Infective Juveniles Of Entomopathogenic Nematodes Research Articles

NEW EXTRACTION METHOD OF ENTOMOPATHOGENIC NEMATODES USING THE BASIC FLOTAC AND THE MINI-FLOTAC TECHNIQUES

The FLOTAC basic technique is a recognised method used mainly in the veterinary field to extract parasites from the faeces of animals found in the stomach and intestine. This study is the first application of the FLOTAC and Mini-FLOTAC techniques to extract infective juveniles of entomopathogenic nematodes from infested Galleria mellonella. The EPN used for this purpose was an endemic Italian nematode belonging to the Rhabitidae family Steinernema carpocapsae. Galleria mellonella larvae were infected and extraction was performed a few days after infection using both techniques. A comparison was then made between the two methods. The results showed that infective juveniles could be extracted from the infected larva as early as three days after infection. The difference between the FLOTAC basic technique and the Mini-FLOTAC technique was not significant in the extraction of entomopathogenic nematodes. Due to its sensitivity and operating easiness the Mini-FLOTAC technique is a promising method for the extraction of entomopathogenic nematodes. It allows faster extraction of nematodes from the inhabited soil and insect host than the conventional White trap method. Key Words: Microbial biocontrol, Entomopathogenic nematodes, Nematode extraction, FLOTAC, Mini-FLOTAC

Novel formulations improve the environmental tolerance of entomopathogenic nematodes

Entomopathogenic nematodes (EPN) are susceptible to environmental factors such as ultraviolet (UV), for which formulations of EPN infective juveniles (IJs) were explored. In the screening test for Steinernema carpocapsae, liquid starch displayed the greatest UV protection, followed by white kaolin clay (WKC), watergel and Barricade. In concentration tests, after 20-min UV S. carpocapsae IJs mixed in WKC caused 100% mortality of Galleria mellonella larvae at ≥5% concentration and had lower IJ mortality than aqueous IJs at 0.5%-10% WKC. Liquid starch showed increased UV tolerance with concentrations up to 30% and 50% for IJ viability and virulence, respectively. The WKC, liquid starch, Barricade and watergel were further compared to water using S. carpocapsae and Heterorhabditis floridensis. H. floridensis was more susceptible to UV radiation than S. carpocapsae. Liquid starch provided the strongest protection of both EPNs in IJ viability and virulence, with more prominent effects for S. carpocapsae than H. floridensis. The WKC and Barricade improved the viability of both EPNs after UV treatment, but only WKC enhanced S. carpocapsae virulence. The number of invading S. carpocapsae decreased as UV exposure time increased for WKC and only decreased at 20-min UV for liquid starch. Very few H. floridensis invaded insects after UV exposure regardless of treatments. In outdoor soil tests, liquid starch and WKC protected S. carpocapsae but were inferior to Barricade and watergel, probably associated with anti-desiccation. Overall, liquid starch and WKC improved the UV tolerance of EPNs, although the effects were less pronounced in soil involving other factors.

Involvement of a novel Pseudomonas protegens strain associated with entomopathogenic nematode infective juveniles in insect pathogenesis.

