Cloning of the cuticular protein genes in Hyphantria cunea and its response to HcNPV stress

This study aimed to clone cuticular protein (CP) family genes from the fall webworm, Hyphantria cunea, characterize their sequence features and spatiotemporal expression patterns, and investigate their transcriptional responses to Hyphantria cunea nucleopolyhedrovirus (HcNPV) infection.

The full-length cDNA sequences of HcCPs genes were cloned using RT-PCR, and their molecular and biological characteristics were analyzed using online bioinformatics tools and software. RT-qPCR was applied to detect the expression profiles of the HcCPs genes across different developmental stages (eggs, 1st–7th instar larvae, pupae and adults), in different tissues (head, cuticle, Malpighian tubules, foregut, midgut, hindgut, silk gland, fat body, testis and ovary), and following exposure to different concentrations of HcNPV.

The open reading frames (ORFs) of the seven H. cunea CP genes were 291, 492, 720, 426, 657, 408 and 742 bp in length, encoding proteins of 96, 163, 239, 141, 218, 135 and 313 amino acids, respectively. Their predicted molecular weights ranged from 10.8 to 32.2 kDa, and theoretical isoelectric points ranged from 5.00 to 9.44. Tertiary structure prediction revealed that HcCP proteins contained mainly of α-helices, β-sheets and random coils. Phylogenetic analysis demonstrated that the HcCPs were most closely related to homologous proteins from other lepidopteran insects. RT-qPCR analysis revealed relatively high expression levels of the HcCP genes in the head, cuticle, testis and during the 5th to 7th larval instars. Their expression levels were also higher in male adults than in female adults. Exposure of H. cunea larvae to different concentrations of HcNPV resulted in time-dependent changes in HcCP expression. Under both high-dose (2 × 10⁵ PIBs/mL) and low-dose (2 × 10³ PIBs/mL) treatments, transcriptional levels initially increased, subsequently decreased, and then increased again. Following exposure to 2 × 10³ PIBs/mL HcNPV, HcCP1, HcCP19, HcPCP36a and HcLCPA2B reached their highest expression levels at 120 h, ranging from 38.94–4994.20-fold relative to the control. In contrast, HcPCP, HcL/PRCP66 and HcLCP30 peaked at 48 h, with expression levels 152.16-, 512.07- and 88.82-fold higher than those of the control, respectively. Following exposure to 2 × 10⁵ PIBs/mL HcNPV, HcCP1 achieved its highest expression level at 24 h (104.12-fold of control). The expression levels of HcCP19, HcPCP36a and HcLCPA2B peaked at 120 h and were 67.93-, 6824.97-and 12.66-fold higher than those of the control, respectively. Meanwhile, HcPCP, HcL/PRCP66 and HcLCP30 reached their highest expression levels at 48 h, at 85.91-, 218.66- and 29.16-fold those of controls, respectively. These findings indicate that HcCP genes are strongly involved in the response of H. cunea to HcNPV infection.

Collectively, these results demonstrate that HcCPs play vital roles in the defense of H. cunea against HcNPV infection and provide a foundation for further elucidating the mechanisms underlying HcNPV infection in this insect.