https://orcid.org/0000-0002-3266-1327 
Figures
Abstract
The reproductive process in various species has undergone evolutionary adaptations at both the physiological and molecular levels, playing a significant role in maintaining their populations. In lepidopteran insects, the spermatophore is a unique structure formed in the female reproductive system, in which sperm storage and activation take place. It is known that the formation of the spermatophore is regulated by seminal fluid proteins derived from males. However, studies investigating the genetic mechanisms behind spermatophore formation in lepidopterans have been limited. In this study, our focus was on SPSL1, a gene that encodes a trypsin-type seminal fluid protein in Spodoptera frugiperda, a pest species with global invasive tendencies. Our findings revealed that SPSL1 expression was predominantly observed in the male reproductive tracts, and the disruption of this gene resulted in male sterility. Surprisingly, fluorescence analysis indicated that the absence of SPSL1 did not affect spermatogenesis or sperm migration within the male reproductive system. However, when females mated with SPSL1-mutant males, several defects were observed. These included disruptions in spermatophore formation, sperm activation in the copulatory bursae, and sperm migration into the spermathecae. Additionally, mass spectrometry analysis highlighted reduced levels of energy-related metabolites, suggesting that SPSL1 plays an essential role in promoting hydrolysis reactions during copulation. Consequently, our study demonstrates that SPSL1 is crucial for male fertility due to its functions in spermatophore formation and sperm activation. This research provides valuable insights into the genetic factors underlying reproductive processes in lepidopteran insects and sheds light on potential strategies for controlling invasive pest populations.
Author summary
In lepidopteran insects, males produce a spermatophore to deliver the sperm during copulation and promote reproductive success. Seminal fluid plays a crucial role in the formation of spermatophore and the regulation of sperm activation. However, little is known about the semen protein molecules involved in these processes. In this work, we used Spodoptera frugiperda as a model to study the function of the seminal fluid protein SPSL1, a trypsin-type serine protease, in the reproductive process. By analyses of insects with a loss-of-function mutation in SPSL1, we found that SPSL1 affects the structure of the spermatophore, sperm activation, sperm distribution, and sperm migration in the female reproductive tract. Our research deepens our understanding of the reproductive process of lepidopterans and demonstrates that SPSL1 is a potential target for novel pest control methods such as sterile insect technology.
Citation: Qian L, Yang X, Xu X, Yang D, Zhu C, Yi M, et al. (2023) SPSL1 is essential for spermatophore formation and sperm activation in Spodoptera frugiperda. PLoS Genet 19(12): e1011073. https://doi.org/10.1371/journal.pgen.1011073
Editor: Subba Reddy Palli, University of Kentucky, UNITED STATES
Received: July 11, 2023; Accepted: November 20, 2023; Published: December 4, 2023
Copyright: © 2023 Qian et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: Y.H received funding from the National Science Foundation of China (32021001 and 31830093). Y.W received funding from the China Postdoctoral Science Foundation Grant (2021M703214). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The successful reproduction of animals relies on various physiological and behavioral processes that occur in a coordinated manner between mating partners [1]. The insects, for instance, transfer seminal fluid proteins (SFPs) from males to females during mating, which serve as both structural components and regulators of reproductive behavior in females [2]. In Drosophila melanogaster, several SFPs have been identified and characterized for their roles in regulating sexual receptivity, oviposition, sperm storage, sperm competition, and mating plug formation in females, including Acp70A/sex peptide, Dup99B, Acp26Aa, Acp36DE, PEBme [3–7]. However, it is important to note that there are significant differences in genetic and molecular mechanisms controlling sexual reproduction among different insect species [8]. While much of the research on SFPs has focused on Drosophila, it is crucial to acquire knowledge about the evolutionary diversity of SFPs in other insect groups.
Lepidoptera is an emerging and noteworthy taxon for reproduction research [9–11]. Understanding the reproductive process in this group is valuable due to the inclusion of both pest species and economically important species [12, 13]. There are substantial differences in reproduction between lepidopteran and dipteran insects, such as sex determination mechanisms, spermatogenesis, and fertilization processes [14–16]. Notably, lepidopteran males exhibit dichotomous spermatogenesis, producing eupyrene sperm (with nuclei) and apyrene sperm (without nuclei) within a single individual male [15]. Both types of sperm are essential for fertilization. Eupyrene sperm carries DNA and fertilizes eggs, while apyrene sperm aids in the transportation of eupyrene sperm to female sperm-storage organs [17–19]. In the reproductive system of female lepidopterans, the spermatophore is a specialized structure formed during mating that serves as a sperm-delivery device. Within the spermatophore, eupyrene sperm bundles dissociate, apyrene sperm gain motility, and are ultimately transferred into the spermatheca for egg fertilization [20, 21]. It has been reported that both types of sperm and SFPs are ejaculated by males and transferred to females, which are crucial for spermatophore formation during copulation [22, 23]. Despite extensive characterization of SFPs in lepidopterans, the understanding of specific factors involved in reproduction remains limited, particularly in regard to the molecular mechanisms underlying processes such as sperm activation and spermatophore formation.
SFPs in lepidopteran insects, including Bombyx mori, Spodoptera litura, Plutella xylostella, have been found to contain numerous trypsin-type serine proteases and play important roles in male reproduction [24–26]. In B. mori, a trypsin-type protease called Initiatorin, also known as Serine Protease 2 (Ser2), has been identified as a sperm activation factor through in vitro enzyme treatment experiments [27]. Recent studies on B. mori have demonstrated that knockout of Ser2 or Serine Protease 1 (Ser1) leads to male sterility [28, 29]. While some studies have explored the reproductive effects of trypsin-type serine proteases in lepidopteran insects, their specific roles in regulating processes like spermatogenesis, sperm activation, and spermatophore formation remain unclear.
