Animals live and evolve in a world dominated by microbes, from which they host a diversity of microbiomes on and in their bodies. It is now widely accepted that microbiomes can be an integral part of the host phenotype, which may influence the health, physiology, development, behavior, and reproduction of the host, and potentially influence the host genome and be a part of the host genome (Schoenmakers et al., 2019; Bellinvia et al., 2020a,b; Rowe et al., 2021). A wide range of studies so far have been performed to reveal the role of microbiomes on host skin, oral, and gut from perspectives of ecology, adaption, and evolution (Dillon and Dillon, 2004; Voirol et al., 2018; Rowe et al., 2021). However, reproductive microbiomes or reproduction-related microbiomes are relatively neglected and less known (Bellinvia et al., 2020a; Rowe et al., 2021). Reproductive microbiomes, including bacteria, fungi, viruses, protozoans, and unicellular algae, living in or on any structure, organ, fluid, or tissue of a host, typically make contact with the gametes or reproductive tract or organs of another individual through mating and spawning (Rowe et al., 2021).

Studies in mammals, particularly human, have demonstrated that a large number of sexually transmitted microbes have negative effects on the health and survival of hosts (Schoenmakers et al., 2019; Rowe et al., 2021). Fitness tests indicated that these microbes often can have significant effects (but not negative always) on the reproductive performance and success of males and females (Schoenmakers et al., 2019; Rowe et al., 2021). Evidence, in male mammals, increasingly recognized that some of these microbes may decrease sperm quality and fertility (Fraczek et al., 2013; Schoenmakers et al., 2019; Rowe et al., 2021) and, in female mammals, demonstrated that some microbes are associated with female health and reproduction, including sexually transmitted infections, preterm birth, infertility, and low fecundity (Lockhart et al., 1996; Ravel et al., 2011; Chen et al., 2017; Schoenmakers et al., 2019; Marconi et al., 2020). Studies in human also found that the typically dominated vaginal microbe, Lactobacillus spp., can be “healthy” vaginal microbiomes, which are oxygen-tolerant anaerobes that exhibit antimicrobial activity against a range of vaginal pathogens, probably by physically barring against pathogen adhesion, production of lactic acid, and stimulating host defense responses (Ravel et al., 2011; Chen et al., 2017; Tachedjian et al., 2017; Younes et al., 2018). Studies in this field are relatively limited in insects. A study in the bedbug showed that exposuring males to polymicrobial mixture (such as Acinetobacter, Alcaligenes, Bacillus, and Staphylococcus) significantly increased sperm mortality (up to 40%) (Otti et al., 2013). In some insects including some lepidopterans, Wolbachia infection may lead to increased survival and reproduction (Chen et al., 2012; Moriyama et al., 2015; Voirol et al., 2018), while some Enterococcus species, such as E. faecalis, may have a negative impact on the fecundity of fruit flies (Akami et al., 2019; Noman et al., 2021). Moreover, studies also found that reproductive-related microbiomes play a diversity of roles in the reproduction process of animals, including insects, which is related to sexual selection, sperm competition, genetic compatibility, sexual conflict, and speciation (Schoenmakers et al., 2019; Rowe et al., 2021). However, most studies examined bacteria effects with only one or a few types of bacteria (Rowe et al., 2021). One possible reason is that these microbes are large in quantity and species, and most of them are hard to cultivate or uncultivable (Bellinvia et al., 2020a). While the study of individual microbes is fundamental and informative, it is important to understand the microbiomes in a community context (Schoenmakers et al., 2019; Rowe et al., 2021).

Recent progress on high-throughput sequencing and bioinformatics has revolutionized our understanding of symbiotic microbiomes. In the field of reproductive microbiomes, a few studies have demonstrated the bacterial communities of the reproductive organs in insects using 16S rDNA sequencing and subsequent bioinformatic analysis (Wang et al., 2014; Otti et al., 2017; Bellinvia et al., 2020a,b). In the citrus fruit fly, Bactrocera minax, sequencing revealed that female ovary has the highest diversity in microbiomes, followed by male testis, and the bacterial diversity of reproductive organs is higher than that of the gut (Wang et al., 2014). Recent studies in the common bedbug, Cimex lectularius, showed that genital microbiomes varied between populations and the sexes and mating changes the genital microbiomes, suggesting that bacteria are sexually transmitted and these bacteria might play an important role in shaping the evolution of reproductive traits (Bellinvia et al., 2020a,b). Future studies based on these new findings and technologies are likely to provide deeper insights in this field by investigating the transmission dynamics between males and females.

