Potato production in tropical and subtropical countries suffers from damage caused by the potato tuber moth (PTM), Phthorimiaea operculella. Development of a germline transformation system and the identification of genes that are differentially expressed within the PTM midgut are the main goals of this research. We tested three components that are critical to genetic transformation systems for insects; promoter activity, marker gene expression, and transposable element function. We compared the transcriptional activities of five different promoters, hsp70, hsp82, actin5C, polyubiquitin and ie1, within PTM embryos. The ie1 promoter flanked with the enhancer element, hr5, showed a very high level of transcriptional activity compared with the other promoters. The expression of the enhanced green fluorescent protein (EGFP) was detected under UV-illumination within the embryonic soma demonstrating that it can be used as an effective marker gene for PTM. The transpositional activities of the Hermes, mariner and piggyBac transposable elements were tested in interplasmid transposition assays. The piggyBac element was shown mobile within the embryonic soma with a transposition frequency of 4.2 X 10-5 transposition/donor plasmid. The piggyBac mobility has been enhanced by incorporating a transactivator plasmid expressing the IE1 protein from the bacoluvirus Autographa californica nuclear polyhedrosis virus. Seven transformation experiments were performed. The experiments failed to produce a transgenic PTM. The insect midgut is a rich region of molecular targets involved in food processing that could be potentially used to design a new control strategy. The suppression subtractive hybridization (SSH) method was used to identify differentially expressed genes from the PTM midgut. From this subtracted library, 2984 clones were collected and screened. Of these clones, 637 clones are candidate differentially expressed genes within the PTM midgut. Sixty-nine cDNA clones were randomly selected for DNA sequencing. Tweleve clones were selected for further analysis using RT-PCR and Northern blot techniques. Eleven of the clones resulted in positive results for midgut expression. Five clones, showing homology with insect immune peptides, were used in the challenge experiment which revealed that these cDNAs are constitutively expressed in the midgut, as well as being up-regulated due to bacterial or viral challenge.

Introduction: The midgut epithelium functions as tissue for nutrient uptake as well as physical barrier against pathogens. Additionally, it responds to pathogen contact by production and release of various factors including antimicrobial peptides, similar to the systemic innate immune response. However, if such a response is restricted to a local stimulus or if it appears in response to a systemic infection, too is a rather underexplored topic in insect immunity. We addressed the role of the midgut and the role of systemic immune tissues in the defense against gut-borne and systemic infections, respectively. Methods: Manduca sexta larvae were challenged with DAP-type peptidoglycan bacteria – Bacillus thuringiensis for local gut infection and Escherichia coli for systemic stimulation. We compared the immune response to both infection models by measuring mRNA levels of four selected immunity-related genes in midgut, fat body, hematopoietic organs (HOs), and hemocytes, and determined hemolymph antimicrobial activity. Hemocytes and HOs were tested for presence and distribution of lysozyme mRNA and protein. Results: The midgut and circulating hemocytes exhibited a significantly increased level of lysozyme mRNA in response to gut infection but did not significantly alter expression in response to a systemic infection. Conversely, fat body and HOs responded to both infection models by altered mRNA levels of at least one gene monitored. Most, but not all hemocytes and HO cells contain lysozyme mRNA and protein. Discussion: These data suggest that the gut recruits immune-related tissues in response to gut infection whereas systemic infections do not induce a response in the midgut. The experimental approach implies a skewed cross-talk: An intestinal infection triggers immune activity in systemic immune organs, while a systemic infection does not elicit any or only a restricted immune response in the midgut. The HOs, which form and release hemocytes in larval M. sexta, i) synthesize lysozyme, and ii) respond to immune challenges by increased immune gene expression. These findings strongly suggest that they not only provide phagocytes for the cellular immune response but also synthesize humoral immune components.