The fall armyworm, Spodoptera frugiperda, is a globally widespread invasive pest that causes significant damage to economically important crops [30]. However, the molecular mechanisms involved in reproduction of S. frugiperda are poorly understood. In this study, we identified and characterized the physiological function of the Serine Protease Snake-like 1 (SPSL1) gene in S. frugiperda. We observed that SPSL1 is predominantly expressed in the internal reproductive organs of males, and loss-of-function mutants of SPSL1 exhibited significantly reduced fertility in males. Fluorescence staining assays revealed that dichotomous spermatogenesis was unaffected in SPSL1-mutant males. However, females mated with SPSL1-mutant males experienced disruptions in spermatophore formation, sperm activation, and sperm migration. Furthermore, mass spectrometry analysis indicated a reduction in energy-related metabolites in the copulatory bursae of females mated with SPSL1-mutant males. Overall, our study contributes to the understanding of specific SFP functions and provides a potential gene target for pest control.
Results
SPSL1 is predominantly expressed in male internal genitalia
Our previous research found that mutation of Ser1 leads to male sterility in B. mori [28], however, how Ser1 is particularly involved in the reproduction process remains unknown. We subsequently identified the gene in the S. frugiperda Genbank RefSeq database annotated as “Serine Protease Snake-like” (accession number: XP_035457022.2) and we propose to rename this gene as SPSL1 (for Serine Protease Snake-like 1). Phylogenetic analysis of a total of 19 trypsin-type serine proteases resulted in five distinct clades and revealed that the SPSL1 protein of S. frugiperda is clustered with the Lepidoptera clade (S1A Fig). The amino acid sequences of SPSL1 are highly conserved among seven species of Lepidoptera including S. frugiperda, S. litura, Trichoplusia ni, Helicoverpa zea, Maniola jurtina, Manduca sexta, and B. mori (S1B Fig). We then performed semi-quantitative RT-PCR analysis of seven different tissues of S. frugiperda adults and found that SPSL1 was predominantly expressed in the internal genitalia of male moths (Fig 1A). We then used qRT-PCR analysis to further detect the expression pattern of SPSL1 during mating and observed that it gradually increased from 1 day before eclosion to 1 day after mating (Fig 1B). Within the male internal genitalia, SPSL1 was highly expressed in single ejaculation ductus, double ejaculation ductus, and accessory glands (Fig 1C and 1D), implying that SPSL1 may have a role in male fertility.
(A) Semi-quantitative RT-PCR analysis of SPSL1 mRNA levels in indicated adult tissues (n = 10). Abbreviations: HE, head; MUS, muscle; FB, fat body; EPI, epidermis; MG, midgut; MT, Malpighian tubules; IG, internal genitalia. The samples other than IG are from the same number of male and female adults. (B) qRT-PCR analysis of SPSL1 mRNA levels in male internal genitalia at indicated times. BE, Before eclosion; NE, New eclosion; BM, Before mating; 2AM, 2h after mating; 24AM, 24h after mating. (C) Schematic of the male reproduction system of S. frugiperda. Abbreviations: TE, testis; VS, vesiculae seminales; VD, vas deferens; DED, double ejaculation ductus; SED, single ejaculation ductus; AG, accessory glands; AD, adeodatus. Scale bar, 1mm. (D) qRT-PCR analysis of SPSL1 mRNA levels in indicated male internal genital tissues. SfGADPH was used as an internal reference. Data are means ± SEM.
SPSL1 mutation impairs male fertility
To explore the biological function of SPSL1, we used the CRISPR/Cas9 system to engineer loss-of-function mutants. We designed two small guide RNAs (sgRNAs) targeting the only exon of SPSL1 (Fig 2A). We confirmed that gene editing was successful by analysis of DNA extracted from approximately 30 randomly selected injected embryos and observed a shorter fragment in injected versus control embryos that is consistent with the expected size of the mutant gene region (Fig 2B). We then amplified and sequenced the SPSL1 gene in four randomly selected representative G0 offspring. This confirmed that large deletions or insertions occurred in SPSL1-mutant individuals (Fig 2C). These results demonstrated that we successfully obtained the loss-of-function mutants of SPSL1. The SPSL1 mutants were viable and grossly normal during all development stages. We then investigated whether the disruption of SPSL1 affects the fertility of S. frugiperda. To do this, we performed fecundity assays. We found that the fertility of the SPSL1-mutant males was dramatically reduced compared to that of the wild-type (WT) males (Fig 2D–2F). The hatching rates of eggs laid by WT females and by SPSL1-mutant females mated with SPSL1-mutant males were only 26.10% and 25.26%, respectively. In contrast, when mated with WT males, 77.87% and 74.85% of eggs laid by WT females and by SPSL1-mutant females were hatched, respectively (Fig 2E). In addition, mating with SPSL1-mutant males significantly reduced the numbers of eggs laid (Fig 2F). When mated with SPSL1-mutant males, WT females and SPSL1-mutant females laid on average 312 and 375 eggs, respectively; when mated with WT males, the numbers were 567 and 551 eggs on average per female, respectively. Taken together, these results demonstrate that SPSL1 is essential for male fertility.
(A) Schematic of SPSL1 gene structure and sgRNA target sites. The gray box indicates the exon. Red arrows indicate the target sites of sgRNA1 and sgRNA2. The target sequence and PAM sequence are highlighted in green and red, respectively. (B) Genomic PCR analysis of SPSL1 mutation at embryo stage. A fragment of around 800 bp was detected in WT. Fragments around 800 bp and 400 bp were detected in SPSL1 mutants. (C) The sequence of the region between sgRNA target sites in the SPSL1 gene for WT and four randomly selected SPSL1-mutant G0 individuals. Dashed lines represent the deleted bases and the lowercase characters in blue represent the inserted bases. The net change in length is given to the right of each sequence (-, deletion; +, insertion). (D) Photographs of eggs produced by different crosses. Eggs were photographed 72 h after spawning. Scale bar, 1mm. (E) The hatching rate for indicated crosses (n > 26. *, p < 0.05; ***, p < 0.001. One-way ANOVA test). (F) The number of eggs laid for indicated crosses (n > 26. *, p < 0.05; ***, p < 0.001. Kruskal-Wallis test). ΔSPSL1 represents SPSL1 mutants.
