PHASMARHABDITIS CALIFORNICA (NEMATODA: RHABDITIDAE) HAS REDUCED ESTABLISHMENT SUCCESS AND PROGENY PRODUCTION IN THE PRESENCE OF PRISTIONCHUS ENTOMOPHAGUS (NEMATODA: DIPLOGASTRIDAE).

Phasmarhabditis (syn. Pellioditis) californica is a facultative parasite that has been marketed as a popular biocontrol agent against pestiferous slugs in England, Scotland, and Wales. The necromenic nematode Pristionchus entomophagus has also been recovered from slugs infected with Ph. californica. In this study, we experimentally investigated the outcome of single and mixed applications of Pr. entomophagus and Ph. californica on the slug Deroceras reticulatum (Müller). Host mortality was comparable for single and mixed applications of Ph. californica, with time to death significantly shorter in both treatment groups compared with controls. However, trials with Pr. entomophagus alone did not cause any significant host mortality relative to controls. Compared with the single Ph. californica applications, mixed applications resulted in 67% fewer infective juveniles establishing in the host, and subsequently far fewer infective juveniles were recovered in the next generation. In contrast, the establishment rate and progeny production in Pr. entomophagus were not impacted by the presence of Ph. californica (i.e., mixed applications). Hence, the presence of Pr. entomophagus had a deleterious effect on the establishment success and progeny production of Ph. californica. Our findings reveal an asymmetrical, antagonistic interaction between Ph. californica and Pr. entomophagus and highlight the importance of understanding the ecological relationships between co-occurring species. A decrease in parasite establishment success and progeny production has the potential to directly impact the persistence, sustainability, and efficacy of Ph. californica as a biological control agent.

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.

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.

Heterorhabditis indica (Nematoda: Rhabditida) a possible new biological control agent against the vector of Chagas disease.

Chagas disease is a zoonosis caused by the protozoan Trypanosoma cruzi and transmitted through the feces of triatomines, mainly in Latin America. Since the 1950s, chemical insecticides have been the primary method for controlling these triatomines, yet resistance has emerged, prompting the exploration of alternative approaches. The objective of this research was to test the capacity of the entomopathogenic nematodes Heterorhabditis indica and its symbiotic bacteria Photorhabdus luminescens, to produce mortality of Triatoma dimidiata a key vector of T. cruzi in Mexico under laboratory conditions. Two bioassays were conducted. In the first bioassay, the experimental unit was a 250 ml plastic jar with 100 g of sterile soil and three adult T. dimidiata. Three nematode quantities were tested: 2250, 4500, and 9000 nematodes per 100 g of sterile soil (n/100 g) per jar, with 3 replicates for each concentration and 1 control per concentration (1 jar with 100 g of sterile soil and 3 T. dimidiata without nematodes). The experimental unit of the second bioassay was a 500 ml plastic jar with 100 g of sterile soil and 4 adult T. dimidiata. This bioassay included 5, 50, 500, and 5000 n/100 g of sterile soil per jar, with 3 replicates of each quantity and 1 control per quantity. Data were analyzed using Kaplan-Meyer survival analysis. Electron microscopy was used to assess the presence of nematodes and tissue damage in T. dimidiata. The results of the first bioassay demonstrated that the nematode induced an accumulated average mortality ranging from 55.5 % (2250 n/100 g) to 100 % (4500 and 9000 n/100 g) within 144 h. In the second bioassay, the 5000 n/100 g concentration yielded 87.5 % mortality at 86 h, but a concentration as small as 500 n/100 g caused 75 % mortality from 84 h onwards. Survival analysis indicated higher T. dimidiata mortality with increased nematode quantities, with significant differences between the 4500, 5000, and 9000 n/100 g and controls. Electron microscopy revealed the presence of nematodes and its presumably symbiotic bacteria in the digestive system of T. dimidiata. Based on these analyses, we assert that the H. indica and P. luminescens complex causes mortality in adult T. dimidiata under laboratory conditions.

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.

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.

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.

Entomopathogenic potential of bacteria associated with soil-borne nematodes and insect immune responses to their infection.

