Sequence and phylogenetic analysis of TaPDK
TaPDK has an 1824-bp open-reading frame that encodes proteins containing 565 amino acid residues with theoretical molecular masses of 57.17 kDa. TaPDK is a serine/threonine-protein kinase, phosphorylating and activating a subgroup of AGC family protein kinases, containing a protein kinase domain (Pkinase, amino acids 103–382) and pleckstrin homology domain (PH, amino acids 460–565). A conserved sequence (SRKVSFVGTAQYVSPE) located in Pkinase domain is the activation loop, and Ser-280 is the activation loop site (Fig. 1A). Furthermore, consistent with our expectation that a phylogenetic analysis based on the PDK sequences collected from various insects grouped TaPDK among the other lepidopterans. In the branches of the Lepidoptera, the relationship between the T. absoluta and H. kahamanoa is the closest (Fig. 1B).
Developmental and tissue-specific expression of TaPDK
The results showed that the expression profiles of TaPDK was significant different among different developmental stages (F19,38 = 41.88, P < 0.0001, Fig. S1A). TaPDK was highest expressed in 5-day-old pupae, then in 6-day-old pupae before the adult emerges and the expression was declined immediately after the eclosion. The expression of TaPDK at other stages was not statistically significant (Fig. S1A). TaPDK was predominantly expressed in three different tissues (F 8,16= 752.8, P < 0.0001, Fig. S1B), with the highest expression levels in the head, followed by the midgut, and then in the abdomen (Fig. S1B).
Knockdown of TaPDK impairs pupal ecdysis
At 1, 2, 3, and 4 days after dsRNA injection, the RNAi silencing efficiency of TaPDK was 78.43% (t = 10.22, df = 4, P = 0.0005), 82.37% (t = 7.94, df = 4, P = 0.0014), 79.91% (t = 11.71, df = 4, P = 0.0003), and 47.14% (t = 4.13, df = 4, P = 0.0145), respectively (Fig. 2A). The survival rate of individuals treated with dsTaPDK was significantly lower than those treated with dsGFP (68.71% vs 92.2%; Chi-square = 14.89, df = 1, P = 0.0001; Fig. 2B); About 22.40% of the pupae showed severe phenotypic changes (cuticular shrinkage) after dsTaPDK injection and eventually all of them blackened and died, 8.89% of the pupae showed abnormal ecdysis of the head and eventually died after the 7 d of dsTaPDK injection, and the remaining 68.71% of the pupal ecdysis normally (Fig. 2C).
Further, the expression levels of TaE75 (t = 202.90, df = 4, P < 0.0001), TaHR3 (t = 11.56, df = 4, P = 0.0003), TaHR38 (t = 130.30, df = 4, P < 0.0001), and TaFTZ-F1 (t = 40.29, df = 4, P < 0.0001) were reduced significantly after the dsTaPDK treatment (Fig. 3B). Similarly, the ecdysteroids titer was reduced significantly compared to the controls treated with dsGFP (5.62 µmol/mL vs 35.08 µmol/mL; t = 35.33, df = 4, P < 0.0001; Fig. 3A). The expression level of TaCDA1 (t = 13.74, df = 4, P = 0.0002), TaCHT5 (t = 32.16, df = 4, P = 0.0014), TaUAP (t = 7.94, df = 4, P = 0.0014), and TaCHS (t = 7.94, df = 4, P < 0.0001) were decreased significantly after the dsTaPDK treatment (Fig. 3D) along with the chitin content (12.40 µmol/mL vs 17.56 µmol/mL; t = 6.53, df = 4, P = 0.0028; Fig. 3C).
Knockdown of TaPDK effects on ovarian development and female fertility
TaPDK silencing caused an observable decrease in female fecundity after the dsTaPDK treatment (t = 29.45, df = 119, P < 0.0001; Fig. 4A). The eggs laid by females in TaPDK-depeletion group exhibited a 33.55% reduction in hatching rate (t = 15.02, df = 111, P < 0.0001; Fig. 4A). The oviposition period of females under dsTaPDK treatment was shortened by 3 days compared with those under the dsGFP treatment (t = 11.64, df = 82, P < 0.0001; Fig. 4B). The mean length of the ovarian tube after TaPDK interference was 3184.3 µm, a 35.41% decrease compared to the control length of 4930.44 µm (t = 12.06, df = 54, P < 0.0001), and the length of the eggs was 410.83 µm, a significant decrease of 13.87% compared to that of the controls, which was 476.97 µm (t = 4.14, df = 96, P < 0.0001; Fig. 4C). Compared with controls, TaPDK knockdown resulted in markedly wrinkled ovarian tubes, with mostly immature white oocytes, and the ovaries remained in the yolk deposition stage (grade II) (Fig. 4F). The expression levels of TaVg and TaVgR were significantly reduced at 1, 2, 3 and 4 days after dsTaPDK injection (Fig. 4E). Similarly, the vitellogenin content was reduced significantly compared to the controls treated with dsGFP (2.20 µg/mL vs 3.60 µg/mL; t = 6.22, df = 4, P = 0.0034; Fig. 4D). The expression levels of TaKr-h1 (t = 73.41, df = 4, P < 0.0001), TaMet (t = 97.48, df = 4, P < 0.0001), and TaJHA (t = 63.71, df = 4, P < 0.0001) reduced significantly in all dsTaPDK-treated individuals (Fig. 4G). Similarly, the JH titer was reduced significantly compared to the dsGFP control (26.42 pg/mL vs 53.84 pg/mL; t = 12.60, df = 4, P = 0.0002; Fig. 4H).