The bioinsecticidal action of entomopathogenic nematodes (EPNs) typically relies on their symbiosis with core bacteria. However, recent studies highlighted the possible involvement of other noncore species. We have recently isolated a novel Pseudomonas protegens strain as a major agent of septicaemia in larvae of the wax moth, Galleria mellonella, infected with a soil-dwelling Steinernema feltiae strain. The actual role of this bacterium in entomopathogenesis was investigated. The association of P.protegens with nematodes appeared to be robust, as supported by its direct and repeated isolation from both nematodes and insect larvae infected for several consecutive generations. The bacterium appeared to be well-adapted to the insect haemocoel, being able to proliferate rapidly after the injection of even a small amount of living cells [100 colony forming units (CFU)] to a larva, causing its fast death. The bacterium also was able to act by ingestion against G.mellonella larvae [median lethal concentration (LC50 )=4.0 × 107 CFU mL-1 ], albeit with a slower action, which supports the involvement of specific virulence factors (e.g. chitinases, Fit toxin) to overcome the intestinal barrier to the haemocoel. Varying levels of bacterial virulence were observed on diverse target Diptera and Lepidoptera. The soil-dwelling bacterium P.protegens appears to have evolved its own potential as a stand-alone entomopathogen, yet the establishment of an opportunistic association with entomoparasitic nematodes would represent a special competitive advantage. This finding contributes to a deeper understanding of the nematode-bacteria biocontrol agent complex and the deriving paradigm of their use as biological control agents. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Insecticidal activities of the local entomopathogenic nematodes and cell-free supernatants from their symbiotic bacteria against the larvae of fall webworm, Hyphantriacunea

The fall webworm (FWW), Hyphantria cunea Drury (Lepidoptera: Erebidae), is an invasive and polyphagous insect pest of many economically important crops such as hazelnuts, apple, and mulberry. Recently, there have been an increasing number of reports about the damaging activities of FWW from hazelnut growing areas of Turkey indicating that currently existing control methods fail to satisfy the expectations of growers. Entomopathogenic nematodes (EPNs) in the Steinernematidae and Heterorhabditidae (Nematoda: Rhabditida) families and the symbiotic bacteria they carry in their intestine have a great potential for the management of many agriculturally important pests. In this study, the symbiotic bacteria of local EPN species (Heterorhabditis bacteriophora AVB-15, Steinernema feltiae KCS-4S, and Steinernema bicornotum MGZ-4S) recovered from the central Anatolia region was characterized using recA gene region as Photorhabdus luminescens, Xenorhabdus bovienii and Xenorhabdus budapestensis. The contact (25, 50, 100, 200 IJs/Petri) and oral efficacies of the infective juveniles (IJs) (25, 50, 100, 200 IJs/leaf) of these EPN isolates determined on 3rd/4th instar larvae, and cell-free supernatants from the identified symbiotic bacteria were evaluated separately on the 3rd and 4th larval instars of FWW in Petri dish environment under laboratory conditions (25 ± 1 °C, 60% of RH). In the Petri dish bioassays of EPN species, the most pathogenic isolate at the 1st DAT and 4th DAT was S. feltiae which caused 50% mortality at the highest concentration (200 IJs/Petri) and the highest mortality rate (97.5%) were achieved at 4th DAT by H. bacteriophora AVB-15 isolate. Surprisingly, the mortality rates were generally higher at the lowest concentrations and 82.5% mortality were reached 4th DAT by S. bicornotum at the lowest concentration (25 IJs/leaf) in the leaf bioassays. Mortality rates were higher in both Petri dish and filter paper efficacies of cell-free supernatants at the 2nd DAT and the highest mortality (87.5%) was reached in the contact efficacy studies when applied X. bovienii KCS-4S strain. The results suggest that the tested EPN species and CFSs have good potential for biological control of the larvae of FWW and can contribute to the IPM programs of FWW. However, the efficacy of both IJs of EPNs and CFSs of their symbiotic bacteria on larvae of FWW requires further studies to verify their efficiency in the field.

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.

Influence of the ascarosides on the recovery, yield and dispersal of entomopathogenic nematodes