Soil-borne nematodes establish close associations with several bacterial species. Whether they confer benefits to their hosts has been investigated in only a few nematode-bacteria systems. Their ecological function, therefore, remains poorly understood. In this study, we isolated several bacterial species from rhabditid nematodes, molecularly identified them, evaluated their entomopathogenic potential on Galleria mellonella larvae, and measured immune responses of G. mellonella larvae to their infection. Bacteria were isolated from Acrobeloides sp., A. bodenheimeri, Heterorhabditis bacteriophora, Oscheius tipulae, and Pristionchus maupasi nematodes. They were identified as Acinetobacter sp., Alcaligenes sp., Bacillus cereus, Enterobacter sp., Kaistia sp., Lysinibacillus fusiformis, Morganella morganii subsp. morganii, Klebsiella quasipneumoniae subsp. quasipneumoniae, and Pseudomonas aeruginosa. All bacterial strains were found to be highly entomopathogenic as they killed at least 53.33% G. mellonella larvae within 72h post-infection, at a dose of 106 CFU/larvae. Among them, Lysinibacillus fusiformis, Enterobacter sp., Acinetobacter sp., and K. quasipneumoniae subsp. quasipneumoniae were the most entomopathogenic bacteria. Insects strongly responded to bacterial infection. However, their responses were apparently little effective to counteract bacterial infection. Our study, therefore, shows that bacteria associated with soil-borne nematodes have entomopathogenic capacities. From an applied perspective, our study motivates more research to determine the potential of these bacterial strains as biocontrol agents in environmentally friendly and sustainable agriculture.

Role of adipokinetic hormone in the Colorado potato beetle, Leptinotarsa decemlineata infected with the entomopathogenic nematode Steinernema carpocapsae.

The effects of the entomopathogenic nematode Steinernema carpocapsae on the Colorado potato beetle (CPB) Leptinotarsa decemlineata and the involvement of adipokinetic hormone (AKH) in the responsive reactions were examined in this study. It was observed that nematode application doubled the amount of AKH (Peram-CAH-I and Peram-CAH-II) in the central nervous system of L. decemlineata, indicating mobilization of anti-stress reactions in the body. Furthermore, the external co-application of Peram-CAH-II with the nematode significantly increased beetle mortality (5.6 and 1.8 times, 1 and 2 days after application, respectively). The mechanism underlying this phenomenon was investigated. As the effect on gut characteristics was equivocal, it was assumed that the nematodes profited from the observed mobilization of metabolites from the fat body into the Peram-CAH-II-induced hemolymph. This phenomenon supplied nematodes with a more nutrient-dense substrate on which they propagated. Furthermore, Peram-CAH-II lowered vitellogenin expression in the fat body, particularly in males, thus limiting the anti-pathogen defense capacity of the protein. However, there could be other possible mechanisms underpinning this chain of events. The findings could be theoretically intriguing but could also aid in developing real insect pest control methods in the future.

The effect of temperature conditioning (9°C and 20°C) on the proteome of entomopathogenic nematode infective juveniles.

Entomopathogenic nematodes (EPN) of the genera Steinernema and Heterorhabditis are parasites which kill and reproduce within insects. While both have life cycles centred around their developmentally arrested, nonfeeding and stress tolerant infective juvenile (IJ) stage, they are relatively distantly related. These IJs are promising biocontrol agents, and their shelf life and stress tolerance may be enhanced by storage at low temperatures. The purpose of this study was to investigate how the proteome of the IJs of two distantly related EPN species is affected by storage at 9°C (for up to 9 weeks) and 20°C (for up to 6 weeks), using label-free quantitative proteomics. Overall, more proteins were detected in S. carpocapsae (2422) than in H. megidis (1582). The S. carpocapsae proteome was strongly affected by temperature, while the H. megidis proteome was affected by both time and temperature. The proteins which increased in abundance to the greatest extent in S. carpocapsae IJs after conditioning at 9°C were chaperone proteins, and proteins related to stress. The proteins which increased in abundance the most after storage at 20°C were proteins related to the cytoskeleton, cell signalling, proteases and their inhibitors, which may have roles in infection. The proteins which decreased in abundance to the greatest extent in S. carpocapsae after both 9°C and 20°C storage were those associated with metabolism, stress and the cytoskeleton. After storage at both temperatures, the proteins increased to the greatest extent in H. megidis IJs were those associated with the cytoskeleton, cell signalling and carbon metabolism, and the proteins decreased in abundance to the greatest extent were heat shock and ribosomal proteins, and those associated with metabolism. As the longest-lived stage of the EPN life cycle, IJs may be affected by proteostatic stress, caused by the accumulation of misfolded proteins and toxic aggregates. The substantial increase of chaperone proteins in S. carpocapsae, and to a greater extent at 9°C, and the general decrease in ribosomal and chaperone proteins in H. megidis may represent species-specific proteostasis mechanisms. Similarly, organisms accumulate reactive oxygen species (ROS) over time and both species exhibited a gradual increase in proteins which enhance ROS tolerance, such as catalase. The species-specific responses of the proteome in response to storage temperature, and over time, may reflect the phylogenetic distance and/or different ecological strategies.

