The S. cerealella strain related to wheat grain was reared in a laboratory under controlled conditions: temperature maintained at 28°C ± 1°C, relative humidity at 75% ± 5%, and a photoperiod of 14:10 h (light:dark). The insects were reared in circular, transparent glass bottles with dimensions of 10 cm in height and a radius of 4 cm. Pupae-containing grains were carefully chosen and transferred to glass tubes for further rearing. Newly emerged adult moths were used to differentiate between males and females for experiments.

Various tissues such as testes, ejaculatory ducts, head, thorax, and fat body, were carefully dissected from adult moths. These tissues were then rinsed with phosphate-buffered saline and rapidly cooled using liquid nitrogen to facilitate RNA extraction. Extraction was carried out utilizing TRIzol reagent (Invitrogen, United Status) following the company’s guidelines. The quality and concentration were evaluated using a Nanodrop spectrophotometer (Thermo Fisher, MA, United Status), while RNA integrity was confirmed on a 1.2% agarose gel. cDNA synthesis was performed using a one-step gDNA removal and cDNA synthesis SuperMix, as per recommended procedures, with 500 ng of RNA template.

The sequence encoding E3 ligase Siah was identified from the testis-specific transcriptome of S. cerealella. Bioinformatic tools such as SMART (http://smart.embl.de/), Pfam (http://pfam.xfam.org/) and NCBI (http://www.ncbi.nlm.nih.gov/) were employed to found the conserved domains in the protein sequence of E3 Siah.

The expression level of tissue-specific ScE3 Siah mRNA was quantified using quantitative real-time PCR (qRT-PCR) conducted on a Bio-Rad iQ2 thermocycler (Bio-Rad, United Status). The cDNA underwent a 1:10 dilution, with 2 μL then applied to a 20 μL reaction mixture comprising 10 μL of SYBR Green Master Mix from Thermo Fisher Scientific (United Status), 0.8 μL each of sense and antisense primers, and 6.4 μL of sterilized ultra-pure water. Three biological replicates were performed, with three technical replicates for each biological sample. The qRT-PCR reaction protocol involved an initial denaturation stage at 95°C for 30 s, followed by 40 cycles comprising denaturation at 95°C for 5 s and annealing/extension at 60°C for 30 s. A melting curve analysis was performed at the end of each reaction. The expression levels were normalized using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Gene-specific primers were designed using Geneious v.10.1.2, and their sequences are provided in Supplementary Table 1. A standard curve was generated to verify primer specificity and efficiency.

The dsRNA was synthesized invitro utilizing the T7 RiboMAX™ Express kit (Promega, United Status) according to the manufacturer’s guidelines. The cDNA template was derived from isolated RNA employing TRIzol Reagent (Invitrogen Life Technologies, United Status) from S. cerealella testis. The target genes, ScE3 Siah and GFP (Green fluorescent protein), underwent amplification using T7GFP and T7 ScE3 Siah primer sets (Supplementary Table 1), resulting in the generation of sense and anti-sense strands individually. The thermal cycling conditions were set as follows: An initial denaturation at 95°C for 5 min, with subsequent 30 cycles comprising denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, concluding with a final extension at 72°C for 10 min. The products were visualized via 1% Agarose gel electrophoresis before purification through ethanol precipitation, and quantified utilizing a UV–visible spectrophotometer. PCR products purified to a concentration of 1 μg/μL were transcribed to yield single-stranded RNAs. Equal volumes of single-stranded RNAs were subjected to annealing through incubation at 70°C for 10 min to produce double-stranded RNA. The dsRNA underwent further purification following the protocol outlined by RiboMAX Express (Promega, United Status), and then quantification via spectrophotometer. Stocks of dsRNA were prepared to a final concentration of 1 μg/μL dsRNA corresponding to the target genes (ScE3 Siah and GFP as a control) was injected into the newly emerged adult male moths. The injection was performed using a fine glass capillary needle, ensuring that the dsRNA was delivered directly into the hemocoel. The injection site was the fifth–sixth internode membrane of the abdomen. The males injected with dsRNA were placed in an artificial climate incubator at a temperature of 28 ± 1°C, a relative humidity of 75 ± 5%, and a photoperiod of L:D = 14:10.

