Master of Science / Department of Biochemistry / Michael R. Kanost / Laccases are multicopper oxidases that catalyze the oxidation of a broad range of substrates, typically phenols and anilines. Research on laccases in fungi, plants, and bacteria has indicated that they have roles in detoxification, pigmentation, wound healing, morphogenesis and lignin synthesis and degradation. However, there has been relatively little investigation on laccases that exist in insects or other invertebrates. Insects have multiple laccase genes, but the function of just one type is known; laccase-2 (Lac2) orthologs are required for tanning of newly synthesized exoskeleton. In the mosquito Anopheles gambiae and other insect species whose genomes have been sequenced, alternative exon splicing may generate two isoforms of Lac2. The objective of this study was to characterize the two isoforms of AgLac2. They are identical in their first 500 residues, but the carboxyl-terminal 262 residues derived from alternative exons are 81% identical. Recombinant Lac2A and Lac2B were expressed and purified. They are both glycoproteins of ~81 kDa, and both can oxidize the laccase substrate ABTS as well as the catechols, N-β-alanyldopamine (NBAD) and N-acetyldopamine (NADA). Lac2A and Lac2B with ABTS have pH optima of 5.0-5.5 and 4.5-5.0, respectively. The pH optima with NBAD and NADA are 5.5-6.5. The Km values (mM) for Lac2A and Lac2B with NBAD are 5.4 ± 2.1 and 5.0 ± 2.6, respectively. The Km values (mM) for Lac2A and Lac2B with NADA are 0.7 ± 0.2 and 1.4 ± 0.5, respectively. Thus, there is little difference between the isoforms in K[subscript]m for these two substrates. The K[subscript]m values do indicate that both isoforms have a greater affinity for the substrate NADA. The kcat values (s[superscript]-1) for Lac2A and Lac2B with NBAD are 14.2 ± 3.5 and 6.0 ± 1.8, respectively. The k[subscript]cat values (s[superscript]-1) for Lac2A and Lac2B with NADA are 2.4 ± 0.2 and 0.5 ± 0.04, respectively. The most apparent difference between the two isoforms detected in the study is that Lac2A was four-fold more active than Lac2B when NADA was used as a substrate. Although the two isoforms are very similar in their amino acid sequences, the differences in catalytic properties may indicate different roles in insect physiology.

Master of Science / Department of Entomology / Srinivas Kambhampati / Termites (Isoptera) are separated into morphologically and behaviorally specialized castes of sterile workers and soldiers, and the reproductive alates. Previous research on eusocial insects has indicated that caste differentiation has a genetic basis. Although much has been studied about the genetic basis of caste differentiation and behavior in the honey bee, Apis mellifera, termites remain comparatively understudied. Therefore, my objective was to compare the gene expression patterns of different castes of the termite Reticulitermes flavipes based on EST analyses and a microarray. Soldier, worker, and alate caste and two larval life stage cDNA libraries were constructed, and ~15,000 randomly chosen clones were sequenced to compile an EST database. Putative gene functions were assigned based on a BLASTX Swissprot search. Categorical expression patterns for each library were compared using the in silico methods of BLAST2GO and r-statistics. I chose 2,240 unique-ESTs based on their putative function and sequence quality, which I used to fabricate a Combimatrix microarray. I used the microarray to compare expression levels between workers and soldiers from Kansas and Florida populations. Seventy to ninety percent of the sequences from the ESTs of each caste and life stages had no significant similarity to those in existing databases. All libraries contained sequences with putative reproductive functions, which was unexpected in the non-reproductive soldier and worker castes. Sequences of interest that showed a putative bias among castes include a viral protein in soldiers and a possible chemosensory protein in alates, which may be involved in termite reproductive functionality or communication. The microarray showed increased expression in the soldier caste of a sequence that matched tropomyosin and an increased expression level of a sequence that matched a PDZ-domain containing protein in some worker samples. This study leads to several candidate genes of potential caste specific function, which can be further tested using functional analysis and between the different castes and life stages of R. flavipes. These genes include the sequences similar to pebIII and RGS-GAIP. I have also expanded upon the available sequences for this termite and utilized the r-statistic in silico method for the first time to putatively compare gene expression in the different castes of a eusocial insect. The in silico analysis allowed us to identify several genes which may show biased expression patterns in the different cDNA libraries and which may reveal caste-specific expression controlling important functions after further analysis. These candidates include: an alate-biased gene, which had a predicted function of neurotransmitter secretion and cholesterol absorption as well as a late larval-biased gene which was predicted to be involved in protein biosynthesis and ligase activity.

