Seminal fluid proteins induce transcriptome changes in the Aedes aegypti female lower reproductive tract.

BACKGROUND: Mating induces behavioral and physiological changes in the arbovirus vector Aedes aegypti, including stimulation of egg development and oviposition, increased survival, and reluctance to re-mate with subsequent males. Transferred seminal fluid proteins and peptides derived from the male accessory glands induce these changes, though the mechanism by which they do this is not known. RESULTS: To determine transcriptome changes induced by seminal proteins, we injected extract from male accessory glands and seminal vesicles (MAG extract) into females and examined female lower reproductive tract (LRT) transcriptomes 24 h later, relative to non-injected controls. MAG extract induced 87 transcript-level changes, 31 of which were also seen in a previous study of the LRT 24 h after a natural mating, including 15 genes with transcript-level changes similarly observed in the spermathecae of mated females. The differentially-regulated genes are involved in diverse molecular processes, including immunity, proteolysis, neuronal function, transcription control, or contain predicted small-molecule binding and transport domains. CONCLUSIONS: Our results reveal that seminal fluid proteins, specifically, can induce gene expression responses after mating and identify gene targets to further investigate for roles in post-mating responses and potential use in vector control.

Proteins, Transcripts, and Genetic Architecture of Seminal Fluid and Sperm in the Mosquito Aedes aegypti.

The yellow fever mosquito, Aedes aegypti,, transmits several viruses causative of serious diseases, including dengue, Zika, and chikungunya. Some proposed efforts to control this vector involve manipulating reproduction to suppress wild populations or to replace them with disease-resistant mosquitoes. The design of such strategies requires an intimate knowledge of reproductive processes, yet our basic understanding of reproductive genetics in this vector remains largely incomplete. To accelerate future investigations, we have comprehensively catalogued sperm and seminal fluid proteins (SFPs) transferred to females in the ejaculate using tandem mass spectrometry. By excluding female-derived proteins using an isotopic labeling approach, we identified 870 sperm proteins and 280 SFPs. Functional composition analysis revealed parallels with known aspects of sperm biology and SFP function in other insects. To corroborate our proteome characterization, we also generated transcriptomes for testes and the male accessory glands-the primary contributors to Ae. aegypti, sperm and seminal fluid, respectively. Differential gene expression of accessory glands from virgin and mated males suggests that transcripts encoding proteins involved in protein translation are upregulated post-mating. Several SFP transcripts were also modulated after mating, but >90% remained unchanged. Finally, a significant enrichment of SFPs was observed on chromosome 1, which harbors the male sex determining locus in this species. Our study provides a comprehensive proteomic and transcriptomic characterization of ejaculate production and composition and thus provides a foundation for future investigations of Ae. aegypti, reproductive biology, from functional analysis of individual proteins to broader examination of reproductive processes.

A mosquito sperm's journey from male ejaculate to egg: Mechanisms, molecules, and methods for exploration.

The fate of mosquito sperm in the female reproductive tract has been addressed sporadically and incompletely, resulting in significant gaps in our understanding of sperm-female interactions that ultimately lead to fertilization. As with other Diptera, mosquito sperm have a complex journey to their ultimate destination, the egg. After copulation, sperm spend a short time at the site of insemination where they are hyperactivated and quickly congregate near the entrance of the spermathecal ducts. Within minutes, they travel up the narrow ducts to the spermathecae, likely through the combined efforts of female transport and sperm locomotion. The female nourishes sperm and maintains them in these permanent storage organs for her entire life. When she is ready, the female coordinates the release of sperm with ovulation, and the descending egg is fertilized. Although this process has been well studied via microscopy, many questions remain regarding the molecular processes that coordinate sperm motility, movement through the reproductive tract, maintenance, and usage. In this review, we describe the current understanding of a mosquito sperm's journey to the egg, highlighting gaps in our knowledge of mosquito reproductive biology. Where insufficient information is available in mosquitoes, we describe analogous processes in other organisms, such as Drosophila melanogaster, as a basis for comparison, and we suggest future areas of research that will illuminate how sperm successfully traverse the female reproductive tract. Such studies may yield molecular targets that could be manipulated to control populations of vector species. Mol. Reprod. Dev. 83: 897-911, 2016 © 2016 Wiley Periodicals, Inc.