Recovery, yield, and dispersal are crucial developmental and behavioral indices for the infective juveniles of entomopathogenic nematodes, which are used as biocontrol agents against a variety of agricultural pests. Ascarosides and isopropylstilbene (ISO) function as nematode pheromones with developmental and behavioral effects. In this study, 11 synthesized ascarosides identified from Caenorhabditis elegans, together with ISO identified from Photorhabdus luminescens, were used to determine their influence on the IJ recovery, growth on agar plates, and dispersal of S. carpocapsae All, H. bacteriophora H06 and H. indica LN2 nematodes. Compared with the controls, significant differences in IJ recovery of three nematode species were detected from the supernatants of their corresponding bacterial cultures with almost all ascarosides or isopropylstilbene (ISO) at 0.04 nM in 6 days. The highest IJ recovery percentages was obtained from ISO and ascr#3 for All strain, ascr#5 and ascr#6 for LN2 strain, and ISO and ascr#12 for H06 strain. The ISO detected from Photorhabdus bacteria also induced IJ recovery of S. carpocapsae All. IJ yields was significantly stimulated by all synthesized compounds for S. carpocapsae All, and by most compounds for H. bacteriophora H06. The higher IJ yields varied with ascarosides. Ascr#7 and DMSO was common for the improved IJ yields of both nematode species. The three nematode species showed marked differences in dispersal behavior. In response to the ascarosides or ISO, S. carpocapsae All IJs actively moved with different dispersal rates, H. indica LN2 IJs in very low dispersal rates, and H. bacteriophora H06 IJs in variable and even suppressed rates on the agar plates at least during the assay period. Based on the synthesized standards, ascr#1, ascr#9 and ascr#10 were detected from three nematode species, ascr#5 and ascr#11 also from S. carpocapsae All and H. bacteriophora H06, and ascr#12 also from H. bacteriophora H06 and H. indica LN2. Ascr#9 was most abundant in three nematode species. Compared with the sterile PBS, significantly more ascr#1, ascr#9 and ascr#10 were detected from S. carpocapsae All and H. indica LN2, but less ascr#5 and ascr#11 from S. carpocapsae All, ascr#1, ascr#5, ascr#11 and ascr#12 from H. bacteriophora H06, in the corresponding bacterial supernatant. It seems that the bacterial supernatants could regulate the ascaroside secretion by the three nematode species. These results will provide useful clues for selecting suitable ascarosides to induce the recovery, improve the yield, and enhance the dispersal of the IJs of these nematodes.

DiI staining of sensory neurons in the entomopathogenic nematode Steinernema hermaphroditum.

Steinernema hermaphroditum entomopathogenic nematodes (EPN) and their Xenorhabdus griffiniae symbiotic bacteria have recently been shown to be a genetically tractable system for the study of both parasitic and mutualistic symbiosis. In their infective juvenile (IJ) stage, EPNs search for insect hosts to invade and quickly kill them with the help of the symbiotic bacteria they contain. The mechanisms behind these behaviors have not been well characterized, including how the nematodes sense their insect hosts. In the well-studied free‑living soil nematode Caenorhabditis elegans, ciliated amphid neurons enable the worms to sense their environment, including chemosensation. Some of these neurons have also been shown to control the decision to develop as a stress-resistant dauer larva, analogous to the infective juveniles of EPNs, or to exit from dauer and resume larval development. In C. elegans and other nematodes, dye-filling with DiI is an easy and efficient method to label these neurons. We developed a protocol for DiI staining of S. hermaphroditum sensory neurons. Using this method, we could identify neurons positionally analogous to the C. elegans amphid neurons ASI, ADL, ASK, ASJ, as well as inner labial neurons IL1 and IL2. Similar to findings in other EPNs, we also found that the IJs of S. hermaphroditum are dye-filling resistant.

Isolation and Characterization of Photorhabdus Spp. (Enterobacteriaceae) Isolated From Heterorhabditis Bacteriophora in Kashere, Nigeria

Photorhabdus bacteria are symbiotically associated with the infective juveniles of entomopathogenic nematodes of the genus Heterorhabditis. These bacteria after infecting a susceptible host, produces a host of lethal toxins that killthe insect hosts within 24 to 72 hours. Consequently they have emerged and are trending as excellent biological control agents against insect pests of agricultural crops. Therefore, this study aim to isolate, identify and test the pathogenicity of Photorhabdus bacteria isolated from Heterorhabditis bacteriophora, entomopathogenic nematode from Kashere, Gombe State, Nigeria. To this end phenotypic and biochemical tests were conducted. The tests conducted showed that the isolate exhibited characteristics similar to those of Photorhabdus bacteria that have been identified. Last instar larvae of G. mellonella exposed to different concentrations of the bacterial isolate showed some level of susceptibility of the larvae to the bacterial isolate confirming its biological control potential.

