Sex peptide receptor is not required for refractoriness to remating or induction of egg laying in Aedes aegypti.

Across diverse insect taxa, the behavior and physiology of females dramatically changes after mating-processes largely triggered by the transfer of seminal proteins from their mates. In the vinegar fly Drosophila melanogaster, the seminal protein sex peptide (SP) decreases the likelihood of female flies remating and causes additional behavioral and physiological changes that promote fertility including increasing egg production. Although SP is only found in the Drosophila genus, its receptor, sex peptide receptor (SPR), is the widely conserved myoinhibitory peptide (MIP) receptor.

An expanded neurogenetic toolkit to decode olfaction in the African malaria mosquito Anopheles gambiae.

Anopheles gambiae uses its sense of smell to hunt humans. We report a two-step method yielding cell-type-specific driver lines for enhanced neuroanatomical and functional studies of its olfactory system. We first integrated a driver-responder-marker (DRM) system cassette consisting of a linked T2A-QF2 driver, QUAS-GFP responder, and a gut-specific transgenesis marker into four chemoreceptor genes (Ir25a, Ir76b, Gr22, and orco) using CRISPR-Cas9-mediated homology-directed repair.

Quantifying Mosquito Host Preference.

The most dangerous mosquito species for human health are those that blood feed preferentially and frequently on humans (anthropophilic mosquitoes). These include prolific disease vectors such as the African malaria mosquito and yellow fever mosquito The chemosensory basis for anthropophilic behavior exhibited by these disease vectors, as well as the factors that drive interindividual differences in human attractiveness to mosquitoes, remain largely uncharacterized. Here, we concisely review established methods to quantify mosquito interspecific and intraspecific host preference in the laboratory, as well as semi-field and field environments.

A Behavioral Assay to Quantify Odor-Guided Thermotaxis with under Semi-Field Conditions.

The African malaria mosquito is strongly attracted to human body odor and skin temperature. Quantitative behavioral assays suitable for use in semi-field environments with this nocturnal mosquito species are essential to gain improved insights into sensory biology, the mechanistic basis of mosquito attraction to humans, and host preference. In this protocol, we describe steps for engineering equipment for a novel behavioral assay for , which we have termed the odor-guided thermotaxis assay (OGTA).

Quantification of Olfactory Preferences under Semi-Field Conditions.

is a highly anthropophilic (human-preferring) malaria vector that prefers to blood feed frequently and selectively on humans. This mosquito species exhibits a strong innate olfactory preference to seek out human scent over other animals, and certain humans over others-key behavioral traits with the potential to drive heterogeneity in biting risk and malaria transmission. Here, we describe the application of a large-scale, semi-field system in Zambia for the quantification of olfactory preferences toward whole body odor sourced from individual humans.

Cuticular profiling of insecticide resistant Aedes aegypti.

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms.

Human scent guides mosquito thermotaxis and host selection under naturalistic conditions.

The African malaria mosquito Anopheles gambiae exhibits a strong innate drive to seek out humans in its sensory environment, classically entering homes to land on human skin in the hours flanking midnight. To gain insight into the role that olfactory cues emanating from the human body play in generating this epidemiologically important behavior, we developed a large-scale multi-choice preference assay in Zambia with infrared motion vision under semi-field conditions. We determined that An.

Cuticular profiling of insecticide resistant .

Insecticides have made great strides in reducing the global burden of vector-borne disease. Nonetheless, serious public health concerns remain because insecticide-resistant vector populations continue to spread globally. To circumvent insecticide resistance, it is essential to understand all contributing mechanisms.

A headspace collection chamber for whole body volatilomics.

The human body secretes a complex blend of volatile organic compounds (VOCs) the skin, breath and bodily fluids, the study of which can provide valuable insight into the physiological and metabolic state of an individual. Methods to profile human-derived volatiles typically source VOCs from bodily fluids, exhaled breath or skin of isolated body parts. To facilitate profiling the whole body volatilome, we have engineered a sampling chamber that enables the collection and analysis of headspace from the entire human body.

Single-Pair and Small-Group Crosses of .

is an emerging model insect species used in genetics studies because of its ease of laboratory rearing, desiccation-resistant eggs, expanding genetic toolkit, and high-quality reference genome. Here, we describe procedures to isolate and sex virgin female and male mosquitoes and establish successful mating crosses. We also detail how to blood feed mosquitoes from these crosses, isolate individual or small groups of females for egg laying, condition these eggs for storage and hatching, and verify female mating status.

Overview of and Use in Laboratory Studies.

The yellow fever mosquito is a prolific disease vector. This mosquito has been the subject of scientific investigation for more than a century. Continued research into biology is crucial for understanding how to halt the suite of major arthropod-borne viral diseases this mosquito transmits.

Batch Rearing .

Standardized rearing methods for the yellow fever mosquito are critical to facilitate controlled laboratory studies. This protocol describes a batch rearing protocol for stocks that yields healthy eggs, larvae, pupae, and adults in the laboratory for long-term colony maintenance and experimental manipulation. Foundational principles for the rearing and containment of these life cycle stages, as well as steps for mating and blood feeding to yield viable eggs for continuous culture or storage, are detailed.

CRISPR-Mediated Genome Engineering in Aedes aegypti.

CRISPR-mediated genome engineering technologies have been adapted to a wide variety of organisms with high efficiency and specificity. The yellow fever mosquito, Aedes aegypti , is one such organism. It is also responsible for transmitting a wide variety of deadly viruses including Dengue, Zika, Yellow fever, and Chikungunya.

