A Mediator subunit imparts robustness to a polyphenism decision.

Polyphenism is a type of developmental plasticity that translates continuous environmental variability into discontinuous phenotypes. Such discontinuity likely requires a switch between alternative gene-regulatory networks, a principle that has been borne out by mechanisms found to promote morph-specific gene expression. However, whether robustness is required to execute a polyphenism decision has awaited testing at the molecular level. Here, we used a nematode model for polyphenism, Pristionchus pacificus, to identify the molecular regulatory factors that ensure the development of alternative forms. This species has a dimorphism in its adult feeding structures, specifically teeth, which are a morphological novelty that allows predation on other nematodes. Through a forward genetic screen, we determined that a duplicate homolog of the Mediator subunit MDT-15/MED15, P. pacificus MDT-15.1, is necessary for the polyphenism and the robustness of the resulting phenotypes. This transcriptional coregulator, which has a conserved role in metabolic responses to nutritional stress, coordinates these processes with its effects on this diet-induced polyphenism. Moreover, this MED15 homolog genetically interacts with two nuclear receptors, NHR-1 and NHR-40, to achieve dimorphism: Single and double mutants for these three factors result in morphologies that together produce a continuum of forms between the extremes of the polyphenism. In summary, we have identified a molecular regulator that confers discontinuity to a morphological polyphenism, while also identifying a role for MED15 as a plasticity effector.

OxyR is required for oxidative stress resistance of the entomopathogenic bacterium Xenorhabdus nematophila and has a minor role during the bacterial interaction with its hosts.

Xenorhabdus nematophila is a Gram-negative bacterium, mutualistically associated with the soil nematode Steinernema carpocapsae, and this nemato-bacterial complex is parasitic for a broad spectrum of insects. The transcriptional regulator OxyR is widely conserved in bacteria and activates the transcription of a set of genes that influence cellular defence against oxidative stress. It is also involved in the virulence of several bacterial pathogens. The aim of this study was to identify the X. nematophila OxyR regulon and investigate its role in the bacterial life cycle. An oxyR mutant was constructed in X. nematophila and phenotypically characterized in vitro and in vivo after reassociation with its nematode partner. OxyR plays a major role during the X. nematophila resistance to oxidative stress in vitro. Transcriptome analysis allowed the identification of 59 genes differentially regulated in the oxyR mutant compared to the parental strain. In vivo, the oxyR mutant was able to reassociate with the nematode as efficiently as the control strain. These nemato-bacterial complexes harbouring the oxyR mutant symbiont were able to rapidly kill the insect larvae in less than 48 h after infestation, suggesting that factors other than OxyR could also allow X. nematophila to cope with oxidative stress encountered during this phase of infection in insect. The significantly increased number of offspring of the nemato-bacterial complex when reassociated with the X. nematophila oxyR mutant compared to the control strain revealed a potential role of OxyR during this symbiotic stage of the bacterial life cycle.

A Nematode Isolate, Oscheius Tipulae, Exhibiting a Wide Entomopathogenic Spectrum and its Application to Control Dipteran Insect Pests.

An entomopathogenic nematode, Oscheius tipulae, was isolated from a soil sample. The identification of this species was supported by morphological and molecular markers. The nematode isolate exhibited pathogenicity against different target insects including lepidopteran, coleopteran, and dipteran insects. The virulence of this nematode was similar to that of a well-known entomopathogenic nematode, Steinernema carpocapsae, against the same insect targets. A comparative metagenomics analysis of these two nematode species predicted the existence of a combined total of 272 bacterial species in their intestines, of which 51 bacterial species were shared between the two nematode species. In particular, the common gut bacteria included several entomopathogenic bacteria including Xenorhabdus nematophila, which is known as a symbiotic bacterium to S. carpocapsae. The nematode virulence of O. tipulae to insects was enhanced by an addition of dexamethasone but suppressed by an addition of arachidonic acid, suggesting that the immune defenses of the target insects against the nematode infection is mediated by eicosanoids, which would be manipulated by the symbiotic bacteria of the nematode. Unlike S. carpocapsae, O. tipulae showed high virulence against dipteran insects including fruit flies, onion flies, and mosquitoes. O. tipulae showed particularly high control efficacies against the onion maggot, Delia platura, infesting the Welsh onion in the rhizosphere in both pot and field assays.

