Symbiotic Bacteria Research Articles

Gut microbiome and bone health: update on mechanisms, clinical correlations, and possible treatment strategies.

The intestinal microbiome is increasingly regarded as a relevant modulator of the pathophysiology of several age-related conditions, including frailty, sarcopenia, and cognitive decline. Aging is in fact associated with alteration of the equilibrium between symbiotic bacteria and opportunistic pathogens, leading to dysbiosis. The microbiome is able to regulate intestinal permeability and systemic inflammation, has a central role in intestinal amino acid metabolism, and produces a large number of metabolites and byproducts, with either beneficial or detrimental consequences for the host physiology. Recent evidence, from both preclinical animal models and clinical studies, suggests that these microbiome-centered pathways could contribute to bone homeostasis, regulating the balance between osteoblast and osteoclast function. In this systematic review, we provide an overview of the mechanisms involved in the gut-bone axis, with a particular focus on microbiome function and microbiome-derived mediators including short-chain fatty acids. We also review the current evidence linking gut microbiota dysbiosis with osteopenia and osteoporosis, and the results of the intervention studies on pre-, pro-, or post-biotics targeting bone mineral density loss in both animal models and human beings, indicating knowledge gaps and highlighting possible avenues for future research.

A conserved bacterial genetic basis for commensal-host specificity.

Animals selectively acquire specific symbiotic gut bacteria from their environments that aid host fitness. To colonize, a symbiont must locate its niche and sustain growth within the gut. Adhesins are bacterial cell surface proteins that facilitate attachment to host tissues and are often virulence factors for opportunistic pathogens. However, the attachments are often transient and nonspecific, and additional mechanisms are required to sustain infection. In this work, we use live imaging of individual symbiotic bacterial cells colonizing the gut of living Drosophila melanogaster to show that Lactiplantibacillus plantarum specifically recognizes the fruit fly foregut as a distinct physical niche. L. plantarum establishes stably within its niche through host-specific adhesins encoded by genes carried on a colonization island. The adhesin binding domains are conserved throughout the Lactobacillales, and the island also encodes a secretion system widely conserved among commensal and pathogenic bacteria.

Genetic Variation in the Atlantic Bobtail Squid-Vibrio Symbiosis From the Galician Rías.

Symbiotic marine bacteria that are transmitted through the environment are susceptible to abiotic factors (salinity, temperature, physical barriers) that can influence their ability to colonize their specific hosts. Given that many symbioses are driven by host specificity, environmentally transmitted symbionts are more susceptible to extrinsic factors depending on conditions over space and time. In order to determine whether the population structure of environmentally transmitted symbionts reflects host specificity or biogeography, we analysed the genetic structure of Sepiola atlantica (Cephalopoda: Sepiolidae) and their Vibrio symbionts (V. fischeri and V. logei) in four Galician Rías (Spain). This geographical location is characterized by a jagged coastline with a deep-sea entrance into the land, ideal for testing whether such population barriers exist due to genetic isolation. We used haplotype estimates combined with nested clade analysis to determine the genetic relatedness for both S. atlantica and Vibrio bacteria. Analyses of molecular variance (AMOVA) were used to estimate variation within and between populations for both host and symbiont genetic data. Our analyses reveal a low percentage of variation among and between host populations, suggesting that these populations are panmictic. In contrast, Vibrio symbiont populations show certain degree of genetic structure, demonstrating that the hydrology of the rías is driving bacterial distribution (and not host specificity). Thus, for environmentally transmitted symbioses such as the sepiolid squid-Vibrio association, abiotic factors can be a major selective force for determining population structure for one of the partners.

Morphological and Molecular Characterization of Endophytic Bacteria Isolated from Bambara Groundnut (Vigna subterranea) Nodules in Daloa, Côte d’Ivoire

A thorough understanding of the diversity, functions and specific interactions of endophytic bacteria present in legumes is essential for improving crop production. This study aimed to identify roots nodule endophytes associated to Bambara groundnut by analyzing their morphological diversity and genetic diversity. A total of 34 bacterial isolates were sampled from different cultivation sites and associated with 5 local varieties of Bambara groundnut. Morphological analysis using macroscopic and microscopic observation revealed that the bacterial isolates were morphologically diverse in terms of colony appearance, shape, colony size, color, opacity and Gram stain result. These bacteria exhibited characters different from those of the symbiotic bacteria. In addition, molecular identification based on sequencing the 16S-rRNA gene and 1081 bp analysis showed the existence of non-symbiotic bacteria in Bambara groundnut nodule. The community of roots nodule endophyte isolated from Bambara groundnut belonged to the genera Pseudomonas, Bacillus, Bacterium, microbacterium, Rahnella, Paenibacillus, Lysobacter with 98 to 99.5% similarity, classified under the Firmicutes, Actinobacteria and Proteobacteria phyla. In Bambara groundnut, the predominant nodule endophytes were Bacillus (56%) and Pseudomonas (17%). Future research could further investigate the ecological implications of these bacterial interactions, and their potential applications in biotechnology and agronomy, particularly in biofertilization and host plant growth promotion.