Master of Science / Department of Entomology / Franklin Arthur / Kun Yan Zhu / Tribolium spp. are major pests in structures used for the processing and storage of grain-based products (e.g., flourmills, warehouses, retail stores). Consumers and regulators have little tolerance for insect-damaged or contaminated food products. The direction and breadth of pest-control strategies in the food industry have changed significantly over the past few years, creating the need to optimize insecticides through improved integrated pest management (IPM) techniques, specifically through the identification of new control agents that are low in mammalian toxicity, as well as any factors that might affect susceptibility to these agents. There is currently renewed interest in developing reduced-risk, low toxicity chemicals that can be effectively utilized in a setting in which grain and other food commodities are vulnerable to insect infestation, as a means of replacing outdated, and at times, less effective methods of insect control. Over the past decade, developed countries have made significant progress toward alternative insect control strategies by employing a variety of applied insecticides. Two classes of insecticide include natural pyrethrum and insect growth regulators (IGRs), which are substances that mimic insect hormones essential to normal development and reproduction. Pyrethrin is a highly efficient, broad spectrum, botanical insecticide that causes a rapid knockdown in exposed insects. Synergists are used to extend the economic usage of natural pyrethrins and because pyrethrum is rapidly metabolized, it is often mixed with a synergist. Methoprene, a juvenile hormone analog, is labeled as an aerosol and surface treatment inside mills, warehouses and other food storage facilities. There is little recent research with large-scale aerosol applications in stored-food facilities; furthermore, there are few published references regarding the efficacy of using methoprene in combination with synergized pyrethrin, in aerosol form. Therefore, the purpose of this research was to evaluate the use of aerosol applications of two aerosol concentrations on flour and finished stored-product packaging materials for the control of Tribolium spp. Results of this research show that T. castaneum are effectively controlled with 1% aerosol application, while the 3% formulation is required to effectively control T. confusum. With regards to the various packaging material surfaces, few differences between the surfaces emerged.

Doctor of Philosophy / Department of Entomology / Ming-Shun Chen / John C. Reese / Keeping in view the important roles of bacteria in almost every aspect of insect’s life, the current study is the first systemic and intensive work on microbes associated with Hessian fly, a serious pest of wheat crop. A whole body analysis of Hessian fly larvae, pupae, or adults suggested that a remarkable diversity of bacteria is associated with different stages of the insect life cycle. The overriding detection of genera Acinetobacter and Enterobacter throughout the life cycle of Hessian fly suggested a stable and intimate relationship with the insect host. Adult Hessian flies have the most dissimilar bacterial composition from other stages with Bacillus as the most dominant genus. Analysis of 5778 high quality sequence reads obtained from larval gut estimated 187, 142, and 262 operational taxonomic units at 3% distance level from the 1st, 2nd, and 3rd instar respectively. Pseudomonas was the most dominant genus found in the gut of all three instars. The 3rd instar larval gut had the most diverse bacterial composition including genera Stenotrophomonas, Pantoea, Enterobacter, Ensifer, and Achromobacter. The transovarial transmission of major bacterial groups provided evidence of their intimate relationship with the Hessian fly. The Hessian fly is known to manipulate wheat plants to its own advantage. This study demonstrated that the combination of a decrease in carbon compounds and an increase in nitrogen compounds in the feeding tissues of Hessian fly-infested plants results in a C/N ratio of 17:1, nearly 2.5 times less than the C/N ratio (42:1) observed in control plants. We propose that bacteria associated with Hessian fly perform nitrogen fixation in the infested wheat, which was responsible for shifting the C/N ratio. The following findings made in the current study i.e. the presence of bacteria encoding nitrogenase (nifH) genes both in Hessian fly and infested wheat, exclusive expression of nifH in infested wheat, presence of diverse bacteria (including the nitrogen fixing genera) in the Hessian fly larvae, presence of similar bacterial microbiota in Hessian fly larvae and at the feeding site tissues in the infested wheat, and reduction in survival of Hessian fly larvae due to loss of bacteria are consistent with this hypothesis. The reduction in Hessian fly longevity after the loss of Alphaproteobacteria in first instar larvae, highest proportion of Alphaproteobacteria in insects surviving after the antibiotic treatments and the nitrogen fixation ability of associated Alphaproteobacteria strongly implies that Alphaproteobacteria are critical for the survival of Hessian fly larvae. This study provides a foundation for future studies to elucidate the role of associated microbes on Hessian fly virulence and biology. A better understanding of Hessian fly-microbe interactions may lead to new strategies to control this pest.

