The sources: where do helminths get their bacteria from?

General research on microbial communities has reported great variability in the taxa associated with plants/animals/environment and described interesting phenomena, such as the ‘founder hypothesis’ (i.e. pre-existing microbial lineages that dominate recolonization), ultimately highlighting the dynamic nature of microbial symbiosis in diverse systems (Litvak & Baumler, Reference Litvak and Baumler2019). The same seems to apply to the bacteriome of helminths. In many instances, bacterial taxa composing microbial communities are variable even among helminths parasitizing the same individual host (Jorge et al., Reference Jorge, Dheilly and Poulin2020, Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b; Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022), and, when disrupted with antibiotics, increased abundance of founder bacteria post-disturbance may follow (Jorge et al., Reference Jorge, Froissard, Dheilly and Poulin2022c). In other cases, there is little diversity in the bacteria composing helminth microbiomes (Brealey et al., Reference Brealey, Lecaudey, Kodama, Rasmussen, Sveier, Dheilly, Martin, Limborg and August2022). The impact of these phenomena on the fitness and evolution of helminths is still unclear.

The extensive range of variability in the bacteriome of helminths leads to questioning the microbial sources of individual helminths in a population (Rosenberg & Zilber-Rosenberg, Reference Rosenberg and Zilber-Rosenberg2021): helminths may horizontally acquire bacteria, from their habitat, be it the external environment when they are in the infectious larval stages, or their surroundings within their host, and their diet, whatever they feed on (host tissue, or even other co-infecting parasites) (Jorge et al., Reference Jorge, Dheilly and Poulin2020, Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b). Helminths may also vertically acquire bacteria, which means bacteria are transmitted among parasite generations (Jorge et al., Reference Jorge, Dheilly and Poulin2020, Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b).

In cases of horizontal transmission, different generations do not share bacteria, but there is consistency in the bacteriome, for example, in populations across different geographical localities, implying a potential role of natural selection in determining the bacteria that colonize the parasite (Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022; Jorge et al., Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b). For example, there is geographical stability in the bacteriome of the trematode Philophthalmus attenuatus: parasites in different localities but at the same life stage share more bacteria than parasites of different life stages in the same locality (Jorge et al., Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b). This suggests that specific bacteria are important in each life stage, but that they are not transmitted from one generation to the other, and rather they are acquired horizontally (from the environment or the parasite's surroundings). Supporting this is the association between different bacteriomes and different genetic lineages of the cestode Schistocephalus solidus (Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022). Thus, a parasite's bacteriome is not simply a random assemblage of the pool of bacteria available in the parasite's habitat, as natural selection may restrict which bacteria will successfully colonize the helminth, although it may also depend on which bacteria were settled in before (Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022; Jorge et al., Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b, Reference Jorge, Froissard, Dheilly and Poulin2022c). Interestingly, Eubothrium cestodes parasitizing salmon were shown to associate with different Mycoplasma lineages than those found in the salmon`s microbiome, suggesting a role of divergent selection for specific Mycoplasma lineages in the cestode parasite and its salmon host (Brealey et al., Reference Brealey, Lecaudey, Kodama, Rasmussen, Sveier, Dheilly, Martin, Limborg and August2022). Yet, the Mycoplasma lineages associated with the cestode and the salmon are phylogenetically very close, suggesting shared ancestry of the specific bacterial lineages between the salmon and cestode (Brealey et al., Reference Brealey, Lecaudey, Kodama, Rasmussen, Sveier, Dheilly, Martin, Limborg and August2022). Fundamentally, in addition to consequences to the parasitized organism (and its bacteriome), the parasite also has a role in the evolution of the bacteria composing its own microbiome, which in turn may interact with the evolution of the parasite (and that of its hosts and their microbiome).

In cases of vertical transmission, if a helminth is associated with a core set of bacteria (or a core microbiome, Neu et al., Reference Neu, Allen and Roy2021) persistent in different habitats (e.g. different host species) and across different life stages of the helminth, then the core bacteriome and the helminth are likely responding to changes as an evolutionary unit (Jorge et al., Reference Jorge, Dheilly and Poulin2020). For example, Coitocaecum parvum trematodes have a core bacteriome that persists over different life stages through different animal hosts and environments, and the main source of these bacteria is the previous life stage (Jorge et al., Reference Jorge, Dheilly and Poulin2020). However, vertical transmission is imperfect, that is, only a proportion of parasite offspring inherit certain bacteria from the parent parasite (Greiman et al., Reference Greiman, Tkach and Vaughan2013).