Measuring insecticide resistance in a vacuum: exploring next steps to link resistance data with mosquito control efficacy.

Insecticide resistance is a great challenge facing mosquito operational control agencies across the United States, where few active ingredients with unique modes of action are available for use, increasing resistance pressure and further hampering resistance management strategies. Emergence and expansion of insecticide resistance in mosquitoes can be detected by resistance monitoring programs; however, there are gaps in our knowledge regarding the link between resistance bioassay results and operational control outcomes. Here, we review both public health and agricultural studies on pesticide resistance bioassays and control outcomes. A discussion on the main gaps in our knowledge of insecticide resistance and a review of resistance management practices is also presented. We conclude with research questions that can advance our understanding of resistance monitoring and control.

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. To test the functional role of SPR in mediating postmating responses in a non-Drosophila dipteran, we generated 2 independent Spr-knockout alleles in the yellow fever mosquito, Aedes aegypti. Although SPR is needed for postmating responses in Drosophila and the cotton bollworm Helicoverpa armigera, Spr mutant Ae. aegypti show completely normal postmating decreases in remating propensity and increases in egg laying. In addition, injection of synthetic SP or accessory gland homogenate from D. melanogaster into virgin female mosquitoes did not elicit these postmating responses. Our results demonstrate that Spr is not required for these canonical postmating responses in Ae. aegypti, indicating that other, as yet unknown, signaling pathways are likely responsible for these behavioral switches in this disease vector.

Associative learning in the dengue vector mosquito, Aedes aegypti: avoidance of a previously attractive odor or surface color that is paired with an aversive stimulus.

Associative learning has been shown in a variety of insects, including the mosquitoes Culex quinquefasciatus and Anopheles gambiae. This study demonstrates associative learning for the first time in Aedes aegypti, an important vector of dengue, yellow fever and chikungunya viruses. This species prefers to rest on dark surfaces and is attracted to the odor of 1-octen-3-ol. After training in which a dark surface alone or a dark surface with odor was paired with electric shock, mosquitoes avoided the previously attractive area. The association was stronger when odor was included in training, was retained for at least 60 min but not for 24 h, and was equal for males and females. These results demonstrate the utility of a bulk-training paradigm for mosquitoes similar to that used with Drosophila melanogaster.

Northeast Regional Center for Excellence in Vector-Borne Diseases' Master of Science training program: a curriculum to support future capacity in public health entomology.

A major lack of expertise in vector biology, surveillance, and control for public health professionals has been acknowledged over the past several decades, especially in light of the introduction of West Nile and Zika viruses to the United States. To address this growing need, the Northeast Regional Center for Excellence in Vector-Borne Diseases (NEVBD) designed a unique educational program to cross-train students in the fundamentals of vector biology and public health. Here, we summarize the formation, evaluation, and outcomes of NEVBD's Master of Science in Entomology: Vector-Borne Disease Biology program and provide details on core competencies to enable adoption and adaptation of the program to other institutions and contexts. A discussion of major barriers to filling the nation's need for public health personnel with medical entomology training, such as financial barriers and recruitment of underrepresented students, is presented. We conclude with considerations for administering these training programs.

Knowledge, attitudes, and practices for tick bite prevention and tick control among residents of Long Island, New York, USA.