SPSL1 is not required for dichotomous spermatogenesis or sperm migration in males
The demonstration that SPSL1-mutant males are sterile indicates that there may be defects in the male reproductive system. Therefore, we first investigated whether the SPSL1-mutant males have gross defects in the genitalia or the reproductive system. However, no obvious defects were detected (S2A–S2B’ Fig), so we searched for anomalies in spermatogenesis. In lepidopteran insects investigated, males have two morphs of sperm, and both are essential for fertility [17–19]. To date, there have been no reports describing the morphology of sperm or the molecular mechanism of spermatogenesis in S. frugiperda. Therefore, to evaluate the sperm morphology and to determine whether SPSL1 is involved in spermatogenesis in S. frugiperda, we performed fluorescence staining of sperm released from the testes on the second day after eclosion.
We observed two morphs of sperm bundles: One type was thick and had a ‘brush-like’ head, and nuclei were neatly arranged near the head (Fig 3A); The other type of sperm bundle was slender, and had irregularly distributed nuclei near the center of the bundle (Fig 3B). We further distinguished the eupyrene sperm bundles and apyrene sperm bundles by using nuclei fluorescence staining of sperm released from double ejaculation ductus, in which apyrene sperm bundles were typically dissociated and nuclei released. We did not observe the slender sperm bundles while only ‘brush-like’ head sperm bundles with neatly arranged nuclei were detected (Fig 3C). This result indicated the ‘brush-like’ head sperm bundles were eupyrene while the others were apyrene. However, we did not observe any obvious differences between WT and SPSL1 mutants in morphs of sperm bundles released from the testes or double ejaculation ductus (Fig 3A–3C), suggesting that the disruption of SPSL1 did not affect spermatogenesis or sperm migration in males.
(A) Representative immunofluorescence images of eupyrene sperm bundles released from the testes of WT and SPSL1-mutant males on the second day after eclosion. (B) Representative immunofluorescence images of apyrene sperm bundles released from the testes of WT and SPSL1-mutant males on the second day after eclosion. (C) Representative immunofluorescence images of eupyrene sperm bundles (green arrow) and apyrene sperm (magenta arrow) released from the double ejaculation ductus of WT and SPSL1-mutant males. Scale bars, 50μm. Blue, Hoechst; Red, F-actin. ΔSPSL1 represents SPSL1 mutants.
SPSL1 mutation leads to defects in spermatophore formation and sperm activation
In lepidopteran females, the spermatophore consists of proteins from the seminal fluid that are transferred from males during copulation [23]. As the SPSL1 mutation does not affect the male reproductive system, we then explored whether the SPSL1 mutation influences spermatophore formation. For this purpose, we evaluated the copulatory bursa before and after mating, and the spermatophore 1 h post copulation (hpc) in WT females mated with WT or SPSL1-mutant males (Fig 4A–4D’). In females mated with WT males, the spermatophore had a balloon-like structure composed of a swollen corpus and a tube-like collum (Fig 4C), similar to that in lepidopteran species previously reported [22, 31]. However, to our surprise, in females mated with SPSL1-mutant males, there were severe defects in spermatophore formation. The membranes of spermatophores in females mated with SPSL1-mutant males were fragile and incomplete, and sperm were observed outside the spermatophores (Fig 4C’ and 4G).
(A, A’) Representative images of the copulatory bursa of virgin WT female. Scale bars, 1mm. (B, B’) Representative images of copulatory bursa of WT females mated with WT or SPSL1-mutant males 1 hour-post copulation (hpc). Scale bars, 1mm. (C, C’) Representative images of spermatophore dissected from the copulatory bursa of females mated WT or SPSL1-mutant males 1 hpc. Scale bars, 1 mm. (D, D’) Schematic of the spermatophore inside in the copulatory bursa of WT females mated with WT or SPSL1-mutant males. scale bars, 1mm. (E) Representative images of sperm bundles released from spermatophores from indicated mating at 1 hpc. The green arrows indicate eupyrene sperm bundles, magenta arrows indicate apyrene sperm. Blue, Hoechst. Scale bars, 50 μm. (F) The frequencies of eupyrene sperm bundles (ESB), eupyrene sperm bundles in dissociation (ESB-D), eupyrene sperm (ES) released from spermatophores. (G) Representative images of sperm bundles released from spermatophore-excluded copulatory bursae at 1 hpc for indicated matings. The green arrows indicate eupyrene sperm bundles. Blue, Hoechst. Scale bars, 50 μm. (H) The frequencies of ESB, ESB-D, ES released from spermatophore-excluded copulatory bursae. (I-K) The numbers of I) ESB, J) ESB-D, K) ES per field of view released from copulatory bursae at 1 hpc for indicated matings. For F and H, 110 fields of view from 22 females mated with WT males and 100 fields of view from 20 females mated with SPSL1-mutant males were analyzed. For I-K, 30 fields of view from 6 females for indicated matings were analyzed. Data are means ± SEM (*p < 0.05; **p < 0.01; ***p < 0.001. two-tailed Student’s t-test). ΔSPSL1 represents SPSL1 mutants.
