Doctor of Philosophy / Department of Biochemistry / Karl J. Kramer / Subbarat Muthukrishnan / Chitin, a linear homopolymer of beta-1,4-linked N-acetylglucosamine, is the second most abundant biopolymer next to cellulose. It is the major structural polysaccharide in the insect’s exoskeleton and gut lining. An extensive study of two of the major genes encoding enzymes involved in chitin metabolism, chitin synthases (CHSs) and beta-N-acetylglucosaminidases (NAGs), was undertaken. CHS genes from the tobacco hornworm, Manduca sexta, and NAG genes from the red flour beetle, Tribolium castaneum, were identified and characterized. In general, chitin deposition occurs in two major extracellular structures of insects, the cuticle that overlays the epidermis, and the peritrophic membrane (PM) that lines the midgut. Only two CHS genes were identified in M. sexta using Southern blot analysis. Extensive expression studies of both M. sexta CHS genes, MsCHS1 and MsCHS2, suggest a strict functional specialization of these two genes for the synthesis of epidermal and PM-associated chitin, respectively. Furthermore, two alternatively spliced transcripts of MsCHS1, MsCHS1a and MsCHS1b, were identified. Analysis of the levels of these transcripts in different tissues and stages of development indicated that the MsCHS1a transcript predominates in the integument during the feeding and pupal stages, whereas the MsCHS1b transcript is more abundantly present in the tracheae, foregut, and hindgut during all developmental stages tested. Four genes encoding putative NAGs (TcNAG1, TcNAG2, TcNAG3, and TcNAG4) were identified by searching the Tribolium genomic database. The full-length cDNAs for all four NAGs were cloned and sequenced, and the exon-intron organizations were determined. Studies on developmental expression patterns of each gene indicated that they are expressed during most developmental stages with TcNAG1 being the predominant one. The function of each NAG was assessed by down regulating the level of each transcript at various developmental stages using RNA interference. Selective knock down of each transcript, without significant reduction in the expression levels of the other NAG transcripts, was verified and the resulting phenotypes were documented. Knockdown of TcNAG1 interrupted larval-larval, larval-pupal, and pupal-adult molting, and the insects were unable to completely shed their old cuticles.

Master of Science / Department of Entomology / David C. Margolies / Yoonseong Park / Phytoseiulus persimilis (Acari: Phytoseiidae) is a specialist predator on tetranychid mites, especially on the twospotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae). The foraging environment of the predatory mites consists of prey colonies distributed in patches within and among plants. Quantitative genetic studies have shown genetic variation in, and phenotypic correlations among, several foraging behaviors within populations of the predatory mite, P. persimilis. The correlations between patch location, patch residence, consumption and oviposition imply possible fitness trade-offs. We used molecular techniques to investigate genetic variation underlying the foraging behaviors. However, these genetic studies require a sufficiently large amount of DNA which was a limiting factor in our studies. Therefore, we developed a method for obtaining DNA from a single mite by using a chelex extraction followed by whole genome amplification. Whole genome amplification from a single mite provided us with a large quantity of high-quality DNA. We obtained more than a ten thousand-fold amplified DNA from a single mite using 0.01ng as template DNA. Sequence polymorphisms of P. persimilis were analyzed for nuclear DNA Inter Transcribed Spacers (ITS1 & ITS2) and for a mitochondrial 12S rRNA. The sequence comparisons among individuals identified a number of polymorphisms in the 12S sequences. The foraging gene (for) associated with rover-sitter behavioral strategies of Drosophila is known to have role in feeding behaviors of honeybee and other arthropods. We surmised that the same or a similar gene may be present in P. persimilis. Among the foraging behavior(s) exhibited by this predatory mite, we were particularly interested in resource/prey-dependent dispersal behavior. We isolated a partial sequence that is presumed to be the orthologue of the foraging (for) gene. We named the putative foraging gene as Ppfor (for Phytoseiulus persimilis foraging gene). We used a fragment of Ppfor gene as a molecular marker between populations and among individuals and, further, to help understand behavioral phenotypes.

