A Single Entomopathogenic Nematode Infection Assay for Larvae

Overview

Journal MethodsX

Date 2025 Jan 27

PMID 39866199

Authors

Sreeradha Mallick

Eric Kenney

Jacob Rashap

Ioannis Eleftherianos

Affiliations

Soon will be listed here.

Abstract

The entomopathogenic nematodes (EPNs) Steinernema carpocapsae and Steinernema hermaphroditum can efficiently infect the fruit fly, Drosophila melanogaster. The EPN infective juvenile (IJ) stage is the free-living and non-feeding stage that seeks out suitable insects to infect. While previous studies have described successful infection of melanogaster larvae with a standard amount of 100 IJs, the pathogenicity of a single IJ nematode towards insects remains poorly understood. Given the variability in pathogenesis among individual IJs, investigating the ability of a single IJ to cause infection in fly larvae addresses a significant gap in our understanding of host-parasite interactions. This protocol aims to evaluate the infection efficiency of a single IJ of S. carpocapsae and S. hermaphroditum. This information contributes towards a better understanding of the EPN-insect interactions and promises to improve the use of EPNs in pest management strategies. The method is summarized below:•Individual third instar wild-type larvae are infected with either a single IJ or 100 IJs of either nematode species. Uninfected control larvae are treated with sterile water.•Larval survival is monitored over time.•Survival curves are generated, and results are statistically analyzed to determine the effect of a single nematode on the fly larval mortality.

References

Jones K, Tafesh-Edwards G, Kenney E, Toubarro D, Simoes N, Eleftherianos I . Excreted secreted products from the parasitic nematode Steinernema carpocapsae manipulate the Drosophila melanogaster immune response. Sci Rep. 2022; 12(1):14237. PMC: 9392720. DOI: 10.1038/s41598-022-18722-7. View

Stock S . Partners in crime: symbiont-assisted resource acquisition in Steinernema entomopathogenic nematodes. Curr Opin Insect Sci. 2019; 32:22-27. DOI: 10.1016/j.cois.2018.10.006. View

Stefanovska T, Luckhart S, Ripa L, Stevens G, Lewis E . Steinernema carpocapsae. Trends Parasitol. 2023; 39(5):400-401. DOI: 10.1016/j.pt.2023.01.002. View

Huynh T, OHalloran D, Hawdon J, Eleftherianos I . The nematode parasite is pathogenic to larvae without activating their immune response. MicroPubl Biol. 2023; 2023. PMC: 10562934. DOI: 10.17912/micropub.biology.000944. View

Ozakman Y, Eleftherianos I . Nematode infection and antinematode immunity in Drosophila. Trends Parasitol. 2021; 37(11):1002-1013. DOI: 10.1016/j.pt.2021.06.001. View

Castillo J, Shokal U, Eleftherianos I . A novel method for infecting Drosophila adult flies with insect pathogenic nematodes. Virulence. 2012; 3(3):339-47. PMC: 3442847. DOI: 10.4161/viru.20244. View

Yadav S, Daugherty S, Shetty A, Eleftherianos I . RNAseq Analysis of the Response to the Entomopathogenic Nematode . G3 (Bethesda). 2017; 7(6):1955-1967. PMC: 5473771. DOI: 10.1534/g3.117.041004. View

Tarasco E, Fanelli E, Salvemini C, El-Khoury Y, Troccoli A, Vovlas A . Entomopathogenic nematodes and their symbiotic bacteria: from genes to field uses. Front Insect Sci. 2024; 3:1195254. PMC: 10926393. DOI: 10.3389/finsc.2023.1195254. View

Cao M, Schwartz H, Tan C, Sternberg P . The entomopathogenic nematode Steinernema hermaphroditum is a self-fertilizing hermaphrodite and a genetically tractable system for the study of parasitic and mutualistic symbiosis. Genetics. 2021; 220(1). PMC: 8733455. DOI: 10.1093/genetics/iyab170. View

10.

Okakpu O, Dillman A . REVIEW OF THE ROLE OF PARASITIC NEMATODE EXCRETORY/SECRETORY PROTEINS IN HOST IMMUNOMODULATION. J Parasitol. 2022; 108(2):199-208. DOI: 10.1645/21-33. View

11.

Lu D, Sepulveda C, Dillman A . Infective Juveniles of the Entomopathogenic Nematode Steinernema scapterisci Are Preferentially Activated by Cricket Tissue. PLoS One. 2017; 12(1):e0169410. PMC: 5207650. DOI: 10.1371/journal.pone.0169410. View

12.

Ratnappan R, Vadnal J, Keaney M, Eleftherianos I, OHalloran D, Hawdon J . RNAi-mediated gene knockdown by microinjection in the model entomopathogenic nematode Heterorhabditis bacteriophora. Parasit Vectors. 2016; 9:160. PMC: 4797128. DOI: 10.1186/s13071-016-1442-4. View

13.

Eleftherianos I, Heryanto C . Molecular Regulators of Entomopathogenic Nematode-Bacterial Symbiosis. Results Probl Cell Differ. 2020; 69:453-468. DOI: 10.1007/978-3-030-51849-3_17. View

14.

Abd-Elgawad M . spp.: An Overview of the Useful Facets of Mutualistic Bacteria of Entomopathogenic Nematodes. Life (Basel). 2022; 12(9). PMC: 9503066. DOI: 10.3390/life12091360. View

15.

Puza V, Machado R . Systematics and phylogeny of the entomopathogenic nematobacterial complexes Steinernema-Xenorhabdus and Heterorhabditis-Photorhabdus. Zoological Lett. 2024; 10(1):13. PMC: 11256433. DOI: 10.1186/s40851-024-00235-y. View

16.

Brivio M, Mastore M . Nematobacterial Complexes and Insect Hosts: Different Weapons for the Same War. Insects. 2018; 9(3). PMC: 6164499. DOI: 10.3390/insects9030117. View

17.

Hartley C, Lillis P, Owens R, Griffin C . Infective juveniles of entomopathogenic nematodes (Steinernema and Heterorhabditis) secrete ascarosides and respond to interspecific dispersal signals. J Invertebr Pathol. 2019; 168:107257. DOI: 10.1016/j.jip.2019.107257. View