# Will anomalies in the field of EPN associated-bacteria lead to a new paradigm?

Jean-Claude Ogier, Sophie Gaudriault

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Jean-Claude Ogier, Sophie Gaudriault. Will anomalies in the field of EPN associated-bacteria lead to a new paradigm?. Journal of Invertebrate Pathology, 2025, 212, pp.108387. 10.1016/j.jip.2025.108387. hal-05162736

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Review article

# Will anomalies in the field of EPN associated-bacteria lead to a new paradigm?
*

Jean-Claude Ogier, Sophie Gaudriault*

DGIMI, Univ Montpellier, INRAE, Montpellier, France

## ARTICLE INFO

**Keywords:**

Anomalies

Paradigm

Entomopathogenic nematodes

Bacterial microbiota

## ABSTRACT

According to the prevailing monoxenic paradigm, entomopathogenic nematodes (EPNs) form specific association with endosymbiotic bacteria. However, many anomalies have been observed over the last 60 years, with bacterial taxa other than the known endosymbiont isolated in culture-based approaches or detected by next-generation sequencing. We have reconsidered the role of this EPN-associated microbiota — known as the second bacterial circle — in the life-cycle of EPNs. In this review, we present arguments supporting a role for certain taxa in the death of the insect and propose hypotheses concerning other properties of the bacteria involved in their interactions with EPNs. Should these functional hypotheses be confirmed, then the bacterial “anomalies” would no longer be regarded as such. Instead, they would form the basis for the establishment of a new paradigm, the polyenic paradigm.

## 1. Anomalies in biological sciences

In *The Structure of Scientific Revolutions*, Thomas Kuhn argued that anomalies are a violation of the expectations induced by the paradigm that governs “normal” science (Kuhn, 2018). According to Kuhn, “normal” science is research solidly based on one or more past scientific achievements in a field. The corresponding scientific community adheres overwhelmingly to a previously established “paradigm”. The paradigm is a coherent “working basis” that serves as a starting point for subsequent work. Empirical analyses sometimes identify anomalies. These anomalies are usually unsolved or partially solved puzzles that call into question the conditions of application of the prevailing paradigm. More often than not, most members of the scientific community regard them as errors, the result of mishandling, and they frequently go unmentioned. However, according to Kuhn, anomalies have also formed the basis of many of the discoveries in any field of study. Indeed, tensions between the accepted paradigm and anomalies can lead to a questioning of the existing science, sometimes laying the foundations for a new paradigm.

Thomas Kuhn is a physicist who has applied his theory principally to major paradigm shifts, such as the Copernican revolution. We do not pretend to envisage paradigm shifts of this scale here. Nevertheless, we thought it would be interesting to try to apply this theory to questions

arising in biological sciences today and relating to the role of microbial communities in the functioning of host-microorganism interactions. Our aim here is to apply the rather exciting epistemological thinking of Thomas Kuhn to EPN-associated bacteria. The current view (the established paradigm) in this field of research is that of monoxenic associations between endosymbiotic bacterial genera, *Xenorhabdus* and *Photorhabdus*, and EPN, *Steinernema* and *Heterorhabditis* respectively. This review provides an overview of current knowledge about other bacteria associated with *Steinernema* and *Heterorhabditis* genera. This review also proposes hypotheses about the role of these other bacteria and various research approaches that should be explored before considering a paradigm shift for bacteria associated with EPNs.

## 2. The monoxenic paradigm for entomopathogenic nematodes

The canonical EPNs, *Steinernema* and *Heterorhabditis*, are soil-dwelling microscopic nematodes that kill a wide range of insects and are, consequently, used in the biological control of insect pests (Dillman et al., 2012; Lacey et al., 2015; Ogier et al., 2023; Ruiu et al., 2024). A specific association between the bacterial genus *Xenorhabdus* and the EPN *Steinernema* has been described in the 1960 s (Poinar and Thomas, 1967), and this was followed by the description, in the 1970 s, of a specific association between the bacterial genus *Photorhabdus* and the