Doctor of Philosophy / Department of Entomology / Michael R. Kanost / James R. Nechols / Cellular encapsulation is the immune response in which insects protect themselves from multicellular parasites such as nematodes or parasitoids. During an encapsulation episode, certain insect hemocytes become attracted to a foreign invader and aggregate on its surface. In short order, the invading entity will become entrapped within a capsule comprised of thousands of hemocytes, thus rendering the parasite harmless to the insect host. Although the process of cellular encapsulation has been known for a great many years, very little knowledge yet exists regarding the biochemistry underlying capsule formation. It would seem likely that cell surface adhesion proteins mediate this immune response. In a series of in vivo encapsulation assays in the tobacco hornworm, Manduca sexta, a collection of anti-hemocyte monoclonal antibodies (mAbs) was screened for their ability to inhibit cellular encapsulation. Two of the mAbs that inhibited this immune response and incidentally specifically bind plasmatocytes, MS13 and MS34, were used to isolate a ≈ 90 kDa protein. Several short peptide sequences contained within this protein were acquired via Edman degradation. Degenerate primers based on two of these peptide sequences and total RNA from M. sexta hemocytes were used to perform RT-PCR and 5´ and 3´ RACE. This resulted in a full-length cDNA sequence of 2426 bp. A 2301 bp open reading frame within this cDNA sequence codes for a protein of 767 residues. This protein, denominated [Beta]Ms1, exhibits significant sequence homology to the [Beta]-subunits of integrins, which are a family of transmembrane, heterodimeric glycoproteins that possess adhesive properties. Analysis of recombinant segments of [Beta]Ms1 showed that the protein produced from the PCR product is the antigen to MS13 and MS34 and that these mAbs bind to the region of the integrin that contains the extracellular binding site. Northern blot analysis of various M. sexta tissues together with immunofluorescence labeling with MS13 and MS34 shows that [Beta]Ms1 is solely expressed in plasmatocytes. The totality of these experiments demonstrates that integrins are essential for the cellular immune response of encapsulation.

Master of Science / Department of Entomology / David C. Margolies / James R. Nechols / The study of how extrinsic factors affect the foraging efficiency and behavior of predaceous arthropods like Phytoseiulus persimilis is important to understand their various processes of acquiring prey, mates, refuges, oviposition sites, and overcoming obstacles posed by the environment. Many intrinsic and extrinsic factors affect predator foraging efficiency and behavior. One of the most influential extrinsic factors may be the host plant on which herbivorous prey are found. Recent studies suggest that plant architecture plays an important role in tritrophic interactions. In this work, I studied the effects of cucumber plant architecture and prey distribution on the foraging efficiency (prey-finding time and prey-consumption rate) and behavior (time allocated between moving, resting and feeding) of P. persimilis. Plant architecture represented differences in leaf number and size; however, all plants had the same total surface area. Plants with 6 small leaves (ca. 82.98 square cm each) were considered as complex architecture, whereas plants with only 2 large leaves (ca. 240.60 square cm each) were considered as simple. The prey distributions were: prey patch on a single basal leaf (closest leaf to the soil) and prey patch on all leaves. The foraging efficiency was assessed by measuring prey-finding time and prey-consumption rate, whereas the behavior was assessed by conducting observational studies on specific foraging activities. When placed either on the top or at the base of the plant P. persimilis encountered prey more rapidly (interval 0-30 minute) on complex and simple plants with prey patches distributed on all leaves. Differences in prey density (number of prey per leaf) had no effect on the prey-finding time of P. persimilis. The predator consumed more eggs on complex plants with prey patches distributed on all leaves. Phytoseiulus perismilis tended to find prey patches more quickly as well as consume more eggs on leaves close to its release point. Furthermore, the predator was observed to lay more eggs on leaves where it had consumed higher number of prey eggs. The dimensions of individual parts of the plant e.g., stem, petiole and leaf, affected the time allocated by P. persimilis between searching, resting and feeding. The predatory mite spent more time foraging on the stems and petioles of the simple plants whereas on complex plants it spent more time foraging on the leaves.