In light of insecticide resistance in vectors and environmental concerns over pesticides, immunological killing of vectors by way of host vaccination with vector antigens has been developed as an alternative vector-borne disease control method. Although this method has proven somewhat successful in ticks, there has been little success in identifying anti-mosquito vaccine antigens. I have identified a number of problems with past anti-mosquito immunization efforts and have applied novel molecular immune strategies to circumvent these problems as well as to elucidate the mediators and the targets involved in anti-mosquito immunity. The aims of this dissertation were threefold. (1) To study and to replicate traditional methods used in generating antimosquito immunity. I identified a possible mosquito midgut-antigen target using previously produced mosquitocidal sera but immunization of rabbits with this semipurified antigen failed to elicit a mosquitocidal immune response. Further immunizations with different preparations of Anopheles gambiae midgut antigens generated significant and specific antibody production, but failed to kill mosquitoes. These data highlighted the difficulty in replicating and dissecting mosquitocidal immunity by traditional midgut protein vaccination. (2) To pan a phage-displayed library of antibody fragments (scFv) on An. gambiae midgut antigens in order to select for midgut-binding scFv clones, and to test these clones for mosquitocidal activity. The methods I used resulted in the identification of a midgut carbohydrate-specific scFv that had no effect on mosquito survival. Although my results suggest that multiple midgut-binding scFv can be selected with slight modifications in selection procedure. (3) To immunize mice with mosquito midgut cDNA, characterize the resulting immune profiles, and test whether they can affect mosquito survival or fecundity. Immunization with midgut cDNA stimulated an immune response that was highly lethal to mosquitoes, but was characterized by weak humoral immunity. A midgut protein boost stimulated a shift to Type II immunity which abrogated the killing effect, but caused a decrease in egg production. These results suggest that mosquito death was primarily due to a cellular immune response against mosquito midgut antigens in vivo. Ultimately, the strategies I employed will help to elucidate mosquito-host immune interactions and will help to discover critical mosquito antigens / acase@tulane.edu

In Colombia, Aedes aegypti is the only known vector of dengue virus transmission, the agent responsible for dengue and dengue hemorrhagic fever. This mosquito is highly adapted to urban areas. Currently, mosquito control and avoidance are the only prophylactic measure available for these diseases In Cali, Colombia, the continued intense use of insecticides by mosquito control programs and the population in general has resulted in the emergence of insecticide resistance. In preliminary studies on Ae. aegypti populations from three localities in Cali during 1996--1999, variation was found, in time and place, and in the types of biochemical mechanisms (esterases and mixed function oxidases) responsible for insecticide resistance. These differences could be a product of distinct genetic populations. Insecticide pressure could induce the selection of insecticide resistance and, depending on the amount of gene flow among populations, could result in genetic differences among populations. To examine if Ae. aegypti in the city of Cali is a population with genetic distinct local populations that can generate differences in traits relevant to dengue transmission, biochemical mechanisms associated with insecticide resistance, vector competence for the dengue-2 virus, and population genetics by RAPD-PCR molecular markers were evaluated in Ae. aegypti populations from 5 localities separated by 4 to 8 km. The results from all the characteristics evaluated in the study demonstrated the presence of genetically differentiated populations in the city. Differential ongoing pressure from insecticides in Cali could be a major selective force that changes the genetic structure of mosquito populations. These results demonstrate that populations of Ae. aegypti in an urban is in continuous change in response to localized selective pressures which differ in time and place. The success of local vector control and severity of dengue transmission can be affected by these population level changes / acase@tulane.edu