<footnote>* This article is part of a special issue entitled: 'EPN 100 years' published in Journal of Invertebrate Pathology.</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: sophie.gaudriault@umontpellier.fr (S. Gaudriault).</footnote>
EPN *Heterorhabditis* (Poinar, 1975; Boemare et al., 1993). *Steinernema* species form specific one-to-one relationships with *Xenorhabdus* spp. whereas *Heterorhabditis* species associate with several *Photorhabdus* species (Púza and Machado, 2024). Both *Xenorhabdus* and *Photorhabdus* are located in the anterior part of the gut in the free-living form of the EPN, the infective juveniles (IJs), and there is a specialized receptacle housing the bacteria in the case of *Xenorhabdus* (Bird and Akhurst, 1983; Ciche and Ensign, 2003; Snyder et al., 2007). *Xenorhabdus* and *Photorhabdus* are injected into the insect by the IJ harboring them (Ciche and Ensign, 2003; Sicard et al., 2004; Snyder et al., 2007). All these features support the concept of a mutualistic symbiosis between the bacterial and nematode partners, which act together to kill the insect host (Heppert et al., 2022). Moreover, under laboratory conditions, the re-association between previously axenized EPNs with their specific endosymbiont is often necessary and sufficient for EPN pathogenesis and normal completion of their entire life cycle (Han and Ehlers, 2000; Sicard et al., 2004). These findings underlie the monoxenic paradigm, — the idea that the EPN-specific microbiota consists of a single bacterial species — a *Xenorhabdus* species for *Steinernema* and a *Photorhabdus* species for a *Heterorhabditis*. Over the last 20 years, the notion of a key role for endosymbiotic bacteria in the main steps of the EPN life cycle has come to predominate (Heryanto and Eleftherianos, 2020; Heppert et al., 2022; Ogier et al., 2023).

### 3. Anomalies in the monoxenic paradigm

Shortly after Poinar and Thomas established their “working basis” for the monoxenic paradigm (Poinar and Thomas, 1967), Lysenko and coworkers failed to isolate the symbiotic bacterium from *S. carpocapsae* IJs (Lysenko and Weiser, 1974). Instead, they identified various Gram-negative bacteria associated with the nematodes. By the 1980 s, N.E. Boemare and coworkers had definitively confirmed that *X. nematophila* was the primary endosymbiont of *S. carpocapsae*. However, they also

consistently observed the presence of other bacterial species, including members of the Enterobacteriaceae and Pseudomonadaceae families, which persisted even after rigorous surface decontamination procedures (Boemare, 1983; Boemare et al., 1983). These studies therefore rapidly challenged the monoxenic paradigm. For the genus *Heterorhabditis*, subsequent studies reported similar findings, identifying dixenic associations involving the primary symbiont *Photorhabdus* alongside other bacterial species, such as *Brucella* (formerly Ochrobactrum) (Babic et al., 2000), *Providencia rettgeri* (Jackson et al., 1995), and *Paenibacillus* (Enright and Griffin, 2004), even after rigorous decontamination protocols. Several studies subsequently reported the presence of various bacterial species other than the known endosymbionts when *Steinernema* or *Heterorhabditis* IJ homogenates were plated on culture media (reviewed by Ogier et al., 2023). These taxa, listed in Fig. 1, were generally ignored or regarded as experimental anomalies (i.e., laboratory contaminants) within the framework of the dominant paradigm, which focused exclusively on primary endosymbionts at the expense of a more complex and dynamic microbial ecology.

