Anthony A. James’s research while affiliated with University of California System and other places

Background Oviposition site selection is an important component of vector mosquito reproductive biology. The Asian Tiger mosquito, Aedes albopictus, is a major and important vector of arboviruses including Dengue. Previous studies documented the preference of gravid females for small, dark-colored water containers as oviposition sites, which they sought during the twilight period (dusk) of their locomotor activity. Vision plays an important role in this behavior, and factors such as the shape, size, and color of the container, light intensity, polarization, spectrum, and other visual cues guide the search for suitable oviposition sites, but the mechanistic factors driving this behavior are unclear. Methodology/Principal findings We blindfolded adult female compound eyes and observed the effects of a lack of vision on the ability to discriminate and utilize preferred oviposition sites. Furthermore, the transcriptomes of blindfolded mosquitoes were screened to identify genes with vision-sensitive expression profiles and gene-editing was used to create non-functional mutations in two of them, rhodopsin-like (mutation designated ‘rho-l△807’) and kynurenine hydroxylase (mutation designated ‘khw’). Behavioral tests of both mutant and control strains revealed that the rho-l△807 mutant mosquitoes had a significant decrease in their ability to search for preferred oviposition sites that correlated with a reduced ability to recognize long-wavelength red light. The khw mutant mosquitoes also had a reduced ability to identify preferred oviposition sites that correlated with reductions in their ability to respond to variations in daily brightness and their ability to discriminate among different color options of the containers and background monochromatic light. Conclusions/Significance This study underscores the importance of visual cues in the oviposition site selection behavior of adult female Ae. albopictus. We demonstrate that wild-type rho-l and kh gene products play a crucial role in this behavior, as mutants exhibit altered sensitivity or recognition of light intensity and substrate colors.

Background Novel technologies are needed to combat anopheline vectors of malaria parasites as the reductions in worldwide disease incidence has stalled in recent years. Gene drive-based approaches utilizing Cas9/guide RNA (gRNA) systems are being developed to suppress anopheline populations or modify them by increasing their refractoriness to the parasites. These systems rely on the successful cleavage of a chromosomal DNA target site followed by homology-directed repair (HDR) in germline cells to bias inheritance of the drive system. An optimal drive system should be highly efficient for HDR-mediated gene conversion with minimal error rates. A gene-drive system, AgNosCd-1, with these attributes has been developed in the Anopheles gambiae G3 strain and serves as a framework for further development of population modification strains. To validate AgNosCd-1 as a versatile platform, it must perform well in a variety of genetic backgrounds. Results We introduced or introgressed AgNosCd-1 into different genetic backgrounds, three in geographically-diverse Anopheles gambiae strains, and one each in an An. coluzzii and An. arabiensis strain. The overall drive inheritance, determined by presence of a dominant marker gene in the F2 hybrids, far exceeded Mendelian inheritance ratios in all genetic backgrounds that produced viable progeny. Haldane’s rule was confirmed for AgNosCd-1 introgression into the An. arabiensis Dongola strain and sterility of the F1 hybrid males prevented production of F2 hybrid offspring. Back-crosses of F1 hybrid females were not performed to keep the experimental design consistent across all the genetic backgrounds and to avoid maternally-generated mutant alleles that might confound the drive dynamics. DNA sequencing of the target site in F1 and F2 mosquitoes with exceptional phenotypes revealed drive system-generated mutations resulting from non-homologous end joining events (NHEJ), which formed at rates similar to AgNosCd-1 in the G3 genetic background and were generated via the same maternal-effect mechanism. Conclusions These findings support the conclusion that the AgNosCd-1 drive system is robust and has high drive inheritance and gene conversion efficiency accompanied by low NHEJ mutation rates in diverse An. gambiae s.l. laboratory strains.