The spermatophore in lepidopteran insects is a reaction vessel for sperm activation [20, 32, 33]. The activation of sperm results in vigorous motility of apyrene sperm and dissociation of eupyrene sperm bundles in the spermatophore [27, 34]. We then investigated whether SPSL1 mutation affects sperm activation in spermatophore and found that, in WT females mated with SPSL1-mutant males, eupyrene sperm bundles failed to dissociate (Fig 4E), and most apyrene sperm were motionless compared to those mated with WT males (S1 and S2 Movies). We then quantified the frequencies of undissociated, dissociating, and dissociated eupyrene sperm bundles and found that the frequencies of dissociating and dissociated eupyrene sperm bundles were reduced in females mated with SPSL1-mutant males compared to those mated with WT males (Fig 4F). We also analyzed sperm activation in spermatophore-excluded copulatory bursae samples and found that the frequencies of undissociated and dissociated eupyrene sperm bundles were increased in those from females mated with SPSL1-mutant males compared to those mated with WT males (Fig 4G and 4H). Moreover, the number of sperm was counted in the copulatory bursae of females mated with WT or SPSL1-mutant males (Fig 4I–4K), and a significant decrease of the number of dissociated eupyrene sperm was detected in the copulatory bursae of females mated with SPSL1-mutant males (Fig 4K). These results showed that SPSL1 is involved in spermatophore formation and sperm activation in the female reproductive system.
SPSL1 is involved in free amino acid and urea regulation in copulatory bursa
During sperm maturation in the lepidopteran model insect Bombyx mori, accumulation of alanine, urea, succinate, and other metabolites result from proteolysis reactions, may provide an energy source for sperm activation [20, 33, 35]. To gain insights into whether SPSL1 is involved in proteolysis reactions in the copulatory bursae, we analyzed key small-molecule metabolites using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Of the 18 metabolites detected, lysine, alanine, aspartate, glutamate, tyrosine, and urea were detected at significantly different levels between WT females that had been mated with WT compared to SPSL1-mutant males (Figs 5 and S3). With exception of lysine, these metabolites were slightly induced after mating with SPSL1-mutant males but were dramatically enriched after mating with WT males (Fig 5A–5F). These results suggest that SPSL1 functions in proteolysis reactions that regulate sperm activation, and the defects in sperm motility and dissociation observed in female copulatory bursae after mating with SPSL1-mutant males are likely due to the reduced amounts of free amino acids and urea that are required for sperm maturation.
(A-F) The relative peak areas of A) alanine, B) aspartate, C) glutamate, D) tyrosine, E) lysine, and F) urea detected in the copulatory bursae of virgin females, WT females mated with WT males, and WT females mated with SPSL1-mutant males. Five biological replicates were performed. Data were normalized to the relative peak area of the virgin females. Data represent means ± SEM. Uppercase letters, p < 0.01; Lowercase letters, p < 0.05. One-way ANOVA test. ΔSPSL1 represents SPSL1 mutants.
SPSL1 mutation leads to failure in sperm migration into the spermatheca
After sperm activation, the apyrene sperm and the dissociated eupyrene sperm move from the copulatory bursa to the spermatheca for storage and fertilization [36, 37]. We next investigated whether SPSL1 mutation affects sperm transfer into spermatheca. We found that at 4 hpc the spermathecae of females mated with WT males contained sperm, whereas the spermathecae of virgin females and females mated with SPSL1-mutant males were empty (Fig 6A–6A”). Quantitative analyses showed that the relative grayscale value and the relative area of the spermathecae of females mated with SPSL1-mutant males were significantly lower than those in females mated with WT males; levels in the former were comparable to those of virgin females (Fig 6B–6D). We also analyzed the outflow from the spermathecae at 4 hpc after fluorescence staining. We observed intertwined eupyrene sperm and apyrene sperm in the spermathecae of females mated with WT males but not in the spermathecae of females mated with SPSL1-mutant males (Fig 6E). To exclude the possibility of delayed transfer of sperm into the spermathecae, the outflow from the spermathecae was analyzed at 24 hpc, and no sperm was detected in the spermathecae of females mated with SPSL1-mutant males (S4 Fig). These data imply that sperm fail to transfer into spermatheca in the absence of SPSL1.
(A-A”) Representative images of spermatheca of A) virgin females, A’) WT females mated with WT males, and A”) WT females mated with SPSL1-mutant males. Scale bars, 1mm. (B) Representative image of spermatheca indicating area used for quantification within red dotted lines. Scale bar, 1mm. (C) The relative grayscale value of spermathecae of virgin females and females for indicated matings. (D) The relative area of spermathecae of virgin females and females for indicated matings. n > 15. Different Uppercase letters, p < 0.01. One-way ANOVA test. (E) Representative images of sperm released from the spermatheca at 4 hpc. Green arrows, eupyrene sperm; Magenta arrows, apyrene sperm. Blue, Hoechst. Scale bars, 20 μm. ΔSPSL1 represents SPSL1 mutants.
Discussion
Trypsin-type serine proteases are known as components transferred from males to females and are crucial for fertility in diverse species [24–26, 38]. Despite our previous work found that the mutation of Ser1 causes male sterility in B. mori [28], how its particular role in the reproductive process of any lepidopteran pest remains incompletely known. The effects on the reproductive tissues and cells induced by the mutation, as well as the biochemical reactions it may involve in are not investigated. In this study, we demonstrated that SPSL1 is involved in the regulation of spermatophore formation and sperm activation in S. frugiperda females. We generated loss-of-function mutants of SPSL1 via CRISPR/Cas9-based genome editing. No gross morphological or developmental defects were observed in the mutant females or males, and fluorescence staining of dichotomous sperm bundles revealed no obvious defects in SPSL1-mutant males. However, fecundity was dramatically reduced in females mated with SPSL1-mutant males, and our analyses indicated that the formation of the spermatophore was disrupted and that activation of both eupyrene sperm and apyrene sperm was severely inhibited. Our mass spectrometry analysis showed that levels of energy-related metabolites were significantly reduced in the copulatory bursae of females mated with SPSL1-mutant males. Furthermore, eupyrene sperm and apyrene sperm were barely detected in the spermathecae of females mated with SPSL1-mutant males, indicating that sperm migration was impeded. We therefore propose that SPSL1 is an integral component for spermatophore formation and sperm activation during copulation in S. frugiperda (Fig 7). Taken together, our study reveals a molecular mechanism in the regulation of spermatophore formation in lepidopteran insects, and gives a panoramic view of how sperm migration has been affected by SPSL1 from male reproductive system to female reproductive system.
