grower-based-in-vivo-propagation-of-entomopathogenic-nematodes

Grower-Based <em>In Vivo</em> Propagation of Entomopathogenic Nematodes

Entomopathogenic nematodes (EPNs) produced through in vitro or in vivo methods are highly effective biocontrol agents for insect pest management. In vivo ...

Southeast Fruit and Tree Nut Research Unit

Environmental factors such as temperature and desiccation impact the survival and efficacy of entomopathogenic nematodes (EPNs). Most studies on environmental tolerance have focused on EPNs applied in aqueous suspension. Another approach for EPN application is via infected host cadavers. Emergence in host cadavers is also more representative of nematodes in natural populations. In prior studies, certain advantages in fitness have been observed with the cadaver application approach relative to aqueous application, yet the impact of environmental stress on these approaches requires investigation. In this study, we compared the effects of various temperatures (heat and cold) and desiccation intervals (48 and 72 hr) on the survival, virulence and reproductive capacity of Heterorhabditis bacteriophora and Steinernema glaseri when applied via cadaver versus aqueous suspension. In the heat tolerance bioassays, following exposure to 30 °C, 35 °C and 37. 5 °C, nematodes (from both species) in the cadaver treatments exhibited higher survival, and reproductive capacity compared with aqueous application. No survival was observed above 37.5 °C regardless of species or application approach. In cold tolerance, no differences were observed between the cadaver and aqueous treatments after a sequence of exposures from 10 °C to -2 °C. In desiccation assays, following exposure to 85% relative humidity for 2 or 3 days, nematodes (from both species) exhibited higher survival and reproduction in the cadaver treatment than in the aqueous treatment, whereas no differences were observed in virulence. This is the first study to find differential stress tolerance among nematodes emerged from infected host cadavers versus those applied in aqueous suspension. Our findings indicate additional advantages when using the cadaver approach for biocontrol applications, and suggest EPNs existing in natural populations may have broader environmental tolerance than those applied via aqueous suspension.

Environmental tolerance of entomopathogenic nematodes differs among nematodes arising from host cadavers versus aqueous suspension.

The pupal soil cell of the pecan weevil, Curculio caryae (Coleoptera: Curculionidae), was reported previously to exhibit antibiosis to an entomopathogenic fungus, Beauveria bassiana. The objectives of this study were to examine 1) if the antimicrobial effect occurs in other insects that form pupal cells, 2) whether the effect extends to plant pathogenic fungi, and 3) identify the source of antibiosis in pupal soil cells of C. caryae. Antibiosis of pupal cells against B. bassiana was confirmed in-vitro in three additional curculionids, Diaprepes abbreviatus, Conotrachelus nenuphar, and Pissodes nemorensis, all of which had fewer fungal colonies relative to controls. Pupal soil cells were found to suppress phytopathogenic fungi in-vitro, including suppression of Alternaria solani by D. abbreviatus pupal cell, and that of Monilinia fructicola by C. caryae. The detection of antibiosis of soil cells formed by surface-sterilized insects using sterile soil implies the antimicrobial effect stemmed from inside the insect. Further, a novel biotic mechanism was identified: a bacterium related to Serratia nematodiphila was isolated from C. caryae pupal soil cells and was found to be associated with antibiosis. The bacterial cultures with or without autoclave had similar effects but were not as potent as pupal soil cells for suppressing B. bassiana. Also, autoclaved soil cells and autoclaved bacterial culture suppressed M. fructicola but were not as inhibitory as non-autoclaved soil cells. This indicates that antibiosis may be due to bacterial metabolites, although other factors may also be involved. Our findings suggest potential to develop the antibiotic compounds as novel bio-fungicides to control plant diseases.

Novel associations in antibiosis stemming from an insect pupal cell.

Toxicity of the metabolites of two bacteria, Photorhabdus luminescens and Xenorhabdus bovienii, symbionts of entomopathogenic nematodes, were tested in the laboratory against the multicolored Asian lady beetle, Harmonia axyridis, the black pecan aphid, Melanocallis caryaefoliae, and the blackmargined aphid, Monellia caryella. Bacterial broth prepared from both P. luminescens and X. bovienii demonstrated high levels of toxicity equivalent to the pyrethroid insecticide bifenthrin and caused higher insect mortality than tryptic soy broth plus yeast extract (TSY) (blank control) against M. caryella; broth culture of P. luminescens was more effective than TSY against M. caryaefoliae. At the levels tested, the metabolites were not toxic to H. axyridis.