### 4. The decisive role of Next-Generation sequencing (NGS) in challenging the monoxenic paradigm

The advent of next-generation sequencing (NGS) technologies revolutionized the description of microbial communities in numerous environments (Berg et al., 2020). The resolution achievable with molecular approaches made it possible for two series of studies using NGS to validate observations from earlier Pasteurian approaches that had been considered anomalies. First, metabarcoding analyses revealed the presence of dynamic bacterial communities in insect cadavers infected with *Heterorhabditis* spp., with taxa such as *Stenotrophomonas* spp. thriving despite the antibiotics produced by the endosymbionts (Wollenberg et al., 2016), which is not surprising given that clinical and environmental strains of *Stenotrophomonas* have been shown to exhibit a

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Bacterial taxa isolated by culture methods from infective juveniles (IJs) or IJ-infested insects from various species of *Steinernema* and *Heterorhabditis*. The colors indicate species belonging to the classes Gammaproteobacteria (dark blue), Betaproteobacteria (brown), Alphaproteobacteria (red), Flavobacteriia (light blue), Actinomycetia (black), Bacilli (green). The references for the identification of EPN-associated bacteria by culture are provided in brackets: (1) Poinar, 1966; (2) Lysenko and Weiser, 1974; (3) Boemare, 1983; Boemare et al., 1983; (4) Gouge and Snyder, 2006; (5) Ortega-Estrada et al., 2012; (6) Aguillera and Smart, 1993; Aguillera et al., 1993; (7) Kary et al., 2017; (8) Kim, 2009; (9) Aryal et al., 2021; (10) Ruiu et al., 2022; (11) Somvanshi et al., 2006, 2007; (12) El-Borai et al., 2005; (13) Quiroz-Castaneda et al., 2015; (14) Jackson et al., 1995; (15) Enright and Griffin, 2004; (16) Babic et al., 2000; (17) Salgado-Morales et al., 2017. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>
wide diversity of antibiotic resistance profiles (Deredjian et al., 2016). Remarkably, *Stenotrophomonas* spp. were also detected on the surfaces of IJs, suggesting that nematodes actively transport these bacteria into insect hosts (Wollenberg et al., 2016).

Ogier et al. (2020) then provided exhaustive information about the EPN-associated microbiota in *S. carpocapsae*. Using metabarcoding, they identified a core microbiota dominated by *X. nematophila*, along with a stable subset of about ten bacterial taxa, including genera such as *Pseudomonas*, *Stenotrophomonas*, *Achromobacter*, *Alcaligenes*, *Brucella* (formerly *Ochrobactrum*), and *Pseudochrobactrum*, etc., which the authors referred to as the Frequently Associated Microbiota (FAM) (Ogier et al., 2020). FAM members have been isolated from different laboratory-reared and field-collected nematodes (Ogier et al., 2020; unpublished data), refuting the idea that they are laboratory-induced anomalies. Moreover, metabarcoding has produced a description of the bacteria associated with *Steinernema glaseri*, *Steinernema weiseri*, *Steinernema feltiae* and *Heterorhabditis bacteriophora*, albeit a less exhaustive list due to the study of a limited number of samples of laboratory-reared nematodes (Ogier et al., 2019, 2020). Fig. 2 illustrates the diversity of the bacteria isolated or identified by NGS methods in *Steinernema* and *Heterorhabditis* in these two series of studies.

Recently, while assembling the genome of *Steinernema hermaphrodитum*, Schwarz and collaborators co-assembled bacterial genomes associated with a mixture of different life stages after careful surface washing with antibiotics. Beyond the endosymbiont *Xenorhabdus griffinae*, the authors also identified eight bacterial genera recurrently

associated with *Steinernema* and *Heterorhabditis: Achromobacter, Serratia, Providencia, Elizabethkingia, Stenotrophomonas, Pseudomonas, Pseudochrobactrum* and *Brucella* (formerly *Ochrobactrum*) (Schwarz et al., 2025). These recent results reinforce the arguments in favor of a polyxenic bacterial association in entomopathogenic nematodes.

## 5. A second bacterial circle with putative roles in the EPN life cycle

As a means of distinguishing between the endosymbiont and the other bacteria associated with EPNs, whether identified by culture or molecular methods, we have previously proposed the classification of these other bacteria as the “second bacterial circle”, a global term highlighting the need to move towards a microbial ecology approach to EPNs (Ogier et al., 2023). We have also formulated several hypotheses concerning their putative roles in the entire parasitic cycle of EPNs. The second bacterial circle may be involved in (i) entry into the living insect, causing infection and death, (ii) reproduction of the nematode in the insect cadaver, and (iii) the transmission and dissemination of the parasitic complex in soils (Ogier et al., 2023). We present arguments to support these hypotheses below, together with an outline of future research directions to explore these aspects.

