ABSTRACT: Diapause is a common feature in several arthropod species that are subject to unfavorable growing seasons. The range of environmental cues that trigger the onset and termination of diapause, in addition to associated hormonal, biochemical, and molecular changes, have been studied extensively in recent years; however, such information is only available for a few insect species. Diapause and cold hardening usually occur together in overwintering arthropods, and can be characterized by recording changes to the wealth of molecules present in the tissue, hemolymph, or whole body of organisms. Recent technological advances, such as high throughput screening and quantification of metabolites via chromatographic analyses, are able to identify such molecules. In the present work, we examined the survival ability of diapausing and non-diapausing females of the two-spotted spider mite, Tetranychus urticae, in the presence (0 or 5°C) or absence of cold acclimation. Furthermore, we examined the metabolic fingerprints of these specimens via gas chromatography-mass spectrophotometry (GC-MS). Partial Least Square Discriminant Analysis (PLS-DA) of metabolites revealed that major metabolic variations were related to diapause, indicating in a clear cut-off between diapausing and non-diapausing females, regardless of acclimation state. Signs of metabolic depression were evident in diapausing females, with most amino acids and TCA cycle intermediates being significantly reduced. Out of the 40 accurately quantified metabolites, seven metabolites remained elevated or were accumulated in diapausing mites, i.e. cadaverine, gluconolactone, glucose, inositol, maltose, mannitol and sorbitol. The capacity to accumulate winter polyols during cold-acclimation was restricted to diapausing females. We conclude that the induction of increased cold hardiness in this species is associated with the diapause syndrome, rather than being a direct effect of low temperature. Our results provide novel information about biochemical events related to the cold hardening process in the two-spotted spider mite.