From a microbial evolution perspective, the transmission mode must contribute to each bacterial lineage's persistence over evolutionary time, avoiding dead ends (Ebert, Reference Ebert2013; Dheilly et al., Reference Dheilly, Poulin and Thomas2015). Thus, there is an important correlation between the mode of bacterial transmission and the ecology of the helminth, including factors such as the helminth population density, fecundity, different life stages and habitats. Horizontal transmission is an effective transmission strategy for the bacteriome of helminths with a large population density, or that have large numbers parasitizing a single individual host, or large numbers in the same environment. In contrast, vertical transmission is a suitable strategy for bacteria persisting over patchy geographical distribution and across different life stages of the parasite. Thus, vertical bacterial transmission is tightly linked to the helminth's reproductive success (Ebert, Reference Ebert2013). Vertical transmission enables bacteria to persist over discrete generations of the parasite and overcome constraints such as helminths with small numbers of offspring and low success in transitioning to the next life stage in a different host species. Clearly, a strategy combining horizontal and vertical transmission enables the exploitation of a larger breadth of possibilities for bacterial persistence (Ebert, Reference Ebert2013) and could contribute to the large variability in the bacteriome composition of helminths. Lastly, the helminth habitat may also play a role in determining bacterial transmission strategies, given that higher vertical transmission rates are more common in terrestrial than aquatic symbiotic microbes (Russell, Reference Russell2019).

The genetic diversity of bacterial lineages and inter-specific association in allele frequencies among the helminth and bacterial alleles may help define the source of specific bacteria in helminths. In horizontal bacterial transmission, high lineage diversity in the bacteriota of a single individual is expected, as different bacterial lineages may colonize an individual helminth over several founding events (Ebert, Reference Ebert2013). Contrastingly, in cases of vertical transmission, specific bacterial genotypes become associated with the genotype of the individuals they inhabit, leading to inter-specific linkage disequilibrium (Ebert, Reference Ebert2013; Hayward et al., Reference Hayward, Poulin and Nakagawa2021; Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022). Ultimately, microbes with strict vertical transmission across many helminth generations may present congruent phylogenies with the parasite (Hayward et al., Reference Hayward, Poulin and Nakagawa2021). However, other factors unrelated to the mode of transmission can lead to interspecific linkage disequilibrium (e.g. selection and spatial structure), and for microbes with mixed transmission modes (vertical and horizontal transmission), inter-specific allelic correlation is expected to be weaker (Brandvain et al., Reference Brandvain, Goodnight and Wade2011; Fitzpatrick, Reference Fitzpatrick2014).

The nature of ecological interactions among helminths and bacteria

The variability and dynamic composition of the bacteriome of helminths reflect the complexity of the symbiotic interactions among helminths and bacteria, and broad generalizations are hardly possible. However, to better understand the ecological impacts of such interactions, it can be helpful to identify shared patterns among case studies. Following Moran et al.'s (Reference Moran, McCutcheon and Nakabachi2008) symbioses’ classifications among microbes and insects, below we propose a system to identify characteristics of obligatory and facultative interactions among bacteria and helminths.

Obligatory mutualism: bacteria that present obligatory mutualism with helminths (also called primary symbionts) are essential to the development of the helminth, which in turn is essential to the microbe's transmission. Obligatory mutualistic bacteria are genus-specific or species-specific, meaning they are only successful in one helminth genus or species and are strictly vertically transmitted. For example, bacteria from the group Candidatus Symbiopectobacterium are strictly maternally transmitted among generations of the nematode Howardula aoronymphium, which has low success in parasitizing its Drosophila host when the association with the bacterium is absent (Martinson et al., Reference Martinson, Gawryluk, Gowen, Curtis, Jaenike and Perlman2020). Some Candidatus Symbiopectobacterium lineages show genomic degradation, a footprint of obligatory symbiotic association due to accumulating deleterious mutations, and are phylogenetically closely related to obligate symbionts of other invertebrates (Martinson et al., Reference Martinson, Gawryluk, Gowen, Curtis, Jaenike and Perlman2020). Few other examples of bacteria–helminth obligatory mutualism are known at present, and their ‘obligatory’ nature has been questioned, such as the case of Xenorhabdus and Photorhabdus gram-negative bacteria associating with Steinernematidae and Heterorhabditidae nematodes (Poinar & Thomas, Reference Poinar and Thomas1966). These bacteria kill the nematode's insect host so that the nematode can feed on the dead insect as it reproduces and grows. The bacteria then infect the nematode juveniles, which are subsequently released to the soil in search of the next insect host (Forst & Clarke, Reference Forst, Clarke and Gaugler2002). However, even though Xenorhabdus and Photorhabdus bacteria are species-specific and vertically transmitted among the nematodes, the bacteria can be cultured in laboratory conditions free of the nematodes, which has led authors to classify the symbiotic relationship as non-obligatory mutualism (Forst & Clarke, Reference Forst, Clarke and Gaugler2002).