### 5.1. Killing the insect

The association of entomopathogenic bacteria other than the

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Bacterial taxa identified by an NGS approach (metabarcoding) applied on infective juveniles (IJ) or IJ-infested insect of various *Steinernema glaseri*, *Steinernema weiseri*, *Steinernema feltiae*, *Steinernema carpocapsae* and *Heterorhabditis bacteriophora* as described by Wollenberg et al., 2016, Ogier et al., 2019 and Ogier et al., 2020.</figcaption></figure>
endosymbiont with IJs provides evidence in favor of a role of these bacteria in killing the insect. Some phylogenetic clusters of plant-beneficial pseudomonads are entomopathogenic, among which *Pseudomonas protegens* and *Pseudomonas chlororaphis* exhibit strong insecticidal activity by injection and oral ingestion (Flury et al., 2016). *P. protegens* and *P. chlororaphis* have been identified in the second bacterial circle of *S. carpocapsae*, *S. weiseri* and *S. glaseri*. Following the injection of $10^2-10^3$ colony-forming unites (CFU) into the hemocoel of *Spodoptera littoralis*, these *P. protegens* and *P. chlororaphis* strains killed 50% of larvae in 19 to 29 h, resulting in an $LT_{50}$ close to that of the endosymbiont (Ogier et al., 2020). Associations of *P. protegens* with *S. feltiae* IJs have also been described, with $10^3$ injected CFU killing almost 100% of larvae for the lepidopterans *Helicoverpa armigera* and *Lymantria dispar*, and the dipterans *Musca domestica* and *Ceratitis capitata* in less than 48 h (Ruiu et al., 2022). *P. protegens* and *P. chlororaphis* harbor a gene encoding the insecticidal FitD toxin responsible for insect pathogenicity once bacteria settled in the hemocoel (Péchy-Tarr et al., 2008; Flury et al., 2016). Interestingly, the *fitD* gene is orthologous to the gene encoding insecticidal Mcf toxins of *Photorhabdus* and *Xenorhabbus* (Ruffner et al., 2015). Although the origin of the ancestral *fitD/mcf* gene is not clear, this highly suggests that *P. protegens*, *Xenorhabdus* and *Photorhabdus* share common niches during their life-cycle, this niche could be the insect cadaver after the EPN infestation.

*Alcaligenes faecalis*, a species associated with IJs of *S. carpocapase*, *S. feltiae* and *H. bacteriophora* (Quiroz-Castañeda et al., 2015; Ogier et al., 2020), is considered as entomopathogenic by some authors (Quiroz-Castañeda et al., 2015). However, we have reservations concerning the possible entomopathogenic status of this taxon. Indeed, according to Bucher's definition (Bucher, 1960, 1973), a bacterium is considered entomopathogenic when it is able to kill an insect host at a dose below $10^5$ CFU. About $10^4-10^5$ CFU of *A. faecalis* are required to kill 100% of larvae for the sensitive insect *Galleria mellonella*, but no mortality was observed with a less sensitive insect, *Spodoptera littoralis* (Quiroz-Castañeda et al., 2015; Ogier et al., 2020).

## 5.2. Other properties of the second bacterial circle

The role of the second bacterial circle in the other steps of the EPN parasitic cycle of EPNs remains a matter of speculation. This role could be investigated by manipulating the microbial community associated with IJs and assessing the impact of these manipulations on various parameters of the EPN parasitic cycle. In our laboratory, we have attempted to reassociate axenic IJs with certain members of the second bacterial circle of *S. carpocapsae*, but have been unsuccessful so far. Another strategy would involve creating a situation of artificial dysbiosis — an imbalance in the IJ associated microbiota. This could be achieved by subjecting laboratory-reared IJs to high temperatures. Dysbiosis can also be induced by axenization and reassociation with the endosymbiont only. The impact of dysbiosis on host phenotypes has been studied in detail for the microbiota of humans, livestock and crops (Zaneveld et al., 2017; Arnault et al., 2023). According to the so-called “Anna Karenina Principle” based on Leon Tolstoy’s assertion and applied to host microbiota, “all healthy microbiota are alike, each disease-associated microbiota is sick in its own way” (Arnault et al., 2023). If this evolutionary principle holds true for the second bacterial circle of EPN, then microbial patterns are likely to be highly variable in dysbiotic situations and their analysis them would require a high statistical power.