Survival and Virulence of Native Strains of <i>Steinernema carpocapsae</i> and <i>Heterorhabditis bacteriophora</i> in Formulations

In this study, entomopathogenic nematodes (EPN) isolated from cotton ecosystem viz., Steinernema carpocapsae (strain APKS2) and Heterorhabditis bacteriophora (strain KKMH1) were evaluated in formulations of alginate gel, talc, sponge and water concentrates. The survival or longevity of infective juveniles (IJs) in these formulations was evaluated under in vitro at storage temperatures of 5 and 25 °C. Simultaneously the virulence of these stored EPN infective juveniles (IJs) was evaluated against the spotted boll worm Earias vitella through in vitro bioassays. The results revealed that S. carpocapsae (APKS2) and H. bacteriophora (KKMH1) formulated in alginate gel survived 70-100% up to 3 months with 55-100% infectivity at 5°C and survived 52-100% with 42.5-100% virulence for 2.5 months at 25°C. In talc formulation, these EPN remained alive to an extent of 55-100% with 70-100% virulence at 5°C and stayed alive 45-100% with 52.5-100% virulence at 25°C for 2 months. In sponge formulation, EPN strains survived 55-100% with 67.5-100% infectivity for 7 weeks at 5°C, but with 50-100% survivals and 62.5-100% virulence at 25°C. EPN strain S. carpocapsae (APKS2) can be stored in water at 5°C up to 8 weeks with viability of 50% and infectivity 60%. At 25°C, it can be stored for 5 weeks with survival of 62% and infectivity of 62.5-100 %. It is inferred from the results that alginate and talc formulations at storage temperature of 5°C is better for long term storage of S. carpocapsae strain APKS2 and H. bacteriophora strain KKMH1. Thus, the results revealed that S. carpocapsae (strain APKS2) and H. bacteriophora (strain KKMH1) can be formulated in sponges for use within 2 months and can be kept as water concentrate if they would be utilized in 2-3 weeks.

Chemotaxis behaviour of Steinernema carpocapsae in response to Galleria mellonella (L.) larvae infected by con- or hetero-specific entomopathogenic nematodes

ABSTRACT Entomopathogenic nematodes (EPNs) infect insects and reproduce within the host cadaver. The nematodes use chemotaxis to assist in host-finding. However, the chemotaxis of nematode species in response to insects previously infected by con- and hetero-specific EPN in a sand context has rarely been reported. Our aim was to address the response of Steinernema carpocapsae All (Sc) to Galleria mellonella cadavers infected by con- or hetero-specific EPNs (Heterorhabditis bacteriophora HB1, S. feltiae SN, S. rarum 17 C&E) in sand via chemotaxis assays. Additionally, the effect of different time points post-infection was also evaluated. Results indicated that previously infected G. mellonella larvae repelled infective juveniles (IJs) of Sc (p < 0.05) regardless of the time point post-infection and EPN species (con- or hetero-specific) and had a lower chemotaxis index as compared to that of no larvae addition in both sides. Moreover, it was found that for each of four tested EPN species, EPNs infected insect cadavers released dimethyl disulphide, which was identified by solid-phase microextraction (SPME) coupled to gas chromatography–mass spectrometry (GC–MS). We found that dimethyl disulphide acted as a repellent to Sc infective juveniles and reached a significant effect at the level of 0.106 mol/L. Our findings could provide a better understanding of movement behaviour of EPN infective juveniles in response to insect resources in the presence of con- or hetero-specific EPNs.

