Genera Steinernema Research Articles

Does the Future of Antibiotics Lie in Secondary Metabolites Produced by Xenorhabdus spp.? A Review.

The over-prescription of antibiotics for treatment of infections is primarily to blame for the increase in bacterial resistance. Added to the problem is the slow rate at which novel antibiotics are discovered and the many processes that need to be followed to classify antimicrobials safe for medical use. Xenorhabdus spp. of the family Enterobacteriaceae, mutualistically associated with entomopathogenic nematodes of the genus Steinernema, produce a variety of antibacterial peptides, including bacteriocins, depsipeptides, xenocoumacins and PAX (peptide antimicrobial-Xenorhabdus) peptides, plus additional secondary metabolites with antibacterial and antifungal activity. The secondary metabolites of some strains are active against protozoa and a few have anti-carcinogenic properties. It is thus not surprising that nematodes invaded by a single strain of a Xenorhabdus species are not infected by other microorganisms. In this review, the antimicrobial compounds produced by Xenorhabdus spp. are listed and the gene clusters involved in synthesis of these secondary metabolites are discussed. We also review growth conditions required for increased production of antimicrobial compounds.

Microbial biopesticides in agroecosystems

Microbial biopesticides include several microorganisms like bacteria, fungi, baculoviruses, and nematode-associated bacteria acting against invertebrate pests in agro-ecosystems. The biopesticide sector is experiencing a significant growth and many discoveries are being developed into new biopesticidal products that are fueling a growing global market offer. Following a few decades of successful use of the entomopathogenic bacterium Bacillus thuringiensis and a few other microbial species, recent academic and industrial efforts have led to the discovery of new microbial species and strains, and of their specific toxins and virulence factors. Many of these have, therefore, been developed into commercial products. Bacterial entomopathogens include several Bacillaceae, Serratia, Pseudomonas, Yersinia, Burkholderia, Chromobacterium, Streptomyces, and Saccharopolyspora species, while fungi comprise different strains of Beauveria bassiana, B. brongniartii, Metarhizium anisopliae, Verticillium, Lecanicillium, Hirsutella, Paecilomyces, and Isaria species. Baculoviruses are species-specific and refer to niche products active against chewing insects, especially Lepidopteran caterpillars. Entomopathogenic nematodes (EPNs) mainly include species in the genera Heterorhabditis and Steinernema associated with mutualistic symbiotic bacteria belonging to the genera Photorhabdus and Xenorhabdus. An updated representation of the current knowledge on microbial biopesticides and of the availability of active substances that can be used in integrated pest management programs in agro-ecosystems is reported her.

A draft genome of Steinernema diaprepesi.

Entomopathogenic nematodes within the genus Steinernema are used as biological control agents against significant agricultural pests. Steinernema diaprepesi is native to Florida and very effective in controlling citrus root weevil, a devastating pest of citrus, ornamental plants, and vegetables. Here, we present the draft genome of Steinernema diaprepesi, which is a valuable tool for understanding the efficacy of this nematode as a biological control agent.

Entomopatogene nematode - prirodni neprijatelji štetnih insekata

In the programmes of biological control, a large number of different beneficial organisms are applied, including certain groups of nematodes. From the aspect of biocontrol measures, the most significant group of nematodes is entomopathogenic nematodes, which represents both natural and ecological solution for pest control management. The protection success relies on the specific-mutualistic relationship between nematodes and bacteria, where nematodes in its intestinal carries cells of a symbiotic bacterium that kills the host insect. Regarding nematode-related biological control, two families draw the most attention, Steinernematidae and Heterorhabditidae. Within family Steinernematidae, the genus Steinernema stands out, while within Heterorhabditidae the most important genus is Heterorhabditis. Entomopatogenic nematodes have been used to control numerous agricultural pests, which include insects of different orders such as: Lepidoptera, Siphonaptera, Diptera, Coleoptera, Blattodea, Orthoptera and Heteroptera. Entomopathogenic nematodes are characterized by numerous traits that make them extremely suitable biological control agents: wide host spectrum, active host seeking, killing the host within 48 hours, easy mass production and field application, long-term efficacy, compatibility with most chemicals and environmentally safe.

