TSSKs are specifically expressed in male testis of C. pomonella
During the larval stage, the C. pomonella, like other Lepidopteran insects, possesses two separate testes. However, as the insect enters the prepupal stage, these testes gradually move closer to each other. By the time the insect reaches the pupal stage, the two testes fuse together to form a single mature testis in the adult stage (Fig. 1A). To investigate the genes that are specifically expressed in the testes of adult males and their role in mating, RNA-seq was conducted on the testes of 2-day-old (presexual maturity, CpA2T) and 5-day-old (postsexual maturity, CpA5T) male of C. pomonella. Other parts of the testes were used as controls (CpA2U and CpA5U, respectively). This analysis identified a total of 7736 DEGs in the comparison of presexual maturity and control samples, with 4240 upregulated and 3496 downregulated genes. Similarly, 7677 DEGs, with 4378 upregulated and 3299 downregulated genes, were identified in the comparison of postsexual maturity and control samples (Fig. 1B; Figure S1A and S1B). Notably, the testes exhibited high expression of serine protease genes (Figure S2), tektin (Figure S3A), cyclin (Figure S3B), ubiquitin (Figure S3C), and tubulin (Figure S3D). These DEGs may serve as potential candidates for modulating the fecundity of male C. pomonella. Furthermore, GO enrichment analysis highlighted the crucial role played by the differential genes (Figure S1C and S1D), while KEGG enrichment analysis showed their close relationship to the metabolic pathway (Figure S1E and S1F), particularly the testicle-specific serine/threonine protein kinase (TSSK), which displayed high expression (Figure S2). Members of the TSSK gene family, including TSSK1, TSSK1a, TSSK2, TSSK2a, and TSSK4, were selected for further investigation to elucidate their role in spermatogenesis. Phylogenetic analysis revealed the clustering of these genes with TSSK from other Lepidoptera species (Fig. 1C; Table S1), and collinearity analysis demonstrated their presence among Lepidoptera insects (Figure S4). Multiple sequence comparisons confirmed the conservation of TSSK residues in the S-TKc region, as well as the adenosine triphosphate (ATP) and substrate binding domains (Fig. 1D). Additionally, RT-qPCR analysis determined the expression levels of TSSKs in the developmental stages and tissues of C. pomonella, showing moderate expression during the pupal stages and increased expression as the insects reached sexual maturity. TSSKs were found to be expressed only in the male testis, with TSSK4 exhibiting the highest expression level (Fig. 1E). These findings suggest that these testis-specifically expressed TSSKs play an important role in the m ale fertility of C. pomonella.
Loss function of TSSKs influence male fertility of C. pomonella
Following a 48-h period of injection, the efficiency of RNAi was observed to increase from 37.32% (Figure S5) to 86.09% (Fig. 2A), suggesting that double injections method enhances the interference efficiency over time. Upon injecting adults subjects with dsTSSKs and conducting subsequent observations after 24 h, it was found that the expression levels of TSSK1, TSSK1a, TSSK2, TSSK2a, and TSSK4 were significantly reduced by 56.13% (p = 0.001)、86.09% (p < 0.001)、42.67% (p = 0.038)、72.08% (p < 0.001), and 75.62% (p < 0.001), respectively, compared to control treatment with dsGFP (Fig. 2A). Although the number of eggs produced by the females mated with dsTSSKs treated males remained unchanged (Fig. 2B), the hatching rate of these eggs was significantly lower compared to dsGFP treatment (Fig. 2C). The sterility rates of TSSK1, TSSK1a, TSSK2, TSSK2a, and TSSK4 knockdown lines were found to be 90.61%, 83.93%, 100%, 100%, and 100%, respectively (Fig. 2C). The unhatched eggs exhibited progressive desiccation during development, with no evidence of reaching the blackhead stage (Fig. 2D). To ascertain whether the underdeveloped eggs were a result of lack of fertilization in the female, a spermatophore examination was conducted, ruling out this possibility (Figure S6). Sperm viability was then assessed in both the dsGFP and dsTSSKs treatment groups, revealing a higher number of dead eupyrene and apyrene sperm bundles in the dsTSSKs treatment group compared to the dsGFP group (Fig. 2E). In males, the number of spermatozoa was reduced by 28.61% (dsTSSK1, p = 0.0014), 63.03% (dsTSSK1a, p < 0.001), 39.00% (dsTSSK2, p = 0.0 030), 46.77% (dsTSSK2a, p < 0.001), and 30.38% (dsTSSK4, p = 0.0024) in the dsTSSKs treatment groups compared to the control (Fig. 2F).