Facultative symbiosis: bacteria that facultatively associate with helminths (also called secondary symbionts) are not essential to the reproduction or development of the helminth and may associate with various helminth species. Thus, there is an important role for horizontal bacterial transmission. These bacteria modulate the phenotype/behaviour of the helminth in order to increase the prevalence and spread of helminth lines containing the symbiotic bacterial lineages. For example, the bacteriome of reproductive morphs of the trematode Philophthalmus attenuatus has been shown to differ from the bacteriome of morphs that do not reproduce (soldiers). When both morphs were treated with antibiotics within the snail host, the development of reproductives was favoured over the development of soldiers, supporting a role of the bacteriome in the formation of different morphs and indicating a potential bacteriome manipulation of the trematode reproductive strategy (increase in lines bearing the reproductive bacteriome) (Jorge et al., Reference Jorge, Dheilly, Froissard and Poulin2022a).

There are two subcategories of facultative symbiosis:

• Facultative mutualism: the phenotypic modulation induced by the bacteria causes a direct benefit to the helminth, in terms of longer life spans or protection from stress, ultimately leading to higher reproductive success. Facultative mutualism may include cases in which the bacteria help protect the helminth against their host's immune response or against other microorganisms that could compete or attack the helminth, as well as benefits in terms of dietary supplementation. For example, electron microscopy has revealed a homogeneous composition of bacteria located within cavities on the surface of two different species of tapeworm, likely providing an increase in food absorption by the worms (Caira & Jensen, Reference Caira and Jensen2021). Moreover, the only known function of these cavities is housing bacteria, suggesting that these structures evolved specifically because the tapeworm benefits from such relationships (Caira & Jensen, Reference Caira and Jensen2021).

• Reproductive manipulation: the phenotypic modulation induced by the bacteria interferes with the helminth's reproduction, favouring helminth lines harbouring the bacteria. In such cases, vertical transmission is possible and would lead to increasing fecundity or reproductive success of helminth lines bearing the bacteria, as opposed to lines free from the bacteria. For example, Neorickettsia bacteria infecting the digenean trematode Plagiorchis elegans have mixed transmission (vertical and horizontal transmission) and are pathogenic to horses (Greiman et al., Reference Greiman, Tkach and Vaughan2013). Even though the trematode is the vector of Neorickettsia to the horse, the trematode cannot reproduce in the horse, thus ruling out a mutualistic relationship (Pusterla et al., Reference Pusterla, Johnson, Chae and Madigan2003; Greiman et al., Reference Greiman, Tkach and Vaughan2013). Neorickettsia rate of transmission during the asexual multiplication phase of P. elegans varies from 11–90%, confirming its imperfect vertical transmission (Greiman et al., Reference Greiman, Tkach and Vaughan2013). However, the effect of Neorickettsia on the trematode's reproductive success in its intermediate hosts (a snail and an arthropod) remains unknown.

From a bacterial evolutionary perspective, selection favouring bacteria with higher fitness does not necessarily incur benefits to the helminth with which they associate (Dheilly et al., Reference Dheilly, Poulin and Thomas2015; Speer et al., Reference Speer, Dheilly and Perkins2020). Indeed, there are cases in which an increase in bacterial fitness may decrease the parasite's fitness, in an antagonistic dynamic. An example is Salmonella bacteria that are shielded from antibiotics when attached to schistosome parasites, however the number of trematodes in a parasitized animal is smaller in co-occurrence with Salmonella than when the bacteria are not associated with the schistosomes (Barnhill et al., Reference Barnhill, Novozhilova, Day and Carlson2011; Zhu et al., Reference Zhu, Chen, Wu, Tang and Wang2017).