Efficacy of Entomopathogenic Nematode, Steinernema dharanaii (TFRIEPN-15) against Termite, Odontotermes obesus (Isoptera: Termitidae)

Entomopathogenic nematodes (EPNs) in the genera Steinernema and Heterorhabditis and their associated bacteria such as Xenorhabdus spp. and Photorhabdus spp. are lethal parasites of soil dwelling and cryptic habit insects. Termites are one of the most severe soil insect pests of number of forestry, agricultural, households items and plantation crops in India and abroad. The efficacy of new native species of entomopathogenic nematode (TFRIEPN-15), Steinernema dharanaii against termites, Odontotermes obesus was tested under laboratory conditions. The termite, Odontotermes obesus was exposed to Infective Juveniles (IJs) from numbers 3 to 40 IJs termite-1. The dose- dependent mortality was noted of the termites. The lowest dose of 3IJs termites-1 caused 26.67% mortality which was significantly superior control treatment. The highest dose of 40IJs termites-1 caused 72.00% mortality was recorded. These experiments have proved that native isolated entomopathogenic nematodes can be used successfully to control termites and other soil insect pests with simple and effective field application techniques achieving 100% mortality of the treated insects.

Biological Control Technology Utilizing Heterorhabditis bacteriophora and Steinernema carpocapsae

Entomopathogenic nematodes (in the genus Steinernema and Heterorhabditis) have been studied and successfully commercialized as biological control agents. These organisms are highly virulent and safe for the non-target environment, animals and humans. For at least 200 target species, the nematode-bacteria complex has the potential to become a mass-marketed agricultural biopesticide. However, before nematodes can be successfully integrated into the agricultural system as a regular-use, “go-to” biopesticide, it is necessary to develop economical manufacturing processes. There are several manufacturing platforms: in vitro solid fermentation; in vitro liquid fermentation; and in vivo production. This review presents an analysis of each approach and discusses the advantages and disadvantages relative to the cost of production, technical expertise required, and quality of the final product.

Hybrid Assembly of the Genome of the Entomopathogenic Nematode Steinernema carpocapsae Identifies the X-Chromosome.

Entomopathogenic nematodes from the genus Steinernema are lethal insect parasites that quickly kill their insect hosts with the help of their symbiotic bacteria. Steinernema carpocapsae is one of the most studied entomopathogens due to its broad lethality to diverse insect species and its effective commercial use as a biological control agent for insect pests, as well as a genetic model for studying parasitism, pathogenesis, and symbiosis. In this study, we used long-reads from the Pacific Biosciences platform and BioNano Genomics Irys system to assemble the most complete genome of the S. carpocapsae ALL strain to date, comprising 84.5 Mb in 16 scaffolds, with an N50 of 7.36 Mb. The largest scaffold, with 20.9 Mb, was identified as chromosome X based on sex-specific genome sequencing. The high level of contiguity allowed us to characterize gene density, repeat content, and GC content. RNA-seq data from 17 developmental stages, spanning from embryo to adult, were used to predict 30,957 gene models. Using this improved genome, we performed a macrosyntenic analysis to Caenorhabditis elegans and Pristionchus pacificus and found S. carpocapsae’s chromosome X to be primarily orthologous to C. elegans’ and P. pacificus’ chromosome II and IV. We also investigated the expansion of protein families and gene expression differences between adult male and female stage nematodes. This new genome and more accurate set of annotations provide a foundation for additional comparative genomic and gene expression studies within the Steinernema clade and across the Nematoda phylum.

A core set of venom proteins is released by entomopathogenic nematodes in the genus Steinernema.

