The gut microbiome of insects plays a crucial role in the ecology of insects and adaptability to different environments [1, 6]. Although H. halys is one of the most important invasive pest species worldwide, research has to date focused mainly on its primary symbiont Pantoea carbekii. Furthermore, studies were mainly performed on laboratory-reared populations [27, 33]. Our study, for the first time, investigated the gut microbiome composition of individuals from natural populations. Overall, we found two main taxa: Pantoea and Commensalibacter which accounted for the majority of the reads. Collections across the year allowed us to show that the gut microbiome has a clear seasonal pattern, with a lower relative abundance of Pantoea in summer compared to winter and a significant increase of Commensalibacter in summer. Overall, we found a higher alpha diversity in the winter months and we could show that the composition of the gut microbiome varies widely across the year.
The primary symbiont Pantoea is present in almost all individuals across the season and accounts for the highest numbers of reads. As showed in previous studies, Pantoea carbekii is the primary symbiont of H. halys and is essential for the fitness and survival of the individuals [14, 27, 33]. Surprisingly, in this study we found three individuals where no Pantoea reads were detected. We do not know if the individuals did not acquire the symbiont or only harbor the symbiont with very low abundance and whether the host suffered any fitness costs. In the winter months where H. halys is in reproductive diapause [26], the relative abundance of Pantoea is higher than in the summer months, when the insects are active. Environmental factors might play a role in the reduction of Pantoea. In the Pentatomid N. viridula, which is also associated with a Pantoea-like symbiont [50], a recent study showed that the symbiont titers decrease at higher temperatures in the laboratory [51]. This could indicate that the lower abundance of Pantoea in the summer months is influenced by the higher temperatures in the field.
The primary symbiont Pantoea carbekii is not the only predominant bacterial genus found in the gut microbiome of H. halys. An interesting finding of our study is that Commensalibacter occurs frequently, especially in the summer months where it is present in 60–100% of all individuals. In one individual all reads are assigned to Commensalibacter and in several individuals collected from May to September, almost all reads were assigned to this taxon. Commensalibacter is widely found in insects [52–54], including other stink bugs as N. viridula [11]. It belongs to the family Acetobacteraceae, which is frequently associated with Hemiptera and generally with insects like honey bees, leafhoppers, and caterpillars having sugar-based diets that lack nitrogen [2, 53]. H. halys preferably feeds on the reproductive parts of plants [26, 55] and might encounter similar nutritional deficits common for a sugar-rich diet which might be compensated by this additional taxon. Most Hemiptera have an acidic anterior midgut region [56] and it was shown that Acetobacteraceae can withstand sugar-rich and acidic environments. Furthermore, in the ant species O. smaragdina it is expected that the higher consumption of carbohydrates results in higher concentrations of Acetobacteraceae [57]. We expect that the environment in the gut of H. halys acts as a chemical filter against pathogenic microbes and supports the growth of Acetobacteraceae [58]. It might be possible that H. halys chooses its feeding hosts based on symbiont acquisition, as it was seen before for other insects [59].
The genus Commensalibacter consists of several species, which can fulfill different functions like amino acid and carbohydrate metabolism [52]. Generally, Acetobacteraceae can be isolated from plants, fruits, herbs, and flowers and do not seem to be fundamental for the survival of insects [53], which explains why we did not detect Commensalibacter in every individual. The low abundance of Commensalibacter in winter might be due to the fact that this symbiont is most probably acquired via the diet and during the overwintering period the bugs reduce or entirely stop the feeding. This could also explain the varying frequency of Commensalibacter in H. halys individuals sampled in the summer months. While some individuals have extremely high frequencies of Commensalibacter, making up all or the majority of all assigned reads, other individuals have only a low number or no reads assigned to this taxon. In the mid of July the first imagines are found in the study region [60]. If Commensalibacter is horizontally acquired by feeding, the newly developed young adults might have not acquired these bacteria yet, while the older, co-occurring individuals from the overwintered generation could feed on a broad variety of plants and hence harbor a high density of Commensalibacter. The role of Commensalibacter in the fitness of H. halys needs to be investigated.
Although the majority of the reads belonged to Pantoea and Commensalibacter we detected many other taxa in lower abundance in different frequencies. Various taxa were already described in literature as part of the gut microbiome of Pentatomids. For example, the Pentatomid N. viridula also harbors bacteria belonging to the genera Enterococcus, Sodalis, Serratia and Bacillus in its gut [11, 30]. Similarly, the two-spotted stink bug Bathycoelia distincta, is frequently associated with Pantoea and Sodalis, whereas Pseudomonas, Serratia, Bacillus, and Lactococcus were also detected in lower frequencies [7]. A study on different true bug species detected a broad variety of different bacterial genera in the guts, that varied widely in relative abundance in different species. For instance, as H. halys other true bugs harbor Commensalibacter, Pantoea, Enterobacteriaceae, Lactococcus, Enterococcus, Wolbachia, Rhizobiaceae, Serratia, and Pseudomonas [61] suggesting that bacteria belonging to these genera are widespread in this insect family. While Yokenella is a genus frequently associated with Pentatomidae and Pentatomorpha [7, 30, 61], we did not find this bacterium in H. halys.
Overall, the Shannon diversity in H. halys is lower compared to the median number of the Shannon diversity in other Hemiptera species [62]. However, we observed high fluctuations of Chao1 index and Shannon diversity across the season. While individuals caught in January and November have a comparably high alpha diversity, single individuals still show low diversities. Individuals caught in the summer months have a lower alpha diversity than those collected during the winter months, which might be explained by the former being young adults that usually are found by the mid of July [60]. We expect that they had a short feeding period before collection and hence, did not acquire different bacteria via the diet. The seasonal fluctuation is in line with other studies that described seasonal differences in the alpha diversity in different insects. Larvae of Spodoptera frugiperda (Lepidoptera: Noctuidae) for instance have significant differences in the core community between the dry and rainy season [63], or in honey bees, in which the microbiome changes mainly in the transition phases from summer to winter and from winter to spring [8]. The higher alpha-diversities of H. halys individuals sampled in winter, might result from a higher feeding activity before the overwintering phase, as the individuals form a fat body before overwintering [64]. Thus, an increased uptake of diverse plant material might result in a higher diversity of the microbial community in the gut.
For the stink bugs Piezodorus guildinii, Euschistus heros, and B. distincta it was shown that the gut microbial community differs geographically [7, 31]. Since we analyzed individuals from a single region, future studies should investigate the microbial community of H. halys in multiple regions. Since invasive species can gain [65, 66] and lose [67] symbionts during the invasion process it might be interesting to investigate if and how microbes influenced the invasion process of this pest species.
The beta diversity among the individuals differed significantly across the season. Changes in beta diversity across the season were by now shown in different species, like honeybees [68] and bark beetles [69]. The beta diversities of spring and fall, summer and winter, and winter and fall are significantly different according to the Bray-Curtis dissimilarity matrix. Given the polyphagous nature of H. halys, we suppose that, as the availability of fruits varies across the seasons, it is one of the main drivers for the variability of the detected bacteria between individuals in the different seasons.
In summary our study shows that H. halys is associated predominantly with two bacteria in the study region in Northern Italy. While the function of the primary symbiont Pantoea for the host biology was investigated in previous studies, knowledge about the second co-occurring bacteria Commensalibacter is scarce. As across the season, dynamic bacterial communities were characterized, further studies about the function of Commensalibacter and the potential interactions about the two taxa should be investigated.