Metabolic disruptions in Biomphalaria glabrata induced by Heterorhabditis bacteriophora HP88: Implications for entomopathogenic nematodes in biological control.

Research on the use of entomopathogenic nematodes (EPNs) as a potential tool for the biological control of invertebrates has been growing in recent years, including studies involving snails with One Health importance. In this study, the effect of exposure time (24 or 48 h) of Heterorhabditis bacteriophora HP88 on the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as well as the concentration of total proteins, uric acid, and urea in the hemolymph of Biomphalaria glabrata, were investigated. The concentrations of these metabolic markers were measured weekly until the end of the third week after exposure. Along with a significant reduction in total protein levels, a significant increase (p < 0.01) in uric acid and urea contents in the hemolymph of B. glabrata exposed to H. bacteriophora was observed. The accumulation of urea in these mollusks could lead to deleterious effects due to its high toxicity, inducing significant cell damage. Variations in transaminase activities were also observed, with snails exposed to EPNs showing significantly higher values (p < 0.01) than individuals in the control group, both for ALT and AST. These results indicate that experimental exposure to infective juveniles of H. bacteriophora causes significant alterations in the metabolic pattern of B. glabrata, compromising the maintenance of its homeostasis. Finally, exposure for 48 h caused more damage to the planorbid in question compared to snails exposed for 24 h, suggesting that the exposure time may influence the intensity of the host's response.

Behavioral and molecular response of the insect parasitic nematode Steinernema carpocapsae to plant volatiles.

Entomopathogenic nematodes (EPNs) use the chemical cues emitted by insects and insect-damaged plants to locate their hosts. Steinernema carpocapsae, a species of EPN, is an established biocontrol agent used against insect pests. Despite its promising potential, the molecular mechanisms underlying its ability to detect plant volatiles remain poorly understood. In this study, we investigated the response of S. carpocapsae infective juveniles (IJs) to 8 different plant volatiles. Among these, carvone was found to be the most attractive volatile compound. To understand the molecular basis of the response of IJs to carvone, we used RNA-Seq technology to identify gene expression changes in response to carvone treatment. Transcriptome analysis revealed 721 differentially expressed genes (DEGs) between carvone-treated and control groups, with 403 genes being significantly upregulated and 318 genes downregulated. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the responsive DEGs to carvone attraction were mainly involved in locomotion, localization, behavior, response to stimulus, and olfactory transduction. We also identified four upregulated genes of chemoreceptor and response to stimulus that were involved in the response of IJs to carvone attraction. Our results provide insights into the potential transcriptional mechanisms underlying the response of S. carpocapsae to carvone, which can be utilized to develop environmentally friendly strategies for attracting EPNs.

Long-term suppression of turfgrass insect pests with native persistent entomopathogenic nematodes.

Entomopathogenic nematodes (EPNs) can control several important turfgrass insect pests including white grubs, weevils, cutworms, and sod webworms. But most of the research has focused on inundative releases in a biopesticide strategy using EPN strains that may have lost some of their ability to persist effectively over years of lab maintenance and / or selection for virulence and efficient mass-production. Our study examined the potential of fresh field isolate mixes of endemic EPNs to provide multi-year suppression of turfgrass insect pests. In early June 2020, we applied isolate mixes from golf courses of the EPNs Steinernema carpocapsae, Heterorhabditis bacteriophora, and their combination to plots straddling fairway and rough on two golf courses in central New Jersey, USA. Populations of EPNs and insect pests were sampled on the fairway and rough side of the plots from just before EPN application until October 2022. EPN populations increased initially in plots treated with the respective species. Steinernema carpocapsae densities stayed high for most of the experiment. Heterorhabditis bacteriophora densities decreased after 6 months and stabilized at lower levels. Several insect pests were reduced across the entire experimental period. In the fairway, the combination treatment reduced annual bluegrass weevil larvae (59 % reduction) and adults (74 %); S. carpocapsae reduced only adults (42 %). White grubs were reduced by H. bacteriophora (67 %) and the combination (63 %). Black turfgrass ataenius adults were reduced in all EPN treatments (43-62 %) in rough and fairway. Sod webworm larvae were reduced by S. carpocapsae in the fairway (75 %) and the rough (100 %) and by H. bacteriophora in the rough (75 %). Cutworm larvae were reduced in the fairway by S. carpocapsae (88 %) and the combination (75 %). Overall, our observations suggest that inoculative applications of fresh field isolate mixes of endemic EPNs may be a feasible approach to long-term suppression of insect pests in turfgrass but may require periodic reapplications.

