Herbivorous insects can host microorganisms, such as bacteria, viruses, fungi, archaea, and protozoa in their digestive tract, referred to as their gut microbiome (Gurung et al., 2019; Muñoz-Benavent et al., 2021). The gut microbiome of insects has been implicated in the transformation of food substrates and acting as a direct dietary source providing essential nutrients lacking in the plant diet (Dillon and Dillon, 2003; Moran et al., 2019). Evidence for an adapted lignocellulosic microbiome is strong for some insect groups, notably termites (König et al., 2013; Jang and Kikuchi, 2020). Studies have shown that bacteria and fungi in insect guts possess enzymes breaking down insoluble plant polymers, including cellulose and hemicellulose, in addition to soluble compounds such as starch and pectin (Watanabe and Tokuda, 2010; Jang and Kikuchi, 2020). Additional roles for the gut microbiome have been inferred from studies of other animal systems and include protection against pathogen infection (Mendes and Raaijmakers, 2015; Raymann and Moran, 2018; Jang and Kikuchi, 2020) and behavioral influences (Davidson et al., 2018, 2020; Jones et al., 2018).

The recruitment of gut microbiomes in insects is not well constrained, with studies on social insects such as bees and termites suggesting direct transfer between individuals [e.g., (Koch and Schmid-Hempel, 2011; Bourguignon et al., 2018)], while other studies indicate that gut microbiomes may reflect recruitment from dietary substrates (Jones et al., 2019). Studies on lepidopterans, for instance, demonstrated a correlation between diet and gut microbial diversity, where the host plants were the main contributors to the herbivorous insects' gut microbiome (Mason et al., 2020). The extent of the effect of diet on gut microbiome differed depending on both the lepidopteran host and host plant species (Jones et al., 2019) and their exposure to environments with soil (Hannula et al., 2019). In herbivorous insects, the gut microbiome has been shown to largely reflect the host insect phylogeny rather than their ecology (McLean et al., 2019). The extent to which the insect gut acts as an environmental filter that shapes the diversity of its microbiome toward taxa that are beneficial symbionts for the insect host appears to depend upon the insect taxa (Moran et al., 2019; Mason, 2020).

Phasmids (stick insects) are non-social folivorous insects that feed almost exclusively on leaves but are able to adapt to foreign diets, that is, plant species that the insects are not usually exposed to or would consume, if the favored host is unavailable (Boucher and Varady-Szabo, 2005; Blüthgen et al., 2006; Shelomi et al., 2014a). The current understanding of gut microbiomes in phasmids is not well constrained. In one study, bacterial gut microbiomes showed no significant differences in composition across several phasmid families (Shelomi et al., 2014a). By sequencing the gut contents, one study found that Proteobacteria was dominant, followed by Actinobacteria and Firmicutes, and Spiroplasma spp. and Sphingobium spp. were the most common microbes isolated (Shelomi et al., 2013). In another study, cultured bacterial strains from phasmid guts affiliated with the Actinomycetales, Enterobacteriales, Lactobacillales, Pseudomonadales, and Xanthomonadales (Shelomi et al., 2015). It has been suggested that the environment and diet can affect the gut microbiome of phasmids (Ignasiak and Maxwell, 2017). Furthermore, gut microorganisms found in phasmids were shown to produce cell wall degrading enzymes including cellulases important to the digestion of plant matter (Shelomi et al., 2014b), and it has also been postulated that some phasmids possess their own genes for breakdown of soluble plant carbohydrates, for example, pectin (Shelomi et al., 2016). For species of phasmids that do not adhere their eggs to the leaf substrate, this stage in life history is unlikely to be a route for microbiome recruitment from the plant substrate.

Here, we set out to further interrogate the hypothesis that different plant diets in phasmids would lead to shifts in the taxonomic composition of the gut microbiome. This is of relevance to Singapore because of the high levels of urbanization with a mixture of native and exotic plant host species and the potential for a shift in plant hosts of herbivores. Specifically, we sought to test the following hypotheses: (a) Does phasmid gut microbiota vary with diet, and (b) is phasmid gut microbiota a subset of food (i.e., plant) or local soil microbiomes? We reared a local species, Gray's Malayan stick insect, Lonchodes brevipes, on two species of host plants. This phasmid is encountered in mangroves and rainforests (Seow-Choen, pers. obs.) and feeds on sea hibiscus (SH), Hibiscus tiliaceus in the wild, and does not adhere their eggs to the leaf substrate. In hobbyist cultures, this phasmid is commonly reared on a widespread non-native ornamental plant, golden penda (GP), Xanthostemon chrysanthus. We compared the gut microbiome of individuals reared on both plants, as well as the microbiome of the host plant leaves and soil because soil is the most microbially emissive component of the habitat.

The high-throughput sequencing of bacterial and fungal diversity in phasmid frass was used as a proxy for gut microbiomes. This approach is congruent with that adopted for most other studies of microbiomes in animal and human systems, for example, Huttenhower et al. (2012). We used this approach to interrogate the central hypothesis that native and introduced plant food sources impacted the composition of phasmid gut microbiomes. Comparison of microbiota associated with food sources and surrounding soils provided triangulation of potential sources and influence on the composition of the gut microorganisms. The DNA yield from samples was used as a general proxy for overall (i.e., bacterial and fungal) microbial biomass, although we recognize that host DNA may also have been a confounding factor. This indicated that while the food substrate and frass had similar levels of biomass (Figure 1A), the soil samples yielded significantly greater microbial biomass per gram of substrate than frass (p = 0.001) and leaves (p < 0.001). Of note was the markedly higher biomass in soil associated with native vs. exotic plants, and this may indicate a more developed rhizosphere microbiome for the native plants that reflects their natural history.

