Wayback: History of Entomopathogenic Nematodes.pptx
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This presentation takes you back through series of developmental stages from the discovery and application of entomopathogenic nematodes for use in agriculture.
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Wayback: History of Entomopathogenic Nematodes.pptx
- 1. Rufus J. Akinrinlola, EPP 520
- 2. Entomopathogenic nematodes • Heterorhabditis spp. and Steinernema spp • Invade and kill host insects • Aided by its symbiotic bacterium • About 100 species of Steinernema and 21 species of Heterorhabditis • Wide host range • Widely used as insect pest control • Compactible with some pesticides Source: Shapiro-Ilan et al., 2016 Gaugler, 1981
- 3. 1923 • The first entomopathogenic nematode was described by Steiner as Aplectana kraussei (now Steinernema kraussei) 1929 • The second entomopathogenic nematode, Neoaplectana glaseri Steiner (1929) (now Steinernema glaseri) • Both were transferred to the Steinernema genus (Wouts et al., 1982) Steinernema carpocapsae was described Steinernema kraussei after original illustration by Steiner 1923. retrieved from Oton, 2014. Glaser, 1931 Scanning electron micrograph of the lip region of Steinernema bibionis. Wouts et al., 1982
- 4. 1932 Steinernema (neoaplectana) carpocapsae control of Japanese beetle • The nematode was applied to 73 plots • Out of the 73 plots, 72 had infected grubs in 2 WKS. • Infection rate was as high as 81% • In a follow up report (Glaser et al., 1940), the nematode persisted for up to 8.5 years in the plot https://extension.entm.purdue.edu/ publications/E-75/E-75.html Glaser, 1932
- 5. 1955 • The third entomopathogenic nematode, Neoaplectana carpocapsae (European population), was isolated from codling moth larva by Jaroslav Weiser (1955) • Fourth DD-136 strain of Steinernematid from codling moth larvae in Eastern North America by Dutky and Hough (1955) • Scientists began intensive search for entomopathogenic nematodes Weiser, 1955; Dutky and Hough, 1955 Sourcescience.com
- 6. 1964 Mass production of EPNs in-vivo • Host: the greater wax moth, Galleria mellonella, • Larvae is soaked in IJS suspension for 2- 7 days • Then nematodes are harvested from the infected cadavers in water • Yields may be up to 200, 000 nematodes per larva. Source: Shapiro-Ilan et al., 2016 Dutky et al., 1964
- 7. 1965 In-vitro mass propagation – dog food medium (DFM) • Nematode is inoculated unto a sterilized dog food plate • The plate is incubated for about 14 days for nematode to reproduce • Yields can be up to 1,800, 000 nematodes per DFM plate • Nematode is then harvested from plates and stored for use Google image House et al., 1965
- 8. 1966 • The bacterium Achromobacter nematophillus was found in the ventricular portion of the nematode intestine – Poinar (1966) • The nematode can penetrate and kill host without the bacteria but it cannot reproduce without the bacteria – Poinar and Thomas (1966) Thomas (left) and Poinar Neoaplectana carpocapsae requires a symbiont bacterium Poinar, 1966; Poinar and Thomas, 1966
- 9. 1975 • Species includes H. bacteriophora • Symbiont bacterium, Xenorhabditis luminescence • Females can be hermaphroditic and dioecious • Kills its host insect within 48 hrs. Genus Heterorhabditis was described https://alchetron.com/Heterorhabditis Poinar, 1975
- 10. A. Electron micrograph Photorhabdus luminescence in the intestine of infective juvenile of H. bacteriophara . B. Galleria mellonella L. larvae infected with X. luminescens glowing in darkness. Source: Poinar and Grewal, 2012. B 1979 • Xenorhabditis luminescence • Intestinal lumen of H. Bacteriophora • In body cavity of host insects • Bacteria have fluoresces ability • Infected cadaver glow in the dark • Bacteria later transferred to genus Photorhabdus Symbiotic bacteria of Heterorhabidits spp - Thomas and Poinar, 1979. A
- 11. 1979 EPNs are non-parasitic to mammals • Rats inoculated no signs or symptoms • Blood and urine samples did not contain nematode • No weight loss in inoculated rats • Most nematodes recovered in fecal samples were dead. Gaugler and Boush, 1979
- 12. 1983 EPN works best in sandier soil • Migration and infectivity increased in silica sand and coarse sandy loam • Can infect host placed 10 cm away • Migration is slow in clay and silt soil types • Host presence stimulates nematode migration Silica sand Sandy loam Georgis and Poinar Jr, 1983.