Parasitic helminths release molecular effectors into their hosts and these effectors can directly damage host tissue and modulate host immunity. Excreted/secreted proteins (ESPs) are one category of parasite molecular effectors that are critical to their success within the host. However, most studies of nematode ESPs rely on in vitro stimulation or culture conditions to collect the ESPs, operating under the assumption that in vitro conditions mimic actual in vivo infection. This assumption is rarely if ever validated. Entomopathogenic nematodes (EPNs) are lethal parasites of insects that produce and release toxins into their insect hosts and are a powerful model parasite system. We compared transcriptional profiles of individual Steinernema feltiae nematodes at different time points of activation under in vitro and in vivo conditions and found that some but not all time points during in vitro parasite activation have similar transcriptional profiles with nematodes from in vivo infections. These findings highlight the importance of experimental validation of ESP collection conditions. Additionally, we found that a suite of genes in the neuropeptide pathway were downregulated as nematodes activated and infection progressed in vivo, suggesting that these genes are involved in host-seeking behavior and are less important during active infection. We then characterized the ESPs of activated S. feltiae infective juveniles (IJs) using mass spectrometry and identified 266 proteins that are released by these nematodes. In comparing these ESPs with those previously identified in activated S. carpocapsae IJs, we identified a core set of 52 proteins that are conserved and present in the ESPs of activated IJs of both species. These core venom proteins include both tissue-damaging and immune-modulating proteins, suggesting that the ESPs of these parasites include both a core set of effectors as well as a specialized set, more adapted to the particular hosts they infect.

Steinernema kandii n. sp. (Rhabditida: Steinernematidae), a new entomopathogenic nematode from northern Benin

SummaryTwo nematode isolates from the genusSteinernemawere collected in northern Benin. Morphological, morphometric, molecular and cross-hybridisation studies placed these nematodes into a new species,Steinernema kandiin. sp., within thebicornutum-group. Phylogenetic analyses based on both ITS and D2-D3 regions of 28S rDNA revealed thatS. kandiin. sp. is different from all knownSteinernemaspecies and sister toS. abbasi(97.3-97.6% ITS nucleotide similarity) andS. bifurcatum(98.3-98.4% D2-D3 similarity).Steinernema kandiin. sp. can be separated from other members of thebicornutum-group by the greater infective juvenile (IJ) max. body diam. of 35 (27-48)μm (type isolate). It differs fromS. abbasiby the greater IJ body length 707 (632-833)μm (type isolate), EP distance 55 (52-60)μm (type isolate), spicule length 67 (57-75)μm (type isolate) and the occurrence of one pair of genital papillae at the cloacal aperture.

Bacteria of the Genus Xenorhabdus, a Novel Source of Bioactive Compounds.

The genus Xenorhabdus of the family Enterobacteriaceae, are mutualistically associated with entomopathogenic nematodes of the genus Steinernema. Although most of the associations are species-specific, a specific Xenorhabdus sp. may infect more than one Steinernema sp. During the Xenorhabdus–Steinernema life cycle, insect larvae are infected and killed, while both mutualists produce bioactive compounds. These compounds act synergistically to ensure reproduction and proliferation of the nematodes and bacteria. A single strain of Xenorhabdus may produce a variety of antibacterial and antifungal compounds, some of which are also active against insects, nematodes, protozoa, and cancer cells. Antimicrobial compounds produced by Xenorhabdus spp. have not been researched to the same extent as other soil bacteria and they may hold the answer to novel antibacterial and antifungal compounds. This review summarizes the bioactive secondary metabolites produced by Xenorhabdus spp. and their application in disease control. Gene regulation and increasing the production of a few of these antimicrobial compounds are discussed. Aspects limiting future development of these novel bioactive compounds are also pointed out.

