The Applied Mosquito Research  program (AMRP) at FMEL conducts research of relevance to Florida mosquito control and public health agencies.  Originally intended to address mosquito control pesticide issues, it has recently been expanded to include issues important to Florida mosquito control in general.

Presently, the AMRP has three principal components:

• Mosquito Identification and DNA Sequence-Based Tools for mosquito and pathogen surveillance.
• Pesticide reduction and non-pesticide mosquito control strategies.
• Pesticide development, testing, and application

Mosquito Identification and DNA Sequence-Based Tools for Mosquito and Pathogen Surveillance.

Staff Scientist: Dr. Lawrence Reeves, Research Assistant Scientist

Project Synopsis: Focus is on the development of DNA barcode-based molecular approaches to surveilling mosquitoes and detecting pathogens, and investigating mosquito biology and ecology and translating these details to improved mosquito control strategies. A secondary focus, in collaboration with Dr. Nathan Burkett-Cadena, is the creation of an updated identification resource for the mosquitoes of Florida.

Primary Goals: Goals include (1) creation of a complete reference database of COI DNA barcoding gene sequences for all Florida and regional mosquito species, collected within the state and region.  A publicly accessible molecular and specimen-based reference collection of sequence data will be created to enhance our ability to identify mosquitoes and recognize introductions of non-native species, and will be a user-friendly alternative to morphology-based ID.  Creation of this database will facilitate next generation DNA sequencing and bioinformatic pipelines for the identification of bulk mosquito collections, environmental samples (e.g., water samples from potential larval habitats), and blood meals (2) which will provide efficient and straightforward methods for operational mosquito surveillance. I provide a DNA barcoding identification service to mosquito control districts to assist in the taxonomic identification of mosquito specimens that cannot be determined through the use of morphological keys (3). Please email lereeves@ufl.edu for details on specimen submission. In addition, Florida is a hotspot for non-native mosquito species, with ten new species detected in the state since 2000. Working with Dr. Burkett-Cadena, I am creating an updated morphological identification resource for the mosquitoes of Florida, including species missing from the current key (4).

Major Results to Date: This program initiated in August 2018 and my lab has begun collecting mosquito specimens for molecular processing. Creation of a complete reference database is prioritized, as specimens used to populate the database are also being used to illustrate the updated Mosquitoes of Florida. His lab is now set up to receive and identify mosquito specimens for mosquito control districts. Major results, below, are derived from work published in 2018, prior the establishment of my program.

Current activity: Since Darsie and Morris published the Keys to the Adult Females and Fourth Instar Larvae of the Mosquitoes of Florida (Diptera, Culicidae) in 2003, at least nine additional mosquito species have been reported from the state, some of them widespread and common. In collaboration with Dr. Nathan Burkett-Cadena, we are creating an updated and photographically illustrated resource for the morphological identification of Florida mosquitoes. While we expect this project to take approximately two years to compile photographs and prepare the text, I am preparing a review of the recently introduced mosquito species to Florida to immediately address the need to morphologically identify species that are not present in the current key. Coincident with this work, I am collecting molecular and morphological specimens to be used toward Goal 1 (above), prioritizing species groups that a preliminary analysis using GenBank-mined barcode sequences indicated were likely to yield undescribed cryptic species.

Pesticide Reduction and Non-Pesticide Mosquito Control Strategies

Staff Scientist: Dr. Dongyoung Shin, Research Assistant Professor

Project Synopsis: This program investigates the genomics and genetics of mosquitoes and determines any interactions between mosquitoes and mosquito-borne diseases at the molecular level.

Primary Goals: Developing novel, non-pesticide control strategies;  investigating the development of insecticide resistance in Florida mosquito populations by investigating interactions between mosquito and mosquito-borne diseases;  characterizing vector capacity (ability to transmit a pathogen) and the genes that are responsible for alteration of vector competence in pesticide-resistant mosquito populations.

Major Results to Date:

• Grated drain covers can serve as a barrier to Culex oviposition in storm drains: Mosquito breeding begins from oviposition. We tested two different physical structures with different types of openings. Most Culex mosquitoes were significantly disrupted from finding oviposition sites if the openings were divided into several small openings. This suggests that physical barriers with grating shape openings over mosquito breeding sites can be used as a method to decrease the number of Culex breeding sites

• Detection of Culex interrogator in multiple Florida counties, a non-native species morphologically similar to native species: Culex interrogator species, known to be abundant in Texas and Central America, were discovered for the first time in Florida. This species, like coronator or Culex declarator, appears to have come from Texas during the warm winter. This route can also be used by other mosquito species carrying vector-bone diseases. Thus, mosquito control needs to focus on this route to prevent invasive species and mosquito-borne diseases. Florida’s mosquito control districts should also be aware of the presence of this mosquito in the state as it is morphologically similar to Cx. restuans and Cx. quinquefasciatus,  important vectors of several arboviruses.

