Multiple matings are a reproductive process that affects the expansion of insect populations. They are classified as monandrous or polyandrous (Newcomer et al., 1999). However, females may be more willing to re-mate with familiar males to avoid any physical damage caused by the genitals during mating (Caesar and Forsman, 2009). Moreover, monandrous multiple matings can provide females with additional benefits (Wang et al., 2018), such as increased lifetime fecundity (Alcock et al., 1978; Ward and Landolt, 1995) and the nutrients from male ejaculates (Arnqvist and Nilsson, 2000). Therefore, most females may obtain physiological or genetic benefits from multiple mating (Jennions and Petrie, 2007). Furthermore, if females receive insufficient ejaculate from one mating, they may mate multiple times to ensure that all their eggs are fertilized (Eberhard, 1996; Simmons, 2019).

Females initiate ovipositioning behavior after mating in sexually reproducing taxa. Seminal fluid proteins (SFPs), which are also known as male accessory gland proteins (Acps), are key factors in sexual reproduction. Insect SFPs are produced in the male reproductive tract (MRT) secretory tissues (testes, seminal vesicles, accessory glands, etc.) (Avila et al., 2011). They are composed of many substances, including polypeptides, lectins, proteases, protease inhibitors, protective proteins similar to antioxidants, and substances that do not encode proteins (Poiani, 2006; Pondeville et al., 2008). SFPs are transferred with sperm to females during mating, and they induce physiological and behavioral changes in females (Neubaum and Wolfner, 1999; Koene et al., 2010), such as decreasing receptivity to re-mating, altering feeding behaviors, and increasing ovulation and egg-laying rate (Avila et al., 2011; Immarigeon et al., 2021). Additionally, the process of coordinates gametes for fertilization are triggered by SFPs in Drosophila melanogaster. (Mann et al., 1982; Bloch Qazi et al., 2003).

Developments in proteomic and RNA interference techniques have advanced the identification and functional analysis of SFPs in many insect species (Avila et al., 2011). SFPs undergo molecular interactions that cause behavioral changes in females post mating and have been shown to be the main functional proteins affecting insect reproduction (Lung and Wolfner, 2001; Mueller et al., 2008). For example, seminal fluid signals encourage females to allocate resources to the ova, resulting in greater egg production in Ephestia kuehniella (Xu and Wang, 2011). RNAi technology has benefited insect system studies. These techniques have shown that double-stranded RNA (dsRNA) or siRNA injections into adult or juvenile insects can successfully knockdown gene transcripts in a diverse array of taxa (Marshall et al., 2009). Furthermore, the downregulation of 15 tested SFP genes substantially decreased defensive first male paternity success in D. melanogaster (Patlar and Civetta, 2022). In addition, knockout of the SFP gene, BmSfp62, led to male sterility in Bombyx mori (Xu et al., 2022), and knockdown of the gene encoding for a protein similar to angiotensin-converting enzyme in Tribolium castaneum males reduced female fecundity after mating (Xu et al., 2013). Thus, it is possible to assess the function of individual proteins and their roles in mediating reproductive physiology using RNAi technology.

Ophraella communa Lesage (Coleoptera: Chrysomelidae) is a specific and effective natural enemy of the invasive common ragweed Ambrosia artemisiifolia L. (Asterales: Asteraceae) (Zhou et al., 2011). This specialist herbivore is native to North America (Futuyma and McCafferty, 1990; Hu and Meng, 2007) and was first discovered in Nanjing, Jiangsu Province, China, in 2001 (Meng and Li, 2005). It has been widely used as a biological control agent of A. artemisiifolia in Canada and China (Mason and Huber, 2002; Zhou et al., 2009; 2010; Guo et al., 2011). Ophraella communa adults mate several times throughout the day and during their lifespan (Meng and Li, 2005; Zheng et al., 2011), and the number of copulation events is positively associated with insect fitness parameters (Zhou et al., 2015). Beetle mating behavior is visible, which means that it can used as a feasible parameter for mating regime studies.

