Doctor of Philosophy / Department of Biochemistry / Subbaratnam Muthukrishnan / In order to grow and develop, insects must undergo a process of molting, wherein the old cuticle is replaced with a new one. A thin envelope layer has been predicted to act as a physical barrier between molting fluid chitinases and the site of new chitin synthesis ensuring selective protection of newly synthesized chitin. The factors that help the new exoskeleton withstand the deleterious effects of chitinolytic enzymes remain poorly understood. In the current study a mechanistic role for two proteins, Knickkopf (Knk) and Retroactive (Rtv), was explored in organization and protection of the newly synthesized procuticular chitin. Our study demonstrated colocalization of molting fluid chitinases (chitinase-5) with chitin in T. castaneum pharate adult elytral cuticle. Presence of chitinases in the new cuticle, disproved the old theory of the envelope being a protective barrier against chitinases. Confocal and transmission electron microscopic imaging of T. castaneum pharate adult elytral cuticle suggested that Knk protein selectively colocalizes with chitin in the new procuticle, organizes chitin into laminae and protects it from the activity of molting fluid chitinases. Down-regulation of Knk expression resulted in reduction of procuticular chitin, disruption of the laminar architecture of the procuticle and severe molting defects that are ultimately lethal at all stages of insect growth. The presence and activity of Rtv protein ensures the trafficking of Knk into the procuticle. Down regulation of Rtv transcripts showed molting defects and a significant decrease in chitin content similar to those following Knk dsRNA treatment. Confocal microscopic analysis revealed an essential role for Rtv in proper trafficking of Knk from epithelial cells to within the newly synthesized procuticule. Once released into the procuticle, Knk organizes and protects chitin from chitinases. The conservation of Knk and Rtv in all insect species suggests a critical role for these proteins in maintenance and protection of chitin in the insect exoskeleton.

Doctor of Philosophy / Department of Biology / Susan J. Brown / RNA interference (RNAi) is a natural gene-silencing phenomenon triggered by dsRNA (dsRNA). While RNAi is an endogenous process that plays essential roles in regulating gene expression it can also be harnessed as a tool for the study of gene function. Introducing dsRNA that is homologous to target mRNA into a cell triggers the RNAi response causing the destruction of the homologous mRNA and a loss of function phenotype. In some organisms, such as the nematode Caenorhabditis elegans, once dsRNA is introduced into the body cavity, the RNAi effect is seen throughout the organism because the dsRNA is taken up by individual cells and is then spread from cell to cell. This process has been termed the systemic RNAi response. For other organisms, such as the fruit fly Drosophila melanogaster, introduction of dsRNA into the body cavity does not result in a systemic RNAi response. This may be due to the cell’s inability to take up dsRNA or spread that dsRNA from cell to cell. For other organisms, including mammals, introduction of dsRNA into the body cavity does not result in a systemic RNAi response because the immune response causes dsRNA destruction before it can be utilized in the RNAi pathway. For organisms that do not exhibit a systemic RNAi response, complex genetic methods are needed to introduce dsRNA into cells to induce the RNAi response. Therefore, one of the challenges in utilizing RNAi as a genetic tool is introducing the dsRNA into individual cells. In recent years, systemic RNAi responses have been documented in both model and non-model organisms, making RNAi an accessible genetic tool. The red flour beetle, Tribolium castaneum is an emerging model organism that has a robust systemic RNAi response. However, the mechanism of systemic RNAi and the specific parameters required to obtain a strong systemic RNAi response in this organism have not been thoroughly investigated. The aim of this work is to provide data that can allow RNAi to be better utilized as a genetic tool in Tribolium and to use this information as a basis for the use of RNAi in other insects in which it can be performed. Specifically we provide data on the essential parameters necessary to achieve an effective systemic response in Tribolium, we describe differences in the systemic RNAi response between Drosophila and Tribolium, we analyze the conservation and function of RNAi machinery genes in Tribolium and we provide information on the genes critical for a systemic RNAi response in Tribolium.

