Global metabolomic analyses of the hemolymph and brain during the initiation, maintenance, and termination of pupal diapause in the cotton bollworm, Helicoverpa armigera

Abstract

A strategy known as diapause (developmental arrest) has evolved in insects to increase their survival rate under harsh environmental conditions. Diapause causes a dramatic reduction in the metabolic rate and drastically extends lifespan. However, little is known about the mechanisms underlying the metabolic changes involved. Using gas chromatography-mass spectrometry, we compared the changes in the metabolite levels in the brain and hemolymph of nondiapause- and diapause-destined cotton bollworm, Helicoverpa armigera, during the initiation, maintenance, and termination of pupal diapause. A total of 55 metabolites in the hemolymph and 52 metabolites in the brain were detected. Of these metabolites, 21 and 12 metabolite levels were altered in the diapause pupal hemolymph and brain, respectively. During diapause initiation and maintenance, the number of metabolites with increased levels in the hemolymph of the diapausing pupae is far greater than the number in the nondiapause pupae. These increased metabolites function as an energy source, metabolic intermediates, and cryoprotectants. The number of metabolites with decreased levels in the brain of diapausing pupae is far greater than the number in the nondiapause pupae. Low metabolite levels are likely to directly or indirectly repress the brain metabolic activity. During diapause termination, most of the metabolite levels in the hemolymph of the diapausing pupae rapidly decrease because they function as energy and metabolic sources that promote pupa-adult development. In conclusion, the metabolites with altered levels in the hemolymph and brain serve as energy and metabolic resources and help to maintain a low brain metabolic activity during diapause.

Figure 1. Principal component analysis (PCA) score plots and altered metabolites levels in the hemolymph.

(A) PCA and (B) altered metabolites levels in the hemolymph of nondiapause- and diapause-destined pupae. The first principal component (PC1) containing valine, threonine, methionine, and phosphoric acid, etc. accounted for 44.0% of total variation, and second principal component (PC2) containing glycine, β-alanine, phenylalanine, and D-gluconic acid accounted for 18.9% of total variation in hemolymph PCA plot. The metabolites were detected in day 1 to 10 pupae. 1) and 2) represent reduced- and elevated-metabolite levels, respectively, in diapause-destined pupal hemolymph. The samples within a treatment group were circumscribed spatially by the dashed line. The relative ratio is the ratio of metabolite peak area to internal standard (sucrose) peak area. All bars represent the mean ± S.D. from four repeats. *, p<0.05; **, p<0.01 (determined by one-way ANOVA). NP, nondiapause pupa; DP, diapause-destined pupa. Arabic number (1, 2, 4, and 10) represent the days after pupation.

Figure 2. Principal component analysis (PCA) score plots and altered metabolites levels in the brain.

(A) PCA and (B) altered metabolites levels in the brain of nondiapause- and diapause-destined pupae. The metabolites were detected in day 1 to 10 pupae. PC1 containing valine, alanine, leucine, isoleucine threonine, methionine, and phosphoric acid, etc. accounted for 52.2% of total variation, and PC2 containing β-alanine and tryptophan accounted for 11.1% of total variation in brain PCA plot. 1) and 2) represent reduced- and elevated-metabolite levels, respectively, in diapause-destined pupal brain. The samples within a treatment group were circumscribed spatially by the dashed line. The relative ratio is the ratio of metabolite peak area to internal standard (sucrose) peak area. All bars represent the mean ± S.D. from four repeats. *, p<0.05; **, p<0.01 (determined by one-way ANOVA). NP, nondiapause pupa; DP, diapause-destined pupa. Arabic number (1, 4, and 10) represent the days after pupation.

Figure 3. Metabolite contents in the hemolymph and brain during diapausing.

Metabolite content in the hemolymph (A) and brain (B) of nondiapause pupae (NP) and diapausing pupae (DP). 1) and 2)represent the significantly reduced-and elevated-metabolite levels, respectively, in the diapause pupal hemolymph or brain (p<0.05; determined by one-way ANOVA). The relative ratio is the ratio of the metabolite peak area to the internal standard (sucrose) peak area. All of the bars represent the mean ± S.D. from four replicates.

Figure 4. Metabolite content in the diapausing pupal hemolymph during diapause termination resulting from injection of 20-Hydroxyecdysone (20E).

Pupae were incubated at 25 °C after injection. The metabolites in the hemolymph were detected at 24, 48, and 72 h after injection. C represents the diapausing pupae not given the 20E injection and used as a control. (A) The PCA analysis for the metabolites in the hemolymph after injection. PC1 containing valine, leucine, proline, and phosphoric acid accounted for 39.1% of total variation, and PC2 containing butanoic acid, phenylalanine, tyrosine, and cholesterol accounted for 21.2% of total variation in brain PCA plot. The samples within a treatment group were circumscribed spatially by the dashed line. (B) Altered metabolite levels during diapause termination. The relative ratio is the ratio of the metabolite peak area to the internal standard (sucrose) peak area. All of the bars represent the mean ± S.D. from four replicates. *, p<0.05; **, p<0.01 (determined by one-way ANOVA).

Figure 5. Possible mechanisms underlying insect pupal diapause.

The increased metabolite levels in the hemolymph serve as energy sources and cryoprotectants for use during a long pupal diapause phase to survive harsh environmental conditions and to sustain pupa-adult development after diapause termination. The decreased metabolite levels in the hemolymph and brain are limiting factors that contribute to repressing brain activity.