Extensive inheritance of gut microbial communities in a superorganismal termite

doi:10.21203/rs.3.rs-1978279/v2

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Extensive inheritance of gut microbial communities in a superorganismal termite

https://doi.org/10.21203/rs.3.rs-1978279/v2

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Mutualistic co-evolution can be mediated by vertical transmission of symbionts between host generations. Organisms exhibit adaptations that ensure optimal microbial inheritance, yet the extent to which this applies to social insects, such as termites that have co-evolved with gut microbes, is poorly resolved. Here, we document consistent vertical transmission across colony generations of fungus-farming termites. Inherited bacteria comprise 44% of the microbiome, over 80 genera, and strains that are specific to termite pedigrees. We show that the superorganism, consisting of reproductives and workers, analogous to gametes and soma of an organism, is adapted to vertically transmit a distinct microbial community with high fidelity. Microbial inheritance is achieved because colony-founding reproductives are endowed with a set of non-random, environmentally-sensitive, and termite-specific gut microbes derived from their colonies of origin. Reproductives biparentally transmit these symbionts to offspring colony workers, where priority effects dictate the composition of the forming colony microbiome. Superorganismal gametes, the reproductives, are thus adapted to secure transmission of entire communities of specific, co-evolved microbes that are critical to the colony microbiome later retained by workers. Extensive vertical transmission aligns with evolutionary patterns of termite-bacterial co-diversification. This colony-level inheritance extends models of transmission from individual organisms to superorganisms, both of which demonstrate adaptations to retain symbiotic fidelity and mixed-mode transmission conducive to mutualism.

symbiosis

vertical transmission

major evolutionary transition

social insects

microbiome

Associations with microbiomes unequivocally impact the ecology and evolution of animal hosts. From humans to insects, animals consistently engage with microbial partners to reap synergistic metabolic, immune, and defensive benefits (Engel & Moran, 2013; Maynard et al., 2012; Rowland et al., 2018). Vertical transmission, the process by which microbes are passed from parents to offspring, ensures inheritance of beneficial microbes (Bright & Bulgheresi, 2010) and provides an initial inoculum that directs microbiome assembly (Coyte et al., 2021; Sprockett et al., 2018). For example, in humans, maternally inherited bacteria govern the early-life microbiome and health outcomes (Olm et al., 2022; Wang et al., 2020). Over evolutionary time, consistent vertical transmission aligns the reproductive interests of hosts and microbes, which is predicted to enhance the exchange of mutualistic benefits and favor host-symbiont co-adaptations (Frank, 1997; Yamamura, 1993). Persistent inheritance of mutualistic microbes requires that hosts have traits that facilitate the passage of microbes and that control the microbial community to promote cooperation (Foster et al., 2017; Frank, 1996; Sharp & Foster, 2022). In the most intimate of mutualisms, hosts integrate bacteria within cells and transmit them via their gametes (eggs) (Kiers & West, 2015; McCutcheon, 2021; Russell et al., 2019). More commonly, animals transmit complex microbiomes externally during birth or early in offspring development, which secures microbial inheritance but also incurs acquisition of environmental microbes through horizontal transmission (Bjork et al., 2019; Leftwich et al., 2020).

Evolutionary histories and co-evolution between social insects and microbial symbionts imply vertical transmission and host adaptations that optimize microbial inheritance (Bourguignon et al., 2018; Brune, 2014; Kwong et al., 2017; Russell et al., 2017; Sanders et al., 2014). In social insects known as superorganisms, these adaptations are predicted to be at the colony-level because natural selection acts on the superorganism as an individual (Boomsma & Gawne, 2018; West et al., 2015; Wheeler, 1911). Adaptations for vertical transmission should thus manifest across the division of reproductive labor, analogous to that of multicellular organisms (Boomsma & Gawne, 2018; Wheeler, 1911). Superorganismal colony life begins with alate reproductives, similar to gametes, who leave their parental colony to found offspring colonies, where they produce workers – the soma of the superorganism (Wheeler, 1911). Workers exchange microbes as a colony-level adaptation for maintaining stable symbiotic communities (Lanan et al., 2016; Nalepa, 2015; Powell et al., 2014). Founding reproductives are thus under extensive selective pressure transmit microbes, as they are the sole colony members to carry microbes that will later populate up to millions of workers. However, these superorganismal traits remain uncharacterized. Assessing adaptations in founding reproductives for vertical transmission requires quantifying transmission of symbionts strains at the level of selection, from parent to offspring colonies. While components of transmission have been examined, such as from parent colonies to reproductives or reproductives to offspring colonies (Diouf et al., 2023; Diouf et al., 2018; Hu et al., 2023; Meirelles et al., 2016; Michaud et al., 2020; Su et al., 2021), complete superorganismal inheritance of gut microbiomes has not been determined.

