In vitro liquid mass production of a South African isolate of Heterorhabditis zealandica

Authors

DOI:

https://doi.org/10.17159/2254-8854/2023/a16150

Keywords:

Entomopathogenic nematodes, biocontrol agents, soy, Photorhabdus thracensis, egg yolk

Abstract

Developing repeatable protocols for the in vitro liquid mass production of entomopathogenic nematodes (EPNs) is a difficult task and depends on the nematode species being cultured. Of critical importance is the establishment of a monoxenic population of nematodes as a stock culture for optimisation and experimental purposes. Establishing a new stock inoculum culture flask of pure infective juveniles (IJs) is challenging, particularly for the Heterorhabditis species, due to their affinity for developing into an amphimictic second generation that does not copulate in liquid culture flasks. Developing mass production protocols for multiple EPNs is advisable because different pest insects are susceptible to different species of EPN. This study attempted to mass-produce a South African isolate of Heterorhabditis zealandica and its symbiotic bacteria, Photorhabdus thracensis, using in vitro liquid culture technology methods previously developed for Steinernema species. The results indicate that the pre-culture protocols developed for Steinernema species are applicable to a H. zealandica isolate. Moreover, the results, in terms of the protein source optimisation experiments, confirm that different EPN species have different culture conditions and nutrient requirements, with H. zealandica seeming to prefer soy-based protein instead of egg yolk, having higher recovery and producing more hermaphrodites, using soy protein. This study illustrates the importance of developing dependable and infallible preculture methods, prior to the flask mass production process.

Downloads

Download data is not yet available.

References

Boemare NE, Akhurst RJ, Mourant RG. 1993. DNA relatedness between Xenorhabdus spp. (Enterobacteriaceae), symbiotic bacteria of entomopathogenic nematodes, and a proposal to transfer Xenorhabdus luminescens to a new genus, Photorhabdus gen. nov. International Journal of Systematic and Evolutionary Bacteriology 43(2):249–255. https://doi.org/10.1099/00207713-43-2-249

Booysen E, Malan AP, Dicks LMT. 2022. Colour of Heterorhabditis zealandica-infected-Galleria mellonella dependent on the Photorhabdus symbiont, with two new nematode-symbiotic associations reported. Journal of Invertebrate Pathology 189:107729. https://doi.org/10.1016/j.jip.2022.107729

Cho CH, Whang KS, Gaugler R, Yoo SK. 2011. Submerged monoxenic culture medium development for Heterorhabditis bacteriophora and its symbiotic bacterium Photorhabdus luminescens: protein sources. Journal of Microbiology and Biotechnology 21(8):869–873.

https://doi.org/10.4014/jmb.1010.10055

Dunn MD, Belur PD, Malan AP. 2020. In vitro liquid culture and optimization of Steinernema jeffreyense, using shake flasks. Biocontrol 65:223–233. https://doi.org/10.1007/s10526-019-09977-7

Dunn MD, Belur PD, Malan AP. 2021. A review of the in vitro liquid mass culture of entomopathogenic nematodes. Biocontrol Science and Technology 31:1–21. https://doi.org/10.1080/09583157.2020.1837072

Dunn MD, Belur PD, Malan SP. 2022. Development of cost-effective media for the in vitro liquid culture of entomopathogenic nematodes. Nematology 24(7):763–775. https://doi.org/10.1163/15685411-bja10166

Ehlers R-U, Niemann I, Hollmer S, Strauch O, Jende D, Shanmugasundaram M, Mehta UK, Easwaramoorthy SK, Burnell A. 2000. Mass production potential of the bacto-helminthic biocontrol complex Heterorhabditis indica-Photorhabdus luminescens. Biocontrol Science and Technology 10(5):607–616. https://doi.org/10.1080/095831500750016406

Ehlers RU. 2001. Mass production of entomopathogenic nematodes for plant protection. Applied Microbiology Biotechnology 56:623–633. https://doi.org/10.1007/s002530100711

Ferreira T, Addison MF, Malan AP. 2014. In vitro liquid culture of a South African isolate of Heterorhabditis zealandica for the control of insect pests. African Entomology 22(1):80–92. https://doi.org/10.4001/003.