The sky is not the limit: Successful foliar application of Steinernema spp. entomopathogenic nematodes to control Lepidopteran caterpillars.

Entomopathogenic nematodes (EPNs) are ubiquitous soil-thriving organisms that use chemical cues to seek and infect soil-dwelling arthropods, yielding various levels of biological control. Going beyond soil application, scientists and practitioners started exploring the option of applying EPNs onto the foliage of crops in attempts to manage leaf-dwelling insect pests as well. Despite some success, particularly with protective formulations, it remains uncertain whether EPNs could indeed survive the phyllospheric environment, and successfully control foliar insect pests. In this context, we tested the potential of commercially produced Steinernema feltiae and S. carpocapsae, two of the most commonly used EPNs in the field of biological control, in controlling Lepidopteran foliar pests of economic importance, i.e. Tuta absoluta and Spodoptera spp. caterpillars as models. We first tested the survival and efficacy of both EPN species against the Lepidopteran caterpillars when applied onto tomato, sweet pepper and lettuce leaves, under controlled conditions and in commercial greenhouse conditions, respectively. Subsequently, we explored the behavioural responses of the EPNs to environmental cues typically encountered in the phyllosphere, and analysed plant volatile organic compounds (VOCs). Our results show that both S. feltiae and S. carpocapsae successfully survived and infected the foliar caterpillars, reaching similar level of control to a standard chemical pesticide in commercial practices. Remarkably, both EPN species survived and remained effective up to four days in the phyllosphere, and needed only a few hours to successfully penetrate the caterpillars. Interestingly, S. feltiae was attracted to VOCs from tomato plants, and tended to prefer those from caterpillar-induced plants, suggesting that the nematodes may actively forage toward its host, although it has never been exposed to leaf-borne volatiles during its evolution. The present study shows the high potential of steinernematids in managing major foliar pests in greenhouses and in becoming a key player in foliar biological control. In particular, the discovery that EPNs use foliar VOCs to locate caterpillar hosts opens up new opportunities in terms of application techniques and affordable effective doses.

Exploring the effects of entomopathogenic nematode symbiotic bacteria and their cell free filtrates on the tomato leafminer Tuta absoluta and its predator Nesidiocoris tenuis.

The use of biocontrol agents, such as predators and entomopathogenic nematodes, is a promising approach for the effective control of the tomato leafminer Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidaean), an oligophagous insect feeding mainly on Solanaceae species and a major pest of field- and greenhouse-grown tomatoes globally. In this context, the effects of two entomopathogenic nematode species Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae), as well as their respective bacterial symbionts, Xenorhabdus nematophila and Photorhabdus luminescens (Enterobacterales: Morganelaceae), which were applied as bacterial cell suspensions and as crude cell-free liquid filtrates on T. absoluta larvae, were investigated. The results showed that of all treatments, the nematodes S. carpocapsae and H. bacteriophora were the most effective, causing up to 98 % mortality of T. absoluta larvae. Regarding bacteria and their filtrates, the bacterium X. nematophila was the most effective (69 % mortality in young larvae), while P. luminescens and both bacterial filtrates showed similar potency (ca. 48-55 % mortality in young larvae). To achieve a holistic approach of controlling this important pest, the impact of these factors on the beneficial predator Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) was also studied. The results demonstrated that although nematodes and especially S. carpocapsae, caused significant mortality on N. tenuis (87 %), the bacterial cell suspensions of X. nematophila and P. luminescens and crude cell-free liquid filtrates had minimum impact on this beneficial predator (∼11-30 % mortality).