In the United States, tick-borne diseases (TBDs) are a major public health concern and a source of significant morbidity. To reduce tick-borne disease incidence, understanding factors associated with increased risk in humans for tick bites is necessary. These risk factors can include TBD knowledge, attitudes about prevention and care, and associated practices of individuals and their communities, including paying for preventive services. Our study focused on Long Island, New York, a region with high endemicity of ticks and TBDs. The purpose of our study was to identify gaps in the knowledge, attitudes, and practices regarding TBDs in residents of this region to inform priorities for TBD interventions. To this end, we performed a knowledge, attitudes, and practices (KAP) survey and collected 803 responses from Long Island residents. Our survey results demonstrated that Long Island residents had a low to moderate level of knowledge regarding ticks and TBDs. Still, residents expressed concern regarding TBDs, with increased levels of concern associated with increased likelihood of paying for tick control and for practicing tick bite prevention strategies. Individuals with pets were more likely to practice these tick bite prevention strategies. Residents with more frequent exposure to ticks, with a history of TBD in their household, and with higher general concern over ticks were also more likely to pay for tick control services. These findings highlight gaps in resident knowledge, differences in attitudes towards tick preventative behavior, and potential factors affecting motivation towards implementing tick control measures, which can inform future public health messaging regarding tick bite prevention.

Jamestown Canyon virus (Bunyavirales: Peribunyaviridae) vector ecology in a focus of human transmission in New Hampshire, USA.

Jamestown Canyon virus disease (JCVD) is a potentially neuroinvasive condition caused by the arbovirus Jamestown Canyon virus (JCV). Human cases of JCVD have increased in New Hampshire (NH) over the past decade, but vector surveillance is limited by funding and person power. We conducted mosquito surveillance with a focus on human JCVD cases south central NH during 2021. Routine surveillance with CDC miniature traps baited with CO2 (lights removed) was supplemented by a paired trapping design to test the collection efficiency of octenol, and New Jersey light traps. We performed virus testing, blood meal analysis, and compared morphological identification with DNA barcoding. Over 50,000 mosquitoes were collected representing 28 species. Twelve JCV-positive pools were derived from 6 species of more than 1,600 pools tested. Of those, Aedes excrucians/stimulans (MLE 4.95, Diptera: Culicidae, Walker, 1856, 1848), and Aedes sticticus (MLE 2.02, Meigen, 1838) had the highest JCV infection rates, and Aedes canadensis (MLE 0.13, Theobold, 1901) and Coquillettidia perturbans (0.10, Diptera: Culicidae, Walker, 1856) had the lowest infection rates. One hundred and fifty-one blood meals were matched to a vertebrate host. All putative vectors fed on the amplifying host of JCV, white-tailed deer (36-100% of bloodmeals). Putative vectors that fed on human hosts included Aedes excrucians (8%), Anopheles punctipennis (25%, Diptera: Culicidae, Say, 1823), and Coquillettidia perturbans (51%). CDC traps baited with CO2 were effective for collecting putative vectors. DNA barcoding enhanced morphological identifications of damaged specimens. We present the first ecological overview of JCV vectors in NH.

Utilization of a zoo for mosquito (Diptera: Culicidae) diversity analysis, arboviral surveillance, and blood feeding patterns.

Zoos provide a unique opportunity to study mosquito feeding ecology as they represent areas where exotic animals, free-roaming native animals, humans, and mosquito habitats overlap. Therefore, these locations are a concern for arbovirus transmission to both valuable zoo animals and human visitors. We sampled mosquitoes in and around The Nashville Zoo at Grassmere in Tennessee, USA, over 4 months in 2020 using 4 mosquito trap methods and 12 sampling locations. Mosquitoes were identified to species, Culex mosquitoes were analyzed for arboviruses, and all engorged mosquitoes were preserved for host usage analysis. We captured over 9,000 mosquitoes representing 27 different species, including a new species record for Davidson County, TN (Culex nigripalpus Theobald). Minimum infection rates for West Nile virus (WNV) (Flaviviridae: Flavivirus), St. Louis encephalitis virus (Flaviviridae: Flavivirus), and Flanders virus (Hapavirus: Rhabdoviridae) were 0.79, 0, and 4.17, respectively. The collection of 100 engorged mosquitoes was dominated by Culex pipiens pipiens Linnaeus (38%), Culex erraticus Dyar and Knab (23%), and Culex pipiens pipiens-Culex pipiens quinquefasciatus hybrids (10%). Host DNA from 84 engorged mosquitoes was successfully matched to a variety of host species (n = 23), with just 8 species belonging to the zoo. Wild birds were the most frequently fed upon host, in particular northern cardinals (Cardinalis cardinalis L. Passeriformes: Cardinalidae), which are competent WNV reservoirs. Taken together, our results demonstrate the utility of zoos as sentinels for emerging pathogens, for studying wildlife and human risk of zoonotic diseases, and for assessing vector diversity.