Characterizing interactions among bacteria and helminths can help understand the ecological impact of the absence of certain interactions or their removal by, for example, antibiotics treatment. If obligatory mutualism is impeded, then both the bacteria and the helminth parasite in question are expected to perish or achieve greatly reduced fitness; in contrast, if an antagonist relationship is impeded, the chances of survival and success of the helminth may increase. Nevertheless, in most bacteria–helminth associations, the nature of the symbiotic relationship is fluid and can be strongly context-dependent. Microbe–microbe interactions are important in microbial communities (Proal et al., Reference Proal, Lindseth and Marshall2017). There is nothing to suggest that, under different circumstances, certain bacterial lineages cannot act as beneficial agents and pathogens to the same helminth species, just as it happens in the human gut microbiome (Schubert et al., Reference Schubert, Sinani and Schloss2015; Sharpton & Gaulkeb, Reference Sharpton and Gaulkeb2015). In addition, mutualism and parasitism are but the ends of an evolutionary continuum (Drew et al., Reference Drew, Stevens and King2021), and defining interactions anywhere along a continuum can be highly subjective (Leung & Poulin, Reference Leung and Poulin2008). Even so, identifying shared patterns among different contexts can be helpful to improve our understanding of the significance of some of these interactions for the evolution of both microbes and helminths, and this is what the aforementioned classification system can be used for.

The diversity of the bacteriome in helminths

Large variability in microbiomes is universally recognized. In humans, increasing sampling efforts inevitably correlate with a decrease in the percentage of common taxa among all people, and currently, fewer than 20 genera are shared by more than 95% of the sampled human populations (Sanna et al., Reference Sanna, Kurilshikov, van der Graaf, Fu and Zhernakova2022). There is an influence of external factors on the composition of the microbiome (e.g. environment and diet), but surprisingly, the heritability of some components of the human microbiome is around 20%, suggesting a role of the genetic makeup of the individual in the composition of its microbiome (Sanna et al., Reference Sanna, Kurilshikov, van der Graaf, Fu and Zhernakova2022). In helminths, both the genotype and the bacteriome of the cestode Schistocephalus solidus correlate with changes in the bacteriome and phenotype of its fish host (Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022). Further research associating the genotype of helminths and their hosts with the diversity of their bacteriome is needed to shed light on the factors underlying bacteriome variability.

There is an important distinction between the core bacteriome and the transient bacteriome in helminths. The core bacteriome refers to specific bacterial lineages present throughout the helminth's life cycle, in which bacterial acquisition via vertical transmission is key (Formenti et al., Reference Formenti, Cortes, Brindley, Cantacessi and Rinaldi2020; Jorge et al., Reference Jorge, Dheilly and Poulin2020; Neu et al., Reference Neu, Allen and Roy2021). Stable bacterial lineages across different geographical localities may also represent a core bacteriome, but in this case, horizontal transmission may tightly interact with natural selection towards keeping specific bacterial lineages associated with specific life stages of the helminth (Jorge et al., Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b; Sheehy et al., Reference Sheehy, Cutler, Weedall and Rae2022). For example, different lineages of Phasmarhabditis nematodes have a core set of bacteria even when originating from different localities and being cultured under different conditions for varying lengths of time (Sheehy et al., Reference Sheehy, Cutler, Weedall and Rae2022). The composition of the core bacteriome is, thus, expected to be relatively stable, probably indicating that either such bacterial lineages play a role in the helminth's ecology and evolution, or they depend on the helminth for their own transmission and survival, or both (Formenti et al., Reference Formenti, Cortes, Brindley, Cantacessi and Rinaldi2020; Jorge et al., Reference Jorge, Dheilly and Poulin2020, Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b; Sheehy et al., Reference Sheehy, Cutler, Weedall and Rae2022).