0114

Ferreira T, Addison MF, Malan AP. 2016. Development and population dynamics of Steinernema yirgalemense (Rhabditida: Steinernematidae) and growth characteristics of its associated Xenorhabdus indica symbiont in liquid culture. Journal of Helminthology 90(3):364–371. https://doi.org/10.1017/S0022149X15000450

Forst S, Nealson K. 1996. Molecular biology of the symbiotic-pathogenic bacteria Xenorhabdus spp and Photorhabdus spp. Microbiology Reviews 60(1): 21–43

Gil G, Choo H, Gaugler R. 2002. Enhancement of entomopathogenic nematode production in in-vitro liquid culture of Heterorhabditis bacteriophora by fed-batch culture with glucose supplementation. Applied Microbiology Biotechnology 58(6):751–755. https://doi.org/

1007/s00253-002-0956-1

Grewal PS, Lewis EE, Gaugler R, Campbell JF. 1994. Host finding behaviour as a predictor of foraging strategy in entomopathogenic nematodes. Parasitology 108(2):207–215. https://doi.org/10.1017s003118200006830x

Hirao A, Ehlers RU. 2009. Influence of cell density and phase variants of bacterial symbionts (Xenorhabdus spp.) on dauer juvenile recovery and development of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida). Applied Microbiology Biotechnology 84:77–85. https://doi.org/10.1007/s00253-009-1961-4

Hirao A, Ehlers RU. 2010. Influence of inoculum density on population dynamics and dauer juvenile yields in liquid culture of biocontrol nematodes Steinernema carpocapsae and S. feltiae (Nematoda: Rhabditida). Applied Microbiology Biotechnology 85: 507–515.

Hominick WM. 2002. Biogeography. In: Gaugler R (ed), Entomopathogenic Nematology. Wallingford: CABI. pp 115–143 .

James M, Malan AP, Addison P. 2018. Surveying and screening South African entomopathogenic nematodes for the control of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Crop Protection 105:41–48.

Kaya HK, Stock S. 1997. Techniques in insect nematology. In: Lacey L (ed), Manual of Techniques in Insect Pathology. San Diego: Academic Press. pp. 281–324. https://doi.org/10.1016/B978-012432555-5/50016

Kaya HK, Aguillera MM, Alumai A, Choo HY, Torre M, Fodor A, et al. 2006. Status of entomopathogenic nematodes and their symbiotic bacteria from selected countries or regions of the world. Biological Control 38:134–155.

Lewis EE, Gaugler R, Harrison R. 1992. Entomopathogenic nematode host finding: response to host cues by cruise and ambush foragers. Parasitology 105:309–315.

Lunau S, Stoessel S, Schmidt-Peisker AJ, Ehlers R-U. 1993. Establishment of monoxenic inocula for scaling up in vitro cultures of the entomopathogenic nematodes Steinernema spp. and Heterorhabditis. Nematology 39:385–399.

Peters A, Ehlers R-D. 1994. Susceptibility of leatherjackets (Tipula paludosa and T. oleracea; Tipulidae: Nematocera) to the entomopathogenic nematode Steinernema feltiae. Journal of Invertebrate Pathology 63:163–171.

Poinar GO. 1975. Description and biology of a new parasitic rhabditoid Heterorhabditis bacteriophora n. gen., n. sp. Nematology 21:463-470.

Poinar GO. 1990. Biology and taxonomy of Steinernematidae and Heterorhabditidae. In: Gaugler R, Kaya HK (eds), Entomopathogenic Nematodes in Biological Control. Boca Raton: CRC Press. pp 23–61.

StatSoft Inc. 2016. Dell Statistica (data analysis software system), version 14. http://www.statsoft.com.

Strauch O, Stoessel S, Ehlers R-U. 1994. Culture conditions define automictic or amphimictic in entomopathogenic rhabditid nematodes of the genus Heterorhabditis. Fundamental and Applied Nematology 17(6):572–582.

Strauch O, Ehlers RU. 1998. Food signal production of Photorhabdus luminescens inducing the recovery of entomopathogenic nematodes Heterorhabditis spp. in liquid culture. Applied Microbiology Biotechnology 50:369–374. https://doi.org/10.1007/s002530051306

Torr P, Spiridonov SE, Heritage S, Wilson MJ. 2007. Habitat associations of two entomopathogenic nematodes: a quantitative study using real-time quantitative polymerase chain reactions. Journal Animal Ecology 76:238–245. https://doi.org/10.1111/j.1365-2656.2006.01196.x

Warrior P. 2000. Living systems as natural crop-protection agents. Pest Management Science 56:681–687. https://doi.org/10.1002/1526-

(200008)56:8<681::AID-PS199>3.0.CO;2-S

Woodring JL, Kaya HK. 1988. Steinernematid and Heterorhabditid Nematodes: A Handbook of Techniques. Southern Cooperative Series Bulletin 331. Fayetteville, Arkansas Agricultural Experimental Station.

Yoo SK, Brown I, Gaugler R. 2000. Liquid media development for Heterorhabditis bacteriophora: lipid source and concentration. Applied Microbiology Biotechnology 54:759–763. https://doi.org/10.1007/s002530000478

Downloads

Published

2023-10-21

How to Cite

1.
Dunn M, Malan A. In vitro liquid mass production of a South African isolate of Heterorhabditis zealandica . Afr. Entomol. [Internet]. 2023 Oct. 21 [cited 2024 Apr. 14];31. Available from: https://www.africanentomology.com/article/view/16150

Issue

Section

Articles