Doctor of Philosophy / Department of Biochemistry / Subbarat Muthukrishnan / The extracellular matrices of cuticle and peritrophic matrix of insects are composed mainly of chitin complexed with proteins, some of which contain chitin-binding domains. This study is focused on the identification and functional characterization of genes encoding proteins that possess one or more copies of the six-cysteine-containing ChtBD2 domain (Peritrophin A motif =CBM_14 =Pfam 01607) in the red flour beetle, Tribolium castaneum. A bioinformatics search of T. castaneum genome yielded previously characterized chitin metabolic enzymes and several additional proteins. Using phylogenetic analyses, the exon-intron organization of the corresponding genes, domain organization of proteins, and temporal and tissue-specificity of expression patterns, these proteins were classified into three large families. The first family includes 11 proteins essentially made up of 1 to 14 repeats of the peritrophin A domain. Transcripts for these proteins are expressed only in the midgut and only during feeding stages of development. We therefore denote these proteins as “Peritrophic Matrix Proteins” or PMPs. The genes of the second and third families are expressed in cuticle-forming tissues throughout all stages of development but not in the midgut. These two families have been denoted as “Cuticular Proteins Analogous to Peritrophins 3” or CPAP3s and “Cuticular Proteins Analogous to Peritophins 1” or CPAP1s based on the number of ChtBD2 domains that they contain. Unlike other cuticular proteins studied so far, TcCPAP1-C protein is localized predominantly in the exocuticle and could contribute to the unique properties of this cuticular layer. RNA interference (RNAi), which down-regulates transcripts for any targeted gene, results in lethal and/or abnormal phenotypes for some, but not all, of these genes. Phenotypes are often unique and are manifested at different developmental stages, including embryonic, pupal and/or adult stages. The experiments presented in this dissertation reveal that while the vast majority of the CPAP3 genes serve distinct and essential functions affecting survival, molting or normal cuticle development. However, a minority of the CPAP1 and PMP family genes are indispensable for survival under laboratory conditions. Some of the non-essential genes may have functional redundancy or may be needed only under special circumstances such as exposure to stress or pathogens.

No description available.

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.

Doctor of Philosophy / Department of Biology / Susan J. Brown / All arthropods share a segmented body plan. Detailed studies on segmentation mechanisms in the long-germ insect Drosophila melanogaster identified a segmentation hierarchy composed of maternal, gap, pair-rule, and segment polarity genes. In this hierarchy, pair-rule genes play an important role to translate gradients of regional information from maternal and gap genes into segmental expression of segment polarity genes. However, our understanding of the role of pair-rule genes in other short-germ insects and basally branching arthropods is still limited. To gain insights into the role of pair-rule genes in short-germ segmentation, I analyzed genetic interactions as well as expression patterns and functions of homologs of Drosophila pair-rule genes in the short-germ insect Tribolium castaneum. Interestingly, despite the pair-rule like expression patterns of Tribolium homologs of almost all eight canonical Drosophila pair-rule genes, only five have a segmentation function. Knock-down of primary pair-rule genes caused asegmental and truncated phenotypes while knock-down of secondary pair-rule genes caused typical pair-rule phenotypes. Epistatic analysis between the genes revealed that primary pair-rule genes form a gene circuit to prepattern a two-segmental unit, and secondary pair-rule genes are downstream targets of the gene circuit. The typical pair-rule phenotypes observed in secondary pair-rule gene RNAi embryos led to a detailed comparative analysis of the role of paired (prd) and sloppy-paired (slp) between Drosophila and Tribolium. This study revealed that prd is functionally conserved while the functional parasegmental register for Tribolium slp is opposite that of Drosophila slp. The fact that the register of slp function has evolved differently in the lineages leading to Drosophila and Tribolium reveals an unprecedented flexibility in pair-rule patterning. Despite this flexibility in pair-rule patterning between Drosophila and Tribolium, segmental expression of engrailed (en) and wingless (wg) at parasegmental boundaries is conserved in both insects. Analysis of double and triple RNAi for pair-rule genes in Tribolium revealed that the primary pair-rule genes even-skipped and runt are redeployed to directly regulate en and wg with prd or slp at parasegmental boundaries. This redeployment of primary pair-rule genes seem to compensate for the apparently fewer number of functional secondary pair-rule genes in Tribolium segmentation.