In comparison, transient bacteriome refers to bacterial lineages that are only present in specific life stages of the helminth, or in specific geographical localities, and can be greatly variable among individual helminths (Formenti et al., Reference Formenti, Cortes, Brindley, Cantacessi and Rinaldi2020; Jorge et al., Reference Jorge, Dheilly and Poulin2020, Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b; Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022). However, the transient bacteriome can still impact the helminth's biology. For example, transient bacterial lineages could correlate with differences in the pathology of virulence of helminths, or even with variability in parasite-induced manipulations of host phenotype and behaviour (Dheilly et al., Reference Dheilly, Poulin and Thomas2015; Poulin et al., Reference Poulin, Jorge and Salloum2022). Transient bacterial lineages depend on horizontal transmission (Formenti et al., Reference Formenti, Cortes, Brindley, Cantacessi and Rinaldi2020). However, as mentioned above, horizontally acquired bacteria do not necessarily represent a random assemblage of the bacterial pool in the helminth's environment, and host-based selective forces are relevant to determining the composition and diversity of the parasite bacteriome (Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022).

Many examples in the literature describe single bacterial taxa interacting with helminths and their hosts (table 1). However, conceptual complexities arise when considering the net effect of many microbial genotypes (i.e. the microbiome), involving interactions among themselves, with the helminths and with the parasitized host (and its microbiome) (Dheilly, Reference Dheilly2014; Theis et al., Reference Theis, Dheilly and Klassen2016). The great taxonomic variability in the bacteriome has led to functional investigations of individual bacterial lineages, with findings converging to the realization that the functions of many lineages are redundant (Speer et al., Reference Speer, Dheilly and Perkins2020). In fact, metabolomics research has shown that microbiomes composed of different taxa may produce similar metabolites (Litvak & Baumler, Reference Litvak and Baumler2019). Furthermore, the many microbial lineages may have a differential contribution to the microbiome (Reynolds et al., Reference Reynolds, Finlay and Maizels2015): a few isolated lineages could have a strong effect, and many individual lineages could have a small effect that results in a stronger combined impact on the ecology and evolution of helminths. Such considerations create a clear distinction in how microbial diversity is defined and studied: taxonomic diversity is concerned with the diversity of lineages composing the microbiome, while functional diversity characterizes the pool of functional traits in a microbiome, regardless of taxonomic diversity (Escalas et al., Reference Escalas, Hale, Voordeckers, Yang, Firestone, Alvarez-Cohen and Zhou2019).

Table 1.

Selected examples of association between bacteria and helminth parasites.

When the nature of the symbiotic relationship was not found in the literature, a suggestion was made based on the current descriptions in the literature, and marked with * to denote that evidence is lacking and that more studies are required.

If knowledge about the microbiome's taxonomy in helminths is still in its infancy, the study of the microbiome's functional diversity in parasitology is even more so. However, the potential of this type of study can already be seen. For example, upon finding differences in lineages of Mycoplasma composing the microbiome of the cestode Eubothrium and its salmon host, Brealey et al. (Reference Brealey, Lecaudey, Kodama, Rasmussen, Sveier, Dheilly, Martin, Limborg and August2022) generated metagenome-assembled-genomes (MAGs) and performed functional annotation by comparison with previously available Mycoplasma genomes. Functional genomic regions coding for different metabolic pathways were present in cestode-associated Mycoplasma vs. salmon-associated Mycoplasma, suggesting adaptations of Mycoplasma to the different environments (i.e. adaptation to the cestode or to live within the fish gut). Nevertheless, the study was limited by the lack of available Mycoplasma genome assemblies in non-mammalian hosts, highlighting the need for further studies to fill this fundamental gap.

Eco-evolutionary impacts: helminths and bacteria associate, but what of it?

The evolution of parasites and their hosts has been much described within the ‘evolutionary arms race’ framework: individuals resistant to a parasitic infection will have better survival compared to susceptible individuals, but as natural selection benefits resistant individuals on the one hand, on the other hand it will also favour parasites with a capacity to bypass the resistance of their hosts (Buckling & Rainey, Reference Buckling and Rainey2002). However, to incorporate the multi-dimensional nature of microbiome–parasite–host interactions, the ‘evolutionary arms race’ framework needs to be expanded (Rafaluk-Mohr et al., Reference Rafaluk-Mohr, Gerth, Sealey, Ekroth, Aboobaker, Kloock and King2022). In short, microbial symbionts have been described as a low-cost source of evolutionary innovation for the organism they associate with, an extra pool of genes providing diversity and a basis over which natural selection may lead to adaptation (Dheilly et al., Reference Dheilly, Poulin and Thomas2015; Martinson et al., Reference Martinson, Gawryluk, Gowen, Curtis, Jaenike and Perlman2020; Poulin et al., Reference Poulin, Jorge and Salloum2022). Symbiotic microbes may provide novel functions to the organisms they associate with, enabling the conquest of different niches and environments (in parasite evolution, this could translate into an increase in the diversity of hosts that can be exploited), but microbes may also manipulate the organisms they associate with (e.g. reproductive manipulation) and become essential to a helminth via evolved dependency (De Mazancourt et al., Reference De Mazancourt, Loreau and Dieckmann2005; Martinson et al., Reference Martinson, Gawryluk, Gowen, Curtis, Jaenike and Perlman2020).