Heterorhabditis bacteriophora (Rhabditida: Heterorhabditidae), isolate HP88, induces reproductive and physiological alterations in Biomphalaria glabrata (Gastropoda: Planorbidae): an alternative for biological control of schistosomiasis.

Heterorhabditis bacteriophora is an entomopathogenic nematode (EPN) that is mutually associated with Photorhabdus luminescens, utilized globally for biological control of numerous organisms. Freshwater snails of the species Biomphalaria glabrata have been incriminated as the main intermediate hosts of Schistosoma mansoni in Brazil, but virtually nothing is known about the susceptibility of these gastropod to EPNs. Information in this respect is relevant for control of these intermediate hosts, and thus of the helminthiases they transmit. This paper for the first time reports the susceptibility of B. glabrata to infective juveniles of H. bacteriophora (isolate HP88) under laboratory conditions. For that purpose, six groups were formed: three Control groups (not exposed) and three Treated groups, in which the snails were exposed to 300 juveniles infecting the nematode over three weeks. The entire experiment was conducted in triplicate, using a total of 270 snails. Significant physiological alterations in B. glabrata were observed in response to the infection by H. bacteriophora HP88, characterized by decreased levels of hemolymphatic glucose as well as reduced contents of glycogen stored in the host's digestive gland. In parallel, the hemolymphatic activity of lactate dehydrogenase increased in the infected snails, indicating that the infection induces breakdown of carbohydrate homeostasis in B. glabrata. Additionally, all the reproductive parameters analyzed were reduced as a consequence of the infection. The results indicate the occurrence of the phenomenon of parasitic castration in the B. glabrata/H. bacteriophora HP88 interface, probably due to the depletion of galactogen in the parasitized organism. Although the infection did not cause lethality in the population of infected snails, H. bacteriophora HP88 compromised the reproductive performance of B. glabrata, suggesting its applicability in programs for biological control of this planorbid.

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.

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.

Tokorhabditis n. gen. (Rhabditida, Rhabditidae), a comparative nematode model for extremophilic living.

Life in extreme environments is typically studied as a physiological problem, although the existence of extremophilic animals suggests that developmental and behavioral traits might also be adaptive in such environments. Here, we describe a new species of nematode, Tokorhabditis tufae, n. gen., n. sp., which was discovered from the alkaline, hypersaline, and arsenic-rich locale of Mono Lake, California. The new species, which offers a tractable model for studying animal-specific adaptations to extremophilic life, shows a combination of unusual reproductive and developmental traits. Like the recently described sister group Auanema, the species has a trioecious mating system comprising males, females, and self-fertilizing hermaphrodites. Our description of the new genus thus reveals that the origin of this uncommon reproductive mode is even more ancient than previously assumed, and it presents a new comparator for the study of mating-system transitions. However, unlike Auanema and almost all other known rhabditid nematodes, the new species is obligately live-bearing, with embryos that grow in utero, suggesting maternal provisioning during development. Finally, our isolation of two additional, molecularly distinct strains of the new genus-specifically from non-extreme locales-establishes a comparative system for the study of extremophilic traits in this model.

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.

Susceptibility of Drosophila suzukii larvae to the combined administration of the entomopathogens Bacillus thuringiensis and Steinernema carpocapsae.

Non-native pests are often responsible for serious crop damage. Since Drosophila suzukii has invaded North America and Europe, the global production of soft, thin-skinned fruits has suffered severe losses. The control of this dipteran by pesticides, although commonly used, is not recommended because of the negative impact on the environment and human health. A possible alternative is the use of bio-insecticides, including Bacillus thuringiensis and entomopathogenic nematodes, such as Steinernema carpocapsae. These biological control agents have a fair effectiveness when used individually on D. suzukii, but both have limits related to different environmental, methodological, and physiological factors. In this work, we tested various concentrations of B. thuringiensis and S. carpocapsae to evaluate their efficacy on D. suzukii larvae, when administered individually or in combination by using agar traps. In the combined trials, we added the nematodes after 16 h or concurrently to the bacteria, and assessed larvae lethality from 16 to 48 h. The assays demonstrated a higher efficacy of the combined administration, both time-shifted and concurrent; the obtained data also showed a relevant decrease of the time needed to kill the larvae. Particularly, the maximum mortality rate, corresponding to 79% already at 16 h, was observed with the highest concentrations (0.564 µg/mL of B. thuringiensis and 8 × 102 IJs of S. carpocapsae) in the concurrent trials. This study, conducted by laboratory tests under controlled conditions, is a good starting point to develop a further application step through field studies for the control of D. suzukii.