An updated antennal lobe atlas for the yellow fever mosquito Aedes aegypti.

The yellow fever mosquito Aedes aegypti is a prolific vector of arboviral and filarial diseases that largely relies on its sense of smell to find humans. To facilitate in-depth analysis of the neural circuitry underlying Ae. aegypti olfactory-driven behaviors, we generated an updated in vitro atlas for the antennal lobe olfactory brain region of this disease vector using two independent neuronal staining methods.

Identification of Essential Oils with Strong Activity against Stationary Phase .

Lyme disease is the most common vector borne-disease in the United States (US). While the majority of the Lyme disease patients can be cured with 2⁻4 weeks antibiotic treatment, about 10⁻20% of patients continue to suffer from persisting symptoms. While the cause of this condition is unclear, persistent infection was proposed as one possibility.

A Peptide Signaling System that Rapidly Enforces Paternity in the Aedes aegypti Mosquito.

Female Aedes aegypti mosquitoes typically mate only once with one male in their lifetime, a behavior known as "monandry" [1]. This single mating event provisions the female with sufficient sperm to fertilize the >500 eggs she will produce during her ∼4- to 6-week lifespan in the laboratory [2]. Successful mating induces lifetime refractoriness to subsequent insemination by other males, enforcing the paternity of the first male [3-5].

Multimodal integration of carbon dioxide and other sensory cues drives mosquito attraction to humans.

Multiple sensory cues emanating from humans are thought to guide blood-feeding female mosquitoes to a host. To determine the relative contribution of carbon dioxide (CO2) detection to mosquito host-seeking behavior, we mutated the AaegGr3 gene, a subunit of the heteromeric CO2 receptor in Aedes aegypti mosquitoes. Gr3 mutants lack electrophysiological and behavioral responses to CO2.

Genomic evolution of the pathogenic Wolbachia strain, wMelPop.

Most strains of the widespread endosymbiotic bacterium Wolbachia pipientis are benign or behave as reproductive parasites. The pathogenic strain wMelPop is a striking exception, however: it overreplicates in its insect hosts and causes severe life shortening. The mechanism of this pathogenesis is currently unknown.

Draft genome sequence of the male-killing Wolbachia strain wBol1 reveals recent horizontal gene transfers from diverse sources.

Background: The endosymbiont Wolbachia pipientis causes diverse and sometimes dramatic phenotypes in its invertebrate hosts. Four Wolbachia strains sequenced to date indicate that the constitution of the genome is dynamic, but these strains are quite divergent and do not allow resolution of genome diversification over shorter time periods. We have sequenced the genome of the strain wBol1-b, found in the butterfly Hypolimnas bolina, which kills the male offspring of infected hosts during embyronic development and is closely related to the non-male-killing strain wPip from Culex pipiens.

A Wolbachia symbiont in Aedes aegypti disrupts mosquito egg development to a greater extent when mosquitoes feed on nonhuman versus human blood.

A vertebrate bloodmeal is required by female mosquitoes of most species to obtain nutrients for egg maturation. The yellowfever mosquito, Aedes aegypti (L.), feeds predominantly on humans, despite having the capacity to use blood from other hosts for this process.

A virulent Wolbachia infection decreases the viability of the dengue vector Aedes aegypti during periods of embryonic quiescence.

A new approach for dengue control has been proposed that relies on life-shortening strains of the obligate intracellular bacterium Wolbachia pipientis to modify mosquito population age structure and reduce pathogen transmission. Previously we reported the stable transinfection of the major dengue vector Aedes aegypti with a life-shortening Wolbachia strain (wMelPop-CLA) from the vinegar fly Drosophila melanogaster. Here, we report a further characterization of the phenotypic effects of this virulent Wolbachia infection on several life-history traits of Ae.

Increased locomotor activity and metabolism of Aedes aegypti infected with a life-shortening strain of Wolbachia pipientis.

A virulent strain of the obligate intracellular bacterium Wolbachia pipientis that shortens insect lifespan has recently been transinfected into the primary mosquito vector of dengue virus, Aedes aegypti L. The microbe's ability to shorten lifespan and spread through host populations under the action of cytoplasmic incompatibility means it has the potential to be used as a biocontrol agent to reduce dengue virus transmission. Wolbachia is present in many host tissues and may have local effects on diverse biological processes.

Stable introduction of a life-shortening Wolbachia infection into the mosquito Aedes aegypti.

Most pathogens require a relatively long period of development in their mosquito vector before they can be transmitted to a new human host; hence, only older insects are of epidemiological importance. The successful transfer of a life-shortening strain of the inherited bacterial symbiont, Wolbachia, into the major mosquito vector of dengue, Aedes aegypti, halved adult life span under laboratory conditions. The association is stable, and the Wolbachia strain is maternally inherited at high frequency.

Host adaptation of a Wolbachia strain after long-term serial passage in mosquito cell lines.

The horizontal transfer of the bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. The experimental transfer of Wolbachia between distantly related host species often results in the loss of infection, presumably due to an inability of Wolbachia to adapt quickly to the new host. To examine the process of adaptation to a novel host, we transferred a life-shortening Wolbachia strain, wMelPop, from the fruit fly Drosophila melanogaster into a cell line derived from the mosquito Aedes albopictus.

Modifying insect population age structure to control vector-borne disease.

Age is a critical determinant of the ability of most arthropod vectors to transmit a range of human pathogens. This is due to the fact that most pathogens require a period of extrinsic incubation in the arthropod host before pathogen transmission can occur. This developmental period for the pathogen often comprises a significant proportion of the expected lifespan of the vector.