ENDOSYMBIONTS OF ENTOMOPATHOGENIC NEMATODES FROM SOUTH ITALY: A PHENOTYPIC STUDY

We examined different Xenorhabdusstrains (five of X. bovieniiand two of X. kozodoii), obtained from EPNisolates belonging to the genus Steinernema(S. feltiae, S. ichnusae, S. apuliae, S. vulcanicum) of different geographicorigin by both genotypic and phenotypic analysis. Common laboratory assays were done for traits such as antibioticresistance, haemolytic activity, lactose utilisation, biofilm production, chosen as the least selectable traits for EPN life-cycle, and thus as (presumably) neutral traits. As selective marker, the activity of the endosymbiont’s toxins was verified inan in vivoassay on G. mellonellalarvae. Genotyping done by 16S partial sequencing was used for identification purposes.Xenorhabdusbovieniiisolates showed a broad phenotypic spectrum; on the other hand, X. kozodoiishowed a less degreeof phenotypic variation, reduced ability of biofilm production and conspicuous β-galactosidase activity. However, all thestrains were able to kill G. mellonellalarvae with high efficiency.

Reproduction of entomopathogenic nematodes of the genera Steinernema Travassos, 1927 (Rhabditida: Steienernematidae) within strawberry blossom weevil larvae Anthonomus rubi Herbst, 1795 (Coleoptera: Curculionidae)

Studies have been conducted on the contamination in the laboratory of larvae of raspberry-straw weevil (Anthonomus rubi Herbst, 1795) with two isolates of entomopathogenic nematodes (ETNs) of the genus Steinernema Travassos, 1927, vchecked the possibility of their reproduction under these conditions, and features of quantitative performance indicators. On the basis of the obtained data, it was established that, regardless of the type of isolate of the EPN, all experimental A. rubi larvae died on the 3rd day after infection, and the yield of larvae nematodes from the dead insects began on day 10—12 and lasted 42—44 days. During this period, on average, from one of the affected specimens of raspberry-straw weevil, from 26965 to 29168 larvae of the EPN was allocated. The total number of larvae of EPN obtained in the infection of 14 larvae of weevil of raspberry-strawberry was 392929 individuals. The migration of newly-formed individuals of the EPN was uneven, the bulk of the larvae (61—66.2%) is released during the second decade of the month, the rest — 24.5%—31.2% in the third decade. In the first and last decades the larvae are significantly less (3.5—5.8%).

Partners in crime: symbiont-assisted resource acquisition in Steinernema entomopathogenic nematodes.

Entomopathogenic nematodes in the genus Steinernema (Nematoda: Steinernematidae) have a mutualistic relationship with Xenorhabdus bacteria (Gram-negative Enterobacteriaceae). This partnership however, is pathogenic to a wide range of insect species. Because of their potent insecticidal ability, they have successfully been implemented in biological control and integrated pest management programs worldwide. Steinernema-Xenorhabdus-insect partnerships are extremely diverse and represent a model system in ecology and evolution to investigate symbioses between invertebrates and microbes. The reproductive fitness of the nematode-bacterium partnership is tightly associated, and maintenance of their virulence is critical to the conversion of the insect host as a suitable environment where this partnership can be perpetuated.

Symbiont-mediated competition: Xenorhabdus bovienii confer an advantage to their nematode host Steinernema affine by killing competitor Steinernema feltiae.

Bacterial symbionts can affect several biotic interactions of their hosts, including their competition with other species. Nematodes in the genus Steinernema utilize Xenorhabdus bacterial symbionts for insect host killing and nutritional bioconversion. Here, we establish that the Xenorhabdus bovienii bacterial symbiont (Xb-Sa-78) of Steinernema affine nematodes can impact competition between S. affine and S. feltiae by a novel mechanism, directly attacking its nematode competitor. Through co-injection and natural infection assays we demonstrate the causal role of Xb-Sa-78 in the superiority of S. affine over S. feltiae nematodes during competition. Survival assays revealed that Xb-Sa-78 bacteria kill reproductive life stages of S. feltiae. Microscopy and timed infection assays indicate that Xb-Sa-78 bacteria colonize S. feltiae nematode intestines, which alters morphology of the intestine. These data suggest that Xb-Sa-78 may be an intestinal pathogen of the non-native S. feltiae nematode, although it is a nonharmful colonizer of the native nematode host, S. affine. Screening additional X. bovienii isolates revealed that intestinal infection and killing of S. feltiae is conserved among isolates from nematodes closely related to S. affine, although the underlying killing mechanisms may vary. Together, these data demonstrate that bacterial symbionts can modulate competition between their hosts, and reinforce specificity in mutualistic interactions.