Symbiotic bacteria play crucial roles in a herbivorous mite host suitability.

The tomato russet mite (TRM), Aculops lycopersici, is a strictly herbivorous and economically significant pest that infests Solanaceae plants, but its host suitability varies, showing high performance on tomatoes. Although symbiotic bacteria have been suggested to play crucial roles in the host adaptation of herbivores, their effects on TRM remain unclear. In this study, using next generation high-throughput sequencing of the bacterial 16S rRNA data, we identified the bacterial diversity and community composition of TRM feeding on tomato, eggplant, and chili. Our results show no significant difference in the bacterial community composition of TRM across three host plants. However, the relative density of Escherichia coli (TRM_Escherichia) showed 9.36-fold higher on tomato than on eggplant and chili. These results align with the observed TRM performance among three host plants. When TRM_Escherichia was reduced using antibiotics, the treated TRM decreased the population density on tomato. However, when we transferred TRM from eggplant to tomato, the population density of TRM increased, coinciding with an increase of the TRM_Escherichia density. These results indicate that TRM_Escherichia may affect the host suitability of TRM. Our fluorescence in situ hybridization (FISH) results further showed that TRM_Escherichia is primarily distributed in the salivary glands. Metagenomic data results suggest that TRM_Escherichia functions in food digestion and energy metabolism. We provided the first comprehensive analysis of TRM bacterial communities. Our findings demonstrate that the symbiotic bacterium TRM_Escherichia may play crucial roles in the suitability of TRM feeding on different Solanaceae hosts. © 2024 Society of Chemical Industry.

Application of Entomopathogenic nematodes Suppresses the Root Knot Nematodes, Agrotis ipsilon, Spodoptera litura in Tobacco

Application of Entomopathogenic nematodes (EPNs) is an efficient and novel alternative for management of plant-parasitic nematode and other insects to improve the productivity of tobacco and to protect the tobacco from the disturbing of underground and leaf chewing insects. In the present work, laboratory and field trials were conducted to control root-knot nematode (M. javanica) by using EPNs and the symbiotic bacterial, and the effect of the application was evaluated in tobacco plant. Approximately after inoculation of sixty days, egg masses and galls on tobacco root and agronomic characters of tobacco were calculated. Results showed that both EPNs and the symbiotic bacterium affected the reproduction of M. javanica population and the gall, egg masses and the root-knot nematodes in the tobacco root were significant reduced. EPNs also showed suppressive effect on S. litura larvae, and Agrotis ipsilon larvae as well. It might be the first study about the use of EPNs in the control of M. javanica in tobacco plant.

A symbiotic mosquito-gut bacterium for flavivirus control.

Dengue virus (DENV) and Zika virus (ZIKV) have emerged as major global public health challenges, causing numerous infections and fatalities each year. However, current measures, including vaccines and treatments, are often limited or ineffective. This highlights the urgent need for novel preventive strategies to control the spread of key mosquito-borne viruses like DENV and ZIKV. In a recent study published in Science, Zhang et al. isolated a bacterium named Rosenbergiella_YN46 from the gut of field-caught Aedes albopictus mosquitoes in Yunnan Province, China. This commentary reviews their findings, published on April19,2024, which describe the symbiotic bacterium Rosenbergiella_YN46 and its ability to block flavivirus transmission, including both DENV and ZIKV. The bacterium shows promising potential for future dengue fever prevention and provides valuable insights into a novel biological approach for controlling mosquito-borne viral diseases.