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.

Behavioral and molecular response of the insect parasitic nematode Steinernema carpocapsae to cues emitted by a host, the red palm weevil, Rhynchophorus ferrugineus.

As the larvae of the date palm pest, the red palm weevil, Rhynchophorus ferrugineus, feeds on the host tissue, they emit a distinctive sound which can be recorded outside of the infected tree. We evaluated the response of infective juveniles (IJs) of the entomopathogenic nematodes Steinernema carpocapsae to the R. ferrugineus larvae and it's sound source, separately. In the presence of the insect larvae, 50.2 % of total IJs moved toward those larvae. Recorded insect larvae sound emitted by the speaker resulted in 7% of total IJs near the sound source. RNA-Seq data indicated that more genes were downregulated in S. carpocapsae IJs exposed to insect and speaker compared to non-stimulated IJs. IJs exposed to insect exhibited more up-regulated genes than IJs exposed to speaker. Enriched pathways and biological processes in IJs were similar for both stimuli. The inhibition of locomotion, regulation of neurotransmitter secretion, response to biotic stimulus, and cellular response to chemical stimuli were enriched with unique GO terms for speaker treatment. The regulation of localization, sodium ion transmembrane transport, regulation of response to stress and response to organic substances were the GO categories enriched unique to insect. The host-parasitic interaction was regulated by the differential expression of Ras/MAP kinase, TGF-beta signaling, insulin signaling, AMPK signaling, PPAR signaling pathways and many developmental pathways. More prominent R. ferrugineus host localization by S. carpocapsae was primarily due to the differential transcriptional regulation of olfactory signal transduction, FOXO-family proteins, calcium signaling, WNT and mTOR signaling pathway. The neural basis for the nematode attraction to insect host is based on the chemosensation and the mechanosensation. Many neuropeptides and neuromodulators are involved in regulating the foraging behavior of S. carpocapsae. The results of this study provide new insights into the molecular mechanisms that allow these nematodes to seek insect hosts. Our finding, especially the molecular ones suggest that chemical cues emitted by the active insect host are stimulants of nematodes attraction. Whereas the sound emitted by the insect has minor effects on the nematode behavior.

Activin and BMP Signaling Activity Affects Different Aspects of Host Anti-Nematode Immunity in Drosophila melanogaster.

The multifaceted functions ranging from cellular and developmental mechanisms to inflammation and immunity have rendered TGF-ß signaling pathways as critical regulators of conserved biological processes. Recent studies have indicated that this evolutionary conserved signaling pathway among metazoans contributes to the Drosophila melanogaster anti-nematode immune response. However, functional characterization of the interaction between TGF-ß signaling activity and the mechanisms activated by the D. melanogaster immune response against parasitic nematode infection remains unexplored. Also, it is essential to evaluate the precise effect of entomopathogenic nematode parasites on the host immune system by separating them from their mutualistic bacteria. Here, we investigated the participation of the TGF-ß signaling branches, activin and bone morphogenetic protein (BMP), to host immune function against axenic or symbiotic Heterorhabditis bacteriophora nematodes (parasites lacking or containing their mutualistic bacteria, respectively). Using D. melanogaster larvae carrying mutations in the genes coding for the TGF-ß extracellular ligands Daw and Dpp, we analyzed the changes in survival ability, cellular immune response, and phenoloxidase (PO) activity during nematode infection. We show that infection with axenic H. bacteriophora decreases the mortality rate of dpp mutants, but not daw mutants. Following axenic or symbiotic H. bacteriophora infection, both daw and dpp mutants contain only plasmatocytes. We further detect higher levels of Dual oxidase gene expression in dpp mutants upon infection with axenic nematodes and Diptericin and Cecropin gene expression in daw mutants upon infection with symbiotic nematodes compared to controls. Finally, following symbiotic H. bacteriophora infection, daw mutants have higher PO activity relative to controls. Together, our findings reveal that while D. melanogaster Dpp/BMP signaling activity modulates the DUOX/ROS response to axenic H. bacteriophora infection, Daw/activin signaling activity modulates the antimicrobial peptide and melanization responses to axenic H. bacteriophora infection. Results from this study expand our current understanding of the molecular and mechanistic interplay between nematode parasites and the host immune system, and the involvement of TGF-ß signaling branches in this process. Such findings will provide valuable insight on the evolution of the immune role of TGF-ß signaling, which could lead to the development of novel strategies for the effective management of human parasitic nematodes.

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.