To better understand host–parasite evolution, two main families of models have been employed, with differences in their underlying assumptions: the matching alleles model, which assumes that a lock-key specificity in alleles of parasite and host is required for infection; and the gene for gene model, which assumes that infection occurs when parasites have more virulence alleles than hosts have resistance alleles (Hamilton et al., Reference Hamilton, Axelrod and Tanese1990; Sasaki, Reference Sasaki2000). Natural systems do not always comply with these assumptions, and as mentioned above, more complex models are required when considering microbiomes. In particular, Kwiatkowski et al. (Reference Kwiatkowski, Engelstadter and Vorburger2012) developed a model incorporating one microbial symbiotic species that may be antagonistic or mutualistic with the parasitized host (not a component of the parasite microbiome). The model revealed that the specificity of the alleles was essential in determining the evolution of the host–symbiont–parasite system, especially for antagonistic species. While such studies are very informative, the models are highly deterministic and consider microbial transmission mostly via perfect maternal inheritance, with limited rates of horizontal transfer and genetic drift (Kwiatkowski et al., Reference Kwiatkowski, Engelstadter and Vorburger2012). Given the highly variable bacteriome of helminths, models with perfect maternal inheritance are restricted to obligate mutualistic relationships, which may have an obvious evolutionary impact, but largely exclude the dynamic nature of the bacteriome and the role it may play in host–parasite co-evolution.

The evolution of each bacterial lineage in the helminth's microbiome depends on its interactions with all other co-occurring lineages, in addition to factors such as the life-history traits of the helminth and the individual bacterium transmission strategies. The combination of all these elements in the parasite will interact with the same level of complexity in the parasitized host, creating eco-evolutionary interdependency. Ultimately, these multi-level interactions represent a paradigm shift in parasitology: the evolutionary arms race of parasites and their hosts needs to incorporate the holobiome dimension, that is, the unit formed by microbiomes and the organisms that they inhabit (Dheilly, Reference Dheilly2014; Theis et al., Reference Theis, Dheilly and Klassen2016).

Where to next?

Currently, partial 16S rRNA metagenomics is the most used approach to characterize the bacteriome of helminths; it has contributed to revealing that the composition of the bacteriome associated with helminths is different from that associated with the organisms that they parasitize (White et al., Reference White, Houlden, Bancroft, Hayes, Goldrick, Grencis and Roberts2018; Hogan et al., Reference Hogan, Walker and Turnbull2019; Jorge et al., Reference Jorge, Dheilly and Poulin2020, Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b, Reference Jorge, Froissard, Dheilly and Poulin2022c; Gobert et al., Reference Gobert, McManus, McMullan, Creevey, Carson, Jones, Nawaratna, Weerakoon and You2022; Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022), identifying vertical and horizontal transmission of bacterial lineages among helminths (Vandekerckhove et al., Reference Vandekerckhove, Willems, Gillis and Coomans2000; Vaughan et al., Reference Vaughan, Tkach and Greiman2012; Greiman et al., Reference Greiman, Tkach and Vaughan2013; Jorge et al., Reference Jorge, Dheilly and Poulin2020, Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b; Hahn et al., Reference Hahn, Piecyk, Jorge, Cerrato, Kalbe and Dheilly2022), and discovering pathogenic bacteria that use helminths as vectors (Pusterla et al., Reference Pusterla, Johnson, Chae and Madigan2003; Greiman et al., Reference Greiman, Tkach and Vaughan2013; Dheilly et al., Reference Dheilly, Ewald, Brindley, Fichorova and Thomas2019a). Partial 16S sequencing has been useful in finding bacteria that are strictly vertically transmitted and in mutualistic associations with helminths (Greiman et al., Reference Greiman, Tkach and Vaughan2013; Martinson et al., Reference Martinson, Gawryluk, Gowen, Curtis, Jaenike and Perlman2020), defining a core bacteriome in a few helminths (Sinnathamby et al., Reference Sinnathamby, Henderson, Umair, Janssen, Bland and Simpson2018; Jorge et al., Reference Jorge, Dheilly and Poulin2020, Reference Jorge, Dheilly, Froissard, Wainwright and Poulin2022b), and revealing great diversity in the composition and abundance of specific bacterial taxa (Palomares-Rius et al., Reference Palomares-Rius, Archidona-Yuste, Cantalapiedra-Navarrete, Prieto and Castillo2016; Mafuna et al., Reference Mafuna, Soma, Tsotetsi-Khambule, Hefer, Muchadeyi, Thekisoe and Pierneef2021). However, this approach ignores the other components of the microbiome (e.g. viruses, protozoa and fungi). Even for the bacteriome, there are recognized constraints to partial 16S rRNA sequencing that mainly derive from the short size of the DNA fragment.