- 13. 1987 Movements in soil vary among EPNs Neoaplectana spp move better in soil than Heterorhabditis spp • N. species (glaseri) reached 90 cm downwards and upwards, while H. species reached 15 cm downwards and up to 45 cm upwards • Neoaplectana spp also performs better in horizontal movement EPNs exhibit better upwards movement in soil Schroeder and Beavers, 1987.
- 14. 1989 and 1989 EPNs exhibit broad host range • In the lab, hosts were up to 250 species of insects • Field host range may be different POINAR, G.O., JR (1986) Entomophagous nematodes, in Biological Plant and Health Protection (FRANZ, J.M., Ed.) Fischer Verlag, Stuttgart, Germany, pp. 95±121. POINAR, G.O., JR (1989) Non-insect hosts for the entomogenous rhabditoid nematodes Neoaplectana (Steinernematidae) and Heterorhabditis (Heterorhabditidae). Revue de NeÂmatologie 12, 423±428. KLEIN, M.G. (1990) Efficacy against soil-inhabiting insect pests, in Entomopathogenic Nematodes in Biological Control (GAUGLER, R. & KAYA, H.K., Eds) CRC Press, Boca Raton, FL, pp. 195±214. Reviewed; Bathon, 1996
- 15. Steinernema spp • More mobile at temp below 7 C • Infective at broader temp range • On average, active between 12 to 32 C temp • Reproduction was low at high temp 1986 EPNs response to soil temperature differently Heterorhabditis spp • Very few were mobile below 9 C • Has narrow infective temp range • Average range 20 to 32 C • Reproduction varies at high temp Google image Molyneux, 1986
- 16. 1989 EPNs host-finding ability can be improved • Repeatedly exposed EPN populations near host • Selected the best host-finding EPNS • Allowed them to reproduce • Repeated the assay 13 times • Host-finding ability increated from 3% to 80% after 13th generation Gaugler et al., 1989
- 17. • 1939 to 1942 • Steinernema glaseri was released to colonize NJ to control Japanese beetle, but the project was due to World War II and development of insecticides • Sites assessed 50 years later, but EPNs were no more there. • Out of 21% samples with EPNs; only 8% had Steinernematids 1992 A failed attempt to colonized New Jersey with EPNs Gaugler et al., 1992
- 18. • Undisturbed habitats of California • Steinernematids were most found • Coniferous forests, oak woodlands, and grasslands • Heterorhabditids were found also • Coastal marshes. • No nematode in desert and redwood regions HABITAT OCCURRENCE (%) Coniferous forest 34 Oak forest 34 Costal marshes 24 Grassland 9 1999 EPNs are found in undisturbed natural habitats Stock et al., 1999.
- 19. 2010 EPNs are compatible with some pesticides • Three EPNs (Heterorhabditis indica, Steinernema carpocapsae and Steinernema glaseri) were tested against 18 insecticides • EPNs were immersed in pesticide solutions, and EPNs’s mortality and infectivity of host was determined. • All the three EPNs tested were compatible with 11 of the 18 insecticides tested Kingpng.com Negrisoli Jr et al., 2010
- 20. 2019-Original paper Boosting of EPN’s dispersal and host-infection efficacy with pheromones Oliveira-Hofman et al., 2019 • Steinernema carpocapsae and S. feltiae were treated woth mercerated host, pheromone, or nothing. • EPNs migration and host-infectivity against Galleria mellonella, Tenebrio molitor L, Curculio caryae, and Hermetica illucens in soil column were assessed • Pheromone or macerated-treated EPNs showed better host finding
- 21. 2019 Boosting of EPN’s dispersal and host-infection efficacy with pheromones • Steinernema carpocapsae and S. feltiae were treated with macerated host, pheromone, or nothing. • EPNs migration and host-infectivity against Galleria mellonella, Tenebrio molitor L, Curculio caryae, and Hermetica illucens in soil column were assessed • Pheromone or macerated-treated EPNs showed better host infectivity Oliveira-Hofman et al., 2019
- 22. 1. Bathon, H. (1996). Impact of entomopathogenic nematodes on non-target hosts. Biocontrol Science and Technology, 6(3), 421-434. 2. Dutky, S. R., Thompson, J. V., & Cantwell, G. E. (1964). A technique for the mass propagation of the DD-136 nematode. Journal of Insect Physiology, 6(4), 417-422. 3. Gaugler, R. (1981). Biological control potential of neoaplectanid nematodes. Journal of Nematology, 13(3), 241. 4. Gaugler, R., & Boush, G. M. (1979). Nonsusceptibility of rats to the entomogenous nematode, Neoaplectana carpocapsae. Environmental Entomology, 8(4), 658-660. 5. Gaugler, R., Campbell, J. F., & McGuire, T. R. (1989). Selection for host-finding in Steinernema feltiae. Journal of Invertebrate Pathology, 54(3), 363-372. 6. Gaugler, R., Campbell, J. F., Selvan, S., & Lewis, E. E. (1992). Large-scale inoculative releases of the entomopathogenic nematode Steinernema glaseri: assessment 50 years later. Biological Control, 2(3), 181-187. 7. Georgis, R., & Poinar Jr, G. O. (1983). Effect of soil texture on the distribution and infectivity of Neoaplectana carpocapsae (Nematoda: Steinernematidae). Journal of Nematology, 15(2), 308. 8. Glaser, R. W. (1931). The cultivation of a nematode parasite of an insect. Science, 73(1901), 614-615. 9. Glaser, R. W. (1932). Studies on Neoaplectana glaseri, a nematode parasite of the Japanese beetle (Popillia japonica). New Jersey Department of Agriculture Circular, 211, 3-24. References