Spermatogenesis and sperm migration are normal in the reproductive system of WT or SPSL1-mutant males (green dot 1 and 2). In the copulatory bursa of females mated with WT males, the spermatophore is formed, and eupyrene sperm bundles are dissociated in the spermatophore during copulation (green dot 3). Apyrene sperm and dissociated eupyrene sperm are then transferred to the spermatheca for fertilization (green dot 4). In the copulatory bursa of females mated with SPSL1-mutant males, the spermatophore is malformed, and eupyrene sperm bundles fail to dissociate during copulation (red dot 3); thus, few sperm are transferred to the spermatheca for fertilization (red dot 4). Abbreviations: TE, testis; DED, double ejaculation ductus; SPE, spermatophore; CB, copulatory bursa; SPA, spermatheca. ΔSPSL1 represents SPSL1 mutants.
How SPSL1 participates in spermatophore formation and sperm activation? Spermatophore is a unique structure in the reproductive system of lepidopterans, and the physiological and biochemical reactions that occur in the spermatophore during copulation are mostly uncharacterized [22, 31]. To our knowledge, SPSL1 is the first SFP identified that is involved in the formation of the spermatophore. We found that lack of SPSL1 leads to incomplete formation of the membrane of the spermatophore during copulation, and therefore sperm were observed outside the spermatophore. The spermatophore is reported as a structure where sperm activation takes place [20], however, our results lead us to ask whether the formation of spermatophore depends on sperm activity. Apyrene sperm have been shown to play a role in the dissociation of eupyrene sperm bundles [17, 18], and we observed that apyrene sperm motility was reduced in the females mated with SPSL1-mutant males. Since alanine and urea are products of the arginine degradation cascade that occurs in the spermatophore [20], we hypothesize that SPSL1 hydrolysates may provide energy for apyrene sperm motility, which promotes eupyrene sperm dissociation and spermatophore formation. It is also possible that SPSL1 or its hydrolysates may act as spermatophore structural proteins and that the failure of spermatophore formation leads to inactivated sperm. These possibilities should be explored in further studies.
In Drosophila, Sex Peptide (also known as Acp70) is a SFP that regulates the post-mating behavior of females including sexual acceptance and oviposition [5, 6]. Genetic and biochemical evidence indicated that the function of Sex Peptide in female Drosophila depends on a processing by a trypsin-mediated cascade [39]. In our study, we observed that the number of eggs laid by females mated with SPSL1-mutant males was significantly lower than the number laid by females mated with WT males. This may be due to decreased migration of sperm or sperm-localized SFPs into spermatheca and therefore failure to stimulate post-mating behavior in females. It is possible that SPSL1 is involved in the regulation of a putative Sex Peptide in S. frugiperda and lack of SPSL1 deactivates the Sex Peptide and leads to defects in oviposition. Efforts to identify substrates of SPSL1 and exploration of their functions will be the focus of further studies.
Materials and methods
Spodoptera frugiperda strains and rearing
The S. frugiperda strains were obtained from Prof. Shutang Zhou (Henan University, College of Life Science) and reared in the laboratory on an artificial diet under conditions of 25 ± 1°C, 60 ± 10% relative humidity, and a 14 h light:10 h dark photoperiod. Moths were fed with 10% sucrose solution after eclosion.
Phylogenetic and amino acid alignment analysis
The phylogenetic tree of the SPSL1 proteins was generated using the neighbor-joining algorithm in MEGA X with a bootstrap of 1000 replications [40]. The evolutionary distances were computed using the Poisson correction method and are in units of the number of amino acid substitutions per site. The sequences were aligned using the ClustalW and the GENEDOC [41]. The protein sequences used to create the diagram were downloaded from National Center for Biotechnology Information and were listed in S1 Table.
RNA isolation, cDNA synthesis, and qRT-PCR
Total RNA was extracted from the reproductive system of the male adults using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s instructions. The cDNAs were synthesized using PrimeScript RT reagent kit (TaKaRa, China) in a 20 μL reaction mixture containing 1 μg of total RNA.
Quantitative real-time RT-PCR (qRT-PCR) was performed using SYBR Green Realtime PCR Master Mix (Thermo Fisher Scientific, USA) on a StepOnePlus Real-Time PCR system (Applied Biosystems, USA). The quantitative variations were evaluated by the relative quantification method (2-ΔΔCt). Sequences of the qRT-PCR primers are listed in S2 Table.
CRISPR/Cas9 genome editing and mutation detection
Two 23-nucleotide single-guide RNAs (sgRNA) targeting SPSL1 were designed according to the GGN19GG rule. The sgRNAs and Cas9 mRNA were synthesized and purified as described [42]. Newly laid eggs were microinjected with 300 ng/μL of Cas9 mRNA and 300 ng/μL of each sgRNA within 3 h of oviposition. About 1.6–2.0 ng of total RNA was injected into each egg. The eggs were incubated at 25°C.
Genomic DNA extracted from injected eggs (n = 30) at 48 h after injection was used as a template to amplify the sgRNA target sites with specific primers (S2 Table). A roughly 800 bp fragment flanking the target sites was amplified by PCR, and the products were directly sequenced or ligated into a pMD-18T vector for sequencing. The mutation of each insect used for experiment was verified.