New York State Tick Blitz: harnessing community-based science to understand range expansion of ticks.

Ticks and tick-borne diseases are an immense public health burden in New York State (NYS), United States. Tick species and associated pathogens are expanding into new areas, shifting threats to human, and animal health across the state. For example, the invasive tick, Haemaphysalis longicornis Neumann (Acari: Ixodidae), was first detected in the United States in 2017 and has since been identified in 17 states, including NYS. In addition, Amblyomma americanum (L.) (Acari: Ixodidae) is a native tick thought to be reestablishing historical populations in NYS. We implemented a community-based science project called the "NYS Tick Blitz" to determine the distribution of A. americanum and H. longicornis in NYS. Community volunteers were recruited, provided with education, training, and materials to conduct active tick sampling during a 2-wk period in June 2021. Fifty-nine volunteers sampled 164 sites across 15 counties, for a total of 179 separate collection events and 3,759 ticks collected. The most frequently collected species was H. longicornis, followed by Dermacentor variabilis Say (Acari: Ixodidae), Ixodes scapularis Say (Acari: Ixodidae), and A. americanum, respectively. Through the NYS Tick Blitz collections, H. longicornis was identified for the first time in Putnam County. We conducted pooled pathogen testing on a subset of specimens, with the highest rates of infection detected for pathogens transmitted by I. scapularis, including Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti. Most participants who completed a follow-up survey (n = 23, 71.9%) were promoters of the NYS Tick Blitz and 50% (n = 15) reported that they enjoyed participating in meaningful science.

Accurate age-grading of field-aged mosquitoes reared under ambient conditions using surface-enhanced Raman spectroscopy and artificial neural networks.

Age-grading mosquitoes are significant because only older mosquitoes are competent to transmit pathogens to humans. However, we lack effective tools to do so, especially at the critical point where mosquitoes become a risk to humans. In this study, we demonstrated the capability of using surface-enhanced Raman spectroscopy and artificial neural networks to accurately age-grade field-aged low-generation (F2) female Aedes aegypti mosquitoes held under ambient conditions (error was 1.9 chronological days, in the range 0-22 days). When degree days were used for model calibration, the accuracy was further improved to 20.8 degree days (approximately equal to 1.4 chronological days), which indicates the impact of temperature fluctuation on prediction accuracy. This performance is a significant advancement over binary classification. The great accuracy of this method outperforms traditional age-grading methods and will facilitate effective epidemiological studies, risk assessment, vector intervention monitoring, and evaluation.

Aedes albopictus host odor preference does not drive observed variation in feeding patterns across field populations.

Laboratory and field-based studies of the invasive mosquito Aedes albopictus demonstrate its competency to transmit over twenty different pathogens linked to a broad range of vertebrate hosts. The vectorial capacity of Ae. albopictus to transmit these pathogens remains unclear, partly due to knowledge gaps regarding its feeding behavior. Blood meal analyses from field-captured specimens have shown vastly different feeding patterns, with a wide range of anthropophagy (human feeding) and host diversity. To address this knowledge gap, we asked whether differences in innate host preference may drive observed variation in Ae. albopictus feeding patterns in nature. Low generation colonies (F2-F4) were established with field-collected mosquitoes from three populations with high reported anthropophagy (Thailand, Cameroon, and Florida, USA) and three populations in the United States with low reported anthropophagy (New York, Maryland, and Virginia). The preference of these Ae. albopictus colonies for human versus non-human animal odor was assessed in a dual-port olfactometer along with control Ae. aegypti colonies already known to show divergent behavior in this assay. All Ae. albopictus colonies were less likely (p < 0.05) to choose the human-baited port than the anthropophilic Ae. aegypti control, instead behaving similarly to zoophilic Ae. aegypti. Our results suggest that variation in reported Ae. albopictus feeding patterns are not driven by differences in innate host preference, but may result from differences in host availability. This work is the first to compare Ae. albopictus and Ae. aegypti host preference directly and provides insight into differential vectorial capacity and human feeding risk.