Toxicity of Photorhabdus luminescens and Xenorhabdus bovienii bacterial metabolites to pecan aphids (Hemiptera: Aphididae) and the lady beetle Harmonia axyridis (Coleoptera: Coccinellidae).

Entomopathogenic nematodes (EPNs) are susceptible to abiotic environmental factors including ultraviolet (UV) radiation, which affects the survival and efficacy. This study evaluated nanoparticle (NP) formulations for protecting Steinernema carpocapsae infective juveniles (IJs) from UV radiation. First, silica-NH NPs at oil-to-water ratios of 2:8, 3:7 and 4:6 were compared with Barricade Fire Gel (1 % and 2 %) and a water control (aqueous IJs) by exposing IJs to UV light (254 nm) for 0, 10 and 20 min. Barricade gel (especially 2 % Barricade) significantly improved IJs viability after UV treatment, while all three NPs had adverse effects on IJ viability after UV radiation. Subsequently, two silica (SiO basic and advanced) and one titania (TiO) based formulations were tested with Barricade (1 % and 2 %) and a water control. The titania-NH NPs provided the highest UV protection, and IJ viability and virulence were not reduced even after 20-min UV. Except TiO, only 2 % Barricade at 10-min UV and SiO basic at 20-min UV had lower IJ mortality than the water control. Only TiO formulated IJs caused higher insect mortality and infection levels than aqueous IJs after UV treatment. The UV tolerance of TiO was further examined by assessing the number of nematodes invading the hosts. Consistent with virulence tests, the number of invading nematodes in titania-NH NPs did not decrease after UV radiation for 10 or 20 min compared with the no-UV control. The anti-UV capability of titania-NH NPs has promise as a tool to enhance biocontrol efficacy of EPNs under field conditions.

Tolerance of Steinernema carpocapsae infective juveniles in novel nanoparticle formulations to ultraviolet radiation.

Entomopathogenic nematodes (EPNs) are roundworms that parasitize insects with the aid of symbiotic bacteria. These nematodes have been used both as model organisms and for biological control of pests. The specialized third stage of an EPN, known as an infective juvenile (IJ) must forage to find a host with strategies varying from species to species (cruising, ambushing, and intermediate). Some IJs move more than others to find a host, despite an increased risk of predation and desiccation. This hints at potential underlying benefits (e.g., increased invasion) for EPNs that move more. We assessed whether EPNs that moved farther down a soil column also exhibit higher levels of invasion when compared to nematodes that remained at or near their point of origin. We found that movers in the cruisier and intermediate species: Steinernema riobrave, Heterorhabditis bacteriophora, and H. indica had higher invasion rates compared to their counterparts that did not move. S. carpocapsae, an ambusher, did not exhibit invasion differences between EPNs that moved versus those that did not. For the three cruiser/intermediate EPNs we tested, our results support our hypothesis that EPNs that tend to move more enjoy related benefits such as increased invasion potential. Further studies are required to explore other parameters that may interact with movement. The results of this study can potentially be used to develop EPN strains that move more and invade more, and thus can potentially be more effective biological control agents.

Movers and shakers: Do nematodes that move more invade more?

Consistent efficacy is required for entomopathogenic nematodes to gain wider adoption as biocontrol agents. Recently, we demonstrated that when exposed to nematode pheromone blends, entomopathogenic nematodes showed increased dispersal, infectivity, and efficacy under laboratory and greenhouse conditions. Prior to this study, the impact of entomopathogenic nematode-pheromone combinations on field efficacy had yet to be studied. Steinernema feltiae is a commercially available entomopathogenic nematode that has been shown to increase mortality in insect pests such as the pecan weevil Curculio caryae. In this study, the pecan weevil was used as a model system to evaluate changes in S. feltiae efficacy when treated with a partially purified ascaroside pheromone blend. Following exposure to the pheromone blend, the efficacy of S. feltiae significantly increased as measured with decreased C. caryae survival despite unfavorable environmental conditions. The results of this study highlight a potential new avenue for using entomopathogenic nematodes in field conditions. With increased efficacy, using entomopathogenic nematodes will reduce reliance on conventional management methods in pecan production, translating into more environmentally acceptable practices.

Enhancing entomopathogenic nematode efficacy with Pheromones: A field study targeting the pecan weevil.

Entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema are biocontrol agents used to control a diverse array of economically important insect pests. Generally, nematodes are applied in aqueous suspensions using standard agricultural equipment (e.g., sprayers and irrigation systems). Formulation and application parameters are critical to the level of biocontrol efficacy achieved by EPNs. Moreover, biocontrol efficacy is mitigated by various abiotic and biotic factors. For example, abiotic factors such as ultraviolet (UV) radiation, desiccation, or temperature extremes can severely limit EPN efficacy in biocontrol. Additionally, biotic aspects, including matching the appropriate EPN species to the target pest, and a suitable application rate, are of extreme importance. EPN formulations can facilitate storage and ease-of-handling during application. Improved formulations may also protect the EPNs from harmful environmental conditions and enhance efficacy and persistence. For example, protective gels or nanoparticle formulations can protect nematodes from UV and desiccation and thereby expand EPN use to aboveground applications. Novel application approaches can also enhance EPN efficacy such as using pheromone boosters to increase nematode movement and infectivity. In this review, we summarize the current status of EPN formulation and application technology and provide examples of novel developments that will help propel EPNs toward future success. Following a brief introduction on current formulation and application practices, we provide information on the basic abiotic and biotic factors that affect efficacy, followed by approaches to improve efficacy within the framework of formulation and application technology.

A century of advancement in entomopathogenic nematode formulation and application technology.

Bovicola caprae, the goat biting louse, is a permanent, obligate ectoparasite of goats. It causes dermatitis, irritation, hypersensitivity and lower productivity in goats. Problems associated with chemical insecticide use such as insecticide resistance and environmental concerns indicate that alternative methods (e.g., biological control) are needed. The objective of this study was to investigate the relative potential of different entomopathogenic nematode (EPN) species to control B. caprae. Five EPN species, Steinernema carpocapsae, S. feltiae, S. riobrave, Heterorhabditis bacteriophora and H. indica were tested in the laboratory. We measured relative EPN host attraction, virulence and reproductive capacity. A series of application rates were used to measure EPN virulence. Infective juvenile (IJ) reproduction was assessed in lice cadavers. Results indicated that all the EPNs tested were attracted, infected and killed adult lice; however, louse survival was dependent on EPN species, rate and exposure time. After 48 h, the lowest B. caprae survival was observed in S. riobrave and S. carpocapsae at 200 IJs/insect, with H. bacteriophora and H. indica exhibiting intermediate levels of virulence. In the reproduction assay, greater numbers of IJs were produced in the S. riobrave treatment followed by S. carpocapsae, H. bacteriophora and H. indica. No IJs were found in S. feltiae treated lice. In conclusion, EPNs can invade and kill B. caprae, with S. riobrave and S. carpocapsae exhibiting the most promise for biocontrol efficacy. Future studies will explore the use of EPNs against B. caprae in live animal applications.

Relative virulence, host finding ability, and reproductive capacity of entomopathogenic nematodes for control of the goat biting louse Bovicola caprae (Phthiraptera: Trichodectidae).

Article	Year
Environmental tolerance of entomopathogenic nematodes differs among nematodes arising from host cadavers versus aqueous suspension. Journal of invertebrate pathology\| PubMed ID: 32763230	2020
Novel associations in antibiosis stemming from an insect pupal cell. Journal of invertebrate pathology\| PubMed ID: 34411606	2021
Toxicity of Photorhabdus luminescens and Xenorhabdus bovienii bacterial metabolites to pecan aphids (Hemiptera: Aphididae) and the lady beetle Harmonia axyridis (Coleoptera: Coccinellidae). Journal of invertebrate pathology\| PubMed ID: 35944664	2022
Tolerance of Steinernema carpocapsae infective juveniles in novel nanoparticle formulations to ultraviolet radiation. Journal of invertebrate pathology\| PubMed ID: 36400242	2023
Movers and shakers: Do nematodes that move more invade more? Journal of invertebrate pathology\| PubMed ID: 38242200	2024
Enhancing entomopathogenic nematode efficacy with Pheromones: A field study targeting the pecan weevil. Journal of invertebrate pathology\| PubMed ID: 38311231	2024
A century of advancement in entomopathogenic nematode formulation and application technology. Journal of invertebrate pathology\| PubMed ID: 40543895	2025
Relative virulence, host finding ability, and reproductive capacity of entomopathogenic nematodes for control of the goat biting louse Bovicola caprae (Phthiraptera: Trichodectidae). Veterinary parasitology\| PubMed ID: 40779826	2025

David I. Shapiro-Ilan

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