Doctor of Philosophy / Department of Biochemistry / Michael R. Kanost / Manduca sexta is a large insect species well-suited for biochemical analysis of proteins in the hemolymph (blood) that respond to infection. Insects lack adaptive immunity and rely entirely on innate immunity to prevent and manage infection. Immune response proteins include proteins that bind pathogens and activate serine proteases, which function in proteolytic cascades that trigger effector responses, such as antimicrobial peptide production and prophenoloxidase activation. Phenoloxidase catalyzes melanin synthesis, which leads to microbial killing. I used MALDI-TOF/TOF mass spectrometry and immunoblotting to identify M. sexta proteins present in putative immune complexes. From analyses of high molecular weight gel filtration fractions of plasma activated by microbial polysaccharides, I detected hemocytin, prophenoloxidase, and cleaved serine protease homologs, suggesting prophenoloxidase and serine protease homologs form large complexes in plasma. I used in vitro bacterial binding assays to identify hemolymph proteins that bind either directly or indirectly to the surface of bacteria or curdlan. Prophenoloxidase, annexin IX, and hemocyte aggregation inhibitor protein were found bound to all the samples tested, indicating they play a role in the early stage of immune response. Serpins regulate specific active proteases by covalently binding and forming serpin-protease complexes. Serpin-1, an abundant plasma protein, has an alternatively spliced ninth exon encoding 12 serpin-1 isoforms that differ in inhibitory selectivity. RT-PCR showed that all 12 isoforms are expressed in hemocytes, fat body, and midgut. Comparisons of naïve and immune-challenged hemocytes and fat body indicated the immune-related upregulation of serpin-1A but not the other isoforms. Using immunoaffinity chromatography I isolated two serpin-1-protease complexes from plasma after activation with bacterial lipopolysaccharide. MALDI-TOF/TOF analysis of these serpin-1-protease complexes identified the digestive enzyme chymotrypsin as a specific target of serpin-1K. Nine out of the twelve serpin-1 isoforms were identified from control plasma at the protein level using 2D-PAGE. Serpin-1 protease complexes were identified by 2D-PAGE analysis: serpin-1A, E and J were found to be complexed with hemolymph proteinase-8 and an unidentified isoform of serpin-1 was complexed with hemolymph proteinase-1. Discovering the serpin-1 isoforms that inhibit specific proteases enhances our understanding of the regulation of proteolytic cascades in M. sexta.

Master of Science / Department of Entomology / Larry L. Buschman, Lawrent Buschman / This study evaluated whether using a cover crop with corn would increase the threat from spider mites in western Kansas because cover crops may serve as a winter host. This study also evaluated whether a cover crop could affect corn rootworm and other ground dwelling arthropods in the cornfield. In the first study, downy brome, Bromus tectorum L., was used as the winter cover crop. There were two trials repeated for three years each. Each trial included: two amounts of irrigation, downy brome, and herbicide to control weeds. In the first trial there were no significant differences in corn rootworm, Diabrotica virgifera LeConte, damage across treatments, because there were no differences in brome residue across the treatments. In the second trial, corn rootworm damage was significantly more in plots with higher amounts of downy brome residue. There were no differences in numbers of spider mites: Banks grass mites, Oligonychus pratensis (Banks) or twospotted spider mites, Tetranychus urticae Koch, across treatments. Spider mite populations appeared to be suppressed by the predatory mite Neoseiulus spp., which also overwintered in the cover crop. Corn rootworm samples taken from a no-till irrigation experiment were variable among irrigation treatments but indicated a trend for rootworm damage to increase with increasing irrigation. In the second study, winter wheat, Triticum aestivum L., was used as the winter cover crop. There were three trials repeated for three years each. Each trial included two amounts of irrigation and winter wheat and three amounts of herbicide to control weeds. Upon completion of the agronomy trials, the plots were split into two subplots and one was tilled. Pitfall traps were installed to capture ground dwelling arthropods: (Coleoptera: Carabidae), wolf spiders (Araneae: Lycosidae) and crickets (Orthoptera: Gryllidae). Four carabid genera were more common under no-till conditions. One was more common in tilled plots. Five carabid genera were more common in plots with a history of high weed densities. Two carabid genera were more numerous in plots with the history of a cover crop. Crickets were more common under no-till conditions. Wolf spiders were more common in no tillage with a history of a cover crop.

