Unraveling the Role of Cuticular Protein 3-like (HvCP3L) in the Chitin Pathway through RNAi and Methoxyfenozide Stress Response in Heortia vitessoides Moore

Abstract

Cuticle proteins (CPs) constitute a multifunctional family; however, the physiological role of Cuticle Protein 3-like (CP3L) in Heortia vitessoides Moore remains largely unclear. In this study, we cloned the HvCP3L gene from the transcriptional library of Heortia vitessoides Moore. RT-qPCR results revealed that HvCP3L exhibited high expression levels during the larval stage of Heortia vitessoides Moore, particularly at the L5D1 stage, observed in both larval and adult heads. Through RNA interference, we successfully silenced the HvCP3L gene, resulting in a significant reduction in the survival rate of Heortia vitessoides Moore, with the survival rate from larvae to adults plummeting to a mere 17.7%, accompanied by phenotypic abnormalities. Additionally, we observed that the knockdown of HvCP3L led to the inhibition of genes in the chitin pathway. Following exposure to methoxyfenozide stress, the HvCP3L gene exhibited significant overexpression, coinciding with phenotypic abnormalities. These findings underscore the pivotal role of HvCP3L in the growth and development of Heortia vitessoides Moore.

Keywords: Heortia vitessoides Moore; RNA interference; chitin pathway; cuticular proteins; growth and development; methoxyfenozide stress.

Figure 1

The amino acid sequence of HvCP3L from Heortia vitessoides Moore. The start codon and the termination codon are marked with underlined, conserved regions are marked with gray shading, “*” Representing termination codons.

Figure 2

Secondary structure prediction of HvCP3L.

Figure 3

Sequence alignment of HvCP3L with insect homologs. The amino acid residues thatare identical in all sequences are marked with dark shading, whereas light shading indicates thatat least 75% amino acids are identical in all sequences. The aligned sequences are the predictedThe aligned sequences are the predicted amino acid sequences of CP3L from Heortia vitessoides Moore (HvCP3L WWZ69572.1), Helicoverpa zea (HzCP3L XP_047031077.1), Achroia grisella (XP_059057921.1), Galleria mellonella (XP_026763387.1), Ostrinia furnacalis (OfCP3LXP_028179309.1), Vanessa cardui (VcCP3L XP_046970535.1).

Figure 4

Phylogenetic analysis of HvCP3L. The predicted amino acid sequences of HvCP3L together with 19 selected CP members were aligned, and a phylogenetic tree was constructed using MEGA7. The CP3L of Heortia vitessoides Moore marked with red underline. GenBank accession numbers are as follows:Trichoplusia ni CP3L (XP_026739799.1); Heortia vitessoides CP3L (WWZ69572.1); Vanessa tameamea CP3L (XP_047536305.1); Zerene cesonia CP3L (XP_038211198.1); Papilio xuthus CP3L (NP_001298774.1); Bombyx mandarina CP3L (XP_028038894.1); Vanessa atalanta CP3L (XP_047536305.1); Spodoptera frugiperda CP3L (XP_035435189.1); Homalodisca vitripennis CP3L (XP_046667001.1); Bemisia tabaci CP3L (XP_018897328.1); Trichogramma pretiosum CP3L(XP_023316065.1); Leptopilina heterotoma CP3L (XP_043472504.1); Anoplophora glabripennis CP3L (XP_018564028.1); Sitophilus oryzae CP3L (XP_030751496.1); Diabrotica virgifera CP3L (XP_050501233.1); Schistocerca nitens CP16.5L (XP_049811031.1); Schistocerca serialis CP21.3L (XP_049952148.1); Hermetia illucens CP21.3L (XP_037907580.1); Topomyia yanbarensis CP21.3L (XP_058835519.1); Malaya genurostris CP21.3L (XP_058461337.1).

Figure 5

Relative expression levels of HvCP3L at different stages: L1–L4, first-to fourth-instar larvae; L5D1-L5D4, 1-to-4-day fifth-instar larvae; PP, pre-pupae; P1–P3, 1-to-3-day-old pupae; A1, 1-day-old adults; A3, 3-day-old adults. Error bars represent mean ± standard error of three biological replicates. Different letters above error bars indicate significant differences (p < 0.05) based on one-way ANOVA and Tukey’s test.

Figure 6

Relative expression levels of HvCP3L in different larval tissues (tissue anatomy for the fifth-instar larvae). Relative expression in larval tissues: HD, head; EP, epidermis; FG, foregut; MG, midgut; HG, hindgut; and FB, fat body. Error bars represent mean ± standard error of three biological replicates Different letters above error bars indicate significant differences (p < 0.05), which were based on one-way analysis of variance (ANOVA) and Tukey’s test.

Figure 7

Relative expression in adult tissues: HD, head; TH, thorax; AB(♂), male abdomen; AB(♀), female abdomen; FO, foot; and WI, wing. Error bars represent mean ± standard error of three biological replicates. Different letters above error bars indicate significant differences (p < 0.05), which were based on one-way analysis of variance (ANOVA) and Tukey’s test.

Figure 8

Changes in mRNA level after treatment with specific RNA interference. Relative transcript levels of HvCP3L in L4 larvae after injection with dsHvCP3L at a concentration of 5.0 µg/µL for 12, 24, 36, 48, 72 h. The sample size was 120 larvae, which were divided into three biological replicates. Error bars represent mean ± standard error of three biological replicates. * p < 0.05, ** p < 0.01. Analysis was performed via one-way analysis of variance (ANOVA), followed by Student’s t-test.

Figure 9

The changes in chitin pathway level after RNA interference of HvCP3L gene. Error bars represent mean ± standard error of three biological replicates. Different letters above error bars indicate significant differences (*p < 0.05, ** p < 0.01), which were based on one-way analysis of variance (ANOVA) and Tukey’s test.

Figure 10

Analysis after interference with HvCP3L and methoxyfenozide treatment. (A) Effects of HvCP3L RNAi on larval-to-pupal and pupal-to-adult transition rates. Rates of insect survival from fifth-instar larval stage to adulthood after dsHvCP3L injection (* p < 0.05, Kaplan–Meier survival analysis with log-rank test). Data are the mean ± standard error of three biological repeats. (B) Phenotypic abnormalities caused by RNAi interference with HvCP3L and methoxyfenozide treatment.

Figure 11

Changes in mRNA level after treatment with methoxyfenozide. The CK indicated that the experimental control was fed without methoxyfenozide. Error bars represent mean ± standard error of three biological replicates. * p < 0.05, ** p < 0.01. Analysis was performed via one-way analysis of variance (ANOVA), followed by Student’s t-test.