Among all the agricultural sectors, the livestock industry is the most economically developed and prolific. Livestock production promotes economic stability globally and provides a significant source of protein in meat and milk. Ticks can transmit pathogens such as Anaplasma marginale, the causative agent of Bovine Anaplasmosis, affecting the cattle industry due to mortality, reduction of meat and milk production, and increased costs for treatment and prevention. Bovine Anaplasmosis results in estimated losses of over 300 million USD per year in the U.S. There are no effective treatments since they act as bacteriostatic antimicrobials, arresting bacterial growth, but do not eliminate them. In subclinical levels the infection can persist. Further, the ability of A. marginale to undergo antigenic variation hinders vaccine development. Therefore, alternatives that target transmission and early establishment of the pathogen are needed. Extracellular vesicles are small membrane blebs secreted by eukaryotic cells that act in cell-to-cell communication. These vesicles can be divided into exosomes and microvesicles, depending on their mechanism of biogenesis and secretion. Recent studies, and my preliminary data, have shown that tick extracellular vesicles carry proteomic material that is conserved among different tick species. This proteomic material can be used as a vaccine candidate for the development of anti-tick vaccines. We hypothesize that these vesicles have potential as vaccine candidates for tick control. To test this hypothesis, we will: 1) Characterize the vesicle populations secreted by Dermacentor andersoni, Amblyomma americanum, and Rhipicephalus microplus ticks, 2) Define the best adjuvant-vesicle combination to trigger strong humoral and cellular responses in cattle, and 3) Determine the effectiveness of extracellular vesicle-derived vaccines for the management of tick populations in semi-field conditions. The emergence of acaricidal resistance in tick populations increases the need to develop new alternative control methods. Current vaccines have shown to be protective only against particular tick populations and species, for this reason artificial vesicles containing conserved proteins among different tick species, present a tempting approach that can be mass-produced and commercialized as an anti-tick vaccine protective against different tick species but specially R. microplus. This technology can be applied to other systems and would have a great impact in animal and human health.

The southern cattle fever tick (SCFT) Rhipicephalus (Boophilus) microplus, is considered the most important ectoparasite of livestock in the world because of high financial losses associated with direct feeding and transmission of the hemoparasites Babesia bovis, B. bigemina, and Anaplasma marginale. Unfortunately, SCFT in many parts of the world have evolved resistance to all market-available pesticides thus driving development of new control technologies. Vaccination against ticks using the tick gut protein Bm86 has been shown to be effective against acaricide-resistant ticks. This technique has been successfully implemented in Puerto Rico for the control of acaricide-resistant R. microplus on dairy and beef cattle. Observations from Puerto Rico indicate a potentially positive interaction between anti-tick vaccination when used in conjunction with systemic acaricide treatment. In this project, controlled animal studies were completed directly comparing efficacy of anti-tick vaccination with and without systemic acaricide. Results show that the Bm86 anti-tick vaccine in combination with the macrocyclic lactone, Moxidectin, expressed a synergistic interaction, providing greater and longer efficacy than either treatment alone.

BACKGROUND:The stable fly, Stomoxys calcitrans, is a major blood-feeding pest of livestock that has near worldwide distribution, causing an annual cost of over $2 billion for control and product loss in the USA alone. Control of these flies has been limited to increased sanitary management practices and insecticide application for suppressing larval stages. Few genetic and molecular resources are available to help in developing novel methods for controlling stable flies.RESULTS:This study examines stable fly biology by utilizing a combination of high-quality genome sequencing and RNA-Seq analyses targeting multiple developmental stages and tissues. In conjunction, 1600 genes were manually curated to characterize genetic features related to stable fly reproduction, vector host interactions, host-microbe dynamics, and putative targets for control. Most notable was characterization of genes associated with reproduction and identification of expanded gene families with functional associations to vision, chemosensation, immunity, and metabolic detoxification pathways.CONCLUSIONS:The combined sequencing, assembly, and curation of the male stable fly genome followed by RNA-Seq and downstream analyses provide insights necessary to understand the biology of this important pest. These resources and new data will provide the groundwork for expanding the tools available to control stable fly infestations. The close relationship of Stomoxys to other blood-feeding (horn flies and Glossina) and non-blood-feeding flies (house flies, medflies, Drosophila) will facilitate understanding of the evolutionary processes associated with development of blood feeding among the Cyclorrhapha.