Southwestern national park service employee risk, knowledge, and concern for triatomine exposure: A qualitative analysis using a novel knowledge, attitudes, and practices survey.

Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a neglected parasitic infection in the United States (US). In the Southwestern US, National Park Service (NPS) employees are a unique population with potential exposure to CD. This population lives in close contact with several species of sylvatic triatomine bugs, the vectors of T. cruzi, that may enter residential buildings at night. Despite the higher potential risk of CD transmission for southwestern NPS employees, the socio-cultural factors that impact autochthonous CD transmission in the US remain unknown. To address this gap, we investigated how NPS employee knowledge and attitudes impact their triatomine preventive behaviors. We distributed a 42-item online questionnaire to NPS employees at four national parks in Arizona and Texas. We detected high self-reported bite exposure in NPS housing, despite moderate- to high-frequency of prevention behaviors. Specific behaviors, such as often or always repairing window screens, were associated with a decreased risk of putative triatomine bug exposure. Additionally, NPS employees had low knowledge of CD. For those with greater knowledge of CD, it was not associated with increased frequency of prevention behavior. We found that increased CD anxiety was associated with increased personal agency to reduce the risk of CD. These results demonstrate the influence of knowledge and attitudes regarding CD on triatomine prevention behavior within a potential high-risk population in the US, and the importance of utilizing strategies beyond provision of education to influence behaviors.

Recording and Analysis of Mosquito Acoustic-Related Mating Behavior.

In mosquitoes, courtship and mating sounds are produced by the movement of the wings during flight. These sounds, usually referred to as flight tones, have been studied using tethered and free-flying individuals. Here, we describe a general approach for recording and analyzing mosquito acoustic-related mating behaviors that can be broadly adapted to a variety of experimental designs.

Acoustic-Related Mating Behavior in Tethered and Free-Flying Mosquitoes.

Acoustics play an essential role in mosquito communication, particularly during courtship and mating. Mosquito mating occurs in flight and is coordinated by the perception of wingbeat tones. Flight tone frequencies have been shown to mediate sex recognition in Aedes, Anopheles, Culex, and Toxorhynchites genera and are thus a conserved feature of mating across the mosquito family (Culicidae). Upon recognizing a flying female, males respond phonotactically by lunging toward the female and initiating a precopulatory courtship flight interaction. During this interaction, males and females often harmonize their flight tones in a behavior known as harmonic convergence, and male acoustics display rapid frequency modulation. These acoustic phenomena have been characterized both in tethered and free-flying mosquitoes using similar audio recording and analysis methods. Further, the manipulation of mosquito acoustic-related mating behavior shows great promise as a tool for reproductive control strategies. In this brief methodological introduction, we provide an overview of the biological and technical concepts necessary for understanding the recording and analysis of mosquito mating acoustics.

Distance to public transit predicts spatial distribution of dengue virus incidence in Medellín, Colombia.

Dengue is a growing global threat in some of the world's most rapidly growing landscapes. Research shows that urbanization and human movement affect the spatial dynamics and magnitude of dengue outbreaks; however, precise effects of urban growth on dengue are not well understood because of a lack of sufficiently fine-scaled data. We analyzed nine years of address-level dengue case data in Medellin, Colombia during a period of public transit expansion. We correlate changes in the spread and magnitude of localized outbreaks to changes in accessibility and usage of public transit. Locations closer to and with a greater utilization of public transit had greater dengue incidence. This relationship was modulated by socioeconomic status; lower socioeconomic status locations experienced stronger effects of public transit accessibility and usage on dengue incidence. Public transit is a vital urban resource, particularly among low socioeconomic populations. These results highlight the importance of public health services concurrent with urban growth.

Effectiveness of a Buffalo Turbine and A1 Mist Sprayer for the Areawide Deployment of Larvicide for Mosquito Control in an Urban Residential Setting.