Toxicological impacts of microplastics on virulence, reproduction and physiological process of entomopathogenic nematodes.

Microplastics have emerged as significant and concerning pollutants within soil ecosystems. Among the soil biota, entomopathogenic nematodes (EPNs) are lethal parasites of arthropods, and are considered among the most effective biological agents against pests. Infective juveniles (IJs) of EPNs, as they navigate the soil matrix scavenging for arthropod hosts to infect, they could potentially encounter microplastics. Howver, the impact of microplastics on EPNs has not been fully elucidated yet. We addressed this gap by subjecting Steinernema feltiae EPNs to polystyrene microplastics (PS-MPs) with various sizes, concentrations, and exposure durations. After confirming PS-MP ingestion by S. feltiae using fluorescent dyes, we found that the PS-MPs reduced the survival, reproduction, and pathogenicity of the tested EPNs, with effects intensifying for smaller PS-MPs (0.1-1 µm) at higher concentrations (105 µg/L). Furthermore, exposure to PS-MPs triggered oxidative stress in S. feltiae, leading to increased reactive oxygen species levels, compromised mitochondrial membrane potential, and increased antioxidative enzyme activity. Furthermore, transcriptome analyses revealed PS-MP-induced suppression of mitochondrial function and oxidative phosphorylation pathways. In conclusion, we show that ingestion of PS-MPs by EPNs can compromise their fitness, due to multple toxicity effects. Our results bear far-reaching consequences, as the presence of microplastics in soil ecosystems could undermine the ecological role of EPNs in regulating pest populations.

The Activin Branch Ligand Daw Regulates the Drosophila melanogaster Immune Response and Lipid Metabolism against the Heterorhabditis bacteriophora Serine Carboxypeptidase.

Despite impressive advances in the broad field of innate immunity, our understanding of the molecules and signaling pathways that control the host immune response to nematode infection remains incomplete. We have shown recently that Transforming Growth Factor-ß (TGF-ß) signaling in the fruit fly Drosophila melanogaster is activated by nematode infection and certain TGF-ß superfamily members regulate the D. melanogaster anti-nematode immune response. Here, we investigate the effect of an entomopathogenic nematode infection factor on host TGF-ß pathway regulation and immune function. We find that Heterorhabditis bacteriophora serine carboxypeptidase activates the Activin branch in D. melanogaster adults and the immune deficiency pathway in Activin-deficient flies, it affects hemocyte numbers and survival in flies deficient for Activin signaling, and causes increased intestinal steatosis in Activin-deficient flies. Thus, insights into the D. melanogaster signaling pathways and metabolic processes interacting with H. bacteriophora pathogenicity factors will be applicable to entomopathogenic nematode infection of important agricultural insect pests and vectors of disease.

Biocontrol potential of six Heterorhabditis bacteriophora strains isolated in the Azores Archipelago.

Entomopathogenic nematodes (EPNs) are closely associated with Popillia japonica and potentially used as their biological control agents, although field results proved inconsistent and evoked a continual pursuit of native EPNs more adapted to the environment. Therefore, we surveyed the Azorean Archipelago to isolate new strains of Heterorhabditis bacteriophora and to evaluate their virulence against the model organism Galleria mellonella under laboratory conditions. Six strains were obtained from pasture and coastal environments and both nematode and symbiont bacteria were molecularly identified. The bioassays revealed that Az172, Az186, and Az171 presented high virulence across the determination of a lethal dose (LD50) and short exposure time experiments with a comparable performance to Az29. After 72 hours, these virulent strains presented a mean determination of a lethal dose of 11 infective juveniles cm-2, a lethal time (LT50) of 34 hours, and achieved 40% mortality after an initial exposure time of only 60 minutes. Az170 exhibited an intermediate performance, whereas Az179 and Az180 were classified as low virulent strains. However, both strains presented the highest reproductive potential with means of 1700 infective juveniles/mg of larvae. The bioassays of the native EPNs obtained revealed that these strains hold the potential to be used in biological control initiatives targeting P. japonica because of their high virulence and locally adapted to environmental conditions.