No description available.

<p>The importance of cryptic female choice, i.e. female post-copulatory influence over male reproductive success, in driving the evolution of male traits remains controversial. The main aim of this thesis was to understand the post-copulatory consequences of sexual interactions and the importance of cryptic female choice in two species of beetle.</p><p>Males of the red flour beetle Tribolium castaneum use their legs to rub the lateral edges of the female elytra during mating. When manipulating female perception of this behaviour, I found that females preferentially use the sperm of males with vigorous leg rubbing when they mate with more than one male. Leg rubbing also appeared to increase female rate of oviposition. Females do not seem to gain any indirect benefits by preferring males with an intense leg rubbing behaviour since this behaviour was found to have very low narrow sense heritability and did not appear to be condition dependent in its expression.</p><p>Males of the bruchid beetle Callosobruchus maculatus have spiny genitalia that harm their mates. Females kick males during copulation and when prevented from kicking, suffered reduced lifetime offspring production as a consequence of more extensive injuries. Males were not able to delay female remating, increase rate of oviposition or increase sperm precedence by inflicting relatively severe injuries to non-kicking females. Hence, the injuries appear to be side effects of male efforts to remain in copula. When copulation duration was manipulated, ejaculate size and female lifetime offspring production increased with the length of copulation. Females reduced their mating rate when they had access to water, suggesting that they obtain water from the large ejaculates and trade-off their need for additional water against the costs of mating. Males may then reduce the benefits of remating by providing their mates with a large amount of water. Females did not increase their remating propensity to avoid inbreeding when they had mated to brothers. Together, these studies reveal the complexity of sexual interactions and the importance of post-copulatory processes for the fitness of both males and females.</p>

The importance of cryptic female choice, i.e. female post-copulatory influence over male reproductive success, in driving the evolution of male traits remains controversial. The main aim of this thesis was to understand the post-copulatory consequences of sexual interactions and the importance of cryptic female choice in two species of beetle. Males of the red flour beetle Tribolium castaneum use their legs to rub the lateral edges of the female elytra during mating. When manipulating female perception of this behaviour, I found that females preferentially use the sperm of males with vigorous leg rubbing when they mate with more than one male. Leg rubbing also appeared to increase female rate of oviposition. Females do not seem to gain any indirect benefits by preferring males with an intense leg rubbing behaviour since this behaviour was found to have very low narrow sense heritability and did not appear to be condition dependent in its expression. Males of the bruchid beetle Callosobruchus maculatus have spiny genitalia that harm their mates. Females kick males during copulation and when prevented from kicking, suffered reduced lifetime offspring production as a consequence of more extensive injuries. Males were not able to delay female remating, increase rate of oviposition or increase sperm precedence by inflicting relatively severe injuries to non-kicking females. Hence, the injuries appear to be side effects of male efforts to remain in copula. When copulation duration was manipulated, ejaculate size and female lifetime offspring production increased with the length of copulation. Females reduced their mating rate when they had access to water, suggesting that they obtain water from the large ejaculates and trade-off their need for additional water against the costs of mating. Males may then reduce the benefits of remating by providing their mates with a large amount of water. Females did not increase their remating propensity to avoid inbreeding when they had mated to brothers. Together, these studies reveal the complexity of sexual interactions and the importance of post-copulatory processes for the fitness of both males and females.

No description available.