Fertility assay
Males on the second day after eclosion were paired with females on the third day after eclosion. Each pair of insects was kept in a plastic bag (25 cm × 35 cm) and fed with 10% sucrose solution. Eggs laid by successfully mated females were manually counted at 48 h-intervals until the death of the female. The hatching rate was calculated by using the number of eggs laid and hatched. The experiment was performed in triplicate, and each replicate used at least 10 pairs of moths.
Fluorescence staining
Sperm bundles were isolated from the testes of male adults on the second day after eclosion. The collected sperm bundles were fixed with immunol staining fix solution (Beyotime, China) for 1 h. After two washes with PBS, samples were incubated in blocking solution (1 × PBS + 0.1% Triton X-100 + 0.5% bovine serum albumin) for 1 h. After a wash with PBS, samples were incubated with TRITC Phalloidin (YEASEN, China) for 1 h, and then with Hoechst (Beyotime, China) for 20 min at room temperature. The samples were further washed two times with PBS and subsequently mounted in antifade medium (YEASEN, China). Sperm isolated from double ejaculation ductus of virgin males (second day after eclosion), and copulatory bursae, spermatophores, and spermathecae of 1 hpc, 1 hpc, and 4 hpc females, respectively, were smeared on a micro-slide and observed by microscopy after Hoechst (Beyotime, China) staining. Images were taken with a Nikon (Japan) Ti-E or an Olympus (Japan) BX53 fluorescence microscope.
Analysis of sperm activation
Apyrene sperm were collected in the spermatophores 1 hpc and imaged using a Nikon Ti-E fluorescence microscope. Eupyrene sperm were collected from spermatophores or spermatophore-excluded copulatory bursae of 1 hpc females and smeared on a square area (1.5 cm × 1.5 cm) of a micro-slide. The samples were imaged using a Nikon Ti-E fluorescence microscopy in 20× fields of view using the five-point sampling method. The occurrence frequency and amount of eupyrene sperm bundles, dissociating eupyrene sperm bundles, and dissociated eupyrene sperm were calculated by analysis of at least 30 fields of 20x view.
LC-MS/MS analysis
The copulatory bursae were dissected from virgin females or mated females 1 hpc on the third day after eclosion and stored at -80°C. Samples were dried at -40°C for over 8 h in a lyophilizer (Labconco, USA). One sample was about 10 mg, and 5 samples from each condition were analyzed. Each sample was grounded in 300 μL 80% methanol (Sigma-Aldrich, USA) and treated with ultrasound at 0°C for 10 min. After centrifugation at 16800 g at 4°C for 5 min, 100 μL of the supernatant was collected and added with 50 μL acetonitrile (Sigma-Aldrich, USA) to precipitate overnight at -80°C. After centrifugation at 16800 g for 10 min, 100 μL of the supernatant was collected for analysis by LC-MS/MS. The analysis was performed on a UPLC instrument combined with a QTRAP 6500+ MS system (AB SCIEX, USA) equipped with an electrospray ionization (ESI) source (AB SCIEX, USA). Instrument control and data acquisition were performed using Analyst 1.6.3 software (AB SCIEX, USA), and data processing was performed using MultiQuant 3.0.2 software (AB SCIEX, USA). The amino acids and urea were separated on an ACQUITY UPLC BEH Amide column (1.7 μm, 2.1 x 100 mm, Waters, USA). Free amino acid mixed standard (AAS18, Sigma Aldrich, USA) and urea standard (U5128, Sigma Aldrich, USA) were used for qualitative analysis. The UPLC methods for different amino acids and urea are shown in S3 Table. The optimized ESI operating parameters for positive mode were: IS, 5500 V; CUR, 35 psi; TEM, 500°C; GS1, 55 psi; GS2, 55 psi. All analytes were detected using multiple reaction monitoring (MRM) mode, and the specific MRM parameters for each analyte are given in S4 Table.
Supporting information
S1 Fig. Phylogenetic analysis and sequence alignment of SPSL1.
(A) Phylogenetic tree of SPSL1 based on the alignment of amino acid sequences of the nineteen insect species using the neighbor-joining algorithm with a bootstrap of 1000 replications. Names and accessions used in the tree are listed in S1 Table. (B) Multiple alignment of the putative SPSL1 protein sequences from the seven lepidopteran insects. The highly conserved regions are highlighted by capital letters.
https://doi.org/10.1371/journal.pgen.1011073.s001
(TIF)
S2 Fig. SPSL1-mutant male does not have gross defects in the reproductive system.
(A, A’) Representative images of the reproductive system of WT and SPSL1-mutant males on the second day after eclosion. Scale bars, 1 cm. (B, B’) Representative images of the single ejaculation ductus (SED) of WT and SPSL1-mutant males on the second day after eclosion. Scale bars, 0.5 cm. ΔSPSL1 represents SPSL1 mutants.
https://doi.org/10.1371/journal.pgen.1011073.s002
(TIF)
S3 Fig. Analysis of free amino acids using LC-MS/MS.
(A-L) The relative peak areas of A) cystine, B) histidine, C) isoleucine, D) leucine, E) proline, F) serine, G) valine, H) arginine, I) glycine, J) methionine, K) phenylalanine, and L) threonine detected in the copulatory bursae of virgin WT females, WT females mated with WT males, and WT females mated with SPSL1-mutant males. Data were normalized to the relative peak area of the virgin females. Five biological replicates were performed. Data represent means ± SEM; Uppercase letters, p < 0.01; Lowercase letters, p < 0.05. One-way ANOVA test. ΔSPSL1 represents SPSL1 mutants.
https://doi.org/10.1371/journal.pgen.1011073.s003
(TIF)
S4 Fig. Mutation of SPSL1 leads to failure of sperm transfer into spermatheca at 24 hpc.