Conserved nuclear hormone receptors controlling a novel plastic trait target fast-evolving genes expressed in a single cell.

Environment shapes development through a phenomenon called developmental plasticity. Deciphering its genetic basis has potential to shed light on the origin of novel traits and adaptation to environmental change. However, molecular studies are scarce, and little is known about molecular mechanisms associated with plasticity. We investigated the gene regulatory network controlling predatory vs. non-predatory dimorphism in the nematode Pristionchus pacificus and found that it consists of genes of extremely different age classes. We isolated mutants in the conserved nuclear hormone receptor nhr-1 with previously unseen phenotypic effects. They disrupt mouth-form determination and result in animals combining features of both wild-type morphs. In contrast, mutants in another conserved nuclear hormone receptor nhr-40 display altered morph ratios, but no intermediate morphology. Despite divergent modes of control, NHR-1 and NHR-40 share transcriptional targets, which encode extracellular proteins that have no orthologs in Caenorhabditis elegans and result from lineage-specific expansions. An array of transcriptional reporters revealed co-expression of all tested targets in the same pharyngeal gland cell. Major morphological changes in this gland cell accompanied the evolution of teeth and predation, linking rapid gene turnover with morphological innovations. Thus, the origin of feeding plasticity involved novelty at the level of genes, cells and behavior.

Multiple origins of obligate nematode and insect symbionts by a clade of bacteria closely related to plant pathogens.

Obligate symbioses involving intracellular bacteria have transformed eukaryotic life, from providing aerobic respiration and photosynthesis to enabling colonization of previously inaccessible niches, such as feeding on xylem and phloem, and surviving in deep-sea hydrothermal vents. A major challenge in the study of obligate symbioses is to understand how they arise. Because the best studied obligate symbioses are ancient, it is especially challenging to identify early or intermediate stages. Here we report the discovery of a nascent obligate symbiosis in Howardula aoronymphium, a well-studied nematode parasite of Drosophila flies. We have found that Haoronymphium and its sister species harbor a maternally inherited intracellular bacterial symbiont. We never find the symbiont in nematode-free flies, and virtually all nematodes in the field and the laboratory are infected. Treating nematodes with antibiotics causes a severe reduction in fly infection success. The association is recent, as more distantly related insect-parasitic tylenchid nematodes do not host these endosymbionts. We also report that the Howardula nematode symbiont is a member of a widespread monophyletic group of invertebrate host-associated microbes that has independently given rise to at least four obligate symbioses, one in nematodes and three in insects, and that is sister to Pectobacterium, a lineage of plant pathogenic bacteria. Comparative genomic analysis of this group, which we name Candidatus Symbiopectobacterium, shows signatures of genome erosion characteristic of early stages of symbiosis, with the Howardula symbiont's genome containing over a thousand predicted pseudogenes, comprising a third of its genome.

Plant defense resistance in natural enemies of a specialist insect herbivore.

Plants defend themselves against herbivores through the production of toxic and deterrent metabolites. Adapted herbivores can tolerate and sometimes sequester these metabolites, allowing them to feed on defended plants and become toxic to their own enemies. Can herbivore natural enemies overcome sequestered plant defense metabolites to prey on adapted herbivores? To address this question, we studied how entomopathogenic nematodes cope with benzoxazinoid defense metabolites that are produced by grasses and sequestered by a specialist maize herbivore, the western corn rootworm. We find that nematodes from US maize fields in regions in which the western corn rootworm was present over the last 50 y are behaviorally and metabolically resistant to sequestered benzoxazinoids and more infective toward the western corn rootworm than nematodes from other parts of the world. Exposure of a benzoxazinoid-susceptible nematode strain to the western corn rootworm for 5 generations results in higher behavioral and metabolic resistance and benzoxazinoid-dependent infectivity toward the western corn rootworm. Thus, herbivores that are exposed to a plant defense sequestering herbivore can evolve both behavioral and metabolic resistance to plant defense metabolites, and these traits are associated with higher infectivity toward a defense sequestering herbivore. We conclude that plant defense metabolites that are transferred through adapted herbivores may result in the evolution of resistance in herbivore natural enemies. Our study also identifies plant defense resistance as a potential target for the improvement of biological control agents.