- 23. 10. Glaser, R. W., McCoy, E. E., & Girth, H. B. (1940). The biology and economic importance of a nematode parasitic in insects. The Journal of Parasitology, 26(6), 479-495. 11. House, H. L., Welch, H. E., & Cleugh, T. R. (1965). Food medium of prepared dog biscuit for the mass- production of the nematode DD136 (Nematoda; Steinernematidae). Nature, 206(4986), 847-847. 12. Molyneux, A. S. (1986). Heterorhabditis spp. and Steinernema (= Neoaplectana) spp.: temperature, and aspects of behavior and infectivity. Experimental Parasitology, 62(2), 169-180. 13. Negrisoli Jr, A. S., Garcia, M. S., & Negrisoli, C. R. B. (2010). Compatibility of entomopathogenic nematodes (Nematoda: Rhabditida) with registered insecticides for Spodoptera frugiperda (Smith, 1797)(Lepidoptera: Noctuidae) under laboratory conditions. Crop Protection, 29(6), 545-549. 14. Oliveira-Hofman, C., Kaplan, F., Stevens, G., Lewis, E., Wu, S., Alborn, H. T., ... & Shapiro-Ilan, D. I. (2019). Pheromone extracts act as boosters for entomopathogenic nematodes efficacy. Journal of invertebrate pathology, 164, 38-42.. 15. Poinar Jr, G. O. (1975). Description and biology of a new insect parasitic Rhabditoid, Heterorhabditis bacteriophora N. Gen., N. Sp.(Rhabditida; Heterorhabditidae N. Fam.). Nematologica., 21(4), 463-470. 16. Poinar Jr, G. O., & Grewal, P. S. (2012). History of entomopathogenic nematology. Journal of Nematology, 44(2), 153. References
- 24. 17. Poinar, G. O., & Thomas, G. M. (1966). Significance of Achromobacter nematophilus Poinar and Thomas Achromobacteraceae: Eubacteriales) in the development of the nematode, DD-136 (Neoaplectana sp. Steinernematidae). Parasitology, 56(2), 385-390. 18. Poinar, G. O., Jr. 1966. The presence of Achromobacter Nematophilus Poinar and Thomas in the infective stage of a Neoaplectana sp. (Stei- nernematidae: Nematoda). Nematologica 12:105–108. 19. Schroeder, W. J., & Beavers, J. B. (1987). Movement of the entomogenous nematodes of the families Heterorhabditidae and Steinernematidae in soil. Journal of Nematology, 19(2), 257. 20. Stock, S. P., Pryor, B. M., & Kaya, H. K. (1999). Distribution of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) in natural habitats in California, USA. Biodiversity & Conservation, 8(4), 535-549. 21. Thomas, G. M., & Poinar JR, G. O. (1979). Xenorhabdus gen. nov., a genus of entomopathogenic, nematophilic bacteria of the family Enterobacteriaceae. International Journal of Systematic and Evolutionary Microbiology, 29(4), 352-360. 22.Wouts, W. M., Mráček, Z., Gerdin, S., & Bedding, R. A. (1982). Neoaplectana STEINER, 1929 a junior synonym of Steinernema TRAVASSOS, 1927 (Nematoda; Rhabditida). Systematic Parasitology, 4(2), 147-154. References
Editor's Notes
- IJSIJs locate host via host stimuli – larva, feces, and carbon dioxide invade through natural openings – mouth, anus, spiracles IJS releases symbiont bacterium into the host hemo-lymph The bacterium multiplies and kills the host by septicemia The nematode will feed o bacteria and degraded host tissue, mature, mate, and reproduce new IJS that will search for new hosts
- The first entomopathogenic nematode was described by Steiner as Aplectana kraussei (now Steinernema kraus- sei) in 1923 and at that time was considered no more than a curiosity
- Glaser and colleagues produced suffi-cient numbers of the nematode for field trials and in the 1930s applied it to 73 dif- ferent field plots in New Jersey to control the Japanese beetle (17,20). Parasitized grubs were recovered from 72 of the 73 plots two weeks after application. Parasit- ism of the grub population by the nema rode in the various plots ranged from 0.3% to 81%. They determined nematode psistence in four of the plots by placing healthy beetle larvae in the plots and later examining them for infection. The nema- tode persisted in the plots for the 8.5 years of their trials (18,20).