Testes from 15 adult moths were collected and dissected into dsGFP and dsScE3 Siah groups in each replicate. They were fixed in aqueous Bouin’s solution for 8 h, followed by dehydration in alcohol, clearing in xylene, embedding in histological paraffin, and slicing into 3 μm thick sections using a Leica RM 2250 microtome. These sections were stained with hematoxylin and eosin (H&E). Observation was recorded using a NIKON Eclipse Ti-SR microscope, and digital images were captured with a digital camera.

The testes of 15 adult moths from each replicate of the dsGFP and dsScE3 Siah groups were collected and immersed in a fixative solution comprising 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.3) for 24 h at room temperature (RT). They were treated with 1% osmium tetroxide in the same buffer for 2 h for post-fixation. After fixation, the specimens were washed with distilled water and stained with a 0.5% aqueous solution of uranyl acetate for 2 h. Dehydration was carried out using a gradient of acetone concentrations (50%, 70%, 90%, and 100%), followed by embedding in Araldite^® resin. Ultrathin sections were prepared and stained with a saturated alcoholic solution of uranyl acetate and lead citrate before examination with a JEOL-1200E transmission electron microscope (TEM). Image acquisition was performed using a MORADA-G2 TEM camera.

Sperm motility and flagellar characteristics, including wavelength, velocity, amplitude, and frequency, were assessed using a Nikon Ti Microscope at either 10x or ×20 magnification. Recording was performed with NIS-Elements Viewer software and a Nikon DS-Ri2 color camera. Image analysis was conducted using ImageJ and FIJI software, along with relevant plugin tools. Sperm velocity was determined by tracking undulating waves and measuring the speed of these waves by clicking on the peak of each wave in every frame utilizing the Manual Tracking Plugin in ImageJ (available at https://imagej.nih.gov/ij/plugins/track/track.html). The instantaneous velocity for each wave was averaged. Amplitude measurements were taken from the approximate midpoint of the wave, from the middle peak to the trough, utilizing the line tool at a 0-degree angle. Wavelength measurements were obtained using the line tool to measure from peak-to-peak or trough-to-trough, depending on the clarity of the wave and ease of measurement. Frequency assessment involved visually estimating a complete cycle of the waveform by selecting a frame within the stack and then identifying where the waveform repeated throughout the stack.

One day post dsRNA injection, males were paired with virgin females emerging within the last 12 h to monitor mating behaviors. Egg-laying paper was placed into the mating tube, and egg counts were recorded after 5 days. Males injected with dsGFP served as controls, with 20 pairs assessed across three replicates. After housing males injected with dsScE3 Siah and dsGFP in an artificial climate incubator for a day, they were separated and paired with virgin females. Mating behaviors were observed, followed by immediate dissection of females to examine and measure spermatophore morphology under a microscope. Twenty spermatophores were analyzed per group, with three biological replicates. Images were captured using LightTools software.

For sperm counts, the method was adopted as previously explained in our work (Yan et al., 2022). Briefly, testis and ejaculatory ducts from dsScE3 Siah and dsGFP injected moths were dissected, cleaned and put in PBS buffer. The dissected tissues were then washed and transferred into PBS buffer. The tissues were gently homogenized, and the resulting mixture was diluted to a final volume of 80 µL with PBS. After the addition of 20 µL of pre-cooled DAPI solution, the mixtures were incubated in the dark at RT for 10–15 min. A small aliquot (2 µL) of the stained solution was then pipetted onto a glass slide and examined using an inverted fluorescence microscope (OLYMPUS, Tokyo, Japan). Sperm cells were identified as fluorescent entities, and their numbers were counted. Each sample was prepared in duplicate using reproductive organs from five individuals per group. Three independent replicates were performed for each experimental group, with male moths injected with dsGFP serving as the control.