Antagonistic effect of symbiotic bacteria from entomopathogenic nematodes and Bacillus spp. against Panax ginseng root rot pathogens

Ginseng root rot is a destructive disease of Panax ginseng that seriously affects the yield and quality of P. ginseng. Based on the broad-spectrum suppression properties of entomopathogenic nematode (EPN) symbiotic bacteria, four symbiotic bacteria were selected: Xenorhabdus bovienii (Xbf), Xenorhabdus nematophila (Xna), Xenorhabdus nematophila Cxrd (Xnc) and Xenorhabdus budapestensis (Xbc). Meanwhile, six beneficial Bacillus strains (Bacillus velezensis S1A; Bacillus amyloliquefaciens H4C; Bacillus subtilis N5D; and Bacillus albus HYS, K8, and N2A) were tested. Their individual and combined effects (both in vitro and in P. ginseng slices) on fungi pathogenic to P. ginseng, Fusarium solani, Fusarium proliferatum and Fusarium oxysporum were evaluated. Xenorhabdus budapestensis had the highest suppression effects (up to 82%) on F. solani and F. oxysporum, and a 60% inhibition rate on F. proliferatum, significantly higher than Xnc, Xbf and Xna. Bacillus velezensis and B. subtilis had the strongest suppression rates on F. solani (40%). The combined effects of Xbc and B. subtilis K8 on F. solani and F. oxysporum were stronger than their individual effects. The P. ginseng slice experiment further confirmed the enhanced inhibition of Fusarium by mixing Xbc with B. subtilis K8. In conclusion, we used EPN symbiotic bacteria to suppress P. ginseng root rot and screened two EPN symbiotic bacteria that were highly effective against Fusarium spp. The combination of Bacillus strains with EPN symbiotic bacteria enhanced the suppression effect. Our results provide new biological materials and directions for biological control of P. ginseng root rot.

Exposure to polypropylene microplastics induces the upregulation of protein digestion-associated genes and microbiome reorganization in the octocoral Junceella squamata

Microplastics, a new type of pollutants found in coral reefs, have attracted increasing attention. However, most of the current research focuses on the scleractinian corals and few reports on Octocorallia. To reveal the impact of microplastic exposure on Octocorallia, we analyzed the transcriptional response of the coral hosts Junceella squamata along with changes to the diversity and community structure of its symbiotic bacteria following exposure to polystyrene microplastics. These results suggest that the microplastics have adverse impacts on nutrient metabolism and absorption in J. squamata. The symbiotic bacteria of J. squamata exhibited a clear response after exposure to microplastics, which may also reflect an adaptation mechanism of corals, and help to maintain the physiological function of coral symbiotic function under the exposure of microplastics. This study has revealed the impact of microplastic exposure on J. squamata, providing new insights for coral protection against the background of increased microplastics pollution.

Testing low-risk bioactive compounds on Halyomorpha halys: an improved pipeline of analyses to investigate their effects on the bacterial endosymbiont Candidatus Pantoea carbekii.

The brown marmorated stink bug Halyomorpha halys has become an invasive insect pest of many crops. A promising control strategy to manage the proliferation of H. halys is based on the suppression of its obligate and vertically transmitted uncultivated symbiotic bacterium Candidatus Pantoea carbekii through surface-sterilization of H. halys eggs. Indeed, the application of antimicrobial formulations on the eggs of H. halys could cause mortality of endosymbiont and consequently of newly emerged nymphs. In this study, a microbial live/dead assay was applied directly on H. halys eggs to evaluate Ca. P. carbekii loss of viability after treatments with seven commercial formulations including fungicides (copper hydroxide, sulphur, sweet orange essential oil) and plant biostimulants (flavonoids and chestnut tannin extract) compared with two disinfectants for civil and industrial use (sodium hypochlorite/hydrated sodium/tetraborate decahydrate and peracetic acid/hydrogen peroxide). Impact of mode of application was also evaluated, as surface treatment of egg masses was performed through spraying and dipping in laboratory conditions. Antimicrobial activity data were finally complemented with observations of egg hatching and vitality of the nymphs. The optimization of live/dead staining is useful for evaluating Ca. P. carbekii mortality directly on eggs, providing a rapid and reliable culture-independent approach. Sodium hypochlorite, copper, sulphur, tannins and sweet orange essential oil showed an antimicrobial effect against Ca. P. carbekii and a H. halys egg hatching reduction and nymph's vitality. The antimicrobial and insecticidal effects of these commercial products should be further studied to assess their in-field efficiency as well as the impact of these substances on non-target organisms. The approach followed in this study could be considered a robust pipeline of analyses to evaluate the effectiveness of antimicrobial eco-friendly compounds in symbiotic control of H. halys. © 2024 Society of Chemical Industry.