Chemical Cues from Entomopathogenic Nematodes Vary Across Three Species with Different Foraging Strategies, Triggering Different Behavioral Responses in Prey and Competitors.

Chemical cues play important roles in predator-prey interactions. Semiochemicals can aid predator foraging and alert prey organisms to the presence of predators. Previous work suggests that predator traits differentially influence prey behavior, however, empirical data on how prey organisms respond to chemical cues from predator species with different hunting strategies, and how foraging predators react to cues from potential competitors, is lacking. Furthermore, most research in this area has focused on aquatic and aboveground terrestrial systems, while interactions among belowground, soiling-dwelling organisms have received relatively little attention. Here, we assessed how chemical cues from three species of entomopathogenic nematodes (EPNs), each with a different foraging strategy, influenced herbivore (cucumber beetle) and natural enemy (EPN) foraging behavior. We predicted these cues could serve as chemical indicators of increased predation risk, prey availability, or competition. Our findings revealed that foraging cucumber beetle larvae avoided chemical cues from Heterorhabditis bacteriophora (active-foraging cruiser EPNs), but not Steinernema carpocapsae (ambusher EPNs) or Steinernema riobrave (intermediate-foraging EPNs). In contrast, foraging H. bacteriophora EPNs were attracted to cues produced by the two Steinernema species but not conspecific cues. Notably, the three EPN species produced distinct blends of olfactory cues, with only a few semi-conserved compounds across species. These results indicate that a belowground insect herbivore responds differently to chemical cues from different EPN species, with some EPN species avoiding prey detection. Moreover, the active-hunting EPNs were attracted to heterospecific cues, suggesting these cues indicate a greater probability of available prey, rather than strong interspecific competition.

Physiological alterations in Pseudosuccinea columella (Mollusca: Gastropoda) after infection by Heterorhabditis baujardi LPP7 (Rhabditida: Heterorhabditidae).

The snail Pseudosuccinea columella participates in the distribution of Fasciola hepatica in the environment by acting as its intermediate host. Therefore, the control of this lymnaeid is one of the ways to prevent hepatic fascioliasis. The objective of this study was to evaluate the susceptibility of P. columella to infective juveniles (IJs) of the entomopathogenic nematode (EPN) Heterorhabditis baujardi in laboratory conditions, as well as to investigate aspects related to the biochemistry and histopathology of snails exposed or not to the EPNs during three weeks. The EPN exposure induced significant reductions in the concentrations of glucose, total proteins and glycogen (gonad-digestive gland complex) in the snails during the onset of the infection, with the levels being restored as the infection progresses. These alterations were accompanied by increased hemolymph activities of aminotransferases and lactate dehydrogenase, as well as the concentrations of uric acid after the first and second weeks of the experiment. The histopathological analyses of the exposed snails revealed cell necrosis at the end of the first week, tissue inflammatory reactions one and two weeks after exposure, and degeneration three weeks afterward in comparison with the unexposed snails. Finally, scanning electronic microscopy revealed proliferation of fibrous connective tissue three weeks after exposure. The results indicate that P. columella is susceptible to H. baujardi. The exposure favored the establishment of a negative energy balance, increased the activity of enzymes related to tissue damages and promoted accumulation of nitrogen compounds in the host snails. Additionally, was observed in P. columella exposed to the EPNs, significant tissue lesions, and demonstrated the strong pathogenic potential of H. baujardi, indicating its possible application for biological control of this snail.

Evaluation of Barricade® to enhance survival of entomopathogenic nematodes on cowhide.