• Identification of the esterase, Temsha-est1, as an indicator of insecticide resistance in Culex nigripalpus: An esterase (Temsha-est1) in a field population of nigripalpus was assessed as an inducible marker of organophosphate insecticide resistance. I developed a single vial bioassay to check the resistance levels of individual mosquitoes using individual mosquito RNA for further analysis. The expression level of Temsha-est1 showed a positive relation with the level of insecticide resistance. This process can be used as a tool for monitoring the level of the development of organophosphate resistance formation.

• Assessment of vectorial capacity in permethrin-selected population of Aedes aegypti: An elevated permethrin resistance level in Aedes aegypti populations collected in Florida cause them to become more susceptible to Dengue virus. The behavioral difference between the permethrin-resistant population and other susceptible populations has also been investigated. We show that in Florida population of aegypti there is a relation between permethrin resistance and target site mutation frequencies by using allele-specific PCR. This is also a good tool for monitoring the level of permethrin resistance of Ae. aegypti in Florida. This work also informs consequences of the elevated insecticide resistant status in various biological aspects of Florida mosquito population.

• Characterization of genes and signaling pathways in mosquito antiviral mechanisms: The genes or pathways that play a role in the mosquito’s defense against mosquito borne-pathogens have been investigated using different mosquito-pathogen models. This information will be used to develop techniques that interfere with the development of mosquito-mediated pathogens. Furthermore, these results can be used to suppress mosquito vector populations in Florida through the manipulation of these functions using molecular biological techniques.

Current Activity:

Ae. aegypti and Ae. albopictus are known as the primary vector of the Zika virus to humans. A laboratory population of Cx. quinquefasciatus collected in Florida was infected with the Zika virus and the transmission ability of the Zika virus was proven by various analyses in mosquito saliva and body tissues. This discovery was significant since this species is one of the most common mosquitoes in Florida and has high host preference to humans. If Cx. quinquefasciatus is found to be an effective vector for Zika virus in the field, control strategies targeting this species should be considered in addition to those implemented against Ae. aegypti and Ae. albopictus. Currently, I am testing other Florida field populations of Cx. quinquefasciatus for their ability to transmit the Zika virus.

I am also combining conventional microbiological and insecticide resistance bioassays with current mid-throughput sequencing technologies to determine whether the microbial communities that inhabit the mosquito guts contribute to insecticide resistance using a permethrin-selected population.

Pesticide Development, Testing and Application

Staff Scientist:  Dr. Liming Zhao, Research Assistant Professor

Project Synopsis: Includes several projects addressing issues related to improved use of pesticides of importance to Florida mosquito control.

Primary Goals: Identifying and investigating the critical metabolic pathways that are part of the response of mosquitoes to arbovirus infection and pesticide exposure through RNA interference (RNAi) and other methods that silence these pathways for application against mosquito larvae and adults. Another goal is to develop novel diagnostic tests for mosquito-borne pathogens to target vector mosquitoes, and to characterize pesticide response mechanisms in mosquitoes that may lead to strategies to reduce the development of insecticide resistance in mosquito populations.

Major Results to Date:

• NADH dehydrogenase genes play a critical role in the development of Aedes taeniorhynchus. The cDNA of a NADH dehydrogenase-ubiquinone Fe-S protein 8 subunit gene from taeniorhynchus Wiedemann has been cloned and sequenced to help understand its role in larval development. The black salt marsh mosquito, Ae. taeniorhynchus, is very common in the eastern coastal areas of the Americas and is responsible for a large part of mosquito insecticide applications in Florida. The black salt marsh mosquito can transmit pathogens to humans and other animals. A mitochondrially encoded NADH dehydrogenase subunit 5 AetNADH5 was highly expressed in different developmental stages of Ae. taeniorhynchus. Our study suggests that AetNDUFS8 and AetNADH5 play an essential role in the development of Ae. taeniorhynchus and will provide information useful for developing dsRNA pesticide for the black salt marsh mosquito control, especially for the large-scale mosquito control as part of the Everglades National Park conservation program in Florida.
• Characterization of gene expression of two detoxification enzymes, glucosyl and glucuronosyl transferases (AaeGGT1 and AaeGGT2) in aegypti, through developmental stages and a time course study in response to larvicide exposure using qualitative real-time polymerase chain reaction (qPCR). Microbial larvicides based on Bacillus thuringiensis Berliner israelensis (Bti) and Saccharopolyspora spinosa, such as VectoBac®12AS and Natular™2EC, have been shown to be effective in reducing larval populations of Ae. aegypti. AaeGGT1 and AaeGGT2 gene expressions were differentially regulated during development of the immature stages. Gene expression of detoxification enzymes, AaeGGT1 and AaeGGT2, were both upregulated in response to treatments with the maximum level of expression occurring 24 h post treated with VectoBac®12AS and Natular™2EC. AaeGGT2 (AAEL014246) was confirmed upregulated gene cause resistance by Faucon group (2017). This information elucidates larvicide-induced changes in the physiology of Ae. aegypti with implications for development of mosquito control strategies for the Florida Mosquitoes Districts.
• Aedes aegypti (L.) is a vector of chikungunya, dengue, yellow fever and Zika viruses. These viruses encounter a variety of induced defense responses from the innate immune system of the mosquito. We cloned defensin A from aegypti using laboratory populations originating from Key West and Orlando, Florida. To characterize inducible immune defensin peptides, we examined the defensin A (DefA) and defensin C (DefC) expression through time course studies using quantitative real-time PCR. Our studies demonstrate that the relative activity of DefA and DefC changed depending on whether Ae. aegypti was infected with CHIKV or ZIKV, suggesting differences in antiviral defense responses. Together, these data show that members of the Ae. aegypti defensin gene family play a role in both Zika and chikungunya antiviral response. We found that DefA and DefC in the adult males consistently had higher expression than adult females of different ages, which may be used for identifying and surveying male adults, such as for male Sterile Insect Technology (SIT), in Florida mosquito control.
• Aedes aegypti (L.) is the primary vector of many emerging arboviruses. Insecticide resistance among mosquito populations is a consequence of the application of insecticides for mosquito control. We used RNA-sequencing to compare transcriptomes between permethrin resistant and susceptible strains of Florida aegypti in response to Zika virus infection. Quantitative real-time PCR analysis was used to validate the Zika-infection response. Our results suggested a highly overexpressed P450, with AAEL014617 and AAEL006798 as potential candidates for the molecular mechanism of permethrin resistance in Ae. aegypti. Our findings indicated that most detoxification enzymes and immune system enzymes altered their gene expression between the two strains of Ae. aegypti in response to Zika virus infection. Understanding the interactions of arboviruses with resistant mosquito vectors at the overexpressed P450, e.g., AAEL014617 and AAEL006798, allows for the possible development of new approaches in mitigating arbovirus transmission. Zika-induced changes in insecticide resistant Ae. aegypti may propose for Florida mosquito control strategies.
• We created a six-step procedure (Zhao’s laboratory protocol) using BLI/BLITZ/antibody-based diagnostic method for detecting arboviruses and malaria (Plasmodium falciparum), employing biotinylated antibodies and the streptavidin (SA) sensors in the BLITZ system.
• Screening new natural products for novel pesticides for mosquito control, I initiated our co-operation with McLaughlin Gormley King (MGK) and Anastasia Mosquito Control District (AMCD) team. The Develop experimental formulations for ULV efficacy against mosquitoes’ indoor/outdoor tests and are of clear benefit to Florida mosquito control.

Current Activity: Ae. aegypti and Ae. albopictus are the primary vectors of the chikungunya and Zika viruses. Currently, I am working on the mosquito innate immunity, including detail research on the leucine-rich repeats (LRR)-protein. Innate immunity, serving as the host’s first line of defense, is a conserved host response that involves the sensing of pathogen-associated molecular patterns through germline-encoded pattern recognition receptors. Mosquitoes entirely depend on their innate immune system to fight infections caused by pathogens such as viruses, bacteria, fungi, and parasites. Finding the exact immune evasion strategies of pathogens will help to create effective ways to control them.

In order to compare lab strains with natural strains of Ae. aegypti and Ae. albopictus in response to arboviruses, we have conducted ZIKV and CHIKV infection with different populations from the fields located in Key West, Miami, Okeechobee, Gainesville, Jacksonville and Orlando.

Larvicides such as VectoBac®12AS and Natular™2EC are being widely used for mosquito control and are likely to also generate mosquito resistance in field populations in the future. It is important to understand the mechanisms leading to the development of resistance. We continue to screen the mosquito mutants with VectoBac®12AS and Natular™2EC in the laboratory (Figure 1). Heavy doses of the larvicide were used to screen the laboratory resistant strains, i.e., 2 x 10^-6 Natular™2EC solution for the screening, and 4.8 x 10³ ITU/L VectoBac®12AS for the screening. The surviving third instar larvae were blood-fed for ovipositions. The eggs will be hatched for further screening.  Fifteen generations of mosquito treated with VectoBac®12AS and Natular™2EC have been processed.

Figure 1. Ae aegypti larvae exposed to pesticides such as VectoBac®12AS and Natular™2EC. Larvae for Vectobac®12AS resistance screening.

Figure 2. Screening resistance Aedes aegypti in the lab. Their eggs were hatched at the same time, on Friday, October 19, 2018. 7 days later, Natular™2EC treated mosquitoes were in pupal stage. However, VectoBac®12AS treated Aedes aegypti delayed in developmental stage, still in 3rd instar larvae and the size of larvae was smaller than Natular™2EC treated mosquitoes, as well as Key West non-treated F20 Ae. aegypti.

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