Here, we elucidate the extent of vertical transmission and potential adaptations for microbial inheritance in termite superorganisms. Termites are an ideal model to characterize superorganismal vertical transmission. Over the past 150 MY, termites have engaged in obligate mutualism with complex gut microbiomes (Bucek et al., 2019). These ancient symbioses have resulted in co-adaptations with microbes for digestion (Arora et al., 2022; Brune, 2014) and co-cladogenesis with symbionts indicative of vertical transmission punctuated by horizonal transmission (Bourguignon et al., 2018; Desai et al., 2010). We assessed vertical transmission from parent to offspring colonies in four distinct termite pedigrees of the fungus-farming termite Macrotermes natalensis (Seite et al., 2022). Fungus-farming termites host a specific (Dietrich et al., 2014), consistent (Otani et al., 2016; Otani et al., 2014), and co-evolved gut microbiome (Hu et al., 2019; Poulsen et al., 2014), and recently, offspring colonies were shown to maintain a substantial portion of microbes inherited from founding reproductives (Diouf et al., 2023). Given their superorganismal life history and symbiotic microbiome, we predicted that (i) the colony microbiome contains a conversed set of microbes that are vertically transmitted from parent to offspring colonies within pedigrees, and that (ii) founding reproductives are uniquely adapted to ensure transmission of this microbial cohort. We used amplicon sequencing of the V3-V4 region of the 16S rRNA bacterial gene to track individual Amplicon Sequence Variants (ASVs) from parental colonies through founding reproductives to offspring colonies (Figure 1A). This allowed us to first assess the extent, consistency, and specificity of vertical transmission across the four pedigrees. Subsequently, we identified host factors and microbial interactions that facilitate selective transmission and support inheritance and then confirmed their impacts with a Random Forest model.

The termite superorganism: a model system for vertical transmission

We determined vertical transmission of the gut microbiome from parent to offspring colonies in four independent pedigrees of fungus-farming termites (Termitidae: Macrotermitinae) (Seite et al., 2022). As superorganisms, fungus-farming termite colonies are founded by a queen and a king (the reproductives) that originate from different mature parental colonies. After a few months, these reproductives have produced their first offspring workers and soldiers that forgo reproduction and support colony growth and homeostasis, while the reproductives gradually transition to exclusively reproduce (Seite et al., 2022). To experimentally track vertical transmission, founding queens from four independent maternal colonies were crossed with founding kings from a paternal colony to establish offspring colonies (Figure 1A). This allowed us to identify microbes transmitted specifically within pedigrees, indicative of vertical transmission (Moeller et al., 2018; Olm et al., 2022), and biparental contribution to inheritance. Offspring colonies were sampled at three months old, prior to acquisition of their fungal cultivar. We invoked a strict definition of inheritance that required an ASV to be present in one of the two parental colonies, in all founding reproductive samples from that parental colony, and in the resulting offspring colonies. Although inherited microbes may be hosted by any termite in the colony, we focused our subsequent analyses on workers and founding reproductives that, respectively, house and transmit most of the colony microbiome (Chouvenc & Su, 2017; Otani et al., 2019) (for quantification of microbial loads and inheritance across all colony members, see Figure 1 – figure supplement 1). Regardless of microbial load and composition, the sequencing effort sufficiently captured the ASV diversity across castes (Figure 1 – figure supplement 2).

Extensive and consistent vertical transmission of a diverse microbiome

The proportion of the microbiome that was vertically transmitted from parent to offspring colonies was, as predicted, extensive and congruent across pedigrees (Figure 1B). The individuals key to microbial inheritance – parental colony workers, founding reproductives, and offspring colony workers – maintained high relative abundances of vertically transmitted ASVs, which accounted for approximately a quarter of all ASVs present (Figure 1B). Parental colony workers transmitted 21.2% relative abundance and 24.1% of ASVs in their microbiome to the founding reproductives and ultimately offspring colonies. Founding reproductives hosted the greatest abundance and percent of vertically transmitted microbes, averaging 57.6% of the relative abundance and 38.6% ASVs. Offspring colony worker microbiomes hosted vertically transmitted ASVs that made up 43.9% of microbial abundance and 18.7% of all ASVs (Figure 1 – source data 1). Offspring colonies also hosted a remarkable diversity of vertically transmitted ASVs, averaging 1,694 ASVs. This finding extends recent insights that founding reproductives vertically transmit bacteria to offspring colonies in another species of fungus-growing termites (Diouf et al., 2023). Our inclusion of parental colonies and the more precise taxonomic classification in our definition led us to quantify less vertical transmission, which was 43.9% of the relative abundance of the microbiome, compared to the 73% previously documented (Diouf et al., 2023). Consistent with previous findings (Michaud et al., 2020), both maternal and paternal reproductives inherit microbes from parental colonies. We further demonstrate these biparental contributions result in offspring colony microbiomes that contain ASVs that are unique to maternal or paternal colonies and others that are shared by both parental colonies (Figure 1). Notably, paternal contributions were conceivably greater than documented here because our experimental design likely underestimated them (Materials and Methods). Extensive superorganismal inheritance of the gut microbiome secures symbiotic benefits and facilitates potential for mutualistic co-evolution, and outbreeding may promote complementarity between maternal and paternal microbiomes, analogous to the genetic contributions of maternal and paternal gametes that form a zygote.