Mating can cause major changes in female physiology and behavior. Studies have shown that mating has a positive effect on female reproductive activities, which may negatively affect female maintenance (such as immunity responses) and survival due to trade-offs on resource utilization by females, or male suppression of female immunity to promote sperm storage and egg fertilization in females (reviewed in Schwenke et al., 2016; Wigby et al., 2019). However, studies have also shown that mating may upregulate the female's immune response due to the transfer of foreign materials and mating caused infections (Delbare et al., 2017; Okada et al., 2017; Oku et al., 2019; Ahmed-Braimah et al., 2021). These different discoveries may stem from different mating systems. Females of polygamous species should have higher postmating immunity if sexually transmitted infection is the major factor driving female postmating costs (Oku et al., 2019). Therefore, it is imperative and informative to study reproductive microbiomes as a whole in a community context in different mating systems (such as polygamy or polyandry) and under different mating conditions (such as repeated mating or multiple mating). Male mating with more than one female is known as polygamy, while female mating with more than one male is known as polyandry. Multiple mating (mating with more than one mate) and repeated mating (mating repeatedly with the same mate) are common in both sexes in insects, birds, and mammals (Andersson, 1994).

As mentioned above, studies have studied the microbiomes in the reproductive organs and guts in mated and virgin insects (Wang et al., 2014; Bellinvia et al., 2020a,b). However, in addition to reproductive organs and guts, symbiotic microbiomes also exist in other tissues or organs, either intracellularly or extracellularly, such as in the fat body and hemolymph (Szklarzewicz and Michalik, 2017). All these symbiotic microbiomes are under the surveillance of insect immune system and thus are likely to be affected by the mating-induced defense responses (Whitlow, 2004; Okada et al., 2017; Oku et al., 2019). Therefore, in this study, we studied the symbiotic microbiomes in the abdomen of Spodoptera frugiperda females by evaluating microbiomes as a whole in a community context. To test the above signified hypotheses, we used virgin females as controls, treat other females to mate once, twice, or thrice with the same (repeated mating) or different males (multiple mating), and then measured the exact changes on microbiomes in female abdomen using 16S rDNA sequencing. The evolutionary significance of reproductive microbiomes and interactions between host physiology and microbial communities were further explored based on these data.

The fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae), is native to tropical and subtropical regions from Argentina to the United States in the Western Hemisphere (Kumar et al., 2009). S. frugiperda is a long-distance migratory pest, but there was no report on its distribution outside the Americas before 2015. This pest was first discovered in Africa in 2016 (Abrahams et al., 2017) and then invaded India and some other areas in Asia in 2018 (FAO, 2019; CABI, 2020). Soon thereafter, this pest was discovered in Yunnan Province of China at the end of 2018 and then quickly spread northward to the vast areas of China (Guo et al., 2019; Xu et al., 2019; Zhang et al., 2019). This pest mainly damages corn and wheat in China, with the annual potential loss being $17,286–52,143 m for corn (Qin et al., 2020) and $15,571–90,143 m for wheat (Xu et al., 2020), respectively. Symbiotic microbiomes play imperative roles in the survival and reproduction of the host, in addition to the interactions of insect pests, host plants, and natural enemies (Ferrari and Vavre, 2011; Perilla-Henao and Casteel, 2016; Beck and Vannette, 2017). Therefore, modifying symbiotic microbiomes is a potential management strategy for the control of agricultural insect pests (Perilla-Henao and Casteel, 2016; Beck and Vannette, 2017).

