Sitodiplosis mosellana, a notorious pest of wheat worldwide, copes with temperature extremes during harsh summers and winters by entering obligatory diapause as larvae. However, the metabolic adaptive mechanism underlying this process is largely unknown. In this study, we performed a comparative metabolomics analysis on S. mosellana larvae at four programmed developmental stages, i.e., pre-diapause, diapause, low temperature quiescence and post-diapause development. In total, we identified 54 differential metabolites based on pairwise comparisons of the four groups. Of these metabolites, 37 decreased in response to diapause, including 4 TCA cycle intermediates (malic acid, citric acid, fumaric acid, α-ketoglutaric acid), 2 saturated fatty acids (palmitic acid, stearic acid) and most amino acids. In contrast, nine metabolites, including trehalose, glycerol, mannitol, proline, alanine, oleic acid and linoleic acid were significantly higher in both the diapause and quiescent stages than the other two stages. In addition to two of them (trehalose, proline), glutamine was also significantly highest in the cold quiescence stage. These elevated metabolites could function as cryoprotectants and/or energy reserves. These findings suggest that the reduced TCA cycle activity and elevated biosynthesis of functional metabolites are most likely responsible for maintaining low metabolic activity and cold tolerance during diapause, which is crucial for the survival and post-diapause development of this pest.

Sitodiplosis mosellana, a periodic but devastating wheat pest that escapes temperature extremes in summer and winter by undergoing obligatory diapause. To determine the roles of small heat shock proteins (sHsps) in diapause of S. mosellana, we characterized two sHsp genes, SmHsp17.4 and SmHsp20.3, from this species. Both SmHsps contained the conserved α-crystallin domain and the carboxy-terminal I/VXI/V motif of the sHsp family. SmHsp17.4 had one intron while SmHsp20.3 had none. Quantitative PCR revealed that SmHsp17.4 expression decreased after diapause initiation, but substantially increased during transition to post-diapause quiescence. In contrast, SmHsp20.3 expression was not affected by entry of diapause, but was clearly up-regulated during summer and winter. Short-term more severe heat-stress (≥35 °C) of over-summering larvae or cold-stress (≤−5 °C) of over-wintering larvae could stimulate higher expression of both genes, and SmHsp17.4 was more responsive to cold stress while SmHsp20.3 was more sensitive to heat stress. Notably, transcription of SmHsp17.4, but not SmHsp20.3, in diapausing larvae was inducible by 20-hydroxyecdysone (20E). Recombinant SmHsp17.4 and SmHsp20.3 proteins also displayed significant chaperone functionality. These findings suggest that both SmHsps play key roles in stress tolerance during diapause; and 20E-regulated SmHsp17.4 was also likely involved in diapause termination.

The heat shock protein 70 (Hsp70) family mediates responses to environmental stress in insects. The wheat midge Sitodiplosis mosellana, a worldwide pest, avoids summer and winter temperature extremes by diapause of the third-instar larvae in the soil. To explore the functions of Hsp70s in this process, we characterized two cytoplasmic Hsp70 genes (SmHsp70A1-1 and SmHsp70A1-2) from this insect. Both SmHsp70s contained three signature motifs of the family and lacked introns. Developmental expression profiling revealed maximal SmHsp70A1-1 expression during early larval stages, while the expression of SmHsp70A1-2 was highest in the pupal stages. The expression of SmHsp70A1-1 was significantly upregulated during diapause, particularly during summer and winter, whereas SmHsp70A1-2 showed marked downregulation and dose-dependent induction by 20-hydroxyecdysone (20E). Furthermore, both genes exhibited similar expression patterns in over-summering and over-wintering larvae under thermal stress, with maximal expression at 40 °C and −10 °C, respectively, but were not significantly induced at prolonged extreme temperatures (50 °C or −15 °C). Knockdown of the two SmHsp70 genes by RNA interference (RNAi) significantly increased the susceptibility of the larvae to cold stress. These results suggest the important role of both SmHsp70 genes in diapause-associated stress tolerance and provide crucial insights into the mechanisms underlying thermal adaptation in S. mosellana.

