Insight into Hypoxia Tolerance in Cowpea Bruchid: Metabolic Repression and Heat Shock Protein Regulation via Hypoxia-Inducible Factor 1

Oxygen is of fundamental importance for most living organisms including insects. Hermetic storage uses airtight containment facilities to withhold oxygen required for development, thus preventing damage by insect pests in stored grain. Cowpea bruchid (Callosobruchus maculatus) ceases feeding and growth when exposed to 2% oxygen. However, although population expansion is temporarily arrested, the bruchids (especially late stage larvae) can survive extended periods of hypoxia and recover development if normoxic conditions resume, an ability rarely found in mammals. To begin to understand fundamental mechanisms that enable insects to cope with oxygen deprivation, we constructed a 3′-anchored cDNA library from 4(th) instar larvae subjected to normoxic and hypoxic treatments (respectively), and performed 454-pyrosequencing. Quality filtering and contig assembly resulted in 20,846 unique sequences. Of these, 5,335 sequences had hits in BlastX searches (E = 10(−6)), constituting a 2,979 unigene set. Further analysis based on gene ontology terms indicated that 1,036 genes were involved in a diverse range of cellular functions. Genes encoding putative glycolytic and TCA cycle enzymes as well as components of respiratory chain complexes were selected and assessed for transcript responses to low oxygen. The majority of these genes were down-regulated, suggesting that hypoxia repressed metabolic activity. However, a group of genes encoding heat shock proteins (HSPs) was induced. Promoter analyses of representative HSP genes suggested the involvement of hypoxia-inducible transcription factor 1 (HIF1) in regulating these hypoxia-induced genes. Its activator function has been confirmed by transient co-transfection into S2 cells of constructs of HIF1 subunits and the HSP promoter-driven reporter.