Differential physiological and microbial responses of the octocoral Junceella squamata to high-temperature and cadmium stress

Global warming and heavy metals have become the major threat to the growth and reproduction of corals. However, unlike scleractinian corals, in the context of widespread coral degradation worldwide, there are few reports on the response of octocorallia corals to high-temperature stress and heavy metals. In the present study, we conducted indoor simulation experiments using Junceella squamata. We evaluated the physiological response of these corals under high-temperature stress at 33 °C and cadmium (Cd) stress by comparing the composition and diversity of their symbiotic bacteria and analyzing differences in their transcriptome. The results show that high-temperature stress has more severe adverse effects than cadmium stress. High-temperature stress disrupts coral symbiotic relationships, leading to an increase in alpha diversity associated with disease-causing bacteria, which may increase the risk of infection and potentially contribute to coral mortality. Meanwhile, cadmium stress increases the instability of the coral holobiont, potentially disrupting DNA stability and RNA transcriptional regulation. However, an increase in Cd-tolerant bacteria may help corals respond to cadmium stress. This study reveals the effects of harmful substances on coral and highlights the urgent need for action to protect octocorals in the face of environmental stress.

Insect Lipid Metabolism in the Presence of Symbiotic and Pathogenic Viruses and Bacteria.

Insects, like most animals, have intimate interactions with microorganisms that can influence the insect host's lipid metabolism. In this chapter, we describe what is known so far about the role prokaryotic microorganisms play in insect lipid metabolism. We start exploring microbe-insect lipid interactions focusing on endosymbionts, and more specifically the gut microbiota that has been predominantly studied in Drosophila melanogaster. We then move onto an overview of the work done on the common and well-studied endosymbiont Wolbachia pipientis, also in interaction with other microbes. Taking a slightly different angle, we then look at the effect of human pathogens, including dengue and other viruses, on the lipids of mosquito vectors. We extend the work on human pathogens and include interactions with the endosymbiont Wolbachia that was identified as a natural tool to reduce the spread of mosquito-borne diseases. Research on lipid metabolism of plant disease vectors is up and coming and we end this chapter by highlighting current knowledge in that field.

The fall armyworm converts maize endophytes into its own probiotics to detoxify benzoxazinoids and promote caterpillar growth

BackgroundThe fall armyworm (FAW, Spodoptera frugiperda) threatens maize production worldwide, and benzoxazinoids (Bxs) are known as the main secondary metabolites produced by maize to defend against FAW. However, we do not yet know whether and in what ways certain endophytes in the digestive system of FAW can metabolize Bxs, thus enhancing the fitness of FAW when feeding on maize.ResultsUsing Bxs as the sole carbon and nitrogen source, we isolated Pantoea dispersa from the guts of FAW. P. dispersa can colonize maize roots and leaves as indicated by GFP-labeling and further successfully established itself as an endophyte in the Malpighian tubules and the gut of FAW after FAW feeding activities. Once established, it can be vertically transmitted through FAW eggs, suggesting the potential that FAW can convert maize-derived endophytes into symbiotic bacteria for intergenerational transmission. The prevalence of P. dispersa in FAW guts and maize leaves was also confirmed over large geographic regions, indicating its evolutionary adaptation in fields. Bxs determination in the gut and frass of FAW combined with bioassays performance on maize bx2 mutants revealed that the colonization of P. dispersa can promote FAW growth by metabolizing Bxs rather than other metabolites. Additionally, genome and transcriptome analyses identified plasmid-borne genes, rather than chromosomes of this species, were crucial for Bxs metabolism. This was further validated through in vitro prokaryotic expression assays by expressing two candidate genes form the plasmid.ConclusionsFAW can convert maize endophytes into its own probiotics to detoxify Bxs and thus enhance caterpillar growth. This represents a novel strategy for lepidopteran pests—transforming allies of the host into its own—thereby shedding light on the rapid spread of FAW and enhancing our understanding of ecological and evolutionary mechanisms underlying the pest-microbe–plant interactions.2f6JcKQYnKTEdqap9saLkxVideo

Chemical interactions between kelp Macrocystis pyrifera and symbiotic bacteria under elevated CO2 condition.

The online version contains supplementary material available at 10.1007/s42995-024-00259-5.

Wolbachia in Antarctic terrestrial invertebrates: Absent or undiscovered?