Entomopathogenic nematodes (EPNs), Steinernema riobrave and Heterorhabditis floridensis are under evaluation for eradication of the southern cattle fever tick, Rhipicephalus microplus infesting nilgai antelope, in South Texas. Cattle fever ticks are a significant threat to the U.S. livestock industry. Although they have been eradicated in the U.S. they frequently re-invade along the Texas-Mexico border. Remotely operated field sprayers have been developed to directly treat nilgai antelope with EPNs as they transit fence crossings and as they contact wetted foliage and soil from the surrounding area. EPNs are known to be susceptible to mortality from ultraviolet light (UV) and desiccation. A sprayable fire gel, Barricade®, has been reported to protect EPNs from UV and desiccation but has not been tested on animal hides. Barricade® at 1 and 2 percent rates was mixed with the water solution of S. riobrave and H. floridensis and applied to cowhides (to mimic direct treatment of nilgai) and filter paper and then these substrates were placed out of doors in 0, 30, 60 or 120 min of sunlight. Wax moth larvae, Galleria mellonella, were exposed to the cowhides and filter paper to determine efficacy of the EPNs. Efficacy of S. riobrave with 1 and 2% Barricade® gel applied to cowhides was significantly improved at 30 and 60 min as compared to the control. At 120 min mortality of the wax moth larvae was near zero for both the control and the treatments. Similar results were found with the filter paper test. In contrast, efficacy of H. floridensis with Barricade® applied to cowhides or filter paper was not significantly improved at 30, 60 or 120 min as compared to the water only control. Barricade® has the potential to improve the efficacy of S. riobrave and other EPNs by reducing mortality and desiccation, especially when used in the remotely operated sprayer developed for treatment of cattle fever tick infested nilgai.

Earthworms and their cutaneous excreta can modify the virulence and reproductive capability of entomopathogenic nematodes and fungi.

Earthworms are ecological engineers that can contribute to the displacement of biological control agents such as the entomopathogenic nematodes (EPNs) and fungi (EPF). However, a previous study showed that the presence of cutaneous excreta (CEx) and feeding behavior of the earthworm species Eisenia fetida (Haplotaxida: Lumbricidae) compromise the biocontrol efficacy of certain EPN species by reducing, for example, their reproductive capability. Whether this phenomenon is a general pattern for the interaction of earthworms-entomopathogens is still unknown. We hypothesized that diverse earthworm species might differentially affect EPN and EPF infectivity and reproductive capability. Here we investigated the interaction of different earthworm species (Eisenia fetida, Lumbricus terrestris, and Perionyx excavatus) (Haplotaxida) and EPN species (Steinernema feltiae, S. riojaense, and Heterorhabditis bacteriophora) (Rhabditida) or EPF species (Beauveria bassiana and Metarhizium anisopliae) (Hypocreales), in two independent experiments. First, we evaluated the application of each entomopathogen combined with earthworms or their CEx in autoclaved soil. Hereafter, we studied the impact of the earthworms' CEx on entomopathogens applied at two different concentrations in autoclaved sand. Overall, we found that the effect of earthworms on entomopathogens was species-specific. For example, E. fetida reduced the virulence of S. feltiae, resulted in neutral effects for S. riojaense, and increased H. bacteriophora virulence. However, the earthworm P. excavates increased the virulence of S. feltiae, reduced the activity of H. bacteriophora, at least at specific timings, while S. riojaense remained unaffected. Finally, none of the EPN species were affected by the presence of L. terrestris. Also, the exposure to earthworm CEx resulted in a positive, negative or neutral effect on the virulence and reproduction capability depending on the earthworm-EPN species interaction. Concerning EPF, the impact of earthworms was also differential among species. Thus, E. fetida was detrimental to M. anisopliae and B. bassiana after eight days post-exposure, whereas Lumbricus terrestris resulted only detrimental to B. bassiana. In addition, most of the CEx treatments of both earthworm species decreased B. bassiana virulence and growth. However, the EPF M. anisopliae was unaffected when exposed to L. terrestris CEx, while the exposure to E. fetida CEx produced contrasting results. We conclude that earthworms and their CEx can have positive, deleterious, or neutral impacts on entomopathogens that often coinhabit soils, and that we must consider the species specificity of these interactions for mutual uses in biological control programs. Additional studies are needed to verify these interactions under natural conditions.