Representative images of sperm released from the spermatheca at 24 hpc. Green arrows, eupyrene sperm; Magenta arrows, apyrene sperm. Blue, Hoechst. Scale bars, 50 μm. ΔSPSL1 represents SPSL1 mutants.
https://doi.org/10.1371/journal.pgen.1011073.s004
(TIF)
S1 Table. List of species and accession number.
https://doi.org/10.1371/journal.pgen.1011073.s005
(XLSX)
S3 Table. The UPLC methods for different amino acids and urea.
A: 95% water, 5% acetonitrile, 10 mM ammonium formate, 0.1% formic acid. B: 95% acetonitrile, 5% water, 10 mM ammonium formate, 0.1% formic acid. The column oven was set at 40°C.
https://doi.org/10.1371/journal.pgen.1011073.s007
(XLSX)
S4 Table. The specific MRM parameters for each analyte.
Q1 is parent ion, Q3 is the daughter ion, CE is the collision energy, DP is the declustering potential. For each compound, the Q1 and the first Q3 were used for quantification, the second as a quality control.
https://doi.org/10.1371/journal.pgen.1011073.s008
(XLSX)
S1 Movie. Behavior of apyrene sperm in the spermatophores 1 hpc of WT females mated with WT males.
Scale bar, 50 μm.
https://doi.org/10.1371/journal.pgen.1011073.s009
(MP4)
S2 Movie. Behavior of apyrene sperm in the spermatophores 1 hpc of WT females mated with SPSL1 mutant males.
Scale bar, 50 μm.
https://doi.org/10.1371/journal.pgen.1011073.s010
(MP4)
Acknowledgments
We thank Shanshan Wang, Lianyan Jing from the Core Facility Centre of the Centre of Excellence of Molecular Plant Sciences (CEMPS) for their technical support of LC-MS/MS analysis.
References
- 1. Van Wielendaele P, Badisco L, Vanden Broeck J. Neuropeptidergic regulation of reproduction in insects. Gen Comp Endocrinol. 2013;188: 23–34. pmid:23454669
- 2. Avila FW, Sirot LK, LaFlamme BA, Rubinstein CD, Wolfner MF. Insect Seminal Fluid Proteins: Identification and Function. Annu Rev Entomol. 2011;56: 21–40. pmid:20868282
- 3. Chapman T, Neubaum DM, Wolfner MF, Partridge L. The role of male accessory gland protein Acp36DE in sperm competition in Drosophila melanogaster. Proc R Soc Lond B Biol Sci. 2000;267: 1097–1105. pmid:10885514
- 4. Chapman T, Herndon LA, Heifetz Y, Partridge L, Wolfner MF. The Acp26Aa seminal fluid protein is a modulator of early egg hatchability in Drosophila melanogaster. Proc R Soc Lond B Biol Sci. 2001;268: 1647–1654. pmid:11506676
- 5. Chapman T, Bangham J, Vinti G, Seifried B, Lung O, Wolfner MF, et al. The sex peptide of Drosophila melanogaster: Female post-mating responses analyzed by using RNA interference. Proc Natl Acad Sci USA. 2003;100: 9923–9928. pmid:12893873
- 6. Liu H, Kubli E. Sex-peptide is the molecular basis of the sperm effect in Drosophila melanogaster. Proc Natl Acad Sci USA. 2003;100: 9929–9933. pmid:12897240
- 7. Avila FW, Cohen AB, Ameerudeen FS, Duneau D, Suresh S, Mattei AL, et al. Retention of Ejaculate by Drosophila melanogaster Females Requires the Male-Derived Mating Plug Protein PEBme. Genetics. 2015;200: 1171–1179. pmid:26058847
- 8. Roy S, Saha TT, Zou Z, Raikhel AS. Regulatory Pathways Controlling Female Insect Reproduction. Annu Rev Entomol. 2018;63: 489–511. pmid:29058980
- 9. Yang X, Chen D, Zheng S, Yi M, Liu Z, Liu Y, et al. BmHen1 is essential for eupyrene sperm development in Bombyx mori but PIWI proteins are not. Insect Biochem Mol Biol. 2022;151: 103874. pmid:36375757
- 10. Yang D, Xu J, Chen K, Liu Y, Yang X, Tang L, et al. BmPMFBP1 regulates the development of eupyrene sperm in the silkworm, Bombyx mori. PLoS Genet. 2022;18: e1010131. pmid:35312700
- 11. Yang X, Chen D, Zheng S, Yi M, Wang S, Liu Y, et al. The Prmt5-Vasa module is essential for spermatogenesis in Bombyx mori. PLoS Genet. 2023;19: e1010600. pmid:36634107
- 12. Goldsmith MR, Shimada T, Abe H. The genetics and genomics of the silkworm, Bombyx mori. Annu Rev Entomol. 2005;50: 71–100. pmid:15355234
- 13. Mitter C, Davis DR, Cummings MP. Phylogeny and Evolution of Lepidoptera. Annu Rev Entomol. 2017;62: 265–283. pmid:27860521
- 14. Yang X, Chen K, Wang Y, Yang D, Huang Y. The Sex Determination Cascade in the Silkworm. Genes (Basel). 2021;12: 315. pmid:33672402
- 15. Phillips DM. Morphogenesis of the lacinate appendages of lepidopteran spermatozoa. J Ultrastruct Res. 1971;34: 567–585. pmid:5555018
- 16. Dallai R, Gottardo M, Beutel RG. Structure and Evolution of Insect Sperm: New Interpretations in the Age of Phylogenomics. Annu Rev Entomol. 2016;61: 1–23. pmid:26982436
- 17. Chen S, Liu Y, Yang X, Liu Z, Luo X, Xu J, et al. Dysfunction of dimorphic sperm impairs male fertility in the silkworm. Cell Discov. 2020;6: 60. pmid:32963806
- 18. Sakai H, Oshima H, Yuri K, Gotoh H, Daimon T, Yaginuma T, et al. Dimorphic sperm formation by Sex-lethal. Proc Natl Acad Sci USA. 2019;116: 10412–10417. pmid:31036645
- 19. Friedländer M. Control of the eupyrene–apyrene sperm dimorphism in Lepidoptera. J Insect Physiol. 1997;43: 1085–1092. pmid:12770480
- 20. Osanai M, Aigaki T, Kasuga H. Energy metabolism in the spermatophore of the silkmoth, Bombyx mori, associated with accumulation of alanine derived from arginine. Insect Biochem. 1987;17: 71–75.
