Aestivation is a state of hypometabolism used by a variety of animals to survive conditions that are too hot or dry to sustain life. Many physiological and molecular changes take place during this dormancy, but little is known of the global control necessary to initiate and maintain these changes. An understanding of this control in the land snail Theba pisana could help with the development of a pest control method for this invasive species, and could also enable a greater understanding of metabolic control in other species including humans, with possible medical implications. Neuropeptides control or influence various complex metabolic events, so this study initially set out to identify the entire set of neuropeptides in T. pisana, some of which may be involved in the control of aestivation. In silico gene data mining of three tissue transcriptomes found a total of 24,920 central nervous system (CNS), 37,661 foot muscle and 40,766 hepatopancreas gene transcripts, which together encode for 5,236 functional protein domains. We predict that as many as 5,504 genes encode for proteins that may be destined for extracellular secretion. Of these, we identified 35 full-length neuropeptide genes encoding precursors that release molluscan-type bioactive neuropeptide products, and are abundantly expressed within T. pisana CNS. These included achatin, allototropin, conopressin, elevenin, FMRFamide, LFRFamide, LRFNVamide, myomodulins, neurokinin Y, PKYMDT, PXFVamide, sCAPamides and several insulin-like peptides. The presence of many of these neuropeptides was confirmed using liquid chromatography-mass spectrometry of neural ganglia. Comparison of the CNS neuropeptidome in active and aestivating states using mass spectral-based analysis, initially revealed 19 differentially produced peptides; 2 were more abundant in active animals and 17 were more abundant in aestivated animals. Using a different extraction method we found a total of 22 peptides differentially produced; 4 upregulated in active animals and 18 upregulated in aestivation. Two peptides upregulated in aestivation, buccalin and sCAP, are known to regulate muscle contractions in a variety of molluscs and for that reason were chosen for further investigation to analyse temporal and spatial expression of their precursor genes and bioactive peptides. One peptide upregulated in active animals, an unknown aestivation-associated peptide (AAP12), was also chosen for further analysis. We found no significant difference in CNS gene expression levels between active and aestivated animals for either buccalin or sCAP, suggesting that regulation may reside at the level of post-translational control of peptide generation. Spatial gene and peptide expression analysis of aestivated snail CNS confirmed that buccalin and sCAP have widespread distribution within neural regions that control several physiological roles. CNS gene expression levels for the AAP12 precursor gene were upregulated in active animals, while the peptide was upregulated in aestivated animals. This study has provided the most comprehensive list of genes and peptides present within the CNS of a land snail. It is also the first detailed molecular analysis of the peptides associated with hypometabolism in a gastropod snail, providing new insight into the molecular basis of aestivation through CNS peptide control.
Submitted in the fulfilment of the requirements of the degree of Doctor of Philosophy, University of the Sunshine Coast, 2015.