The microbiomes associated with animal reproduction are opportunistic and/or sexually transmittable, which may affect host immune defense and future colonization of other microorganisms. Only a few studies have studied the microbiomes in the reproductive organs and guts in insects (Wang et al., 2014; Otti et al., 2017; Bellinvia et al., 2020a,b) and tested the effect of mating on the composition of microbiomes (Bellinvia et al., 2020a,b). In this study, we considered the symbiotic microbiomes in the abdomen as a whole in a community context and tested the diversity and abundance of microbiomes under different mating conditions (Figure 1).

Beta-diversity analysis based on Bray–Curtis distance showed significant variances in the composition of OTUs in females with different mating statuses (Figures 2D–F), suggesting that mating significantly affected the composition of microbiomes in the abdomen of S. frugiperda females. The composition of genital microbiomes of females should be more affected by the sexual transmission of bacterial strains, via male genitalia and the ejaculate, and by bacteria transferred from the cuticle to the reproductive duct during and after mating (Lange et al., 2013; Reinhardt et al., 2015). Moreover, these invading bacteria may enter the hemolymph and other organs via reproductive duct and copulatory wounds (Lange et al., 2013; Reinhardt et al., 2015; Bellinvia et al., 2020a). Previous studies in a number of insect species have found pronounced differences in the microbiomes of reproductive organs between females and males (Wang et al., 2014; Otti et al., 2017; Bellinvia et al., 2020a,b). Bellinvia et al. (2020a,b) further demonstrated that bacteria are sexually transmitted in the common bedbug, C. lectularius. However, results in C. lectularius show that mating did not cause significant effect on alpha diversity of bacteria in the reproductive organs (Bellinvia et al., 2020b). In this study, alpha diversity indicated that the OTU diversity was significantly different in S. frugiperda females with different mating statuses, and mated females showed significant lower diversity than virgin females (Figure 2A) and in more details, virgin females have the highest diversity, followed by one-time mated females and females mated with multiple males, while females mated repeatedly showed the lowest diversity (Figures 2B,C). On the one hand, mating is likely to promote the diversity of microbes due to sexual transmission (Bellinvia et al., 2020a,b). On the other hand, mating may upregulate female immune responses (Oku et al., 2019), particularly in females with polygamous mating systems (Okada et al., 2017; Oku et al., 2019), which may affect the composition and diversity of microbes inside their bodies. In our study, females mated repeatedly with the same mates showed the lowest diversity (Figures 2B,C), which may be due to lower sexual transmission as only mated with one male but higher immune response from repeated matings.

In this study, the obtained 2005 OTUs were classified into 33 phyla, with Proteobacteria, Firmicutes, and Bacteroidota being the first three dominant phyla (Figure 3). Matings further increased the abundance of Proteobacteria (up to 90%) but decreased the abundance of Firmicutes and Bacteroidota (Figure 3). In the citrus fruit fly, Bactrocera minax, 16S rDNA sequencing revealed that Proteobacteria were dominant in gut and reproductive organs (Wang et al., 2014). Other studies have also found an abundance of Proteobacteria in various insect species, such as the sawfly Cephalcia chuxiongica (Yu et al., 2021), the desert locust Schistocerca gregaria (Dillon et al., 2010), the Lutzomyia sand fly (Sant'anna et al., 2012), and two ground beetles (Lundgren et al., 2007). Proteobacteria may play important roles in insects, such as help insects to fix nitrogen and preventing the establishment or proliferation of pathogenic bacteria (Dillon and Dillon, 2004; Dixon and Kahn, 2004; Behar et al., 2005). OTUs clustering into the genus (Figure 4A) also revealed significant differences in females from different mating types. For example, Morganella and Serratia are the most dominant genus in multiple mated females, while in other mating types their abundances are much lower. Further, Enterococcus mundtii showed a higher abundance in virgin females than in females from the other mating types, while Morganella morganii and Serratia marcescens showed higher abundances in multiple mated females than in females from the other mating types (Figure 4B). Species from Enterococcus may play a protective role against insect pathogens in the lepidopteran intestine (Voirol et al., 2018). For example, E. faecalis found in the gypsy moth can acidify its local environment to facilitate it colonize alkaline niches, which may help to host against pathogenic toxins that are activated in alkaline conditions, such as those from Bacillus thuringiensis (Broderick et al., 2004). E. mundtii is a highly abundant bacterium in the gut of S. littoralis, which is known to produce antimicrobial compounds that act against gram-positive pathogens (such as Listeria), but not against resident gut bacteria (Shao et al., 2017; Voirol et al., 2018). M. morganii is a lethal pathogen of Mexican fruit fly larvae (Salas et al., 2017).