Characterization of bacterial communities associated with the pinewood nematode insect vector Monochamus alternatus Hope and the host tree Pinus massoniana.

BACKGROUND: Monochamus alternatus Hope is one of the insect vectors of pinewood nematode (Bursaphelenchus xylophilus), which causes the destructive pine wilt disease. The microorganisms within the ecosystem, comprising plants, their environment, and insect vectors, form complex networks. This study presents a systematic analysis of the bacterial microbiota in the M. alternatus midgut and its habitat niche. METHODS: Total DNA was extracted from 20 types of samples (with three replicates each) from M. alternatus and various tissues of healthy and infected P. massoniana (pines). 16S rDNA amplicon sequencing was conducted to determine the composition and diversity of the bacterial microbiota in each sample. Moreover, the relative abundances of bacteria in the midgut of M. alternatus larvae were verified by counting the colony-forming units. RESULTS: Pinewood nematode infection increased the microbial diversity in pines. Bradyrhizobium, Burkholderia, Dyella, Mycobacterium, and Mucilaginibacter were the dominant bacterial genera in the soil and infected pines. These results indicate that the bacterial community in infected pines may be associated with the soil microbiota. Interestingly, the abundance of the genus Gryllotalpicola was highest in the bark of infected pines. The genus Cellulomonas was not found in the midgut of M. alternatus, but it peaked in the phloem of infected pines, followed by the phloem of heathy pines. Moreover, the genus Serratia was not only present in the habitat niche, but it was also enriched in the M. alternatus midgut. The colony-forming unit assays showed that the relative abundance of Serratia sp. peaked in the midgut of instar II larvae (81%). CONCLUSIONS: Overall, the results indicate that the bacterial microbiota in the soil and in infected pines are correlated. The Gryllotalpicola sp. and Cellulomonas sp. are potential microbial markers of pine wilt disease. Additionally, Serratia sp. could be an ideal agent for expressing insecticidal protein in the insect midgut by genetic engineering, which represents a new use of microbes to control M. alternatus.

Prevalence and parasite load of nematodes and trematodes in an invasive slug and its susceptibility to a slug parasitic nematode compared to native gastropods.

The invasive slug Arion vulgaris (Gastropoda: Arionidae) is an agricultural pest and serious nuisance in gardens of Central and Northern Europe. To investigate if the success of A.vulgaris in Norway can be attributed to a release from parasites, we compared the prevalence and parasite load of nematodes and trematodes in A. vulgaris to that of three native gastropod species, A. circumscriptus, A. fasciatus and Arianta arbustorum, in SE Norway. We found A. vulgaris to have the highest prevalence of both parasite groups (49% nematodes, 76% trematodes), which does not support the parasite release hypothesis, but rather points to A. vulgaris as a potentially important intermediate host of these parasites. For trematodes the number of individuals (parasite load) did not differ among host species; for nematodes it was higher in A. vulgaris than A. fasciatus. To further compare the parasite susceptibility of the surveyed gastropods, we exposed A. vulgaris, A. fasciatus, and A. arbustorum to a slug parasitic nematode, Phasmarhabditis hermaphrodita, in the laboratory. This nematode is commercially available and widely used to control A. vulgaris. The non-target species A. fasciatus was most affected, with 100% infection, 60% mortality and significant feeding inhibition. A. vulgaris was also 100% infected, but suffered only 20% mortality and little feeding inhibition. The load of P. hermaphrodita in infected specimens was not significantly different for the two Arion species (median: 22.5 and 45, respectively). Only 35% of A. arbustorum snails were infected, none died, and parasite load was very low (median: 2). However, they showed a near complete feeding inhibition at highest nematode dose, and avoided nematode-infested soil. Our results indicate that A. vulgaris may be less susceptible to P. hermaphrodita than the native A. fasciatus, and that non-target effects of applying this nematode in fields and gardens should be further investigated.