Aedes albopictus is an important vector of arboviruses and prefers small containers of stagnant water as oviposition sites. One of the mechanisms mosquitoes use to search for suitable oviposition sites is relying on odor cues from prospective sites and their surroundings. The genetic and molecular bases of this behavior are not known for Ae. albopictus. Oviposition site-searching behavior can be separated into 2 stages: container location and water detection. We applied a glue compound to the antennae and the maxillary palps of adult females to mask their ability to detect molecules that may guide them to preferred oviposition sites. Treatment of the antennae significantly reduces the location index (P < 0.001), indicating a decreased ability to find oviposition sites, whereas no significant difference was observed in mosquitoes with maxillary palps treated with the same glue compound (P > 0.05). The detection time, measured as the duration from contact with the water surface to the deposition of the first egg, was extended in mosquitoes with treated antennae or maxillary palps, supporting the conclusion that olfaction is involved in the detection of oviposition site. Transcriptomic analysis identified differentially expressed olfactory-related genes, including obp67, obp56d-like, obp19d-like and obp67-like. RNA interference (RNAi)-mediated knockdown of obp67 and obp56d-like significantly affected the location index and detection time, respectively. Cas9/guide RNA-mediated knockout of obp56d-like resulted in a prolonged detection time, compared with the wild type (P < 0.05). These findings help to elucidate aspects of the olfactory mechanisms involved in Ae. albopictus oviposition site selection, and provide a basis for the development of mosquito surveillance and control strategies.

Sustainable reductions in African malaria transmission require innovative tools for mosquito control. One proposal involves the use of low-threshold gene drive in Anopheles vector species, where a ‘causal pathway’ would be initiated by (i) the release of a gene drive system in target mosquito vector species, leading to (ii) its transmission to subsequent generations, (iii) its increase in frequency and spread in target mosquito populations, (iv) its simultaneous propagation of a linked genetic trait aimed at reducing vectorial capacity for Plasmodium, and (v) reduced vectorial capacity for parasites in target mosquito populations as the gene drive system reaches fixation in target mosquito populations, causing (vi) decreased malaria incidence and prevalence. Here the scope, objectives, trial design elements, and approaches to monitoring for initial field releases of such gene dive systems are considered, informed by the successful implementation of field trials of biological control agents, as well as other vector control tools, including insecticides, Wolbachia, larvicides, and attractive-toxic sugar bait systems. Specific research questions to be addressed in initial gene drive field trials are identified, and adaptive trial design is explored as a potentially constructive and flexible approach to facilitate testing of the causal pathway. A fundamental question for decision-makers for the first field trials will be whether there should be a selective focus on earlier points of the pathway, such as genetic efficacy via measurement of the increase in frequency and spread of the gene drive system in target populations, or on wider interrogation of the entire pathway including entomological and epidemiological efficacy. How and when epidemiological efficacy will eventually be assessed will be an essential consideration before decisions on any field trial protocols are finalized and implemented, regardless of whether initial field trials focus exclusively on the measurement of genetic efficacy, or on broader aspects of the causal pathway. Statistical and modelling tools are currently under active development and will inform such decisions on initial trial design, locations, and endpoints. Collectively, the considerations here advance the realization of developer ambitions for the first field trials of low-threshold gene drive for malaria vector control within the next 5 years.

The vast majority of all global species have circadian rhythm cycles that allow them to adapt to natural environments. These regular rhythms are regulated by core clock genes and recent studies have also implicated roles for microRNAs in this regulation. Oviposition is an important circadian behavior in the reproductive cycle of insect vectors of diseases, and little is known about the rhythm or its regulation in mosquitoes. Aedes albopictus is a diurnal mosquito that transmits arboviruses and is the major cause of outbreaks of dengue fever in China. We analyzed the oviposition rhythm patterns of A. albopictus under different light/dark conditions and show that the mosquitoes have an oviposition peak between zeitgeber time 9 (ZT 9) and ZT 12. Furthermore, the antagomir‐mediated knockdown of expression of the microRNA miR‐2940‐1 affected the oviposition rhythm of A. albopictus . These data support the conclusion that miR‐2940‐1 is involved in the regulation of oviposition rhythm in A. albopictus and provide a foundation for using oviposition rhythms as a new target for vector mosquito control.