The development of long-range sequencing technologies such as Nanopore and PacBio has promoted and simplified full-length 16S rRNA gene sequencing (Callahan et al., Reference Callahan, Wong, Heiner, Oh, Theriot, Gulati, McGill and Dougherty2019; Johnson et al., Reference Johnson, Spakowicz and Hong2019). These longer DNA fragments provide improved resolution for bacterial taxonomic classification down to lineage levels, opening possibilities such as identifying lineages on the lower side of the divergence and abundance scale and undertaking phylogenetic assessments of more closely related lineages (Frank et al., Reference Frank, Pan, Tooming-Klunderud, Eijsink, McHardy, Nederbragt and Pope2016; Johnson et al., Reference Johnson, Spakowicz and Hong2019; Brealey et al., Reference Brealey, Lecaudey, Kodama, Rasmussen, Sveier, Dheilly, Martin, Limborg and August2022; Luo et al., Reference Luo, Kang and Schonhuth2022). However, microbiomes are composed of a number of non-bacterial organisms that are excluded by 16S amplicon-based technologies.

Amplicon-based sequencing of the internal transcribed spacer or the 18S rRNA genes can be useful to characterize the eukaryotic members of the microbiome, but is also limited in terms of taxonomic resolution, is targeted to specific components of the microbiome, and has significant challenges given the evolutionary proximity of the eukaryotic components of the microbiome with the organisms harbouring the microbiome (Hu et al., Reference Hu, Liu and Lie2015; Popovic et al., Reference Popovic, Bourdon, Wang, Guttman, Voskuijl, Grigg, Bandsma and Parkinson2018; Campo et al., Reference Campo, Bass, Keeling and Bennett2019). In the case of RNA and DNA viruses, non-amplicon-based metatranscriptomics and metagenomics are necessary, in particular for the genomic discovery and characterization of highly variable viruses in the microbiome (Dheilly et al., Reference Dheilly, Lucas, Blanchard and Rosario2022; Lee et al., Reference Lee, Zoqratt, Phipps, Barr, Lal, Ayub and Rahman2022).

Moving away from targeted sequencing, long-range sequencing methods have been facilitating the generation of lineage-resolved MAGs in complex microbial communities, with potential functional annotation of such metagenomes (Zimmermann et al., Reference Zimmermann, Obeng and Yang2020; Bickhart et al., Reference Bickhart, Kolmogorov and Tseng2022; Jin et al., Reference Jin, You, Zhao, Li, Ma, Kwok, Xu and Sun2022). Methods such as high-fidelity sequencing can result in continuous reads that are 10,000 base-pairs long, potentially spanning the full length of shorter microbial genomes (Bickhart et al., Reference Bickhart, Kolmogorov and Tseng2022; Feng et al., Reference Feng, Cheng, Portik and Li2022), and accelerating approaches such as shotgun metagenome profiling and the generation of MAGs. However, metagenome profiling and functional characterization of helminth microbiomes are currently capped by the lack of information in databases that are directly applicable to microbial lineages in helminths (Brealey et al., Reference Brealey, Lecaudey, Kodama, Rasmussen, Sveier, Dheilly, Martin, Limborg and August2022), stressing the importance of increasing the number of studies on this specific subject. Given the potential redundant functions of different bacterial lineages (Speer et al., Reference Speer, Dheilly and Perkins2020), increasing microbiome functional characterizations will lead to a better understanding of the fundamental contribution of the whole microbiome to the interaction with the parasite and with the host (fig. 2).