The Joint Action of Entomopathogenic Nematodes Mixtures and Chemical Pesticides on Controlling Helicoverpa armigera (Hübner)

Chemical pesticides are characterized by the rapid impact effect in reducing pest population, while, microbial pesticides need along the latent period to cause a limited reduction of the pest population. Low efficiency of biocides may be due to low compatibility with agrochemicals or poor application of biocide, so, the study carried out to evaluate the possibility of mixing five common chemical insecticides and nematicides with five compatible entomopathogenic nematodes (EPNs). For the control of tomato fruit borer (Helicoverpaarmigera),bioassay revealed that pesticide chlorpyrifos (Pestban 48% EC), chlorpyrifos (Tafaban 48% EC) and flubendiamide (Takumi 20% WG) were applied at the recommended dose. Flubendiamide was less toxic to EPNs infective juveniles compared to fenamiphos (Dento 40% EC). Moreover, steinernematid species were more sensitive than heterorhabditid species to pesticides recording 49.45% and 43.76%, respectively, after 7 days of exposure. The joint action of tested chemical pesticides with IJs of EPNs in controlling the 5th instar larvae of tomato fruit wormshowed an additive or antagonistic reaction with no evidence of synergistic action. Antagonism reaction was recorded with all fenamiphos combinations;the combination ofabamectin and S. feltiae as well as H. bacteriophora (Ba-1), in addition to, flubendiamide combinations with S. feltiae and H. bacteriophora (Ba-1). While, an additive effect was observed in flubendiamide combinations with S. glaseri, S. carpocapsae and H. bacteriophora (HP 88). In the greenhouse experiment, the application of EPNs alone caused mortality ranged from 28 to 36% for 5th instar larvae. Whereas, the highest larval mortality was observed in descending order for combinations between H. bacteriophora (HP88 strain) with fenamiphos (64.0%), chlorpyrifos (54%) and abamectin (54.0%), while, local isolate, H. bacteriophora (Ba-1 strain) achieved mortality ranged from 40 to 50 % with the tested pesticides. Overall, results indicate the feasibility of the integrated use of these nematode species and chemical pesticides in crop protection.

Activity of Steinernema colombiense in plant-based oils.

Entomopathogenic nematodes (EPNs) are excellent biological control agents. Although traditionally EPN application targeted belowground insects, their aboveground use can be supported if combined with adjuvants. We hypothesized that EPN infective juveniles (IJs) could be combined with plant-based oils as adjuvants, without decreasing their efficacy against insect larvae under various scenarios. Specifically, our objectives were to evaluate the activity of Steinernema colombiense (Nematoda: Steinernematidae) when mixed with two plant-based oils (coconut and olive oils) and maintained at different temperatures and times, or combined with entomopathogenic fungi. First, we evaluated how these oils affected IJ survival and virulence against last instar Galleria mellonella (Lepidoptera: Pyralidae) larvae when maintained at five different temperatures (4, 8, 14, 20, and 24°C) and five incubation times (1, 3, 7, 14, and 21 days), using water as control treatment. Second, we evaluated virulence when combined with these two oils as well as with water (control) and combined with the entomopathogenic fungi (EPF), Beauveria bassiana (Hypocreales: Clavicipitaceae). Infective juvenile survival was higher in coconut than olive oil and water mixtures up to 7 days at 4°C. Conversely, olive oil supported higher larval mortality than coconut oil at 4 to 20°C and 14 days. Similarly, the number of days needed to kill insect larvae increased at extreme temperatures (4 and 24°C) after 14 days. Finally, the EPN + EPF combination showed an additive effect compared to EPN and EPF single treatments. Our findings indicate that our plant-based oil mixtures maintain viable IJs at moderate temperatures and up to 7 to 14 days, and can be used in single EPN mixtures or combined with EPF.

Effect of an Alltech soil health product on entomopathogenic nematodes, root-knot nematodes and on the growth of tomato plants in the greenhouse.