We then assessed the specificity of the microbiome inherited by quantifying the number of ASVs that were uniquely inherited in individual maternal pedigrees or shared between pedigrees. In each maternal pedigree, on average almost 1,000 unique ASVs were transmitted to founding reproductives, of which 84% were maintained in offspring colonies (Figure 2A). In contrast, only 364 ASVs were ubiquitous to all pedigrees (Figure 2A), and others were shared between two or three pedigrees (Figure 2 – figure supplement 1A and B). The remarkable extent of pedigree specificity, a robust indicator of vertical transmission (Moeller et al., 2018; Olm et al., 2022), confirms that a substantial portion of the microbiome is passed from parent to offspring colonies. Ultimately, the observed pedigree specificity is an estimate because ASVs themselves still do not reflect true bacterial strain diversity (Strube, 2021). However, this is unproblematic for comparisons of the extent and consistency of transmission, as ASVs have previously been used to assess patterns of vertical transmission (Bjork et al., 2019; Bourguignon et al., 2018; Moeller et al., 2018).

Our evaluation of the taxonomic consistency of the communities that were vertically transmitted across pedigrees revealed that colonies inherit a diverse and predictable set of microbes. We found genus-level composition of the inherited microbiomes to be nearly identical across pedigrees (Figure 2B). Specifically, 85 genera were inherited by all pedigrees (Figure 2 – source data 1), and any two pedigrees shared on average 95.2% bacterial genera (Figure 2 – figure supplement 1C). Further, pedigree-specific ASVs accounted for many of these genera (Figure 2 – source data 1), and ASV diversity within genera was comparable across pedigrees (Figure 2B). Thus, pedigrees inherit a diverse and congruent set of microbes, including multiple taxa that are consistently associated with fungus-growing termites (Otani et al., 2014) and known to be maintained in offspring colonies (Diouf et al., 2023). Microbiome consistency in transmission ensures that offspring colonies have predictable access to beneficial co-adapted symbionts.

The microbial endowment of founding reproductives secures transmission

Founding reproductives are the only avenue for offspring colonies to obtain gut microbes that are absent in the environment. The richness, abundance, and consistency of gut microbes inherited imply that reproductives transmit a specific and optimal bacterial community (Figures 1B and 2A). The bacteria endowed in founding reproductives could be randomly assembled, based on the prevalence of microbes within parental colonies, as previously observed for transmission of termite protists (Michaud et al., 2020). Alternatively, the founding reproductives may be adapted to selectively transmit a set of key microbial symbionts.

By comparing a null model of inheritance to the observed inheritance, we elucidated that founding reproductives selectively transmit non-random microbial communities. The null model considers an ASV’s probability of vertical transmission based on its abundance within maternal colony workers, and then selects the same number of ASVs that were observed to be transmitted from parent colony to founding reproductive or founding reproductive to offspring colony (see Materials and Methods for details). Comparison of the null model to the patterns observed revealed that significantly fewer genera were inherited by founding reproductives from maternal colonies than expected by chance (Figure 3A; permutation tests: p < 0.0001, for each pedigree). Most genera within reproductives were inherited by offspring colonies, so that offspring worker communities also contained fewer genera than predicted by the null model (permutation tests: p < 0.0001, for each pedigree). Inheritance is thus not function of ASV prevalence in parental colonies alone, but selects a non-random subset of microbial genera, implying adaptations that ensure inheritance of specific microbes.

We then went on to test whether transmission to founding reproductives impacts the relative abundances of inherited bacterial genera. Differential abundance analysis comparing the microbiota in maternal colony workers to that in founding reproductives revealed four genera that were always less abundant in reproductive guts. These genera were then either never inherited by offspring colonies or only inherited in one pedigree (Figure 3B). In contrast, the thirteen genera that increased in abundance in founding reproductives completed vertical transmission to offspring colonies across all four pedigrees (Figure 3B; Figure 3 – source data 1). These genera thus appear critical for the fungus-farming termite host, which is consistent with findings that several genera have diversified with the termite host (Bourguignon et al., 2018) and are members of the core microbiome (Otani et al., 2014). The subsequent transfer to offspring colonies did not drastically alter the inherited microbiome, but some inherited genera either significantly increased or decreased in relative abundance within offspring colony workers (Figure 3 – figure supplement 1; Figure 3 – source data 1).