Transmission Success of Entomopathogenic Nematodes Used in Pest Control.

Entomopathogenic nematodes from the two genera Steinernema and Heterorhabditis are widely used as biological agents against various insect pests and represent a promising alternative to replace pesticides. Efficacy and biocontrol success can be enhanced through improved understanding of their biology and ecology. Many endogenous and environmental factors influence the survival of nematodes following application, as well as their transmission success to the target species. The aim of this paper is to give an overview of the major topics currently considered to affect transmission success of these biological control agents, including interactions with insects, plants and other members of the soil biota including conspecifics.

The pathogenicity and reproduction capability of five epn strains on white grubs (Alissonotum impressicolle Arrow) in the laboratory conditions

Five entomopathogenic nematode strains, included 3 strains of the genus Steinernema, e.g. S-PQ16, SS-CP12 and S-TX1, and two strains of genus Heterorhabditis indica, e.g. H-CB3452 and H-KT3987 were evaluated on virulence and reproduction capacity on white grubs of black scarab (Alissonotum impressicolle Arrow) a serious pest damaging in the soils of sugarcane and many economic crops in Western Highland, particular Lam Dong province. The experiments were evaluated the pathogenicity virulence of EPN strains through establishing index as lethal concentration of 50 mortality percentage of host insects (LC50). While the reproduction capacity of EPN strains were established yield of infective juveniles (IJs) that produced inside insect cadavers. The bioassays on virulence of S-PQ16, S-CP12, S-TX1, H-CB3452 and H-KT3987 were revealed the mortality of white grubs as 93.3%, 86.7%; 93.3%; 86.7% and 73.3%, respectively, at the highest concentration of 5,000 IJs/insect. The 50% mortality of five indigenous strains was high levels with LC50 values ranged between 1,362 and 2,725 IJs. These values are also similar with our results previously on white grubs with some other EPN indigenous strains. It is also suitable with some evaluation bioassays of EPN on white grubs reported from China and Australia. The IJs yields from the insect cadavers were obtained up to 31×103 IJs with the strain S- CP12; 59.7×103 IJs with the S-PQ16 strain and 73.5×103 IJs with the strain S-TX1. Particularly, the highest yields were obtained from two strains of Heterorhabditis indica, viz. 125.1×103 IJs with H-KT3987 strain and 112.6×103 IJs with H-CB3452 strains. Respect to high virulence and also high reproduction capacity all these EPN strains should be satisfied the biological agents that can be used for biological control of white grubs of pests are living in the soil environment.

Entomopathogenic Nematodes and Fungi Virulence to Cowpea Curculio (Coleoptera: Curculionidae) Larvae1

Abstract Cowpea curculio, Chalcodermus aeneus (Boheman), is the major pest of southern peas or cowpea, Vigna unguiculata (L.). Throughout the southeastern United States, current management recommendations for cowpea curculio rely on insecticide applications; however, resistance has been observed. Entomopathogenic biocontrol agents, specifically nematodes in the genera Steinernema and Heterorhabditis and fungi in the genera Beauveria and Metarhizium, have shown efficacy with other curculio pests. We conducted laboratory bioassays to assess the virulence of entomopathogenic agents as a first step to provide alternative control strategies against cowpea curculio. Using controlled environmental conditions, we tested the effects of nematodes (Steinernema carpocapsae Weiser, Steinernema feltiae Filipjev, Heterorhabditis bacteriophora Poinar, and Heterorhabditis indica Poinar) and two fungi (Beauveria bassiana Balsamo and Metarhizium brunneum Petch) on survival and infectivity of cowpea curculio. Heterorhabditis...