An organic product that consists of proprietary blend of fermentation and plant extracts with micronutrients (ACS 5075, Alltech®, Inc., Nicholasville, KY USA) was evaluated against four strains of entomopathogenic nematodes (EPN): Steinernema feltiae (SB12(1), a wild enviroCORE strain and a commercial form e-NEMA), Steinernema carpocapsae (e-NEMA), and Heterorhabditis bacteriophora. The effects on egg hatching and survival of root-knot nematodes (RKN) were also examined. The sensitivity to the product was tested by estimating mortality and survival of EPN infective juveniles (IJ) after 24-hr treatment with four different concentrations of product (4, 7, 8, and 10%) compared with the control in a 96-well plate. There was no significant difference in survival of IJ with 4% product compared to the control. A twofold reduction in survival was observed when the EPN were exposed to the product at 7%. A 10.5% RKN egg hatching was observed when RKN were exposed to 3% ACS 5075 concentration compared to 100% hatching in the untreated. A significant (p < 0.05) reduction in juvenile survival was observed at 0.5% treatment, however, it dropped to 0 when 1% and above was used for M. javanica. Juveniles did not survive with all treatment concentrations in the case of M. incognita. The improvement in tomato plant growth and development were also observed when plants were treated with 1 and 3% ACS 5075. The product shows potential as a sustainable soil health alternative causing no harm to beneficial nematodes (EPN) at concentrations below 4%, while is promising against plant parasitic nematodes (PPN) and toward plant growth promotion.

Pheromone extracts act as boosters for entomopathogenic nematodes efficacy.

Inconsistency in entomopathogenic nematode (EPN) efficacy is still one of the biggest challenges for the wider adoption of EPNs as biocontrol agents. Previous studies demonstrated that extracts from EPN-infected hosts enhance dispersal and efficacy, two key factors in success of EPNs. Some active components in the insect host cadavers responsible for dispersal, ascarosides, have been identified as nematode pheromones. We hypothesized that pheromone extracts increase dispersal of EPN infective juveniles (IJs) leading to increased efficacy. First, we determined whether pheromone extracts improved IJ movement/dispersal in soil columns baited with Tenebrio molitor larvae. We found that pheromone extracts induced higher numbers of Steinernema carpocapsae and Steinernema feltiae IJs to move towards T. molitor larvae in the bottom of the column compared to IJs treated with infected cadaver macerate and water, positive and negative controls, respectively. Furthermore, the number of S. carpocapsae IJs that invaded T. molitor larvae was higher for the pheromone extract treatment than the controls. S. feltiae IJs that were pretreated with pheromone extracts and macerate (positive control) infected T. molitor at the same rate but invasion was superior to IJs that were treated with water. Consistent with the soil column tests, both S. carpocapsae and S. feltiae IJs treated with pheromone extracts performed better in killing larvae of two economically important insect larvae, pecan weevil, Curculio caryae, and black soldier fly, Hermetia illucens, in greenhouse tests compared to IJs treated with water. We demonstrated pheromone-mediated behavioral manipulation of a biological control agent to enhance pest control potential. Conceivably, nematodes can be exposed to efficacy-enhancing pheromones prior to field application.

Chemical cues linked to risk: Cues from below‐ground natural enemies enhance plant defences and influence herbivore behaviour and performance