Another possible field of investigation concerns the link between the second bacterial circle and the fitness of nematodes in situations closer to those encountered in nature than those used in the laboratory. We have already shown that in natural soil conditions, the endosymbiont is rarely dominant in insect cadavers infested with EPNs (Cambon et al., 2020). We also know that in certain nematode bacterium pairs, such as *S. glaseri* and *X. poinarii*, the association with the endosymbiont is not very robust (Akhurst, 1986; Ogier et al., 2014), suggesting that other

bacterial taxa might be involved in assisting the nematode to complete its life-cycle.

## 5.3. Nature of the nematode- second bacterial circle association

Another key point is the nature of the association between the nematode and its second bacterial circle. This relationship may be mutualistic or based on simple vectorization. We have identified three main questions that should be addressed as a matter of priority.

Where are the members of the second bacterial circle located? Some bacteria have been visualized in the intercuticular area of the IJs (Bonifassi et al., 1999; unpublished data), suggesting that this area may allow transient compartmentation of the second bacterial circle. Since some taxa such as soil-associated *Pseudomonas* exhibit a broad spectrum of antimicrobial activities (Pronk et al., 2022), their intercuticular location may prevent direct competition with the endosymbiont. The taxonomic identification of these intercuticular bacteria, by FISH for example, is required, but this approach has not yet been successfully applied to IJs. Other members may be present at very low quantities in the nematode gut. In this context, the challenge is survival in the presence of antimicrobial molecules, for which the endosymbionts have a high production potential (Shi et al., 2022). This survival can be achieved by a spatial compartmentation inside the gut, or by exhibiting low sensitivity to the endosymbiont-produced antimicrobials. For example, *Brucella* (formerly *Ochrobactrum*) and *Achromobacter* are known for their strong resistance to antimicrobial (Furlan and Stehling, 2017).

How are the members of the second bacterial circle transmitted? This question has been addressed for the endosymbionts in several studies with fluorescently labeled *Xenorhabdus* (Sicard et al., 2004; Snyder et al., 2007; Chaston et al., 2013) or *Photorhabdus* (Ciche and Ensign, 2003; Ciche et al., 2008). Similar studies should be conducted on members of the second bacterial circle.

Is there a specific association between EPNs and certain intra-specific clades of taxa belonging to the second bacterial circle? Indeed, unlike the association between EPNs and endosymbionts (Púza and Machado, 2024), the members of the second bacterial circle belong to species found in many environments. Addressing this question would involve the identification of specific phylogenetic clades within the second bacterial circle species associated with certain EPN species or genera and suggestive of co-evolution with EPNs.

# 6. Conclusion

Associations between EPNs and multiple bacterial species should now be viewed as a rule rather than an anomaly. EPN research is therefore entering a new polygenic paradigm thanks to methodological advances, such as the advent and increasing use of NGS approaches, together with a conceptual change, with biologists increasingly adopting a more holistic view of the host-symbiont interactions. While the functional hypotheses proposed here have yet to be supported experimentally, we are convinced of the as yet misunderstood role of the second bacterial circle. This role is probably difficult to uncover under standardized and optimized laboratory conditions. This is why we propose more ecologically relevant experimental setups, such as the use of mesocosms, which better reflect the complexity of natural environments.

## Funding source

Financial support for the preparation of this article was provided by a rolling grant from the Plant Health and Environment Department of INRAE.

## Declaration of competing interest

The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.

Acknowledgements

We thank Alain Givaudan for his careful reading of the manuscript.

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