Small heat shock proteins (sHsps) play crucial roles in organismal adaptation to stress tolerance. Sitodiplosis mosellana, a devastating insect wheat pest, undergoes long obligatory larval diapause to survive temperature extremes during summer and winter. To elucidate the function of sHsps in this process, two sHsp-encoding genes (SmHsp22.2 and SmHsp26.7) were characterized from S. mosellana, and their responsiveness to diapause and thermal stress, as well as their roles in cold stress, was analyzed. Both SmHsp22.2 and SmHsp26.7 possessed the canonical α-crystallin domain and lacked introns. Quantitative PCR indicated significant upregulation of SmHsp22.2 and SmHsp26.7 during diapause, especially in summer and winter. Notably, SmHsp22.2 exhibited higher expression in summer relative to winter, whereas SmHsp26.7 showed the opposite profile. Moreover, short-term heat shock (≥35 °C) in over-summering larvae or cold shock (≤−10 °C) in over-wintering larvae was found to trigger transcriptional upregulation of both genes, while prolonged temperature extremes (i.e., 45–50 °C or −15 °C) did not elicit a comparable response. RNA interference-mediated knockdown of both genes significantly increased the mortality of S. mosellana larvae under cold stress. These findings indicate the importance of both SmHsps in diapause and environmental adaptation in S. mosellana.

20-hydroxyecdysone (20E) is essential for insect development and diapause. Ecdysone receptor (EcR) and ultraspiracle (USP) proteins are crucial regulators of 20E signaling. To explore their potential roles in the development of Sitodiplosis mosellana, a major wheat pest that undergoes obligatory diapause as a larva, one SmEcR and two SmUSPs (SmUSP-A and SmUSP-B) from this species were isolated and characterized. The deduced SmEcR and SmUSP-A/B proteins contained a conserved DNA-binding domain with two zinc finger motifs that bind to specific DNA sequences. Expression of SmEcR and the SmUSPs was developmentally controlled, as was 20E induction. Their transcription levels increased as the larvae entered pre-diapause, followed by downregulation during diapause and upregulation during the shift to post-diapause quiescence, which is highly consistent with ecdysteroid titers in this species. Topical application of 20E to diapausing larvae also elicited a dose-dependent expression of the three genes. Expression of SmEcR and SmUSPs decreased markedly during the pre-pupal stage and was higher in adult females compared to males. These findings suggested that 20E-induced expression of SmEcR and SmUSPs has key roles in diapause initiation and maintenance, post-diapause quiescence, and adult reproduction, while the larval–pupal transformation may be associated with a decrease in their expression levels.

The mason bee Osmia excavata Alfken (Hymenoptera: Megachilidae) is an essential pollinator for rosaceous fruit trees in northern China, but its populations are declining because of severe infestations by the mite Chaetodactylus hirashimai Kurosa (Acari: Sarcoptiformes). This mite feeds on pollen within mason bee nest tubes, thereby impairing larval development. However, the effects of different floral resources on the fitness of C. hirashimai remain unclear. Herein, we evaluated the development, survival, and reproduction of C. hirashimai reared on four diets (cherry pollen, cherry bee bread, apple pollen, or apple bee bread), by using an age-stage, two-sex life table. Chaetodactylus hirashimai successfully completed its life cycle on all diets. Cherry pollen supported shorter pre-adult development, longer adult longevity, and higher fecundity than apple pollen. Bee bread diets resulted in significantly lower pre-adult mortality (11.76–14.81%), and higher fecundity (270–278 eggs/female) and net reproductive rate (R₀ = 115–120 offspring/individual), than raw pollen (44.64–44.83% mortality; 117–179 eggs/female; R₀ = 40–50 offspring/individual). No significant differences in the intrinsic rate of increase (r) and finite rate of increase (λ) were observed among diets. Our findings demonstrated that cherry pollen is a highly favorable resource for C. hirashimai, and bee bread further enhances mite population growth. This study highlights the ecological conflict between cherry pollination and mite infestation, and these life-table results can provide preliminary laboratory support for subsequent research on integrated mite control in fruit orchards.