The ejaculate substance stored by T. castaneum females increased by 33% in doubly mated females compared to that in singly mated females, indicating that the spermatheca was filled to only two-thirds of its capacity following insemination by the first male (Lewis and Jutkiewicz, 1998). Multiple matings by O. communa may have a cumulative effect that is similar to that in T. castaneum and involves SFPs transferred by mating. The high reproductive ability of the beetle is considered to be not only related to the physiological basis of the females but also to the regulation of the SFPs supplied by the males. However, the mechanism controlling male-mediated reproduction regulation remains unclear, especially whether insects maximize reproduction by obtaining more seminal proteins through multiple matings. Thus, we sampled mating and non-mating bursa copulatrix of females, and used transcriptomic and proteomic approaches to construct an O. Communa transferable SFPs database. In previous study, Gao et al. successfully identified a subset of transcriptional profiles of the testes and accessory glands of male O. communa. Following screening of this database, two SFP genes whose expression levels were modified in the bursa copulatrix were obtained—namely, carboxypeptidase B (EC 3.4.17.2, Cpb) and carbonic anhydrase (EC 4.2.1.1, Ca) genes.

Cpb is a carboxypeptidase that contains the peptidase_M14 domain and requires divalent metal ions such as Zn²⁺, for the specific hydrolysis of arginine and lysine residues at the C-terminus. It is a crucial enzyme in the digestive tract of insects and contributes to insect metamorphosis, development, and resistance (Barrett et al., 2012). Furthermore, Cpb activity has been reported in the male reproductive system of Bombyx mori (Aigaki et al., 1988). Ca is a metalloenzyme that combines Zn²⁺ with its active centers. It was first discovered in human red blood cells (Meldrum and Roughton, 1933), and was subsequently reported in algae, fungi, and bacteria (Smith et al., 1999). Ca reversibly catalyzes the reaction between CO₂ and HCO₃ ⁻, and this reaction is involved in various physiological functions in organisms, enabling them to maintain their physiological activities (Mirjafari et al., 2007). A novel pH/HCO₃ ⁻ dependent regulatory mechanism mediated by Ca is reported to be involved in the motility control in flatfish sperm (Inaba et al., 2003).

We hypothesized that SFP genes play a reproductive regulatory role in multiple matings of O. communa adults. To test this hypothesis, we created different mating regimes to explore the functions of OcCpb and OcCa in multiple matings between beetle adults. Technical methods, such as gene cloning, quantitative real-time PCR (qPCR), and RNAi were used in this study.

Mature females exhibited physiological ovulation behavior without mating, which provides a physiological basis for the strong reproductive ability of O. communa. Our data demonstrated that multiple matings promotes oogenesis, which supports the hypothesis that multiple matings brings direct benefits to females (Arnqvist and Nilsson, 2000; Caspers et al., 2014). This finding is also consistent with the results for multiple matings in other insects, such as Colaphellus bozoringi (Liu et al., 2013), Galerucella birmanica (Wang et al., 2018) and Chrysochus asclepiadeus (Schwartz and Peterson, 2006), where fecundity almost doubled on average when the organisms were allowed to mate repeatedly compared to that in individuals that mated once. Female lifetime fecundity generally increases with mating frequency, and these increases are considered to be due to the sufficient amount of sperm or SFPs in the male ejaculate for fertilization (Arnqvist and Nilsson, 2000; Avila et al., 2011). After females are stimulated by SFPs, ovarian oogenesis is accelerated, and the number of eggs laid by females increases (Wolfner, 2002; Tseng et al., 2007). SFPs are necessary for the efficient utilization of stored sperm, with the few sperm stored in the absence of SFPs not used to fertilize eggs in D. melanogaster females (Xue and Noll, 2000). The reproductive success of both sexes is adversely affected when SFPs are absent from the ejaculate (Avila et al., 2011). Therefore, O. communa adults have a high reproductive capacity, which is related to both the physiological foundation of females and the regulation of SFPs in males.

Many SFPs have been identified in insects, and most SFPs identification studies have examined RNA or proteins found in the tissues of the MRT (Avila et al., 2011). The expression patterns of genes encoding 13 of the 14 proteins identified by South et al. (South et al., 2011) were highly male-biased, with undetectable transcript levels in the female tissues. Furthermore, genes encoding 15 SFPs were highly expressed in Callosobruchus maculatus male abdomens, but were only negligibly expressed in females. In our study, the expression patterns of OcCpb and OcCa were also highly male-biased (Figure 2). These results were similar to the expression profiles for SFP genes in T. castaneum and C. maculatus. Thus, combined with the findings reported by Gao et al. (2020) reported, two lines of evidence suggest that OcCpb and OcCa with male-specific gene expression levels represent putative SFPs: 1) they were identified in mating bursa copulatrix but were not detected in the NM bursa copulatrix and 2) the qPCR revealed low expression levels in females.