Interactions between a host organism and its associated microbiota, including symbiotic bacteria, play a crucial role in host adaptation to changing environmental conditions. Antarctica provides a unique environment for the establishment and maintenance of symbiotic relationships. One of the most extensively studied symbiotic bacteria in invertebrates is Wolbachia pipientis, which is associated with a wide variety of invertebrates. Wolbachia is known for manipulating host reproduction and having obligate or facultative mutualistic relationships with various hosts. However, there is a lack of clear understanding of the prevalence of Wolbachia in terrestrial invertebrates in Antarctica. We present the outcomes of a literature search for information on the occurrence of Wolbachia in each of the major taxonomic groups of terrestrial invertebrates (Acari, Collembola, Diptera, Rotifera, Nematoda, Tardigrada). We also performed profiling of prokaryotes based on three marker genes and Kraken2 in available whole genome sequence data obtained from Antarctic invertebrate samples. We found no reports or molecular evidence of Wolbachia in these invertebrate groups in Antarctica. We discuss possible reasons underlying this apparent absence and suggest opportunities for more targeted future research to confirm bacteria's presence or absence.

Pathobiont and symbiont contribute to microbiota homeostasis through Malpighian tubules-gut countercurrent flow in Bactrocera dorsalis.

Host-gut microbiota interactions are more complex than good or bad. Both gut symbiotic bacteria and pathobionts can provide essential functions to their host in one scenario and yet be detrimental to host health in another. So, these gut-dwelling bacteria must be tightly controlled to avoid harmful effects on the host. However, how pathobionts and other symbiotic bacteria coordinate to establish a host immune defense system remains unclear. Here, using a Tephritidae fruit fly Bactrocera dorsalis, we report that both pathobionts and other gut symbiotic bacteria release tyramine, which is recognized by the host insects. These tyramines induce the formation of insect-conserved Malpighian tubules-gut countercurrent flow upon bacterial infection, which requires tyramine receptors and aquaporins. At the same time, pathobionts but not gut symbiotic bacteria induce the generation of reactive oxygen species, which are preserved by the countercurrent flow, promoting bacteria elimination through increasing gut peristalsis. More importantly, our results show that the Malpighian tubules-gut countercurrent flow maintains proper microbiota composition. Our work suggests a model where pathobiont-induced reactive oxygen species are preserved by Malpighian tubules-gut countercurrent flow involving both pathobionts and symbiotic bacteria. Furthermore, our work provides a Malpighian tubules-gut interaction that ensures efficient maintenance of the gut microbiota.

Identification and characterization of a surfactin from Pseudomonas gessardii: A symbiotic bacterium with potent anticancer activity

Prasiola japonica, traditionally used as food and folk medicine in South Korea, exerts pharmacological properties, including antioxidant, anti-inflammatory, antidiabetic, and anticancer effects. In this study, we explored symbiotic microbes associated with P. japonica and identified Pseudomonas gessardii as a nonpathogenic symbiotic bacterium through 16 S rDNA sequencing. Bioactivity-guided fractionation of P. gessardii ethanol extracts, utilizing a series of non-polar to polar solvents, led to the isolation of a single bioactive compound (SF10) from the ethyl acetate fraction. Structural analysis using LC-MS and NMR spectroscopy identified SF10 as surfactin C15, a lipopeptide consisting of 7 amino acids and a β-hydroxy fatty acid chain containing 15 carbon atoms. This represents the first discovery of surfactin production in P. gessardii, expanding known surfactin-producing genera beyond Bacillus. In HT-29 colorectal cancer cells, surfactin C15 demonstrated significant anticancer activity through multiple mechanisms: inhibition of cancer stem cell marker CD133 expression, upregulation of pro-apoptotic factors (CHOP, PUMA, DR5), and modulation of cell cycle regulators (CDKN1A,CCNE1, CDK5). Furthermore, surfactin C15 induced necrotic cell death, confirmed by increased lactate dehydrogenase release and flow cytometry analysis showing dose-dependent increases in necrotic cell populations. This study reveals a novel source of surfactin with unique cancer cell-targeting properties, particularly through its ability to induce necrosis in colorectal cancer cells, suggesting potential therapeutic applications in cancer treatment.

Soybean Bradyrhizobium spp. Spontaneously Produce Abundant and Diverse Temperate Phages in Culture.

