Effects Of Diapause And Nutritional Transfer On Cold Tolerance Of Nasonia Vitripennis And Its Molecular Mechanism

Parasitoids offer tremendous potential as biological control agents, and boosting cold hardiness in parasitoids is a goal that is particularly attractive for increasing shelf life, shipment and establishment of biological control agents. Cold tolerance of parasitoids is a plastic trait influenced by both endogenous and exogenous factors, including nutrition, diapause status, and environmental conditions. The host and host diets could impact cold hardiness by modulating physiological and biochemical conditions in parasitoids that feed on the host, thus affect cold hardiness of the parasitoid; but to date little information on this subject is available. Several recent studies demonstrated that certain dietary supplements can enhance cold tolerance. The experiments we present here extend this idea by testing the hypothesis that manipulating the host diet can also affect cold tolerance in parasitoid that feed upon the host. And this effect was evaluated from biology, physiology and metabolomics.In this study we investigated how the parasitoid’s diapause status, the host’s diapause status, and manipulations of the host diet can influence low temperature tolerance of the parasitoid by using the ectoparasitoid Nasonia vitripennis and one of its favored hosts, the flesh fly Sarcophaga crassipalpis as a system. And, we defined alterations in metabolite profiles of N. vitripennis caused by diapause in the parasitoid, diapause of the host, and augmentation of the host’s diet with proline. The transcript encoding PEPCK, a putative key metabolic player for insect diapause, was cloned from N. vitripennis, and expression of pepck in association with diapause and thermal stress was examined. These results provide references for exploring molecular physiological mechanisms of parasitoid diapause and cold hardiness, and also suggest techniques that could be exploited for improving shelf-life, long-distance shipment and application of parasitoids for release in biological control. Our results demonstrated that:1. Larval diapause in N. vitripennis dramatically increased cold tolerance, and the level of cold tolerance of diapausing larvae was elevated with increased diapause age, indicating a developmental progression of cold tolerance that increases during early diapause. The diapause status of the host also exerted a positive, although less dramatic, effect on enhancing parasitoid cold tolerance.2. We proposed and verified our hypothesis of the tritrophic effect on insect cold tolerance the tritrophic effect on insect cold tolerance. Augmenting the host fly’s diet with supplements of putative cryoprotectants(alanine, proline and glycerol) enhanced cold tolerance in parasitoids that fed on the flies. The most pronounced improvement in cold tolerance was noted in parasitoids fed on fly hosts that had received a diet augmented with proline.3. Metabolic profiles of diapausing and nondiapausing parasitoid were significantly differentiated, with pronounced distinctions in levels of multiple cryoprotectants, amino acids, and carbohydrates. Accumulation of high levels of pipecolic acid, proline and trehalose were observed during parasitoid diapause, and the three metabolites can be recognized as major biomarkers that distinguish diapausing parasitoids from their nondiapausing counterparts. The dynamic nature of diapause was underscored by a shift in the wasp’s metabolomic profile as the duration of diapause increased, a feature especially evident for increased concentrations of a suite of cryoprotectants. Metabolic pathways involved in amino acid and carbohydrate metabolism were distinctly enriched during diapause in the parasitoid. Higher cryoprotectants accumulated and decreased energy metabolism may account for increased cold tolerance in the diapausing parasitoid.4. The host flesh fly accumulated more cryoprotectants during diapause, accompanied with decreased citric acid cycle and elevated gluconeogensis and pyruvate metabolism. Host diapause status elicited a pronounced, although less dramatic effect, on metabolic signatures of the parasitoid, noted by higher cryoprotectants and elevated compounds derived from glycolysis, as well as changes in multiple amino acid and lipid metabolism. Shifts in metabolic signatures of the host fly may influence the metabolomic profiles of the parasitoid indirectly, and thus increase parasitoid cold tolerance.5. Proline supplementation of the host diet did not translate directly into elevated proline in the parasitoid but resulted in an alteration in the abundance of many other metabolites, including elevated concentrations of cryoprotectants and essential amino acids, and reduction in metabolites linked to energy utilization, lipid and amino acid metabolism. Thus, the enhanced cold tolerance of N. vitripennis associated with proline augmentation of the host diet appears to be an indirect effect caused by the metabolic perturbations associated with diet supplementation.6. The pepck transcript isolated from N. vitripennis encodes mitochondrial PEPCK,and the deduced amino acid sequence of pepck is highly conserved. Expression of pepck was down-regulated during diapause of the parasitoid, which is consistant with the metabolic result. The expression proflie of pepck remained unaffected by heat shock, but pepck was down-regulated significantly after cold stress. The different expression patterns of pepck in response to diapause and environmental stress between N. vitripennis and other insects suggest a species-specific responsive mechanism, and underscore the rich diversity of mechanisms that subserve insect diapause and cold hardening.