Abstract Chemical cues are essential for many ecological interactions. Previous studies of chemically mediated multitrophic interactions have typically focused on responses to cues from plants or herbivores above‐ground. It is increasingly clear, however, that below‐ground cues and those produced by organisms at higher trophic levels also have ecological importance. Prey animals often avoid predator odours to improve survival, and previous research has documented enhanced plant resistance following contact with below‐ground natural enemies, though the ecological basis was unknown. Here, we investigated plant and insect responses to chemical cues from below‐ground natural enemies and explored the ecological significance of these cues for multitrophic interactions. More specifically, we examined the influence of odours emitted by entomopathogenic nematodes (EPNs), a natural enemy of insect herbivores, on the performance and behaviour of their insect prey and the defence responses of nearby plants. Our findings revealed that EPN‐infected insect cadavers emit a characteristic blend of volatile compounds with bioactivity in plants and insects. EPN chemical cues influenced both performance and preference of a specialist herbivore, Colorado potato beetle (CPB, Leptinotarsa decemlineata), feeding on its host plant, potato (Solanum tuberosum). CPB larvae consumed less leaf tissue and gained less mass feeding on plants exposed to EPN cues compared to control plants. Female CPBs laid fewer eggs on plants with EPN cues than on controls, indicating deterrence by EPN cues or EPN‐altered plant defences. Plant defences were enhanced by exposure to live EPNs or EPN chemical cues. Potato plants exposed to EPN infective juveniles induced higher amounts of the defence hormone salicylic acid (SA) and had higher expression of the pathogenisis‐related gene PR‐1(PR4) in foliar tissue. Exposing plants to EPN cues primed induction of SA and jasmonic acid in response to feeding damage by CPB larvae. These findings suggest that herbivores avoid cues from their EPN natural enemies and plants respond to the beneficial nematodes by enhancing systemic defences that reduce herbivore performance. This work has important implications for the chemical ecology of tritrophic interactions as we report that the third trophic level can play direct and indirect roles in plant defence. A plain language summary is available for this article.

Host-Specific Activation of Entomopathogenic Nematode Infective Juveniles.

Entomopathogenic nematodes (EPNs) are potent insect parasites and have been used for pest control in agriculture. Despite the complexity of the EPN infection process, hosts are typically killed within 5 days of initial infection. When free-living infective juveniles (IJs) infect a host, they release their bacterial symbiont, secrete toxic products, and undergo notable morphological changes. Collectively, this process is referred to as “activation” and represents the point in a nematode’s life cycle when it becomes actively parasitic. The effect of different host tissues and IJ age on activation, and how activation itself is related to virulence, are not well understood. Here, we employed a recently developed bioassay, which quantifies IJ activation, as a tool to address these matters. Appreciating that activation is a key part of the EPN infection process, we hypothesized that activation would positively correlate to virulence. Using the EPNs Steinernema carpocapsae and S. feltiae we found that EPN activation is host-specific and influenced by infective juvenile age. Additionally, our data suggest that activation has a context-dependent influence on virulence and could be predictive of virulence in some cases such as when IJ activation is especially low.

Parameters affecting plant defense pathway mediated recruitment of entomopathogenic nematodes

ABSTRACTEntomopathogenic nematodes are natural enemies and effective biological control agents of subterranean insect herbivores. Interactions between herbivores, plants, and entomopathogenic nematodes are mediated by plant defense pathways. These pathways can induce release of volatiles and recruit entomopathogenic nematodes. Stimulation of these plant defense pathways for induced defense against belowground herbivory may enhance biological control in the field. Knowledge of the factors affecting entomopathogenic nematode behaviour belowground is needed to effectively implement such strategies. To that end, we explore the effect of elicitor, elicitor dose, mechanical damage, and entomopathogenic nematode release distance on recruitment of entomopathogenic nematode infective juveniles to corn seedlings. Increasing doses of methyl jasmonate and methyl salicylate elicitors recruited more entomopathogenic nematodes as did mechanical damage. Recruitment of entomopathogenic nematodes was higher at greater release distances. These results suggest entomopathogenic nematodes are highly tuned to plant status and present a strategy for enhancing biological control using elicitor-stimulated recruitment of entomopathogenic nematodes.