Some common gut bacterial inhabitants can be beneficial or detrimental depending on the community composition of the gut. Serratia spp. is one of such bacteria, which is a genus known to be pathogenic in many animals including insects (Chadwick et al., 1990; Ishii et al., 2014; Momose et al., 2018; Voirol et al., 2018). For example, the SM1 strain of S. marcescens was highly toxic to the black-winged termite, Odontotermes formosanus, probably through the production of insecticidal protease and metalloproteinases (Fu et al., 2021). Serratia spp. are commonly reported in lepidopterans, while their roles are unclear in terms of detrimental or beneficial effects (Broderick et al., 2004). Future studies should evaluate whether such bacteria exert no negative effects or provide benefits on insect health depending on the composition of the entire insect microbial community and whether they switch from a symbiont to a systemic pathogen when the environmental condition is altered, such as postmating.

Functional prediction by FAPROTAX also disclosed a most variable on the function of human pathogens all in S. frugiperda females from different mating types, where females mated with multiple males showed higher abundance on this function than females from other mating types (Figure 5A, Supplementary Figure 3).

Due to the turnover on beneficial and detrimental microbiomes between virgin and multiple mated females found by taxonomy assignment and functional prediction by FAPROTAX, the possible pathogens and beneficial bacteria were further determined based on evidence from previous studies. The FAPROTAX and evidence-based double checking ensure the credibility of the functional evaluation of bacteria. Three insect pathogenic species and 14 pathogenic species from human and other animal taxa were verified by previous published studies (Table 1). Twelve beneficial bacterial species from four genera, Lactobacillus, Buchnera, Proteus, and Enterococcus, were also verified by published studies (Table 1). Buchnera has been implicated in various nutritional and non-nutritional functions in insects, such as provisioning of essential amino acids and nitrogen recycling (Douglas, 1998; Dillon and Dillon, 2004). Studies in human found that the Lactobacillus spp. are beneficial vaginal microbiomes, which may function in barring against pathogen adhesion, production of lactic acid against the colonization of pathogens, and stimulating host defense responses (Ravel et al., 2011; Chen et al., 2017; Tachedjian et al., 2017; Younes et al., 2018). The function of Lactobacillus spp. in insects is still unclear and thus warrants further studies.

Statistics showed that females mated with multiple males have the highest abundance on pathogens (more than five times than that of females from the other mating types) (Figure 5B) while they have the lowest abundance on beneficial bacteria (Figures 4B, 5C) in comparison with that of females from the other mating types. Moreover, LDA demonstrates that the Enterococcus mundtii (a beneficial bacterium) is one of the important biomarkers in virgin females, whereas Morganella morganii and Serratia marcescens (pathogens) are two of the important biomarkers in multiple mated females (Figure 6), further supporting the hypothesis that promiscuity turned over the abundance of beneficial and detrimental microbiomes in S. frugiperda females.

However, whether and how these possible pathogens and beneficial bacteria affect the health and reproduction of S. frugiperda still needs to be validated by further studies. Moreover, future studies by comparing microbiomes of reproductive organs and non-reproductive organs of both sexes under different mating conditions are expected to achieve deeper insight in this field.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

L-YZ and HYu collected the insects. L-YZ, D-YF, and JX designed the study. L-YZ, HYu, and D-YF dissected the insects and extracted DNA. L-YZ, SY, HYe, and JX analyzed the data. L-YZ, JX, and HYe wrote the paper. All authors read and approved the final manuscript.

This study was jointly supported by projects from Science and Technology Planning Project in Key Areas of Yunnan Province (202001BB050002), Basic Research Projects of Yunnan Province (202201AS070025), Joint Special Project of Yunnan Province for Agricultural Basic Research (2018FG001-002), and National Natural Science Foundation Program of P.R. China (31760635, 31560606).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.