Overwintering strategy and mechanisms of cold tolerance in the codling moth (Cydia pomonella)

Abstract

Background: The codling moth (Cydia pomonella) is a major insect pest of apples worldwide. Fully grown last instar larvae overwinter in diapause state. Their overwintering strategies and physiological principles of cold tolerance have been insufficiently studied. No elaborate analysis of overwintering physiology is available for European populations.

Principal findings: We observed that codling moth larvae of a Central European population prefer to overwinter in the microhabitat of litter layer near the base of trees. Reliance on extensive supercooling, or freeze-avoidance, appears as their major strategy for survival of the winter cold. The supercooling point decreases from approximately -15.3 °C during summer to -26.3 °C during winter. Seasonal extension of supercooling capacity is assisted by partial dehydration, increasing osmolality of body fluids, and the accumulation of a complex mixture of winter specific metabolites. Glycogen and glutamine reserves are depleted, while fructose, alanine and some other sugars, polyols and free amino acids are accumulated during winter. The concentrations of trehalose and proline remain high and relatively constant throughout the season, and may contribute to the stabilization of proteins and membranes at subzero temperatures. In addition to supercooling, overwintering larvae acquire considerable capacity to survive at subzero temperatures, down to -15 °C, even in partially frozen state.

Conclusion: Our detailed laboratory analysis of cold tolerance, and whole-winter survival assays in semi-natural conditions, suggest that the average winter cold does not represent a major threat for codling moth populations. More than 83% of larvae survived over winter in the field and pupated in spring irrespective of the overwintering microhabitat (cold-exposed tree trunk or temperature-buffered litter layer).

Figure 1. Fresh mass, dry mass, total lipid mass.

Gradual losses of FM, DM and total lipid mass (all masses are in mg) in caterpillars of Cydia pomonella during their overwintering in the field in 2011/2012. Each point is the mean ± S.D. (n = 10 individuals). Black symbols are for larvae that were analyzed at the beginning of November, while red symbols are for larvae, in which gradual loss of FM was measured in approximately 14 d-intervals throughout the cold season and their DM and total lipids were analyzed in April (see text for details). The larvae were located on tree trunks (see text for details) and the course of ambient temperatures was recorded in 2 h-intervals.

Figure 2. Glycogen.

Seasonal whole-body and tissues changes of glycogen contents in field-sampled caterpillars of Cydia pomonella during 2010/2011. Each point is the mean ± S.D. (whole body, n = 5 individuals; tissues, n = 3 replicates, 3 individuals each). Influence of sampling date on glycogen content was tested by ANOVA followed by Bonferroni's post hoc test (means flanked with different letters are significantly different).

Figure 3. Sugars and polyols.

Seasonal changes in concentrations of selected sugars and polyols in hemolymph (A), fat body (B), and body wall (C) of field-sampled caterpillars of Cydia pomonella during 2010/2011. The areas showing concentrations of individual compounds are stacked and the total concentration of all sugars and polyols is shown as a broken line. See Dataset S1 for details.

Figure 4. Glutamine.

Seasonal whole-body and tissues changes of glutamine concentrations in field-sampled caterpillars of Cydia pomonella during 2010/2011. Each point is the mean ± S.D. (n = 3 replicates, 3 individuals each). Influence of sampling date on glutamine concentration was tested by ANOVA followed by Bonferroni's post hoc test (means flanked with different letters are significantly different).

Figure 5. Free amino acids.

Seasonal changes in concentrations of selected amino acids in hemolymph (A), fat body (B), and body wall (C) of field-sampled caterpillars of Cydia pomonella during 2010/2011. The areas showing concentrations of individual compounds are stacked and the total concentration of free amino acids is shown as a broken line. See Dataset S1 for details.

Figure 6. Hemolymph metabolom.

Principal component analysis showing the association between sampling date (red circles) and the concentration of 52 different metabolites (eigenvectors) in the hemolymph of field-sampled caterpillars of Cydia pomonella during 2010/2011. The numbers coding for metabolites are decoded in Dataset S1. The eigenvectors of alanine (6), fructose (44), and mannitol (46) extend beyond the circle delimiting 90% fit of the model. The metabolites (42–47) most characteristic for winter (January) sample are enclosed by a dashed line ellipse.

Figure 7. Osmolality and SCP.

Seasonal changes of hemolymph osmolality and whole-body supercooling point (SCP) of field-sampled caterpillars of Cydia pomonella during 2010/2011. Each point is the mean ± S.D. (osmolality, n = 10 individuals; SCP, n = 8 individuals). Influence of sampling date on parameter was tested by ANOVA followed by Bonferroni's post hoc test (means flanked with different letters are significantly different). Inset shows that Pearson's correlation between osmolality and SCP was relatively tight (close to statistical significance).

Figure 8. Cold tolerance.

Survival at subzero temperatures in supercooled and partially frozen states in the field-sampled caterpillars of Cydia pomonella during 2010/2011. Each point is the percentage of survivors in a sample of n larvae (n = flanking number). Supercooled larvae were exposed either to −5°C for 14 d or to −15°C for 7 d. Partially frozen larvae were exposed to −5°C for 1 h.