In addition, we employed the CRISPR/Cas9 gene editing system to induce mutation in the TSSK genes (Fig. 2G), which led to deletions at the desired location (Fig. 2H and I). The mutation rate of the TSSK genes varied from 9.38–12.46% (Table S3), and these genetic modifications did not have any impact on the insect′s developmental period (Table S4). When TSSKs−/− males were mated with wt females, the egg-laying levels were similar to the results of RNAi (Fig. 2J). However, a significant decrease in hatchability, consistent with the results of RNAi approach, was observed (Fig. 2K). Most embryos derived from TSSKs−/− males failed to develop properly, even after seven days following ovulation. Consequently, TSSKs−/− males exhibit normal sexual behavior but are ultimately sterile.
Lnc117962 is specifically expressed in male testis of C. pomonella
Distinctive expression patterns of lncRNAs have been observed in testis, accessory gland, seminal vesicles and vas deferens., indicating the presence of tissue-specific lncRNAs (Fig. 3A). By analyzing the Venn diagram, it was found that 6361 lncRNAs were specifically expressed in the testis (CpT1D vs CpT1DC). Furthermore, the testis exhibited specific expression of 7390 lncRNAs in CpT3D compared to CpT3DC, with 4430 lncRNAs being expressed in both CpT1D vs CpT1DC and CpT3D vs CpT3DC (Fig. 3B). Twelve lncRNAs with differential expression (log2fold change ≥ 4 threshold) were identified, and their tissue-specific expression patterns in different developmental stages were confirmed using RT-qPCR. The RT-qPCR results revealed that all 12 lncRNAs were highly expressed in the testis, while exhibiting negligible expression in the vas deferens, accessory glands, and seminal vesicles (Fig. 3C). Notably, lnc117962showed the highest expression level in the testes, with subsequent decreased expression as sperm matured and shifted. Furthermore, fluorescent in FISH revealed a localization signal in the testes of C. pomonella (Fig. 3D). Results from Gene Ontology (GO) pathway analysis suggest that lnc117962 has the potential to modulate various gene enrichments associated with ATP activity and serine/threonine protein kinase pathways. This indicates that lnc117962 might play a crucial role in regulating the expression of TSSKs and could be significant in the post-transcriptional control of male fertility (Figure S7).
Loss function of lnc117962 influences male fertility of C. pomonella
The role of lnc117962 in the regulation of male fertility was investigated using RNAi technology. Silencing lnc117962 with ds117962 resulted in a 74.83% efficacy after 48 h (Fig. 4A). Assessment of TSSK family gene expression levels following ds117962 treatment showed reductions of 71.94%, 42.87%, 47.96%, 91.58%, and 63.36% compared to dsGFP control (Fig. 4B). Overexpression of lnc117962 with pcDNA3.1-lnc117962 resulted in a 2.47-fold increase in lnc117962 expression compared to pcDNA3.1-GFP (Fig. 4C). Additionally, TSSK1, TSSK1a, and TSSK4 expression decreased by 39.28%, 26.61%, and 19.93%, respectively, while TSSK2a expression increased by 1.26-fold with no significant change in TSSK2 expression (Fig. 4D). Fertility response to dslnc117962 treatment was assessed based on fertilization and hatching rates, showing no significant difference in egg laying but a 28.5% reduction in hatching rate (Fig. 4E, 4F). Male longevity was unaffected (Figure S8). The dslnc117962 treatment group exhibited more dead sperm in eupyrene and apyrene sperm bundles compared to the control group (Fig. 4G), suggesting lnc117962 involvement in C. pomonella male spermatogenesis and its role in male fertility.