- 21. Osanai M, Aigaki T, Kasuga H, Yonezawa Y. Role of arginase transferred from the vesicula seminalis during mating and changes in amino acid pools of the spermatophore after ejaculation in the silkworm, Bombyx mori. Insect Biochem. 1986;16: 879–885.
- 22. Osanai M, Kasuga H, Aigaki T. The spermatophore and its structural changes with time in the bursa copulatrix of the silkworm, Bombyx mori. J Morphol. 1987;193: 1–11. pmid:29929344
- 23. Happ GM. Maturation of the Male Reproductive System and its Endocrine Regulation. Annu Rev Entomol. 1992;37: 303–320. pmid:1539938
- 24. Wu L-J, Li F, Song Y, Zhang Z-F, Fan Y-L, Liu T-X. Proteome Analysis of Male Accessory Gland Secretions in the Diamondback Moth, Plutella xylostella (Lepidoptera: Plutellidae). Insects. 2023;14: 132. pmid:36835702
- 25. Mamtha R, Kiran T, Chandramohan V, Gowrishankar BS, Manjulakumari D. Genome-wide identification and expression analysis of the mating-responsive genes in the male accessory glands of Spodoptera litura (Lepidoptera: Noctuidae). J Genet Eng Biotechnol. 2023;21: 11. pmid:36723695
- 26. Dong Z, Wang X, Zhang Y, Zhang L, Chen Q, Zhang X, et al. Proteome profiling reveals tissue-specific protein expression in male and female accessory glands of the silkworm, Bombyx mori. Amino Acids. 2016;48: 1173–1183. pmid:26822097
- 27. Nagaoka S, Kato K, Takata Y, Kamei K. Identification of the sperm-activating factor initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori. Insect Biochem Mol Biol. 2012;42: 571–582. pmid:22575161
- 28. Xu X, Wang Y, Chen J, Du X, Yao L, Xu J, et al. Mutation of Serine protease 1 Induces Male Sterility in Bombyx mori. Front Physiol. 2022;13. pmid:35222089
- 29. Xu X, Wang Y, Bi H, Xu J, Liu Z, Niu C, et al. Mutation of the seminal protease gene, serine protease 2, results in male sterility in diverse lepidopterans. Insect Biochem Mol Biol. 2020;116: 103243. pmid:31541694
- 30. Savary S, Willocquet L, Pethybridge SJ, Esker P, McRoberts N, Nelson A. The global burden of pathogens and pests on major food crops. Nat Ecol Evol. 2019;3: 430–439. pmid:30718852
- 31. Watanabe M, Sasaki N. Pattern of sperm storage and migration in the reproductive tract of the swallowtail butterfly Papilio xuthus: cryptic female choice after second mating. Physiol Entomol. 2010;35: 328–333.
- 32. Aigaki T, Kasuga H, Nagaoka S, Osanai M. Purification and partial amino acid sequence of initiatorin, a prostatic endopeptidase of the silkworm, Bombyx mori. Insect Biochem Mol Biol. 1994;24: 969–975. pmid:7703988
- 33. Osanai M, Isono M. Dissociation of eusperm bundles by acids, especially by succinate accumulated in the spermatophore of the silkmoth, Bombyx mori. Invertebr Reprod Dev. 1997;31: 99–108.
- 34. Osanai M, Kasuga H, Aigaki T. Physiological role of apyrene spermatozoa of Bombyx mori. Experientia. 1987;43.
- 35. Kasuga H, Aigaki T, Osanai M. System for supply of free arginine in the spermatophore of Bombyx mori. Insect Biochem. 1987;17: 317–322.
- 36. Watanabe M, Hachisuka A. Dynamics of eupyrene and apyrene sperm storage in ovipositing females of the swallowtail butterfly Papilio xuthus (Lepidoptera: Papilionidae). Entomol Sci. 2005;8: 65–71.
- 37. Hague NL, Dickinson JL, Shepherd JG. Transfer, subsequent movement, and fate of sperm in the tobacco hornworm moth, Manduca sexta. Physiol Entomol. 2021;46: 218–229.
- 38. Bi H, Xu X, Li X, Wang Y, Zhou S, Huang Y. CRISPR/Cas9-mediated Serine protease 2 disruption induces male sterility in Spodoptera litura. Front Physiol. 2022;13. pmid:35991171
- 39. LaFlamme BA, Ravi Ram K, Wolfner MF. The Drosophila melanogaster Seminal Fluid Protease “Seminase” Regulates Proteolytic and Post-Mating Reproductive Processes. PLoS Genet. 2012;8: e1002435. pmid:22253601
- 40. Felsenstein J. Phylogenies and the Comparative Method. Am Nat. 1985;125: 1–15.
- 41. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22: 4673–4680. pmid:7984417
- 42. Wang Y, Li Z, Xu J, Zeng B, Ling L, You L, et al. The CRISPR/Cas System mediates efficient genome engineering in Bombyx mori. Cell Res. 2013;23: 1414–1416. pmid:24165890