Testes dissected from male moths injected with dsScE3 Siah and dsGFP were placed onto a glass slide immersed in PBS, followed by fixation using a solution containing 4% paraformaldehyde (Beyotime, China) in PBS for 1 h. After fixation, the samples underwent three washes with PBS, each lasting for 5 min, then Actin proteins were stained with TRITC Phalloidin (YEASEN, China) for 1 h, while nuclei were stained with Hoechst for 10 min. Afterward, the samples underwent three washes with PBS, were smeared onto a microslide, and observed under the microscope (Olympus, BX53).

The ATP concentration was determined using a luciferase-based ATP bioluminescence assay ATPlite kit (Perkin Elmer, United States) following the manufacturer’s protocol. Mammalian cell lysis solution (50 µL) was mixed with sperm cell suspension (100 µL) from dsGFP and dsScE3 Siah injected adult males in each well of a microplate. Substrate solution (50 µL) was added to each well, and the plate was shaken and incubated in darkness. Luminescence was measured with an Envision multimode plate reader (Perkin Elmer United Status). The experiment was triplicated with biological replicates, utilizing a total of 10 insects from each group.

Data analyses were conducted utilizing GraphPad Software, version 6.0 (GraphPad Software Inc., United Status), expressing the data as mean ± standard error mean (SEM). Statistical comparison between different groups were made using Student’s t-test and one-way analysis of variance (ANOVA) with significance set at p < 0.05.

The S. cerealella ScE3 Siah protein sequence comprises 305 amino acids and exhibits significant similarity to other insect E3 Siah proteins. It possesses conserved domains referred to as RING (Really Interesting New Gene) and SINA (Seven in Absentia) (Figure 1A). Furthermore, investigating the expression pattern of the ScE3 Siah in various adult tissues using qRT-PCR, we found widespread and relatively uniform expression across the spectrum of adult tissues including head, thorax, testis, fat bodies, and ejaculatory ducts (Figure 1B).

To clarify the biological function of ScE3 Siah in adult male moth reproduction, dsRNA was synthesized specifically designed to suppress ScE3 Siah gene expression. ScE3 Siah expression level were assessed via qRT-PCR from day 1 to day 3 after microinjection (Figure 1C). The results showed a significant reduction in ScE3 Siah expression within 3 days compared to the control group injected with dsGFP, indicating successful utilization of RNAi to silence the expression of ScE3 Siah in male moths (Figure 1C).

Male moths injected with dsScE3 Siah displayed normal behavior and successfully mated with female partners, exhibiting mating behavior indistinguishable from that of dsGFP moths. However, females mating with dsScE3 Siah-injected males laid significantly fewer eggs compared to those that mating with dsGFP-injected males (Figure 1D). Over 5 days, females mated with dsGFP-injected males laid an average of 61 eggs, while those mated with dsScE3 Siah-injected males laid only 51 eggs (Figure 1A). These results highlight ScE3 Siah crucial role in male fertility, directly impacting moth reproductive success.

We examined the morphology of the testes of adult male moths microinjected with dsScE3 and dsGFP. Histological examination via paraffin section and hematoxylin eosin staining revealed that the structure and morphology of the testes in male moths injected with dsGFP exhibited normal sperm morphology and a well-organized distribution of cysts and sperm bundles. Spermatozoa were loosely bundled and displayed a filamentous spiral arrangement (Figure 2A'). In contrast, the dsE3 Siah phenotypes led to significant alterations in the shape and morphology of the adult testes. These testes exhibited notable signs of cellular stress, with disrupted distribution and organization of spermatocytes, spermatids, cysts, and sperm bundles, ultimately affecting sperm numbers (Figure 2B').