Contrasting alpha diversity estimates were observed for bacteria and fungi across the sample types. Bacterial richness estimates (Chao1) were significantly higher in the frass of phasmids fed on native (sea hibiscus) vs. exotic (golden penda) leaf substrates (Figure 1B; Mann–Whitney U = 0, n₁ = 16, n₂ = 19, p < 0.001). Similarly, fungal richness was significantly higher in the frass of phasmids reared on native vs. exotic leaves (Figure 1C; Mann–Whitney U = 0, n₁ = 16, n₂ = 19, p < 0.001). The Shannon's index estimates that consider richness and evenness were not significantly different for bacteria between native and exotic substrates (Figure 1D), whereas for fungi estimates, richness and evenness were significantly higher for the native substrate (Figure 1E; Mann–Whitney U = 2, n₁ = 11, n₂ = 19, p < 0.001). The leaves of native and exotic plants displayed similar bacterial richness to that of frass while the soil as expected supported a markedly greater diversity of bacteria (Thompson et al., 2017). In contrast, both leaves and soil supported greater fungal richness than frass, and this reflected the importance of the phyllosphere as a reservoir of fungal diversity (Xu et al., 2022).

Beta diversity patterns measured by Hellinger distances revealed that gut bacterial assemblages were more similar to each other than their associated leaf or soil assemblages regardless of the leaf diet, suggesting that bacteria in the gut had undergone environmental filtering and recruitment (Figure 2A). In contrast, the fungal assemblages in frass from the different diets displayed less overlap with each other but more overlap with the corresponding leaf substrate, thus suggesting recruitment from the leaf substrate may be important for fungi (Figure 2B). For both bacteria and fungi, the frass assemblages displayed no overlap in overall similarity with soil assemblages. This raises the interesting question of what the recruitment sources are for gut bacteria and fungi in phasmids. The leaves and soil were unable to fully explain the observed diversity in frass and so additional sources might include vertical transmission by phasmids, or other aeolian or environmental sources. Interestingly, the results also revealed that bacteria and fungi in soil respond differently to native and exotic plants, with bacteria assemblages remaining essentially unchanged while a significant shift in fungal assemblages occurred in soils planted with native vs. exotic plants (Figures 2A, B). The altered soil mycobiome with exotic plants may thus have undetermined impacts on the functioning of the soil and phyllosphere. This has potential implications for horticultural management and conservation.

Taxonomic diversity estimation revealed that frass bacterial assemblages were enriched in taxa within Alpha- and Gamma-proteobacteria. The most frequently encountered bacterial families were Pseudomonadaceae and Enterobacteriaceae for the native plant-fed insects and Comamonadaceae and Enterobacteriaceae in the exotic plant-fed insects (Figure 3A; Supplementary Figure 1). Interestingly, while Enterobacteriaceae is abundant in both groups of frass samples, in SH, the family consisted almost entirely of the genus Klebsiella, whereas in GP, Salmonella was also present. Patterns in diversity for fungi were less evident (Figure 3B; Supplementary Figure 2) due to the high taxonomic diversity and large numbers of unclassified taxa. Many of the fungal genera (e.g., Aspergillus and Penicillium) include known cellulose-degrading taxa (Vázquez-Montoya et al., 2020), although whether genetic signals represent vegetative cells rather than dormant spores requires these data to be interpreted with some caution. In order to further interrogate the likelihood that gut microbiomes of insects reared on native and exotic plant feed had distinct assembly patterns, we conducted differential abundance analysis to identify bacterial and fungal families that differed significantly (i.e., were enriched) between frass samples from phasmids fed on the two plant hosts (Figure 4A). Notably, the strong differential abundance pattern for Cladosporium species (enriched in SH frass and virtually absent in GP frass) may indicate the effect of selection pressure due to the gut environment affected by the different diets (Figure 4B). The core ASVs shared between the sample type (frass, leaf, or soil) and associated host plant (golden penda or sea hibiscus) varied greatly, with the greatest number of core ASVs shared between the leaves of the two host plant species (Figure 5), while the majority of core ASVs were only found in the respective host plant leaves and frass of insects feeding on sea hibiscus, that is, not shared between communities in different sample types (Figure 5).

Overall, this study shows that community structure was driven by host selection instead of dispersal from the environment, as the environments (soil/leaf) were not as different as the frass associated with two source plant species. Nonetheless, the sample types (i.e., frass, leaf, and soil), the associated host plant species, and the interaction term of the two factors were all statistically significant in structuring the bacterial and fungal communities observed in this study (manyglm, all p < 0.001). Furthermore, the microbiomes varied significantly in response to a native or exotic plant diet, with fungi accounting for more variation than bacteria. While the gut communities of some herbivorous insects are characterized by transient and food-derived bacteria (Whitaker et al., 2016; Hernández-García et al., 2017), other studies do not find a strong correlation between diet and the gut microbiome (Mason and Raffa, 2014; Chaturvedi et al., 2017). A previous study suggests that phasmids are well adapted to changes in diets and may not be reliant on their gut microbiome for specialized digestion of plants (Shelomi et al., 2014b). Our study provides additional insight that indicates a shift in diet, resulting in measurable changes to the insect gut microbiome that includes known cellulolytic taxa. The data also indicate that exotic plants result in altered soil and herbivorous insect microbiomes and this is relevant to the conservation and management issues where factors that potentially affect insect herbivory are important (Bahrndorff et al., 2016). Finally, the characterization of the Lonchodes brevipes microbiome also adds to the growing global inventory of animal microbiomes (Douglas, 2019).

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.

ET and SP conceived the project, interpreted the findings, and wrote the manuscript. YL and YP performed the specimen collections and DNA extractions. BW performed PCR amplification and sequencing. KL processed the sequencing data. All authors read and agreed on the final manuscript.