Aedes albopictus is an Asian vector of dengue that has expanded its range over the past decade to include the Americas. As a potential dengue vector in the Western hemisphere, it is important to identify factors that affect this immigrant species and its interactions with the indigenous Ae. aegypti The effect of nutrients and Ascogregarina taiwanensis infections on the bionomics and vector potential of a New Orleans strain of Aedes albopictus were evaluated. Larvae were infected with oocysts and reared under optimal and deficient nutrient conditions using leaf litter as a detritus source. Mortality, development time, wing length and reproductive potential were measured for infected and uninfected groups. Parasitism and low nutrients caused a 35% increase in the rate of larval mortality and doubled the development time, of females. Parasitized adults were 5% smaller and produced 23% fewer eggs. While small body size is correlated with a lowered reproductive capacity, ascogregarine infections caused an additional decrease in fecundity Large, ascogregarine-infected females fed on blood infected with Dirofilaria immitis, had a higher (70%) filaria yield and a lower (22%) post blood meal mortality than single infected females. In large females it is possible that coinfection enhanced the immune response allowing fewer filaria to develop with less host mortality. In small females the lower vector potential may have resulted from low food reserves that limited filarial development, while further reductions in coinfected females may have been caused by ascogregarine damage to tubules Aedes aegypti cross infected with A. taiwanensis showed larval mortality, reduced size and extended development time. Responses varied according to the geographic origin of host and parasite strains, but indicated that the geographic expansion of Ae. albopictus may be aided by ascogregarine infections of the non natural host, Ae. aegypti The combined effects of nutrient levels and parasitism therefore regulate the population density of tire pile Ae. albopictus so the carrying capacity of the habitat is not exceeded. Thee factors also influence the vector capacity of this species for D. immitis. Finally, ascogregarines may play a role in the expansion of Ae. albopictus into habitats occupied by Ae. aegypti / acase@tulane.edu

<p>Gustation, also known as taste perception, is critical for the survival of most animal species. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of sugars, bitter or toxic compounds, and pheromones. However, with a few notable exceptions, the functions of most GRs involved in feeding are unknown. Our research has focused on a cluster of highly-related Drosophila Grs, known as the Gr64 family, that have been shown to be critical for the perception of multiple sugars. Furthermore, we have demonstrated that another gene related to the Gr64 genes, Gr61a, is a sugar receptor that is narrowly tuned to a subset of pyranose sugars and may (along with the Gr64 genes) be indispensable for early fly development. </p><p>As a complementary approach to our behavioral analysis, we have examined the expression pattern of the Drosophila sugar receptors using knock-in driver alleles created by homologous recombination. As expected, most of these drivers have shown strong expression in various taste tissues. Intriguingly, some of these knock-in alleles also show expression in the maxillary palp and antenna, tissues previously thought to be involved only in olfaction. These expression patterns raise interesting questions about the true range of function of these chemosensory receptors and whether or not they might be involved in olfaction as well as gustation.</p> / Dissertation