Effect of Immersion Time on Efficacy of Entomopathogenic Nematodes against Engorged Females of Cattle Fever Tick,Rhipicephalus(=Boophilus)microplus

Abstract. Immersion was used to test adults of six species of entomopathogenic nematodes for their effects on virulence and reproduction of engorged females of southern cattle fever tick, Rhipicephalus microplus (Canestrini) (Deutch strain). The nematodes were in the genera Steinernema (Rhabditida: Steinernematidae) -- Steinernema carpocapsae (Weiser) (All strain), Steinernema feltiae Filipjev (SN strain), and Steinernema riobrave Cabanillas, Poinar, and Raulston (355 strain), and Heterorhabditis (Rhabditida: Heterorhabditidae) -- Heterorhabditis bacteriophora Poinar (VS strain), Heterorhabditis floridensis Nguyen, Gozel, Koppenhofer, and Adams (K22 strain), and Heterorhabditis indica Poinar, Karunakar, and David (HOM1 strain). Five engorged female ticks were immersed in a suspension of 5,000 infective juvenile nematodes per milliliter for various times (0.5 to 30 minutes), and the number of ticks killed over time, egg mass weight, reproductive index, and percentage of inhibition of oviposition were calcu...

Xenocyloin Derivatives from Liquid Cultures of Xenorhabdus bovienii SN52

Members of genus Xenorhabdus are symbiotically associated with entomopathogenic nematodes of genus Steinernema. Herein, we describe the isolation and purification of four novel metabolites, xenocyloins G-J (1–4), from liquid cultures of Xenorhabdus bovienii SN52 by extensive column chromatography and semi-preparative HPLC. Chemical structures of the xenocyloin derivatives were determined based on comprehensive NMR spectra and HR-ESI-MS analyses. All compounds exhibited significant collagen-induced anti-platelet aggregation activities, among which xenocyloin H (2) exhibited the strongest activity with an inhibition rate of 96.0 ± 0.1% at a concentration of 50 μM. The xenocyloin G (1) and H (2) exhibited anti-platelet activities with IC50 values which were 31.7 ± 4.4 μM and 27.5 ± 3.5 μM respectively; whilst under similar conditions the IC50 value for aspirin was 289.5 ± 15.7 μM, nearly ten times less than 1 and 2.

Comparative Transcriptomics of Steinernema and Caenorhabditis Single Embryos Reveals Orthologous Gene Expression Convergence during Late Embryogenesis.

Cells express distinct sets of genes in a precise spatio-temporal manner during embryonic development. There is a wealth of information on the deterministic embryonic development of Caenorhabditis elegans, but much less is known about embryonic development in nematodes from other taxa, especially at the molecular level. We are interested in insect pathogenic nematodes from the genus Steinernema as models of parasitism and symbiosis as well as a satellite model for evolution in comparison to C. elegans. To explore gene expression differences across taxa, we sequenced the transcriptomes of single embryos of two Steinernema species and two Caenorhabditis species at 11 stages during embryonic development and found several interesting features. Our findings show that zygotic transcription initiates at different developmental stages in each species, with the Steinernema species initiating transcription earlier than Caenorhabditis. We found that ortholog expression conservation during development is higher at the later embryonic stages than at the earlier ones. The surprisingly higher conservation of orthologous gene expression in later embryonic stages strongly suggests a funnel-shaped model of embryonic developmental gene expression divergence in nematodes. This work provides novel insight into embryonic development across distantly related nematode species and demonstrates that the mechanisms controlling early development are more diverse than previously thought at the transcriptional level.