Founding reproductive also shape the ASV-level composition of the inherited microbiome. If vertical transmission was random and abundance driven, ASVs with higher abundance within genera would have a greater chance of transmission to founding reproductives and then offspring colony workers. However, this is not the case for transmission from maternal colony workers to founding reproductives. ASV abundance in maternal colonies had a significant negative effect on the probability of transmission to founding reproductives (Figure 3C). This suggests that reproductives secure a diverse inoculum of ASVs within key genera rather than allowing competitive exclusion to drive transmission. However, ASV abundance in the founding reproductives positively impacted whether ASVs would establish within offspring colony workers, such that inherited ASVs were more abundant than those that were not inherited (Figure 3C; Figure 3 – source data 2). Thus, vertically transmitted microbes become abundant in founding reproductive and establish in the offspring colony microbiome. The selective processes in transmission of ASVs and genera emphasize that the reproductives are adapted to carry a specific inherited microbiome to the next generation.

Inheritance grants microbes priority effects

Once founding reproductives have passed the microbiome onto their offspring workers, they gradually specialize on hosting very few bacterial genera (Otani et al., 2019; Poulsen et al., 2014). Founding reproductives thus vector inherited microbiomes to secure the emerging functions of the workers, who perform digestion and provide defense for superorganism. Priority effects, in which early-arriving microbes dictate community assembly, could both enhance establishment of inherited microbes and facilitate uptake of beneficial non-inherited microbes in the colony microbiome (Debray et al., 2022).

Inherited ASVs indeed generate priority effects by gaining higher abundances within genera than those that are not inherited. Relative abundance of ASVs within founding reproductives and offspring colonies was significantly related to whether an ASV was inherited or not (Figure 4A). Ultimately, this means that inherited ASVs gained higher relative abundances within the superorganism microbiome (Figure 4A; Figure 4- source data 1). Larvae and the maturing reproductives in offspring colonies also showed priority effects of inherited ASVs (Figure 4 – figure supplement 1). Initial inoculation may thus promote the predictable presence of inherited microbes in a community that also contains horizontally transmitted (non-inherited) bacteria, which jointly are likely needed to optimize performance of the mature colony superorganism.

The observed priority effects imply that microbial interactions within the gut underlie the propensity for inherited ASVs to establish and proliferate. To shed light on community dynamics, we performed network analysis of the positive (co-occurrence of two ASVs) and negative (presence of an ASV associated with absence of another ASV) interactions (Kurtz et al., 2015). This revealed that inheritance itself did not significantly affect microbial interactions (positive interactions linear model (LM): F_1,829=0.9817, p=0.3221; negative interactions LM: F_1,829=1.468, p=0.2260), implying that we cannot detect cooperation or competition between microbes, although they are known to be essential to microbiome functions (Coyte et al., 2021; Coyte et al., 2015). However, when we assessed network assortativity further understand ecological interactions amongst inherited and non-inherited taxa, we uncovered that ASVs with positive network interactions, meaning they co-occur, tended to share the same mode of inheritance (Figure 4B). We then compared this to the inheritance predicted by the null model (Materials and Methods), and assortativity was significantly higher than expected by chance for ASVs with positive interactions (Figure 4C), but not for the disassortative trend for negative interactions. Positive assortativity is often found among related microbes (Hall et al., 2019; Kurtz et al., 2015) and may contribute to network structure, but it may also reflect priority effects, as co-occurring inherited ASVs direct the establishment of horizontally acquired environmental ASVs.

Microbial taxonomy and acquisition by founding reproductives are major drivers of inheritance

We confirmed that vertical transmission by founding reproductives and ASV taxonomy strongly impact the probability of inheritance with a Random Forest (RF) model. The RF model assessed the predictive power of a set of experimental variables for the observed inheritance and the null model previously described, where the chance of vertical transmission was relative to ASV abundance. The experimental variables included bacterial taxonomy, abundance in maternal colonies, and network features. The predictive power of the RF model using the observed data was 2.0 to 2.5-fold higher than the null model, highlighting the impact of variables aside from abundance, such as microbial taxonomy (Figure 5A). Consistent with founding reproductives shaping the inherited microbiome, transmission to founding reproductives was more predictable than transmission to offspring colonies (Figure 5A). Although factors aside form ASV abundance have additional predictive power for transmission (Figure 5A), abundance of ASVs in maternal colonies was a strong indicator that strains would be vertically transmitted to offspring colony workers (Figure 5B, right), allowing for the establishment of the core colony microbiome.

Phylum and genus were the most powerful predictors of inheritance, based on the average predictive power of taxonomic levels (Figure 5C). This suggests that an interplay between basal traits of phyla and more recently evolved traits of genera affects inheritance. The largely anaerobic non-spore forming phyla Synergistetes (Jumas-Bilak & Marchandin, 2014), Bacteroidetes (Bergey’s Manual of Systematic Bacteriology, 2010), and Proteobacteria (Beskrovnaya et al., 2021) are strongly predicted to be represented in the inherited microbiome. Conversely, spore-formers like Actinobacteria and Firmicutes with more flexible respiration (Bergey's Manual of Systematic Bacteriology, 2012; Bergey’s Manual of Systematic Bacteriology, 2009) are predicted to not be inherited, except for the Candidate Phylum TG3 that includes obligate anaerobes (Bergey’s Manual of Systematic Bacteriology, 2010) (Figure 5D). Differential inheritance of bacterial phyla may be a consequence of their respective environmental resistance. These patterns are reminiscent of findings in the human microbiome, which has spore-forming bacteria as consistently late-colonizers (Egan et al., 2021; Nayfach et al., 2016). For example, we observed Bacteroidetes has a higher propensity for vertical transmission, as has been found in humans (Olm et al., 2022) and in mice (Moeller et al., 2018), and less so for the transmission of Firmicutes, also similar to what has been documented in humans (Olm et al., 2022). The genera with the strongest predictive power included six that were previously identified as differentially enriched in founding reproductives (Figure 3B) and that are core gut microbiome members of fungus-growing termites (Otani et al., 2014), i.e., well beyond the single Macrotermes species we explored here.