Through the use of natural history records, published literature, and personal sampling (2011-2016) a total of 454 caddisfly species represented by 24 families and 93 genera were documented from the south-central United States. Two Hydroptilidae species were collected during the 2011-2016 collection efforts that are new to the region: Hydroptilia scheringi and Mayatrichia tuscaloosa. Eightteen species are endemic and 30 are considered species of concern by either federal or state agencies. The majority of each of these groups is Hydroptilidae, or microcaddisflies. Trichoptera community structure, by minimum number of species, was analysed in conjunction with large-scale geographical factors to determine which factor illustrated caddisfly community structure across the region. Physiographic provinces compared to other geographic factors analyzed best-represented caddisfly communities with a minimum of 10 or more species. Statistically, Hydrologic Unit Code 4 (HUC 4) was the most significant geographical factor but low number of samples representing this variable rendered it less representative of caddisfly community structure for the study area.

Ces dernières années, l’insécurité alimentaire et la pauvreté de la population djiboutienne en particulier rurale ne cessent d’accroitre à cause de la croissance démographique et l’élévation des prix des denrées alimentaires mondiales. De ce fait, le gouvernement djiboutien s’est orienté vers le développement de l’agriculture durable par le biais de la phoeniciculture. Cette dernière s’adapte bien aux conditions pédoclimatiques difficiles de la République de Djibouti. Des introductions massives de palmier dattier ont eu lieu pour restaurer les anciennes palmeraies traditionnelles (polycultures et en étage) et la création ou l’extension des nouvelles palmeraies, orientées vers la monoculture. Cependant, le pays connait des contraintes importantes dans le domaine phytosanitaire : l’absence d’infrastructure, l’insuffisance de savoir-faire et l’inexistence d’études sur les ravageurs de cette culture. Dans un but ultime de protéger et d’augmenter les rendements phoenicicoles djiboutiens, ce présent travail évalue la susceptibilité des palmeraies aux insectes ravageurs et aux ennemis naturels. Pour atteindre cet objectif, une comparaison de l’entomofaune des deux modèles de palmeraies, traditionnelle et nouvelle, a été réalisée à l’aide de pièges composés placés sous la couronne du palmier dattier. L’expérience a été menée du 20 juin au 2 septembre 2018, dans six vergers repartis sur trois sites. Les prélèvements dans ces palmeraies ont montré l’existence d’une diversité d’insectes d’une part, et d’autre, ils ont révélé la présence et l’abondance de ravageurs spécialistes dans les palmeraies monoculturales. Il est ajouté que les ennemis naturels ont été observés en abondance dans les palmeraies traditionnelles mais leur diversité spécifique n’a pas démontré de patron. Enfin, cette étude contribue à la connaissance de la diversité des insectes et l’existence des ravageurs dans les palmeraies djiboutiennes. Ces ravageurs peuvent avoir des impacts sur le rendement suivant le modèle d’habitat de palmeraie, d’où l’intérêt d’élaborer de moyens de lutte adéquats. / In recent years, food insecurity and poverty, especially among the rural populations of Djibouti, have been increasing due to population growth and rising world food prices. As a result, the government of Djibouti seeks to develop sustainable agriculture through date farming. The cultivation of date palms adapts well to the difficult pedoclimatic conditions of the Republic of Djibouti. Massive introductions of date palm have taken place in recent years to restore the older traditional palm groves (polycultures) and to create and/or extend new palm groves oriented towards monoculture. However, the country faces major phytosanitary challenges: poor infrastructure, insufficient knowledge, a lack of research on the pests of this crop. With the ultimate aim of protecting and increasing Djibouti's date yields, this present work seeks to assess the susceptibility of palm groves to insect pests. To achieve this objective, a comparison of the insect fauna of the two types of palm groves was carried out using composite traps placed within the crown of the date palm. The experiment was carried out from 20 June to 2 September 2018, in six orchards on three sites. The samples documented the existence and diversity of insects in these palm groves, as well as the presence and abundance of specialist date palm pests in monocultural palm groves. Natural enemies were also particularly abundant in the traditional palm groves, but species-level diversity did not show any correlations with the type of palm grove. This study contributes to the knowledge of insect diversity and potential pest species in the palm groves of Djibouti. These pests can have an important impact on date yields depending on the type of palm grove and this study thus underlines the need to develop adequate means of pest control.