Among social insects, colony-level variation is likely to be widespread and has significant ecological consequences. Very few studies, however, have documented how genetic factors relate to behaviour at the colony level. Differences in expression of the foraging gene have been associated with differences in foraging and activity of a wide variety of organisms. We quantified expression of the red imported fire ant foraging gene (sifor) in workers from 21 colonies collected across the natural range of Texas fire ant populations, but maintained under standardized, environmentally controlled conditions. Colonies varied significantly in their behaviour. The most active colonies had up to 10 times more active foragers than the least active colony and more than 16 times as many workers outside the nest. Expression differences among colonies correlated with this colony-level behavioural variation. Colonies with higher sifor expression in foragers had, on average, significantly higher foraging activity, exploratory activity and recruitment to nectar than colonies with lower expression. Expression of sifor was also strongly correlated with worker task (foraging vs. working in the interior of the nest). These results provide insight into the genetic and physiological processes underlying collective differences in social behaviour. Quantifying variation in expression of the foraging gene may provide an important tool for understanding and predicting the ecological consequences of colony-level behavioural variation.

Aedes aegypti, the principal vector of yellow fever and dengue fever, is responsible for more than 30,000 deaths annually. Compounds such as carbon dioxide, amino acids, fatty acids and other volatile organic compounds (VOCs) have been widely studied for their role in attracting Ae. aegypti to hosts. Many VOCs from humans are produced by associated skin microbiota. Staphyloccocus epidermidis, although not the most abundant bacteria according to surveys of relative 16S ribosomal RNA abundance, commonly occurs on human skin. Bacteria demonstrate population level decision-making through quorum sensing. Many quorum sensing molecules, such as indole, volatilize and become part of the host odor plum. To date, no one has directly demonstrated the link between quorum sensing (i.e., decision-making) by bacteria associated with a host as a factor regulating arthropod vector attraction. This study examined this specific question with regards to S. epidermidis and Ae. aegypti. Pairwise tests were conducted to examine the response of female Ae. aegypti to combinations of tryptic soy broth (TSB) and S. epidermidis wildtype and agr- strains. The agr gene expresses an accessory gene regulator for quorum sensing; therefore, removing this gene inhibits quorum sensing of the bacteria. Differential attractiveness of mosquitoes to the wildtype and agr- strains was observed. Both wildtype and the agr- strain of S. epidermidis with TSB were marginally more attractive to Ae. aegypti than the TSB alone. Most interestingly, the blood-feeder treated with wildtype S. epidermidis/TSB attracted 74% of Ae. aegypti compared to the agr- strain of S. epidermidis/TSB (P ≤ 0.0001). This study is the first to suggest a role for interkingdom communication between host symbiotic bacteria and mosquitoes. This may have implications for mosquito decision-making with regards to host detection, location and acceptance. We speculate that mosquitoes “eavesdrop” on the chemical discussions occurring between host-associated microbes to determine suitability for blood feeding. We believe these data suggest that manipulating quorum sensing by bacteria could serve as a novel approach for reducing mosquito attraction to hosts, or possibly enhancing the trapping of adults at favored oviposition sites.

Pesticides currently in widespread use often lack species specificity and also become less effective as resistance emerges. Consequently, there is a pressing need to develop novel agents that are narrowly targeted and safe to humans. A cell-based screening platform was designed to discover compounds that are lethal to mosquito (Anopheles and Aedes) cells but show little or no activity against other insect (Drosophila) or human cell lines. Mosquito-specific, aqueous-stable cytotoxins were recovered at rare frequencies. Three of these were profiled for structure-activity relationships and also assessed in whole-animal toxicity assays. In at least one test case, species-specific cytotoxicity seen in culture effectively translated to the whole-animal level, with potent toxicity against Anopheles yet none against Drosophila. Therefore, this initiative has the potential to advance novel mosquitocidal agents and, in a broader sense, could establish a versatile platform for developing customized pesticides that selectively target other disease vectors as well.

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4–PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.

Efficient integration of functional genes is an essential prerequisite for successful gene delivery such as cell transfection, animal transgenesis, and gene therapy. Gene delivery strategies based on viral vectors are currently the most efficient. However, limited cargo capacity, host immune response, and the risk of insertional mutagenesis are limiting factors and of concern. Recently, several groups have used transposon-based approaches to deliver genes to a variety of cells. The piggyBac (pB) transposase in particular has been shown to be well suited for cell transfection and gene therapy approaches because of its flexibility for molecular modification, large cargo capacity, and high transposition activity. However, safety considerations regarding transposase gene insertions into host genomes have rarely been addressed. Here we report our results on engineering helper-independent pB plasmids. The single-plasmid gene delivery system carries both the piggyBac transposase (pBt) expression cassette as well as the transposon cargo flanked by terminal repeat element sequences. Improvements to the helper-independent structure were achieved by developing new plasmids in which the pBt gene is rendered inactive after excision of the transposon from the plasmid. As a consequence, potentially negative effects that may develop by the persistence of an active pBt gene posttransposition are eliminated. The results presented herein demonstrate that our helper-independent plasmids represent an important step in the development of safe and efficient gene delivery methods that should prove valuable in gene therapy and transgenic approaches.

