An Integrated Approach Toward Identifying Resistance to Cotton Fleahopper (Pseudatomoscelis seriatus) in Upland Cotton

Cotton fleahoppers (Pseudatomoscelis seriatus) are an early season pest of upland cotton. Feeding damage is characterized by death and abscission of developing cotton floral buds, called squares, and is thought to result from infection of the plant tissue with the bacterial pathogen, Pantoea ananatis, which is vectored by the insect during feeding. Heavy infestations cause delayed maturity and can result in substantial yield loss. Cotton fleahoppers are primarily controlled by chemical insecticides, and thus there exists a strong need to identify resistance in the available upland germplasm for resistance breeding purposes. To that end, three integrated projects were designed to identify and characterize host plant resistance in the available upland germplasm: (1) field evaluation of candidate germplasm to identify resistance and introgression of the resistance trait through backcross breeding, (2) characterization of resistance identified in the first objective through assays of feeding behavior and morphological analysis of the plants and cotton fleahoppers, and (3) RNA-seq transcriptome analysis of plant response to herbivory in one susceptible and three resistant genotypes identified in the first objective. Germplasm obtained from a previous cotton fleahopper breeding effort at Texas A&M and from the Texas A&M AgriLife Research Cotton Improvement Lab at College Station was screened for resistance by estimating percent square loss in three years of field tests in College Station and Corpus Christi, TX and included two high-yielding breeding lines and 18 lines derived from crosses of Pilose (a densely pubescent cultigen resistant to cotton fleahopper) with ‘Deltapine50,’ ‘All-Tex Atlas,’ and ‘TAM 96 WD- 69s’. Field evaluations identified resistance to cotton fleahoppers in lines derived from crosses with Pilose. Field evaluations of backcross progeny lines identified one line, 12525, with high resistance to cotton fleahoppers in both College Station and Corpus Christi and good yield and fiber traits. Behavioral assays examined the interactions of adult cotton fleahoppers with excised cotton squares. Behavior was categorized as walking, resting, probing, feeding or cleaning. Analysis revealed significant differences among parental and backcross progeny lines in time cotton fleahoppers spent feeding, indicating non-preference as a mechanism of resistance. Morphological analysis of square structure, in which square width and length and depth of the developing ovary were measured, indicated variation in depth of the developing ovary may contribute to resistance to cotton fleahoppers; squares with greater ovary depth may escape direct penetration by the proboscis of a feeding cotton fleahopper. RNA-seq transcriptome profilining examined the effects of cotton fleahopper herbivory on gene expression. Analysis revealed differential expression of transcripts associated with three regulators of the hypersensitive response (HR)—myb transcription factor, alternative oxidase (AOX), and BAX inhibitor-1— and indicated the difference between susceptible types (plants that shed squares) and resistant types (plants that retain squares) may lie in regulation of HR-associated lesion formation. Together, the projects presented in this dissertation indicate that the relationship between cotton fleahopper and upland cotton is complex and involves several host plant resistance mechanisms that can be exploited in future efforts to breed for resistance to this insect in cotton

Microplitis croceipes (Hymenoptera: Braconidae): A Life History Study and in vitro Rearing

Microplitis croceipes (Hymenoptera: Braconidae) is an endoparasitoid and potential biological control agent of the tobacco budworm, Heliothis virescens (Lepidoptera: Noctuidae), an agricultural pest. The first objective of the following research was to amend current larval life history descriptions of M. croceipes. Larval head capsule width measurements were used to distinguish instar, and exuvium in abdominal cavities of post-egression hosts were indicative of a molt during parasitoid egression. Data revealed the larvae of M. croceipes pass through five instars, rather than three, as is indicated in the literature. The second objective was to investigate the suitability of potential artificial diets to be used in in vitro rearing of M. croceipes larvae. Three concentrations each of glucose, trehalose, and protein, as well as a combination diet (derived from initial diet trials) were tested. Growth, molting, and death were noted for each diet, and data indicated that diet had a significant effect for each performance measure (p = 0.0000, p < 0.0001, p < 0.0001, respectively). Data also indicated that trehalose and protein were more vital to larval parasitoid development (growth and molting) than was glucose, but no larvae were reared passed the second instar on an artificial diet. The final goals of the research were to evaluate the plausibility of rearing M. croceipes larvae to adulthood in vitro and to investigate post-egression host defensive behavior. Larvae were dissected from their hosts just prior to egression and placed in a cell culture plate in previously collected host hemolymph. Larvae were able to initiate pre-egression behavior in an in vitro environment, and a small percentage (6.67%) exhibited ecdysial splitting of the cuticle, however, no larvae were able to make the final molt in vitro. Post-egression hosts exhibited defensive behavior that may suggest they play a role in protecting pupating parasitoids. When the parasitoid exuvium was pulled from the egression wound in the host, hemolymph loss occurred and duration of the defensive behavior significantly decreased (p < 0.0001), indicating the exuvium acted to plug the egression wound, which prevented the host from bleeding to death and made it possible for the host to exhibit defensive behavior