Activated entomopathogenic nematode infective juveniles release lethal venom proteins

Entomopathogenic nematodes (EPNs) are unique parasites due to their symbiosis with entomopathogenic bacteria and their ability to kill insect hosts quickly after infection. It is widely believed that EPNs rely on their bacterial partners for killing hosts. Here we disproved this theory by demonstrating that the in vitro activated infective juveniles (IJs) of Steinernema carpocapsae (a well-studied EPN species) release venom proteins that are lethal to several insects including Drosophila melanogaster. We confirmed that the in vitro activation is a good approximation of the in vivo process by comparing the transcriptomes of individual in vitro and in vivo activated IJs. We further analyzed the transcriptomes of non-activated and activated IJs and revealed a dramatic shift in gene expression during IJ activation. We also analyzed the venom proteome using mass spectrometry. Among the 472 venom proteins, proteases and protease inhibitors are especially abundant, and toxin-related proteins such as Shk domain-containing proteins and fatty acid- and retinol-binding proteins are also detected, which are potential candidates for suppressing the host immune system. Many of the venom proteins have conserved orthologs in vertebrate-parasitic nematodes and are differentially expressed during IJ activation, suggesting conserved functions in nematode parasitism. In summary, our findings strongly support a new model that S. carpocapsae and likely other Steinernema EPNs have a more active role in contributing to the pathogenicity of the nematode-bacterium complex than simply relying on their symbiotic bacteria. Furthermore, we propose that EPNs are a good model system for investigating vertebrate- and human-parasitic nematodes, especially regarding the function of excretory/secretory products.

Compatibility of acaricides with entomopathogenic nematodes (Steinernema and Heterorhabditis)

To expand our knowledge about the compatibility of entomopathogenic nematodes (EPN) to pesticides, we studied the compatibility of EPN infective juveniles (IJ) to five acaricides under laboratory conditions. Four EPN species were used: Steinernema feltiae, S. carpocapsae, S. kraussei and Heterorhabditis bacteriophora. The results of our laboratory investigation showed that H. bacteriophora was the most tolerant EPN species and the most sensitive species was S. feltiae. Our observations showed that S. feltiae can be mixed with only two acaricides. The active substance (a.s.) fenpyroximate proved to be the most suitable for mixing with EPN. Fenpyroximate was only lethal to S. feltiae (44% mortality) at 25°C. The mortality of EPN was highest in the a.s. abamectin and pyrethrin. The mortality of EPN was influenced by species, exposure time, a.s. and temperature. The combined use of EPN and acaricides could represent an advantage in integrated plant protection programmes.

Temperature-dependent changes in the host-seeking behaviors of parasitic nematodes.

BackgroundEntomopathogenic nematodes (EPNs) are lethal parasites of insects that are of interest as biocontrol agents for insect pests and disease vectors. Although EPNs have been successfully commercialized for pest control, their efficacy in the field is often inconsistent for reasons that remain elusive. EPN infective juveniles (IJs) actively search for hosts to infect using a diverse array of host-emitted odorants. Here we investigate whether their host-seeking behavior is subject to context-dependent modulation.ResultsWe find that EPN IJs exhibit extreme plasticity of olfactory behavior as a function of cultivation temperature. Many odorants that are attractive for IJs grown at lower temperatures are repulsive for IJs grown at higher temperatures and vice versa. Temperature-induced changes in olfactory preferences occur gradually over the course of days to weeks and are reversible. Similar changes in olfactory behavior occur in some EPNs as a function of IJ age. EPNs also show temperature-dependent changes in their host-seeking strategy: IJs cultured at lower temperatures appear to more actively cruise for hosts than IJs cultured at higher temperatures. Furthermore, we find that the skin-penetrating rat parasite Strongyloides ratti also shows temperature-dependent changes in olfactory behavior, demonstrating that such changes occur in mammalian-parasitic nematodes.ConclusionsIJs are developmentally arrested and long-lived, often surviving in the environment through multiple seasonal temperature changes. Temperature-dependent modulation of behavior may enable IJs to optimize host seeking in response to changing environmental conditions, and may play a previously unrecognized role in shaping the interactions of both beneficial and harmful parasitic nematodes with their hosts.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0259-0) contains supplementary material, which is available to authorized users.