Our quantification of extensive inheritance of gut bacteria in termite superorganisms revealed that inheritance critically relies on the adaptations of founding reproductives. The founding reproductives are endowed with a consistent and specific microbial community they vertically transmit from parent to offspring colonies. As predicted, we elucidate adaptations that support transmission of the colony microbiome because natural selection acts on the superorganism as in individual (Boomsma & Gawne, 2018; West et al., 2015). Mechanistically, the microbial endowment of founding reproductives may be driven by diet and physiology, both which influence termite microbiome composition (Brune, 2014; Brune & Dietrich, 2015) and are known to differ between workers and founding reproductives (Eggleton, 2006; Seite et al., 2022). Superorganismal inheritance is then completed when the first workers of the offspring colony inherit the diverse inoculum, which directs the assembly of the complex microbial community as the colony matures. The workers maintain these key inherited microbes, consistent with workers microbiomes of other termite species containing microbes derived from founding reproductives (Chouvenc & Su, 2017; Diouf et al., 2023). The magnitude of inheritance and adaptations to ensure it likely extend to other superorganismal termites, bees, and ants that also host co-evolved mutualistic gut microbiomes (Brune, 2014; Kwong et al., 2017; Russell et al., 2017).

The extent and patterns of inheritance were remarkably consistent across pedigrees. The congruent patterns, despite the relatively small subset of samples from each pedigree, underline that our findings are robust. Further, the large number of ASVs that were exclusive to each of the four maternal pedigrees validates vertical transmission. Pedigree-specificity implies adequate taxonomic resolution to detect inheritance and to test our predictions, and, generally, ASV-level transmission specificity from parents to offspring is a recognized indicator of inheritance (Moeller et al., 2018; Olm et al., 2022). Even so, the sequence length of ASVs provides an estimate of microbial strain diversity (Strube, 2021), and strain-tracking through metagenomics would further elucidate association specificity and deduce symbiont functions (Ellegaard & Engel, 2016, 2019). The inherited microbiota includes many of the bacteria that are co-adapted to fungus-farming termites (Hu et al., 2019; Poulsen et al., 2014) and consistently present in developing and mature colonies (Dietrich et al., 2014; Diouf et al., 2023; Otani et al., 2014). Ultimately, obligate dependence of termites on their microbiome and co-evolutionary history of symbioses dictate that transmission involves adaptive traits.

The termite superorganism manages the microbiome through extensive inheritance, yet only a portion of the extraordinary diversity of parental colony gut microbiomes is vertically transmitted. This implies that symbiotic assembly continues during colony development. Our assessment was performed at the earliest stages in colony life, before environmental acquisition of the Termitomyces fungal cultivar (Aanen et al., 2002), which complements the gut microbiome in symbiotic digestion of plant substrates (Poulsen et al., 2014). With establishment of the fungal cultivar, acquisition of complementary symbionts is expected. Subsequently, the superorganism microbiome should continue to differentiate based on termite division of labor (Otani et al., 2019; Sinotte et al., 2020). The reproductive microbiomes drastically simplify to contain only a few microbial genera as reproductives mature (Otani et al., 2019; Poulsen et al., 2014; Shimada et al., 2013), underlining that the diverse microbiome they carry during colony founding is an adapatation to allow for vertical transmission. However, this can only take place when the superorganism has successfully established complex microbial communities within workers, whom secure colony homeostasis and maintain the microbial inoculum for future founding reproductives.