Master of Science / Department of Entomology / Srinivas Kambhampati / Termites (Isoptera) are eusocial insects and exhibit highly complex eusocial behavior. Eusociality is characterized by the presence of castes (workers, soldiers, reproductives), polyphenisms (same genotype exhibiting multiple phenotypes), flexible developmental pathways, complex communication, cooperative brood care, construction and maintenance of complex nests, and division of labor. Previous studies on honey bees implicated several genes in caste-specific behavior; here, we investigate if orthologs of such genes are present in termites and if so, whether they are expressed differentially among the castes. A candidate gene approach using degenerate primers was used to amplify three candidate genes in the termite Reticulitermes flavipes. Quantitative real time PCR analysis revealed differential expression among termite workers, soldiers, and alates, with a general pattern of higher expression in alates. These results provide information on three novel genes in the termite R. flavipes.

Doctor of Philosophy / Department of Entomology / Kun Yan Zhu / Chitin metabolism represents an attractive target site for combating insect pests as insect growth and development are strictly dependent on precisely toned chitin synthesis and degradation and this process is absent in humans and other vertebrates. However, current understanding on this process and the involved enzymes is rather limited in insects. In this study, two chitin synthase genes (AgCHS1 and AgCHS2 or AgCHSA and AgCHSB), and 20 chitinase and chitinase-like genes (groups I-VIII) presumably encoding the enzymes for chitin biosynthesis and degradation, respectively, were identified and characterized in African malaria mosquito, Anopheles gambiae. Immunohistochemistry analysis and developmental stage- and tissue-dependent transcript profiling by using reverse transcription PCR, real-time quantitative PCR, and in situ hybridization revealed new information on these genes. Current understanding on chitin synthases is extended by the expression profiles such as the localization of AgCHS1 and AgCHS2 transcripts in eggs, AgCHS2 transcripts in the posterior larval midgut, AgCHS1 and AgCHS2 proteins in the compound eyes, and AgCHS2 enzyme in pupal inter-segments. Chitinase and chitinase-like genes are highly diverse in their gene structure, domain organization, and stage- and tissue-specific expression patterns. Most of these genes were expressed in several stages. However, some genes are stage- and tissue-specific such as AgCht8 mainly in pupal and adult stages, AgCht2 and AgCht12 specifically in foregut, AgCht13 exclusively in midgut. Functional analysis of each chitin synthase gene was conducted by using the chitosan/dsRNA nanoparticle-based RNA interference (RNAi) through larval feeding. The repression of the AgCHS1 transcripts which are predominantly expressed in carcass initiated from the mosquito larval feeding of dsRNA suggests the systemic nature of RNAi in mosquito larvae. In addition, silencing of AgCHS1 increased larval susceptibilities to diflubenzuron, whereas silencing of AgCHS2 enhanced the peritrophic matrix disruption and thus increased larval susceptibilities to calcofluor white or dithiothreitol. Furthermore, a non-radioactive method was adapted and optimized to examine the chitin synthase activity in mosquitoes. By using this method, diflubenzuron and nikkomycin Z show limited in vitro inhibition on chitin synthase at high concentration in cell free system, whereas no in vivo inhibition was observed.