Taken together, these observations reveal that dsE3 Siah is essential for spermatogenesis, providing evidence of impaired dichotomous spermatogenesis.

We then employed fluorescence staining with phalloidin and Hoechst to examine the structure of dichotomous sperm bundles in the adult testes of S. cerealella. Males injected with dsGFP, the eupyrene sperm displayed a thick, brush-like head with neatly arranged nuclei near the head (Figure 3A, dsGFP). However, in males injected with dsScE3 Siah, abnormalities in eupyrene sperm were evident, with nuclei showing a diffuse distribution within the sperm bundles (Figure 3A, dsE3). Additionally, a disrupted cytoskeleton was observed, with hollow bundles featuring distinctive grooves at the various points along the bundles (Figure 3A, dsE3). The helical structure of the sperm bundles was disrupted in dsScE3 Siah phenotypes.

For apyrene sperm bundles, those from dsGFP-injected male moth testes exhibited a slender morphology with nuclei irregularly distributed near the bundle’s center (Figure 3B, dsGFP). No notable differences were observed in the apyrene sperm bundles between the two groups, however, some intermediate changes in the structure of apyrene sperm has been observed (Figure 3B, dsE3).

To further investigate the role of ScE3 Siah in the structural changes of dichotomous sperm, we used transmission electron microscopy (TEM) to analyze the ultrastructural alterations in apyrene and eupyrene sperm in the testes of male moths injected with dsGFP and dsScE3 Siah. Eupyrene sperm have a distinct head and flagellum. The head contains the nucleus, while the flagellum is structured with a 9 + 9 + 2 microtubule arrangement, along with mitochondrial derivatives. Additionally, plasma membrane extensions form reticular and lacinate appendages on both the head and flagellum. On the other hand, apyrene sperm lack a nucleus but still have a flagellum with the same 9 + 9 + 2 microtubule structure. The flagellum is associated with two mitochondrial derivatives that run parallel to the axoneme. We observed disrupted and defective ultrastructure in the eupyrene sperm of dsScE3 Siah injected male testes (Figure 4A, dsE3) compared to those of dsGFP injected males, which exhibited normal axonemes and mitochondrial derivatives in eupyrene sperm (Figure 4A, dsGFP). Conversely, no obvious changes were observed in the structure of apyrene sperm between the dsGFP and dsScE3 Siah phenotypes (Figure 4B). However, we did observe a significant decrease in the number of both apyrene and eupyrene sperm within bundles in the dsScE3 Siah phenotypes.

The mean number of eupyrene sperm per bundle was significantly reduced in dsScE3 Siah injected male moths compared to dsGFP injected males, with counts of 189 and 254, respectively (Figure 4C). Similarly, the mean number of apyrene sperm per bundle was significantly lower in the dsScE3 Siah phenotypes, with 262 sperm counted, compared to 485 sperm in the dsGFP phenotypes (Figure 4D). These findings indicate that ScE3 Siah leads to a significant reduction in the number of dichotomous sperm and induces aberrant morphological and structural changes specifically in eupyrene sperm.

As we observed abnormalities and defective structures in dichotomous sperm within the testes of male moths following ScE3 Siah knockdown, we proceeded to evaluate the process of sperm migration from the testes to females. Male moths injected with dsScE3 Siah and dsGFP were mated with normal females, and upon completion of mating, the females were dissected to examine their spermatophores.

We observed that females mated with dsGFP injected males had normal and filled spermatophores, indicating successful sperm transfer (Figure 5A, dsGFP). In contrast, females mated with males injected with dsScE3 Siah exhibited empty and abnormal spermatophores, with a significant reduction in the number of sperm (Figure 5A, dsE3). A total of 30 females from each group were dissected after mating, and the frequency of abnormal spermatophores in the dsScE3 Siah injected group was significantly higher than that in the dsGFP group (Figure 5B). Among 30 females mated with dsScE3 Siah injected males exhibited 24 abnormal spermatophore, whereas female mated with dsGFP injected male had 3 abnormal spermatophore (Figure 5B). These findings reveal that ScE3 Siah knockdown results in defects in the sperm transfer process from male to female moths.