Cpb and Ca have been identified as SFPs in O. communa, but their reproductive functions in insects have rarely been reported. Previous studies have reported that Cpb plays an important role in the degradation of dietary proteins in the insects intestines (Bown and Gatehouse, 2004). When the activity of Cpb was inhibited, the development of parasites in the mosquito midgut was blocked in Anopheles gambiae (Lavazec et al., 2007). In this study, we determined that OcCpb and OcCa mediates the female fertility (Figure 4). A non-specific carboxylesterase with proteolytic activity is present in the secretions compartmentalized closer to the D. melanogaster male genital opening in the ejaculatory duct and is transported almost immediately after mating begins. (Meikle et al., 1990). Zymogen Cpb, synthesized and secreted in the male reproductive organs, was reported to be catabolically activated post mating and in the female reproductive organs to initiate the molecular pathway of the fertilized egg in the silkworm (Sakakura et al., 2022). It appears that OcCpb influences fertilization egg formation by the same mechanism and involved in the regulation of reproduction in insects. Ca catalyzes the reversible hydration of carbon dioxide and bicarbonate anion. It was confirmed that HCO₃ ⁻ involved in fertilized egg formation by inducing intracellular alkalinization and by directly accelerating sperm movement (Wennemuth et al., 2003). In recent years, research on the relationship between Ca and fertilization has mainly been conducted in higher animals. There are 14 subtypes of this protein, which are mainly expressed in the testes and epididymis of rats, rabbits, cows, and humans (Ekstedt et al., 2003). Human sperm and egg fuse to generate a new individual strongly depends on Ca activity (José et al., 2015). It is also important to mention that Ca can create both physical and functional metabolons with a variety of different anion exchangers, which further complicates the role of Ca in fertilization (Ali Akbar et al., 1998; Del Prete et al., 2014). Hence, OcCa may indirectly affect fertilization through bicarbonate ions. To the best of our knowledge, this study is the first study to identify Cpb and Ca as SFP genes that play a regulatory role in insects’ reproduction.

The mating bursa copulatrix contains a newly transferred spermatophore. It also includes sperm encapsulated by SFPs, which is similar to those in ground crickets (Allonemobius socius) (Marshall et al., 2009). The spermatophore in the bursa copulatrix is an active substance that acts as a signaling factor and stimulates hormonal effects in females (Wolfner, 2002; Fiumera et al., 2007; Mueller et al., 2008). SFPs can induce gene expression post mating (McGraw et al., 2008; Amaro et al., 2021). Acp levels continue to increase within the D. melanogaster female genital tract throughout mating (Lung and Wolfner, 1999). Similarly, the expression of OcCpb and OcCa upregulates in the mating bursa copulatrix. The phenomenon that OcCpb and OcCa upregulation in the bursa copulatrix with different mating events does not show a linear increase in mating frequency, we believe it is most likely due to the inconsistency of each mating time interval. Moreover, new SFPs were transferred to females and previous SFPs were consumed when there were two mating events. Some SFPs transferred from the D. melanogaster male enter the female’s circulatory system whereas others are confined within the reproductive tract (Lung and Wolfner, 1999; Neubaum and Wolfner, 1999). Given this, we hypothesized that the SFP genes related to multiple mating to promote egg production should be downregulated due to the SFPs consumption by females during the interval between two mating. Interestingly, the expression of OcCpb and OcCa in mating bursa copulatrix was lower in the 3 h than at the 15 min, which were consistent with our expectations. Similar trends are seen with Acp26Aa, Acp26Ab, and Acp62F (Monsma et al., 1990; Lung and Wolfner, 1999). Moreover, levels of two D. melanogaster SFPs (ovulin and sex peptide) decline in the mated female with time since mating (Sirot et al., 2009). It may be the result from the decrease or termination of SFP entry into the female genital tract or hemolymph in post mating along with removal of SFP from the general circulation as a result of metabolism or receptor binding at their target tissues (Lung and Wolfner, 1999). Downregulation of SFP genes occur with each mating events, which continuously stimulates the female to respond. It seems to imply that OcCpb and OcCa have a cumulative effect and play a reproductive regulatory role in multiple matings.

In summary, we have demonstrated that multiple matings promotes oogenesis and identified that Cpb and Ca are putative seminal fluid protein genes in O. communa. Although these genes in the bursa copulatrix did not show a linear increase with mating frequency, they were upregulated during different mating events and gradually downregulated after finishing copulation. Additionally, OcCpb and OcCa mediate oogenesis and female fertility, which provided insights into the relevance of them involvement in multiple matings. Further studies are required to identify the various roles played by OcCpb and OcCa in regulating male reproduction, especially regarding SFP synthesis and secretion.