<p>The initiation of metamorphosis causes the cessation of the larval growth period which determines the final body size of adult insects. Because larval growth is roughly exponential, differences in timing the initiation of metamorphosis can cause large differences body size. Although many of the processes involved in metamorphosis have been well characterized, little is known about how the timing of the initiation of metamorphosis is determined. </p> <p>Using different strains from <italic>Tribolium castaneum<italic>, <italic>Tribolium freemani<italic>, and <italic>Manduca sexta<italic> and varied nutritional conditions, I was able to document the existence of a threshold size, which determines when the larva becomes competent to metamorphose. Threshold size, however, does not dictate the exact timing of initiation. The exact timing for the initiation of metamorphosis is determined by a pulse of the molting hormone, ecdysone, but only after threshold size has been reached. Ecdysone pulses before the larva attains threshold size only cause the larva to molt to another larval instar. These results indicate the timing of metamorphosis initiation is controlled by two factors: (1) attainment of threshold size, at which the larva becomes competent to initiate metamorphosis and (2) the timing of an ecdysone pulse after attaining threshold size. </p> <p>I hypothesize the attainment of threshold size, and therefore competence to metamorphose, is mediated by the effect of changing juvenile hormone concentrations caused by the increase in size of the larva. While the larval body grows nearly exponentially, the corpora allata, which secretes juvenile hormone, grows very little if at all. The difference in relative growth causes juvenile hormone concentrations to gradually become diluted. When juvenile hormone concentrations fall below a threshold, changes in protein-protein binding occur that can cause changes in signaling networks and ultimately gene expression. These changes make the larva competent for metamorphosis. </p> <p>I have demonstrated that only threshold size is consistently correlated with body size; other growth parameters such as growth rate, duration of instars, or number of instars do not consistently correlate with variation in body size. Using the black mutant strain of <italic>M. sexta<italic> I have shown that lower juvenile hormone titers correlate with lower threshold sizes. My hypothesis is consistent with the large body of literature indicating the involvement of juvenile hormone. I also hypothesize that the diversity of metamorphosis types in holometabolous insects can be explained by heterochronic shifts in the timing of threshold size and other developmental events related to metamorphosis. The heterochronic shifts affect not only the morphology of organs, but can also affect the overall phenotypic response of the larva to changes in the environment. The different phenotypic responses among species may make the more or less suited for certain types of niches.</p> / Dissertation

Conservation of biodiversity is a major issue in ancient temperate rainforests of British Columbia. While significant efforts have been made to document species of birds and mammals, little is known about canopy arthropod communities in British Columbia. Microhabitats (suspended soils) within canopy systems of temperate rainforests support diverse microarthropod communities, dominated by mites of the suborder Oribatida. These oribatid mite communities are distinct from forest floor communities, contribute significantly to overall forest biodiversity, and are functionally important components of forest ecosystems. I used the suspended soil system in western redcedar trees in the Walbran Valley to study the relative importance of local environmental factors versus regional spatial factors contributing to the structure of arboreal oribatid mite communities. and compare these patterns to forest floor oribatid mite communities at the same study location. An array of sampling methods was used to complete these objectives: substrate core samples. bark scrapings, colonisation of litterbags, litterfall collection, and colonisation of artificial canopy habitats. This study identified 138 species of oribatid mites associated with ancient western redcedar trees of the Walbran Valley on Vancouver Island. The documented patterns of oribatid mite diversity in this rainforest demonstrated higher local species richness (alpha diversity) in terrestrial (forest floor) habitats, and greater beta diversity in arboreal (suspended soil) habitats. The oribatid mite community within suspended soils are formed primarily by dispersal patterns and colonisation within the canopy system. with limited colonisation from the forest floor. The factors, which shape canopy oribatid mite communities within suspended soils, are related to habitat availability, moisture limitation and random dispersal events of individual species. My results suggest the arboreal oribatid mite community experiences regional dispersal limitation associated with physical tree-to-tree dispersal barriers. However, factors other than physical dispersal barriers. such as aggregation and niche partitioning, likely also limit the local distribution of species in both arboreal and terrestrial habitats. Stochastic dispersal dynamics within the canopy are crucial to understanding oribatid mite community structure in suspended soils. yet the relative importance of regional dispersal processes is dependent on deterministic factors relating to the environmental tolerances of individual species at the local scale.