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

Single-strand conformation polymorphism (SSCP) analysis was examined in a 303-bp region of the 16S and 12S mitochondrial rDNA genes to study haplotype frequencies among populations of Gulf Coast ticks collected from Refugio Co., TX, Payne Co., OK, and two sites in Osage Co., KS. Seven haplotypes were identified from the 16S rDNA gene fragment, whereas only two haplotypes were detected from the 12S fragment. Only the results from the 16S rDNA fragment are discussed. Haplotype diversity was greatest in Kansas (site 1), where three of the four haplotypes detected were unique to this site. All Gulf Coast tick populations shared the fourth haplotype. Two haplotypes were determined for Texas and Oklahoma populations, one of which appeared only in Texas, whereas the other was shared. Nei’s haplotype diversity (h) indicated that the Texas population was relatively homogeneous (15%), whereas the remaining populations were heterogeneous (42-59%), although the Bonferroni confidence interval found no significant differences (P < 0.05). Nucleotide sequencing of the seven haplotypes and subsequent phylogenetic analysis using neighbor joining showed a monophyletic relationship among these haplotypes. One haplotype, shared by both Oklahoma and Kansas (site 2), was basal to the remaining haplotypes and formed a distinct clade. Two haplotypes, both from Kansas (site 1), formed a unique clade, whereas the remaining four haplotypes were unresolved polytomies.

Bacteria were isolated and cultured from the red imported fire ant (Solenopsis invicta) midgut. The small-subunit ribosomal RNA gene, (16s rRNA gene, approximately 1500 bp) was amplified from bacterial genomic DNA using the polymerase chain reaction and consensus sequence primers. Restriction fragment length polymorphism analysis revealed 10 unique profiles, indicating that at least 10 different bacteria are present in red imported fire ant midguts. The 16s rRNA gene sequence was determined for these isolates and queried against the NCBI genetic database. The results identified all isolates to at least the genus level. Antibiotic resistance profiles and biochemical activities were also determined for these species. This work provides the basis for a wider characterization of bacterial distributions in fire ant colonies and provides strains suitable for genetic manipulation to develop novel methods of fire ant control.

Insertional mutagenesis can be achieved by a variety of approaches, including both random and targeted methods. In contrast to chemical mutagenesis, insertional mutagens provide a molecular tag, thereby allowing rapid identification of the mutated genomic region. Integration into defined genomic locations has great utility for both gene insertion and mutagenesis. Our laboratories have explored targeted integration through the use of transposases coupled to defined DNA-binding domains. This technology holds great promise for targeted insertional mutagenesis by biasing integration events to regions recognized by the chosen DNA-binding domain. Herein, we provide a brief background on targeted transposon integration and detailed protocols for testing chimeric transposases in both mammalian cell culture and insect embryos.

Honey Bee Gene Regulation and the Transcriptional Effects of a Pheromone and a Parasite. (May 2008) Lara Elizabeth Butler, B.S., University of the Ozarks Co-Chairs of Advisory Committee: Dr. C. Coates Dr. T. Pankiw The European honey bee, Apis mellifera, is a primarily beneficial insect for mankind. It has been utilized by humans for thousands of years for the products and services it provides. Crop pollination and honey production are two of the most economically beneficial activities of the honey bee. Though they have been important for many centuries and immeasurable amounts of effort have been expended investigating the methods and means to harness their natural abilities, a far lesser amount of attention has been directed towards exploring their molecular makeup. These experiments involve identifying modification of gene transcription as a result of exposure to a pheromone or a parasite. This data will provide information on the general types of transcripts involved in the biochemical response of the honey bee to the two stimuli and will also provide specific candidates for further investigation of their potential role in downstream behavioral events.

We constructed non-toxic albumin nanoparticles with a plasmid coding for Flt intraceptor comprising domains 2–3 linked to the endoplasmic retention sequence KDEL (pCMV.Flt23K). The nanoparticles entered into the Balb/C mice corneal cytoplasm as determined by confocal microscopy and transmission electron microscopy. FITC-conjugated nanoparticles were injected into the cornea, and fluorescence was observed for 4 weeks. Flt23K was expressed by naked plasmids in corneas for 8 days and by the nanoparticles for 5 weeks. Mouse corneas injected with pCMV.Flt23K-loaded nanoparticles 3 weeks prior to mechanical-alkali trauma developed a mean percentage of corneal neovascularization of 35.0±6.0% as compared to naked pCMV.Flt23K (58.3±8.7%), empty pCMV nanoparticles (55.4 ± 2.7%), or saline (61.6±10.5%) (p’s <0.05) (n = 7) two weeks after injury. Experiments are being conducted for permanent, stable production of Flt23K (and also soluble Flt-1) with the non-viral integrating vector, piggyBac, to allow for longer and more pronounced suppression of corneal neovascularization.