Fig. 2.

Circular plot representing the diversity of functional domains and microbial taxa associated with different individual helminths of the same species in a population. From the centre out: first layer represents data associated with the helminths (e.g. phenotypic data, here represented by arbitrary letters – A and B); second layer represents the relative abundance of major functional domains (or gene ontology terms, such as in Ugarte et al., Reference Ugarte, Vicedomini, Bernardes and Carbone2018), with different domains represented by bars with different colours (from F1 to F7), and arbitrary functional domains named in the bottom left legend. Not all domains are necessarily present/detected in all individual helminths, and their relative effect in each individual can be different (different height of bars); third layer represents the relative abundance of microbial taxa (different coloured sections within a bar, arbitrarily represented in the bottom right legend) associated with each functional domain in each individual (from F1 to F7). There is diversity in the taxa associated with each functional domain among helminth individuals, due to functional redundancy.

In parallel to functional profiling based on MAGs, metabolomics approaches can provide a snapshot of the small molecules in a system, helping characterize function and responses to experimental manipulations of the microbiome (Whitman et al., Reference Whitman, Sakanari and Mitreva2021; Bauermeister et al., Reference Bauermeister, Mannochio-Russo, Costa-Lotufo, Jarmusch and Dorrestein2022). Metabolomics combined with the sequencing-based characterization of the components of the microbiome can provide powerful insights into the ecological function of microbes in association with helminths and their host.

In addition to microbiome functional descriptions, differential abundances of individual taxa within microbial communities are relevant to the net effect of the microbiome in the parasite–host interaction (Reynolds et al., Reference Reynolds, Finlay and Maizels2015; Gaulke et al., Reference Gaulke, Martins, Watral, Humphreys, Spagnoli, Kent and Sharpton2019; Poulin et al., Reference Poulin, Jorge and Salloum2022). Increasing the number of quantitative microbiome characterizations with techniques such as quantitative polymerase chain reaction, flow cytometry and microbiome profiling poses its own challenges (Galazzo et al., Reference Galazzo, van Best and Benedikter2020), but is essential to advancing our understanding of the differential prevalence and contribution of microbial lineages to the eco-evolutionary dynamics of parasite–host interactions. The use of fluorescence in situ hybridization, immunofluorescence and electron microscopy to visualize and localize larger microbial symbionts associated with helminths is also very informative, leading to a better understanding of the nature of the microbe–parasite association and mode of transmission (Plotnikov & Korneva, Reference Plotnikov and Korneva2008; Tropini et al., Reference Tropini, Earle, Huang and Sonnenburg2017; Jenkins et al., Reference Jenkins, Brindley, Gasser and Cantacessi2019; Caira & Jensen, Reference Caira and Jensen2021).

As sequencing costs decrease and bioinformatic resources are further developed, a considerable methodological challenge to advancing research in the microbiome of helminths lies in the input DNA requirements, in terms of quality and quantity of DNA per sample (Petrone et al., Reference Petrone, Munoz-Beristain, Glusberger, Russell and Triplett2022). Most new approaches do not rely on polymerase chain reactions, which eliminates the issue of amplification bias (McLaren et al., Reference McLaren, Willis and Callahan2019; Petrone et al., Reference Petrone, Munoz-Beristain, Glusberger, Russell and Triplett2022), and facilitates the inclusion of non-bacterial components of the microbiome. However, due to the nature of the samples and the fact that not all microbial lineages can be cultured, obtaining large volumes of biological material may not be viable. Thus, for research on the microbiomes of helminths to benefit from deeper sequencing methods and MAGs, it will be necessary to optimize and benchmark laboratory protocols to improve the DNA/RNA quality and quantity retained. Developing and following best-practice guidelines, such as the recommendations of the Parasite Microbiome Project (Dheilly et al., Reference Dheilly, Bolnick and Bordenstein2017, Reference Dheilly, Martinez Martinez and Rosario2019b; Formenti et al., Reference Formenti, Cortes, Brindley, Cantacessi and Rinaldi2020), will be essential to both be able to embark on these extraordinary research avenues and to form an active community to share experiences and move the field forward.