The field-testing and eventual adoption of genetically-engineered mosquitoes (GEMs) to control vector-borne pathogen transmission will require them meeting safety criteria specified by regulatory authorities in regions where the technology is being considered for use and other locales that might be impacted. Preliminary risk considerations by researchers and developers may be useful for planning the baseline data collection and field research used to address the anticipated safety concerns. Part of this process is to identify potential hazards (defined as the inherent ability of an entity to cause harm) and their harms, and then chart the pathways to harm and evaluate their probability as part of a risk assessment. The University of California Malaria Initiative (UCMI) participated in a series of workshops held to identify potential hazards specific to mosquito population modification strains carrying gene-drive systems coupled to anti-parasite effector genes and their use in a hypothetical island field trial. The hazards identified were placed within the broader context of previous efforts discussed in the scientific literature. Five risk areas were considered i) pathogens, infections and diseases, and the impacts of GEMs on human and animal health, ii) invasiveness and persistence of GEMs, and interactions of GEMs with target organisms, iii) interactions of GEMs with non-target organisms including horizontal gene transfer, iv) impacts of techniques used for the management of GEMs and v) evolutionary and stability considerations. A preliminary hazards list (PHL) was developed and is made available here. This PHL is useful for internal project risk evaluation and is available to regulators at prospective field sites. UCMI project scientists affirm that the subsequent processes associated with the comprehensive risk assessment for the application of this technology should be driven by the stakeholders at the proposed field site and areas that could be affected by this intervention strategy.

Transposon-mediated transgenesis has revolutionized both basic and applied studies of mosquito vectors of disease. Currently, techniques such as enhancer traps and transposon tagging, which rely on remobilizable insertional mutagenesis, are only possible with transposon-based vector systems. Here, we provide general descriptions of methods and applications of transposon-based mosquito transgenesis. The exact procedures must be adapted to each mosquito species and comparisons of some differences among different mosquito species are outlined. A number of excellent publications showing detailed and specific protocols and methods are featured and referenced.

Transposon-mediated transgenesis of mosquito vectors of disease pathogens followed the early success of transgenesis in the vinegar fly, Drosophila melanogaster The P transposable element used in Drosophila does not function canonically in mosquitoes, and repeatable, routine transgenesis in mosquitoes was not accomplished until new transposable elements were discovered and validated. A number of distinct transposons were subsequently identified that mediate the introduction of exogenous DNA in a stable and heritable manner in mosquito species, including members of the genera Aedes, Anopheles, and Culex The most versatile element, piggyBac, is functional in all of these mosquito genera, as well as in many other insects in diverse orders, and has been used extensively outside the class. Transposon-mediated transgenesis of recessive and dominant marker genes and reporter systems has been used to define functional fragments of gene control sequences, introduce exogenous DNA encoding products beneficial to medical interests, and act as "enhancer traps" to identify endogenous genes with specific expression characteristics.

Background Worldwide invasion and expansion of Aedes albopictus , an important vector of dengue, chikungunya, and Zika viruses, has become a serious concern in global public health. Chemical insecticides are the primary means currently available to control the mosquito populations. However, long-term and large-scale use of insecticides has selected for resistance in the mosquito that is accompanied by a genetic load that impacts fitness. Results A number of laboratory strains representing different resistance mechanisms were isolated and identified from laboratory-derived, deltamethrin-resistant Ae. albopictus recovered in previous work. Resistance levels and fitness costs of the strains were evaluated and compared to characterize the evolution of the resistance genotypes and phenotypes. The heterozygous F1534S mutation (1534 F/S ) in the voltage gated sodium channel ( vgsc ) gene product (VGSC), first detected in early stages of resistance evolution, not only confers high-level resistance, but also produces no significant fitness costs, leading to the rapid spread of resistance in the population. This is followed by the increase in frequency of homozygous F1534S (1534 S/S ) mosquitoes that have significant fitness disadvantages, prompting the emergence of an unlinked I1532T mutation with fewer side effects and a mating advantage better adapted to the selection and reproductive pressures imposed in the experiments. Metabolic resistance with no significant fitness cost and mediating a high-tolerance resistance phenotype may play a dominant role in the subsequent evolution of resistance. The different resistant strains had similar vector competence for dengue virus type-2 (DENV-2). Furthermore, a comparative analysis of vectorial capacity revealed that increased survival due to deltamethrin resistance balanced the negative fitness cost effects and contributed to the risk of dengue virus (DENV) transmission by resistant populations. The progressive evolution of resistance results in mosquitoes with both target-site insensitivity and metabolic resistance with lower fitness costs, which further leads to resistant populations with both high resistance levels and vectorial capacity. Conclusions This study reveals a possible mechanism for the evolution of deltamethrin resistance in Aedes albopictus . These findings will help guide practical strategies for insecticide use, resistance management and the prevention and control of mosquito-borne disease.