The insignificance of the overall fitness of dslnc117962 knockdown males was anticipated and evaluated using a mating competitiveness assay (Fig. 4H). The results indicated that dslnc117962 knockdown males displayed the capacity for courtship, mating, and successful competition with wt females. Specifically, the egg hatch rate was found to be 52.47% ± 15.96% for a pair of one wt male, one dslnc117962 knockdown male and one wt female, compared to 25.52% ± 9.55% for a pair of one wt male and one wt female, and 75.26% ± 4.28% for a pair of one dslnc117962 treated male and one wt female, confirming the mating competitiveness (0.84) of dslnc117962 knockdown males (Table 1). Furthermore, the mating performance of lnc117962 knockdown males was assessed through field cage experiments conducted in the first two weeks of June 2023, under controlled humidity and temperature conditions (Figure S9). Results obtained from the apple orchard (Fig. 4J) indicated no significant disparity in egg production between 10 wt females mated with 10 dslnc117962 knockdown males and those mated with 20 wt males (Fig. 4K). However, the hatch rates were 39.91% ± 7.46% and 83.13% ± 2.7%, respectively (Fig. 4L). Notably, the hatching rate of the F1 generation was determined to be 67.56 ± 5.41% (p = 0.0029), suggesting that the RNAi treatment predominantly affected the parents and had a lasting impact on the offspring (Figure S10). These results indicate that lnc117962 regulates the activity of TSSKs through post-transcriptional mechanisms, exerting a suppressive effect on the population development of C. pomonella.
Table 1
Effect of dslnc117962 knockdown males on the mating competitiveness of C. pomonella.
Matching ratio
(wtM♂: lnc117962KDM♂: wtF♀)
|
Egg laid per female
|
Hatching rate
(%)
|
competition mating
index (C)
|
1:0:1
|
103.33 ± 18.67 a
|
75.26 ± 4.28 a
|
0.84
|
1:1:1
|
101.00 ± 17.59 a
|
54.47 ± 15.96 b
|
0:1:1
|
105.40 ± 22.64 a
|
25.52 ± 9.55 c
|
Lnc117962 knockdown males (lnc117962KDM), wild-type males (wtM) and wild-type females (wtF) were introduced for mating in the ratios 0:1:1, 1:0:1 and 1:1:1, respectively. A total of 15 replicate experiments were evaluated. The table shows mean ± standard deviation (SD) data. Letters following data indicate significant differences analyzed by one-way analysis of variance (ANOVA) using Duncan's test (p < 0.05). |
Lnc117962 regulates TSSKs through competitive bindings to miR-3960
In order to further elucidate the regulatory mechanism of lnc117962 on TSSKs gene expression, we predicted its potential miRNA partner. The miR-3960 emerged as the top candidate among the predicted targets (Table S5 and Figure S11), suggesting its possible interaction with lnc17962. The resemblance in their seed sequences indicates that miR-3960 could plausibly act as a target of lnc117962 (Fig. 5A). To confirm this interaction, a dual luciferase assay demonstrated a 44.33% reduction in luciferase activity in cells transfected with miR-3960 and pmirGLO-lnc117962 mimics compared to mimic negative control (Fig. 5B). Additionally, computational tools including miRanda, PITA, and RNAhybrid were utilized to predict the binding potential of miR-3960 to the 3’UTR of TSSKs, revealing it capability to interact with the 3'UTR of TSSKs (Table S6). Further analysis was conducted to ascertain if lnc117962 functions as a "sponge" or decoy ceRNA of miR-3960. The synthesis of agomir/antagomir-miR-3960 and subsequent RT-qPCR assessment of miR-3960, lnc117962, and TSSKs revealed noteworthy. Following the injection of agomir-miR-3960, the expression level of miR-3960 increased by 3.4 times, along with a significant increase in the expression level of lnc117962, TSSK1a, and TSSK2a, while TSSK1, TSSK2, and TSSK4 levels decreased (P < 0.05, Fig. 5C). Conversely, antagomir-miR-3960 injection led to reduced expression levels of miR-3960, lnc117962, TSSK1, TSSK1a, TSSK2a, and TSSK4 (P < 0.05, Figure. 5D). These findings indicate that lnc117962 acts as a ceRNA by suppressing miR-3960, thereby positively regulating the expression of the TSSK2a (Figure. 5E). However, further research is required to explore the regulatory mechanisms of TSSK1, TSSK1a, TSSK2, and TSSK4.