The observed inheritance across colony generations extends models of transmission from individual organisms to superorganisms. Mixed-mode transmission, the predominate strategy organisms use to assemble adaptive microbiomes (Bruijning et al., 2021; Leftwich et al., 2020) also occurs in the superorganismal termites. The termite colony microbiome contains both vertically and horizontally transmitted microbes. This mixed-mode transmission on proximate timescales aligns with evolutionary patterns of termite-bacteria cladogenesis (Bourguignon et al., 2018). Superorganimsal transmission of specific microbial phyla is also comparable to that of other organsims like humans and mice (Moeller et al., 2018; Olm et al., 2022). Further, organismal vertical transmission via gametes ensures high-fidelity host-microbe interactions in the most intimate mutualisms (Kiers & West, 2015; Russell et al., 2019). Analogous adaptations have occurred through the superorganismal gametes, founding reproductives, to ensure inheritance. While in organsims, transmission via gametes typically selects for to a single strain of microbes (Frank, 1996; Perreau & Moran, 2022), founding reproductives are themselves mutlicellular organsims that can carry a cohort of microbes. This enables reliable transmission of a microbial community key to superorganismal fitness. However, within this diverse vertically transmitted community, conflicts between microbes and with the host are predicted to arise (Frank, 1996; Sharp & Foster, 2022). Compartmentalization can mitigate these conflicts (Chomicki et al., 2020), which occurs at two levels of organizaition in superorgansims. First, the microbiome is partitioned in the regions of the termite gut (Brune & Dietrich, 2015), and second across the division of labor within the colony (Sinotte et al., 2020). The higher-level organization of superorgansims thus favors vertical transmission of a diverse microbial cohort. Overall, the mixed-mode transmission and adaptations for inheritance in termite superorganisms mirror traits employed by organismal hosts to favor mutualistic cooperation.

Termite collection and pedigree establishment

Termites of the genus Macrotermes natalensis were sampled from four maternal colonies and a paternal colony, after which founding reproductives (i.e., alates) were crossed to establish offspring colonies. Details of the experimental design can be found in (Seite et al., 2022). These offspring colonies were sampled at three months old before they naturally acquire their fungal cultivars (Aanen et al., 2002). Termites from maternal colonies and female founding reproductives were collected in 2016 when the crosses were established. However, workers from paternal colonies and male founding reproductives were sampled in 2018. The reduced paternal inheritance observed, in comparison to maternal inheritance, may be due to the time that elapsed between establishing crosses for pedigrees and when colonies were sampled for amplicon sequencing. The gut microbiome composition of fungus-farming termites has been shown to shift slightly across years of colony life (Otani et al., 2016).

Gut dissections, DNA extraction, and quantification of microbial load

Guts of reproductive termites were dissected aseptically and stored in RNAlater® (Sigma-Aldrich, Germany), and sterile castes (workers, larvae, soldiers) were dry frozen. All samples were stored at -80°C. The termites from sterile castes were briefly rinsed in 70% EtOH and sterile dH₂O (1 minute each) to reduce surface contaminants, dissected aseptically, and pooled. Termite gut samples, cellular mock community DNA standards (Zymobiomics, Nordic BioSite ApS, Denmark), and two negative controls were included for DNA extraction and sequencing (see Figure 1 – source data 2 for sample information, including the number of guts pooled per sample). Offspring colony workers were pooled from 2-3 colonies within pedigrees because colony sizes were too small at 3 months to obtain enough individuals per colony. DNA was extracted using a modified DNeasy Blood and Tissue kit protocol (Qiagen, Germany) (Hu et al., 2019). The guts of queens from parental colonies were divided into three extractions due to tissue size and later pooled in silico.

Relative microbial loads per termite gut were determined by quantifying copies of the V2 region of the 16S rRNA marker gene using droplet digital PCR (BioRad, Denmark) (Zhukova et al., 2017). Non-template controls were over 100-fold less that the lowest quantification of termite gut extracts. In the final analysis, any dilution for ddPCR, the total volume of the elute from DNA extraction, and the number of termites per DNA extract were corrected for to determine gene copies per gut (Figure 1 – source data 2).

Amplicon sequencing, quality control, and taxonomic assignment

DNA was sent to BGI-Hong Kong for paired-end HiSeq2500 amplicon sequencing with 341F/806R primers targeting the V3-V4 region of the 16S rRNA gene. All analyses were performed in R (v.3.6.1) (Team, 2017). We utilized the dada2 pipeline (v.1.12.1) (Callahan et al., 2016) with default parameters and the following adjustments to increase the stringency of the analysis: we set the truncLen parameter in filterAndTrim to c(270, 260), maxEE to c(1,1) and truncQ to 1, and increased the minOverlap parameter in mergePairs to 20. We obtained a total of 50,016 Amplicon Sequence Variants (ASVs) in 4,403,068 non-chimeric clean paired-end sequences, of which 19,909 ASVs in 4,262,444 reads remained after removal of taxa with <25 reads across the full dataset. This removal of taxa of low prevalence or relative abundance is commonplace in microbiome studies to avoid contaminants and many microbial dimensions, focusing on the most relevant microbes in the dataset. Rarefaction plots indicated that we captured sufficient community diversity to assess bacterial inheritance (Figure 1 – figure supplement 2).

We assigned ASVs with taxonomic ranks using default parameters in the assignTaxonomy function in dada2. Taxa were classified with the Dict_db v.3.0 database (Mikaelyan et al., 2015), after which ASVs without genus-level taxonomic assignments were reclassified with SILVA v.132 (Quast et al., 2013). The cellular mock community validated the detection of all eight expected taxa.

