The effect of within-instar development on tracheal diameter and hypoxia-inducible factors α and ß in the tobacco hornworm, Manduca sexta.

As insects grow within an instar, body mass increases, often more than doubling. The increase in mass causes an increase in metabolic rate and hence oxygen demand. However, the insect tracheal system is hypothesized to increase only after molting and may be compressed as tissues grow within an instar. The increase in oxygen demand in the face of a potentially fixed or decreasing supply could result in hypoxia as insects near the end of an instar. To test these hypotheses, we first used synchrotron X-ray imaging to determine how diameters of large tracheae change within an instar and after molting to the next instar in the tobacco hornworm, Manduca sexta. Large tracheae did not increase in diameter within the first, second, third, and fourth instars, but increased upon molting. To determine if insects are hypoxic at the end of instars, we used the presence of hypoxia-inducible factors (HIFs) as an index. HIF-α and HIF-ß dimerize in hypoxia and act as a transcription factor that turns on genes that will increase oxygen delivery. We sequenced both of these genes and measured their mRNA levels at the beginning and end of each larval instar. Finally, we obtained an antibody to HIF-α and measured protein expression during the same time. Both mRNA and protein levels of HIFs were increased at the end of most instars. These data support the hypothesis that some insects may experience hypoxia at the end of an instar, which could be a signal for molting. SUMMARY STATEMENT: As caterpillars grow within an instar, major tracheae do not increase in size, while metabolic demand increases. At the same life stages, caterpillars increased expression of hypoxia inducible factors, suggesting that they become hypoxic near the end of an instar.

Identification of a novel metalloproteinase and its role in juvenile development of the tobacco hornworm, Manduca sexta (Linnaeus).

Matrix metalloproteinases (MMPs) are a class of zinc-dependent endopeptidases that are highly conserved across numerous taxa, from bacteria to humans. Recently, MMPs have been identified in several insect species and are hypothesized to function in immunity and development. In this study, we identify a putative MMP and correlate its proteolytic activity and gene and protein expression in the tracheae with developmental stage. Ms-MMP gene expression increases 10-fold during molting, which is accompanied by an increase in both protein expression and gelatinolytic activity. To directly test the hypothesis that Ms-MMP plays a critical role in juvenile development of Manduca sexta, we injected a broad-spectrum MMP inhibitor and recorded its effects on growth and development. Inhibition of MMPs caused a delay in juvenile development and decreased growth rates. Understanding the function of MMPs will help us better understand molting and control of body size in insects. Furthermore, elucidating functions for MMPs in lower taxa may yield critical information about the evolution of the numerous MMPs found in vertebrates.

Population structure and linkage disequilibrium in Lupinus albus L. germplasm and its implication for association mapping.

White lupin (Lupinus albus L.) has been around since 300 B.C. and is recognized for its ability to grow on poor soils and application as green manure in addition to seed harvest. The seed has very high levels of protein (33-47 %) and oil (6-13 %). It also has many secondary metabolites that are potentially of nutraceutical value to animals and humans. Despite such a great potential, lupins role in modern agriculture began only in the twentieth century. Although a large collection of Lupinus germplasm accessions is available worldwide, rarely have they been genetically characterized. Additionally, scarce genomic resources in terms of recombinant populations and genome information have been generated for L. albus. With the advancement in association mapping methods, the natural populations have the potential to replace the recombinant populations in gene mapping and marker-trait associations. Therefore, we studied the genetic similarity, population structure and marker-trait association in a USDA germplasm collection for their current and future application in this crop improvement. A total of 122 PI (Plant Inventory) lines were screened with 18 AFLP primer pairs that generated 2,277 fragments. A subset of 892 polymorphic markers with MAF >0.05 (minor allele frequency) were used for association mapping. The cluster analysis failed to group accessions on the basis of their passport information, and a weak structure and low linkage disequilibrium (LD) were observed indicating the usefulness of the collection for association mapping. Moreover, we were also able to identify two markers (a p value of 1.53 × 10(-4) and 2.3 × 10(-4)) that explained 22.69 and 20.5 % of seed weight variation determined using R (LR) (2) . The implications of lack of geographic clustering, population structure, low LD and the ability of AFLP to map seed weight trait using association mapping and the usefulness of the PI collections in breeding programs are discussed.

Root response to Fusarium solani f. sp . glycines: temporal accumulation of transcripts in partially resistant and susceptible soybean.

Sudden death syndrome (SDS) of soybean is a complex of root rot disease caused by the semi-biotrophic fungus Fusarium solani f. sp. glycines (Fsg) and a leaf scorch disease caused by toxins produced by the pathogen in the roots. Development of partial rate-reducing resistance in roots to SDS was studied. The recombinant inbred line 23 (RIL23) that carried resistance conferred by six quantitative trait loci (QTL) derived from cultivars 'Essex' x 'Forrest' was compared to the susceptible cultivar Essex. Roots of RIL23 and its susceptible parent Essex were inoculated with Fsg. Transcript abundance (TA) of 191 ESTs was studied at five time points after inoculation. For most of the genes, there was an initial decrease in TA in the inoculated roots of both genotypes. By days 7 and 10 the inoculated roots of Essex failed to increase expression of the transcripts of defense-related genes. In RIL23 inoculated roots, the TA of 81 genes was increased by at least two-fold at day 3 (P=0.004), 88 genes at day 7 (P=0.0023) and 129 genes at day 10 (P=0.0026). A set of 35 genes maintained at least a two-fold higher abundance at all three time points. The increase in TA in RIL23 was in contrast to that observed in Essex where most of the ESTs showed either no change or a decreased TA. The ESTs with an increased TA had homology to the genes involved in resistance (analogs), signal transduction, plant defense, cell wall synthesis and transport of metabolites. Pathways that responded included the protein phosphorylation cascade, the phospholipase cascade and the phenolic natural products pathways, including isoflavone and cell wall synthesis.