The control of medically important container-inhabiting mosquitoes is an ongoing challenge for mosquito control operations. Truck-mounted application equipment is a common option for rapid areawide larvicide deployment utilized by mosquito control operations. We tested the effectiveness of two truck-mounted sprayers (A1 Super Duty + Buffalo Turbine CSM3), for the deployment of water-dispersible biopesticides (VectoBac WDG:VectoLex WDG 50:50). Sixty residences within four residential neighborhoods in New Jersey were treated in 2019 and 2020. Three empty bioassay cups were placed in specific locations on each property (front yard/ back yard/ side of house), with an additional cup placed in an adjacent catch basin. This approach was replicated in two untreated control neighborhoods. Following larvicide application, cups were subjected to bioassays wherein larval mortality was tracked through adult eclosion. Overall, average larval mortality rates were 56% higher in treated cups compared against untreated controls. Mortality rates were affected by cup location, with 39% mortality in bioassay cups from back yards, 54% in those from the sides of houses, 73% in front yards, and 76% from cups in catch basins. Mortality did not differ significantly between the four treated neighborhoods, nor by the type of sprayer used. Our research shows that truck-mounted sprayers can be an effective method for larvicide deployment in residential neighborhoods, but effectiveness may depend upon the location of the target treatment area in relation to residences and other geographic obstacles.

Quantitative age grading of mosquitoes using surface-enhanced Raman spectroscopy.

Mosquito-borne pathogens, including malaria, Zika, dengue, and chikungunya continue to be a major public health concern globally. Based on the understanding that only older female mosquitoes are infectious and represent a risk to human health, scientists have sought to age-grade mosquitoes for decades. To date, however, no reliable, cost-effective and practical methods exist to age older mosquitoes despite the tremendous epidemiological value of this approach. This study is the first attempt to develop a surface-enhanced Raman spectroscopic (SERS) method to age mosquitoes. The water extracts of Aedes aegypti mosquitoes aged 0-22 days were mixed with silver nanoparticles. The SERS spectra, which were analysed by principal component analysis and partial least square (PLS), demonstrated the capability of this approach to predict the calendar age of mosquitoes between 0 and 22 days with the coefficient of correlation (R) = 0.994 and 0.990 for PLS model calibration and validation, respectively. Spectral analysis with both SERS and infrared spectroscopy revealed the key biological sources leading to changes in spectra allowing mosquito age-grading is adenine-containing compounds and proteins. In addition, we evaluated the impact of two arthropod-borne pathogen deactivating pre-treatments (bleach and ethanol) on the discrimination capability of the SERS approach. The result shows the ethanol treatment has the potential to enhance the discrimination capability and the safety of the approach. This study represents the first step towards developing the SERS approach as a quick, reliable and field-deployable method for mosquito age-grading, which would significantly improve the effectiveness of vector-borne disease monitoring and prevention.

Male mating history and body size influence female fecundity and longevity of the dengue vector Aedes aegypti.

Male reproductive success is dependent on insemination success and reproductive output. During mating, male mosquitoes transfer not just sperm, but also seminal fluid proteins that may have profound effects on mated female biology and behavior. In this study, we investigated the role of male body size and mating history on semen depletion, female longevity, and reproductive success in Aedes aegypti L. Small and large males were mated in rapid succession with up to five females. Our results indicate that large males had greater mating capacity than small males. A reduction in fecundity by >50% was observed in females that were fourth to mate with small males in comparison with females that mated earlier in sequence. For females mated to large males, this reduction became evident for females that mated fifth in sequence. No loss of fertility (measured as hatch rate) was observed in females that were third-fifth in mating sequence compared with females mated to virgin males. When females were maintained on a low-quality (5% sucrose) diet, those mated to virgin males had a greater longevity compared with females mated third in sequence. We conclude that small males experience more rapid seminal depletion than large males, and discuss the role of semen depletion in the mated female. Our results contribute toward a better understanding of the complexity of Ae. aegypti mating biology and provide refined estimates of mating capacity for genetic control efforts.