Soybean bradyrhizobia (Bradyrhizobium spp.) are symbiotic root-nodulating bacteria that fix atmospheric nitrogen for the host plant. The University of Delaware Bradyrhizobium Culture Collection (UDBCC; 353 accessions) was created to study the diversity and ecology of soybean bradyrhizobia. Some UDBCC accessions produce temperate (lysogenic) bacteriophages spontaneously under routine culture conditions without chemical or other apparent inducing agents. Spontaneous phage production may promote horizontal gene transfer and shape bacterial genomes and associated phenotypes. A diverse subset (n = 98) of the UDBCC was examined for spontaneously produced virus-like particles (VLPs) using epifluorescent microscopy, with a majority (69%) producing detectable VLPs (>1 × 107 mL-1) in laboratory culture. Phages from the higher-producing accessions (>2.0 × 108 VLP mL-1; n = 44) were examined using transmission electron microscopy. Diverse morphologies were observed, including various tail types and lengths, capsid sizes and shapes, and the presence of collars or baseplates. In many instances, putative extracellular vesicles of a size similar to virions were also observed. Three of the four species examined (B. japonicum, B. elkanii, and B. diazoefficiens) produced apparently tailless phages. All species except B. ottawaense also produced siphovirus-like phages, while all but B. diazoefficiens additionally produced podovirus-like phages. Myovirus-like phages were restricted to B. japonicum and B. elkanii. At least three strains were polylysogens, producing up to three distinct morphotypes. These observations suggest spontaneously produced phages may play a significant role in the ecology and evolution of soybean bradyrhizobia.

Bacterial communities in Asecodes hispinarum (Hymenoptera: Eulophidae) and its host Brontispa longissima (Coleoptera: Chrysomelidae), with comparison of Wolbachia dominance.

The endoparasitoid Asecodes hispinarum (Bouček) (Hymenoptera: Eulophidae) serves as an effective biological control agent against Brontispa longissima (Gestro) (Coleoptera: Chrysomelidae), a notorious palm pest. Endosymbionts found in parasitoids and their hosts have attracted significant attention due to their substantial influence on biocontrol efficacy. In this study, we employed 16S rRNA sequencing, polymerase chain reaction, and fluorescence in situ hybridization to assess the symbiotic bacteria composition, diversity, phylogeny, and localization in A. hispinarum and its host B. longissima. Our findings showed significant differences in the richness, diversity, and composition of symbiotic bacteria among different life stages of B. longissima. Notably, the bacterial richness, diversity, and composition of A. hispinarum was similar to that of B. longissima. Firmicutes and Proteobacteria were the dominant phyla, while Wolbachia was the dominant genera across the parasitoid and host. It was discovered for the first time that Wolbachia was present in A. hispinarum with a high infection rate at ≥ 96.67%. Notably, the Wolbachia strain in A. hispinarum was placed in supergroup A, whereas it was categorized under supergroup B in B. longissima. Furthermore, Wolbachia is concentrated in the abdomen of A. hispinarum, with particularly high levels observed in the ovipositors of female adults. These findings highlight the composition and diversity of symbiotic bacteria in both A. hispinarum and its host B. longissima, providing a foundation for the development of population regulation strategies targeting B. longissima.

To be a good killer: Evaluation of morphometry and nematodes-bacteria complex effect on entomopathogenic nematodes virulence against wireworms

Entomopathogenic nematodes (EPNs) have emerged as a promising tool for controlling soil-dwelling crop pests. However, their efficacy varies according to EPN populations and targeted hosts. Wireworms are polyphagous insects causing significant crop losses, especially since the ban on pesticides previously used for their control. They are highly resistant to EPN populations and require high number of infective juveniles (IJs) to achieve optimal mortality rates. In this research, we collected and compared the virulence of 16 EPN populations, of foreign origin, purchased or collected from wireworms infested fields. Then, we have tested two hypotheses: (i) smaller nematodes induce heightened mortality rates against wireworms; (ii) virulence levels can be linked to nematodes-bacteria complex. Mortality rates scaled from three to 43 % after 56 days of continuous exposure across the 16 tested EPN populations (Heterorhabditis spp and Steinernema spp.). Morphometric analysis of IJs revealed both intra- and interspecific variations in length and diameter among populations. Interestingly, while EPN length influence mortality at three days post-inoculation. We found leaner IJs (< 25 µm) to induce higher mortality rates at 56 days post-inoculation. To better determine the structure and dimensions of the primary entry routes utilized by EPNs, we provide optical microscope micrographs of wireworm Agriotes spp. spiracle, anal sclerotized coating anus and anal muscles. Symbiotic bacteria of each EPN population were identified, and a biochemical characterization was performed using Analytical Profile Index tests. The symbiotic bacteria belong to the species Photorhabdus antumapuensis, P. laumondii subsp. laumondii, P. thracensis, Xenorhabdus bovienii and X nematophila. Bacteria biochemical profiles did not reflect the differences in virulence of nematodes-bacteria complex against wireworms. These findings highlight the importance of considering EPN morphometry and intraspecific variability in designing applications to control wireworms.