO2 and a skin odor mimic produced the highest capture (14% of the total insects in the semi-field cage), but its capture efficiency was just 5% (of mosquitoes encountering the trap). Mosquitoes often had multiple encounters with a trap that did not result in capture; they crossed over the trap entrance without being captured or landed on the sides of the trap. Understanding these behaviors and the factors that induce them has the potential to suggest improvement in trap design and therefore capture efficiency.

Investigating Male Aedes aegypti (Diptera: Culicidae) Attraction to Different Oviposition Containers Using Various Configurations of the Sound Gravid Aedes Trap.

Aedes aegypti (Linnaeus), the primary vectors of the arboviruses dengue virus and Zika virus, continue to expand their global distributions. In efforts to better control such species, several mosquito control programs are investigating the efficacy of rearing and releasing millions of altered male Aedes throughout landscapes to reduce populations and disease transmission risk. Unfortunately, little is known about Ae. aegypti, especially male, dispersal behaviors within urban habitats. We deployed Sound-producing Gravid Aedes Traps (SGATs) in Cairns, northern Australia, to investigate male Ae. aegypti attraction to various oviposition container configurations. The traps were arranged to include: 1) water only, 2) organically infused water, 3) infused water and L3 larvae, 4) infused water and a human-scented lure, and lastly 5) no water or olfactory attractant (dry). Our data suggest that males were more attracted to SGATs representing active larval sites than potential larval sites, but were equally attracted to dry SGATs relative to those containing water and/or infusion. Additionally, we found that female Ae. aegypti were equally attracted to wet SGATs, with or without infusion, but not dry ones. These results suggest that male Ae. aegypti within northern Australia are more attracted to active larval sites and equally attracted to dry containers as wet or infused ones. Additionally, female Ae. aegypti are unlikely to enter dry containers. Such findings contribute to our understanding of potentially attractive features for local and released Ae. aegypti throughout the northern Australian urban landscape.

Small Hive Beetle (Coleoptera: Nitidulidae) and the Yeast, Kodamaea ohmeri: A Facultative Relationship Under Laboratory Conditions.

The small hive beetle, Aethina tumida Murray, is a pest of honeybees, Apis mellifera L. (Hymenoptera: Apidae). We investigated the significance of its association with the yeast, Kodamaea ohmeri (Etchells & Bell) (Ascomycota: Saccharomycotina), in laboratory experiments. The mean (± SEM) viability of A. tumida eggs was 84 (± 3)%; the viability was not affected if eggs were separated from clutches or if mucilage containing K. ohmeri was removed from the egg surface. Life tables of conventional (= K. ohmeri contaminated) A. tumida and K. ohmeri-free A. tumida revealed no differences in stage-specific mortality between the treatments; in both cases, the highest mortality occurred in the first larval instar. There was no significant difference in the initial egg production of conventionally reared and K. ohmeri-free A. tumida under laboratory conditions. The volatile profiles of pollen dough (Bee Build) fed on by conventional and K. ohmeri-free A. tumida larvae were qualitatively and quantitatively different; the volatiles produced by pollen dough fed on by conventional A. tumida were more attractive to adult beetles. There was a clear difference between growth of K. ohmeri on pollen dough substrate in the presence and absence of A. tumida. Results suggest that this association is facultative for A. tumida under laboratory conditions but has benefit for the yeast associate, K. ohmeri. A clearer understanding of the nature of this fungus-insect association is essential for the development of management strategies for this pest, especially in the development of fermentate-based attractants in trapping systems.

Associations Between the Small Hive Beetle and the Yeast Kodamaea ohmeri Throughout the Host Life Cycle.

The small hive beetle, Aethina tumida Murray (Coleoptera: Nitidulidae), is a pest of colonies of social bees, including the honeybee Apis mellifera L. (Hymenoptera: Apidae). We investigated A. tumida oviposition behavior and development and found that it laid eggs in clutches that ranged in size (3-75 eggs per clutch) and that when fed on hive products in laboratory culture (27°C; RH 65%; 12:12 (L:D) h) it completed three larval instars before pupation. The yeast Kodamaea ohmeri (Etchells & Bell) Y. Yamada, T. Suzuki, M. Matsuda & K. Mikata (Ascomycota: Saccharomycotina) is associated with A. tumida, but the exact nature of this relationship is unknown. We examined the association in host eggs, larvae, pupae, and adults to establish its extent and potential specificity and determined the likely mechanism of vertical transmission. K. ohmeri was detected in egg mucilage and on host cuticle and from internal preparations of A. tumida at every stage of development. Based on colony forming unit (CFU) counts, the K. ohmeri densities varied significantly between developmental stages; the highest internal density was recorded in third instar larvae. Presence of K. ohmeri within adult A. tumida was not affected by contamination of the cuticle by the yeast during the larval and pupal stages nor by the mated status of the adult. This deepened understanding of A. tumida ovipositional behavior and larval development along with a better understanding of the relationship between K. ohmeri and its host is important for the development of management strategies for this important pest.