Negative control samples lacked DNA and failed library preparation according to BGI-Hong Kong’s sequencing service, suggesting the very low levels or absence of contaminants. The package Decontam was run to identify potential contaminant taxa (v.1.10.0) (Davis et al., 2018) despite the lack of negative controls. Mature queen samples from parental colonies contained exceptionally low numbers of 16S rRNA copies, while workers, founding reproductives, kings, and mature colony queens contained 10³ higher concentrations of 16S rRNA copies (Figure 1 – source data 2), creating a broad variation in bacterial loads for package to effectively run. The Decontam package suggested 1.3% potential contaminant reads across samples. We did not remove them from the dataset considering: 1) we removed taxa with <25 reads across the dataset to reduce the influence of contaminants, 2) the worker and founding reproductive samples on which the majority of analysis were based on carry high microbial loads (Figure 1 – source data 2), minimizing any effect of contaminants, and 3) the potential contaminants represented a very low number of total reads and some were identified taxa were common in fungus-farming termite microbiomes (Otani et al., 2014).

Inheritance criteria

Superorganismal termite colonies are the unit upon which natural selection acts (Boomsma & Gawne, 2018). Therefore, we included all castes (i.e., king reproductive, queen reproductive, workers, and larvae) in our assessment of inheritance despite differences in the diversity and microbial loads that individual castes harbor (Otani et al., 2019). We used the most stringent classification possible and hence considered ASVs (100% identical OTUs) independently. To be considered inherited, an ASV had to be found in the parental colony and all founding reproductives of that pedigree (i.e., colony). Subsequently, the ASV had to be present in at least one offspring colony sample in the same pedigree. The number of ASVs, ASV abundance, and the percent of ASVs that were inherited were determined for each sample (Figure 1 – source data 1).

Statistical analyses

To test whether the relative abundance of an ASV predicts how frequently it was inherited, we normalized for the relative abundance of the genus it belonged to. To do that, we subtracted the mean relative abundance of the given genus from the relative abundance of the given ASV. First, to determine if abundance in the transmitter impacted inheritance, we fitted a linear mixed model to predict whether ASVs were inherited or not with the transformed ASV abundances, while controlling for termite pedigree and bacterial genus as random effects. We did this for ASVs that were transmitted from maternal colonies to founding reproductives, and ASVs transmitted from founding reproductives to offspring colonies (Figure 3C). Second, to determine if inherited ASVs were more abundant in the receiver of each step, we repeated this analysis with ASVs received by founding reproductives and by offspring colony workers, respectively (Figure 4A). The models were run using the lme function in nlme. We then created density plots to visualize the distribution of inherited versus environmentally acquired ASVs. The normalized abundance values were log-transformed to clearly see the contrasting distribution of the inherited and non-inherited ASVs. To allow for logarithmic transformation, we increased the normalized abundance values by a factor of 10^5, then maintained the sign in relation to the mean (Webber, 2013), which was performed as follows: y(x) = sign(x)*log(1+abs(x)/10^-5). The result of the data with this transformation is what is presented in Figures 3C and 4A, while the x-axis is labelled in accordance with the non-transformed normalized abundance values.

We utilized permutation tests to measure any statistical difference between patterns of observed inheritance and the null model (described below). This included the number of inherited genera and the assortativity coefficient of inheritance for the positive and negative interactions in the networks. The permutation was manually calculated by determining the proportion of simulations above or below the observed value. For example, if 96 of the 100 iterations of the simulation were above the observed value, the p value was 0.04.

To assess increases and decreases in relative abundance of genera throughout inheritance, we used ALDEx2 (v. 1.24.0) (Gloor et al., 2016), with the generalized linear model function and pedigree as a fixed effect. We performed analyses that identified genus-level enrichment or depletions from both maternal colony workers to founding reproductives, and from founding reproductives to offspring colony workers.

Abundance-driven null model

To assess if the propensity for inheritance could be explained by ASV abundance, we simulated an abundance-driven null model. The null model selected the same number of ASVs as was found to inherited by founding reproductives or offspring colonies within a termite pedigree from those present in the respective maternal colony workers, weighed by their average relative abundance in these workers. We ran 100 iterations of the null model for each stage of inheritance and pedigree.

In comparing the null model to the observed inheritance, we first calculated the number of genera predicted or observed to be transmitted to founding reproductives and offspring colonies within each pedigree (Figure 3A). We used permutation tests to compare the outcome of the null model to observed values for each stage of transmission and for each pedigree.