Upon observing morphological changes of the spermatophores and considering the significance of sperm in moth reproduction, we conducted a quantitative analysis of dichotomous sperm in various tissues of male moths injected with dsGFP and dsScE3 Siah, as well as in mated females. Our findings revealed a significant reduction in the number of both apyrene and eupyrene sperm in the testes, ejaculatory ducts, and spermatophores of moths injected with dsScE3 Siah compared to those injected with dsGFP (Figure 5C). In the testes, the number of apyrene sperm decreased significantly from 78.33 × 10³ in the dsGFP group to 61.83 × 10³ in the dsScE3 Siah group (p < 0.001). Similarly, the eupyrene sperm count decreased from 4.23 × 10³ to 2.41 × 10³ (p < 0.01) in the dsGFP and dsScE3 Siah groups, respectively. In the ejaculatory ducts, the apyrene sperm count decreased from 43 × 10³ in the dsGFP group to 32.33 × 10³ in the dsScE3 Siah group, while the eupyrene sperm count decreased from 2.65 × 10³ to 1.81 × 10³ (p < 0.05) in the respective groups.

Analysis of spermatophores from females mated with dsScE3 Siah males revealed a significant reduction in both apyrene and eupyrene sperm numbers compared to those mated with dsGFP males. The apyrene sperm count decreased from 89.37 × 10³ to 42 × 10³ (p < 0.0001), while the eupyrene sperm count decreased from 1.117 × 10³ to 0.63 × 10³ (p < 0.01) in females mated with dsGFP and dsScE3 Siah males, respectively.

As the knockdown of ScE3 Siah resulted in reduced sperm count and structural disruptions in dichotomous sperm, inhibiting their transfer from male to female moths, we conducted further analysis to assess the motility of dichotomous sperm in female reproductive tracts after mating with dsGFP and dsScE3 Siah injected males. Our investigation revealed a significant reduction in major sperm motility parameters due to the silencing of ScE3 Siah (Figures 6A–D).

Specifically, the knockdown of ScE3 Siah led to a substantial decrease in the wave velocities of dichotomous sperm, dropping from 27.30 μm/s to 13.91 μm/s (p < 0.0001) compared to the dsGFP phenotypes (Figure 6A). Additionally, the dsScE3 Siah resulted in a reduction in the amplitude of short waves (SW) from 5.73 μm to 4.407 μm compared to dsGFP, while the amplitude of long waves (LW) remained relatively unchanged, with values of 14.64 μm for dsGFP and 13.62 μm for dsScE3 Siah (p > 0.05) (Figure 6B).

Moreover, in the dsScE3 Siah group, both the wavelengths of SW and LW exhibited a decrease compared to the dsGFP control group (Figure 6C). Specifically, the average wavelength of SW decreased from 14.38 μm in dsGFP to 5.63 μm in dsScE3 Siah (p < 0.0001), while for LW, the wavelength decreased from 22.68 μm in dsGFP to 13.02 μm in dsScE3 Siah (p < 0.0001).

The frequencies of both SW and LW were significantly reduced in the dsScE3 Siah group (Figure 6D). The frequency of SW decreased from 3.09 Hz in dsGFP to 1.66 Hz in dsScE3 Siah (p < 0.0001), whereas for LW, the frequency decreased from 1.49 Hz in dsGFP to 0.76 Hz in dsScE3 Siah (p < 0.0001).

Since the silencing of ScE3 Siah caused decreased in sperm motility parameters and adenosine triphosphate (ATP) is considered the major source for metabolic functioning of sperm and related activities, we conducted measurements of sperm ATP content. Surprisingly, our analysis revealed that silencing ScE3 Siah had no apparent impact on the profiling of sperm ATP content (Figure 6E).