Network construction

We calculated ASV association networks for the offspring colony worker samples to infer ecological relationships using the SpiecEasi package v.1.1.0 (Kurtz et al., 2015) and the “mb” algorithm, lambda.min.ratio = 1e-3, nlambda = 300. Prior to network calculation, following good network practices, we removed rare ASVs with less than 200 reads across all offspring colony samples. To improve visualizations, we only included nodes with six or more connections in the Figure 4B. We handled graphs using igraph v.1.2.5, network v.1.16.0 and plotted them using ggnet v.0.1.0 and ggnetwork v.0.5.8. To understand ecological interactions amongst and between inherited and non-inherited taxa, we calculated the assortativity coefficient for the variable ‘inheritance’, using the function with the same name within igraph. This metric ranges from -1, if all interactions are disassortative and between an inherited and a non-inherited ASVs, to 1 if all interactions are between inherited ASVs or between non-inherited ASVs. Thus, assortativity indicates a preference for nodes in the network to attach to others that are similar in inheritance. We did so for the full networks and for subsets of networks with only positive or negative associations. For the null model, we calculated assortativity using the same network and assigned nodes to be inherited or non-inherited according to the results of the abundance-driven null model. We then compared positive assortativity values between the observed inheritance network to those of the null model using a permutation test.

We then tested if interactions between ASVs in the network could be explained by whether taxa were inherited or non-inherited, where positive interactions indicate potential cooperation and negative interactions indicate potential competition. To do this, we created a linear model where all interactions, positive or negative, were the response variables and the fixed effects were whether the ASV was inherited or not, the log of ASV abundance, and the interaction between the two. The latter was not significant and was consequently removed from the final model.

Random Forest models to identify major features predicting inheritance

We fitted Random Forest classifiers (RF) implemented in python’s scikit-learn⁶⁶ to uncover which experimental characteristics predict inheritance. We focused on the impact of bacterial taxonomy, abundance in maternal colonies, and network features on the propensity for inheritance of an ASV. This was done in six different models based on dependent variables: observed and null model inheritance to founding reproductives, offspring colonies, and overall. Each ASV was classified eight times (once per stage of inheritance and per pedigree) with each taxonomical level recorded (the null model was assigned a dummy binary variable), which, along with stage of inheritance, network features and abundance (normalized as above) were the independent variables. ASVs containing missing data in any features were removed.

The contribution of each predictor to inheritance was estimated in utilizing Gini importance values and SHAP values (Shapley Additive exPlanations) (Lundberg et al., 2020). We randomly split each dataset into a training set comprising 80% of the ASVs and a test set comprising 20%. We used the training dataset to form a 10-fold cross-validated grid search for the best number of features used in each split, while fixing the number of trees used to 500. Using the best model, we predicted the test’s ASV inheritance status. We used a game theory approach to unlock the predictive potential of each feature (i.e., taxonomy, transmission stage, abundance, and network properties) in each ASV via SHAP values.

We used SHAP values as 1) an indication of overall importance, and 2) to compare the feature importance between the null models and the observed inheritance models. We normalized the SHAP values from each model by retrieving the difference between the SHAP values for binomial predictors (true / false), and the SHAP value difference between predictor values above the median and below the median for continuous predictors. To determine the overall predictive power of the different models (Figure 5A), predictive power ratios between observed data and the null model were calculated as the sum of the absolute values of the SHAP of each feature from the observed data, divided by the sum of the absolute values of the SHAP for each feature from the null data. The process was performed for each model pair (i.e., founding reproductive, offspring colonies, and complete inheritance). Then, to compare the importance of individual features in the null model and the observed inheritance model (Figures 5B-E), we calculated the difference between the normalized SHAP values of the observed inheritance model minus the null model. SHAP values close to 0 hold little predictive power, positive values indicate a prediction that variable leads to inheritance, and negative values indicate a prediction that variable reduces chance of inheritance.

Data availability

Amplicon sequences are available from the SRA archive in GenBank (BioProject PRJNA860052). ASV tables, taxonomy, metadata, and ASVs identified as potential contaminants are available through Figshare (Project: Extensive inheritance of gut microbial communities in a superorganismal termite)

Code availability

Code for analyses is available at https://github.com/vsin632/vertical_transmission.

Acknowledgments

We thank Jacobus J. Boomsma, Robert R. Dunn, Kasun H. Bodawatta, and members of the Social and Symbiotic Evolution Group at the University of Copenhagen for constructive comments. We are grateful to David Sillam-Dussès and Alain Robert for field assistance and Sylvia Mathiasen for laboratory support. This work was funded by a Ph.D. stipend from the Department of Biology, University of Copenhagen to V.M.S., the International Human Frontier Science Program RGP0060/2018 to M.V.-C., and a European Research Council Consolidator Grant (ERC-CoG 771349) to M.P.

Author Contributions: The study was conceptualized and designed by J.R.-H., V.M.S, M.V.-C and M.P. The biological samples were established and supplied by M.V.-C. Laboratory methods were performed by J.R.-H and V.M.S. The data was analyzed by J.R.-H., S.A.-S., and V.M.S., and then curated by J.R.-H. and V.M.S. The original draft was written by V.M.S., J.R.-H., M.P., and figures visualized by V.M.S. and J.R.-H. Further editing of the manuscript was performed by S.A.-S. and M.V.-C. The project was supervised by M.P. and M.V.-C.

Competing Interest Statement: The authors declare no competing interests.

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There is NO Competing Interest.

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Extensive inheritance of gut microbial communities in a superorganismal termite

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