- It was not until Jaroslav Weiser (1955) (Fig. 1) described a European population of Neoaplectana carpo- capsae from codling moth larvae and Dutky and Hough (1955) isolated the DD-136 strain of an undescribed steinernematid from codling moth larvae in Eastern North America that serious studies on the pathogenic- ity and life history of entomopathogenic nematodes began. In 1965, cultures of S. carpocapsae were obtained from Weiser and using morphology and hybridization studies, it was shown that the Czechoslovakian strain of S. carpocapsae and the North American DD-136 nema- tode were conspecific (Poinar, 1967).
- Abstract : Stock cultures of the nematode, DD-136, and its associated bacteria may be maintained on autoclaved cubes of pork kidney on peptone glucose medium, and on reconstituted whole egg solids. Mass rearing in larvae of the greater wax moth, Galleria mellonella, is described and yields up to 200, 000 infective stage nematodes per larva. Survival times of wax moth larvae exposed to the nematode are given. Storage procedures are outlined. H.E.W. ISSN : 0022-1910 Shapiro-Ilan, D. I., Morales-Ramos, J. A., & Rojas, M. G. (2016). In vivo production of entomopathogenic nematodes. In Microbial-Based Biopesticides (pp. 137-158). Humana Press, New York, NY.
- The technique is simple. 20 ml. of the dog food is mixed with 20 ml. of distilled wat er in a 9-cm Petri- dish. This is then autoclaved for 15 min at l5lb. pressure and cooled overnight under sterile conditions. The nematodes for inoculum are rinsed five times with 0·1 per cent formalin to remove most of the contaminants. Each plate is inoculated wit h 1- 2 x 103 nematodes. A few days incubation at about 24° C gives rise to large numbers of nematodes, indicated by the shiny grey a ppearance of the culture. When t he nematodes become heavily concentrated in the culture they align and remain m.otionless until disturbed. They may be gathered from 8 to 14 days after inoculation, but some samples may t ake up to 20 days to develop to this stage. When the inoculum is too sparse (1- 5 x 102 nematodes per plate), collection may be delayed a week or more. The noma- todes are washed from the medium into a large container and are allowed to settle. The suspended debris from the medium is decanted and discarded. The nematodes are then stored in 0 ·1 per cent formalin at 5°-10° C in litre flasks.
- Most infective juveniles of DD-136 (Neoaplectana, Steinernematidae) were found to contain cells of Achromobacter nematophilus Poinar & Thomas in the ventricular portion of their intestinal lumen. In two instances, anterior intestinal cells of the infective juveniles were found to contain bacterial cells, presumably those of A. nematophilus. When the infective stage penetrated into the body cavity of a suitable host, the bacteria were released through the anus and multiplied rapidly in the host's body, resulting in a fatal septicemia. The symbiotic bacterium (under the name, Achromobacter nematophilus)associated with S. carpocapsae was de- scribed by Poinar and Thomas (1965). By culturing Galleria mellonella, Neoaplectana sp. (DD-136) and Achromobacter nematophilus separately under axenic conditions in the laboratory, it was possible to study the relationship between the bacterium and nematode during nematode parasitism of the insect. The infective-stage juveniles of the nematode were able to penetrate and kill the insect host without the presence of A. nematophilus or any other bacterium. However, without accompanying bacteria the nematode was unable to reproduce. Only when A. nematophilus or a possible replacement, such as Pseudomonas aeruginosa, was added to the blood did reproduction occur. The relationship between A. nematophilus and the nematode is considered a mutualistic one, since the bacterium lives and is protected inside the intestine of the free-living stage of the nematode and is transported and released by the nematode to the haemolymph of a host insect. The nematode, in turn, is dependent on the bacterium for reproduction.
- heterogonic cycle with both hermaphroditic and dioecious females; males with a well developed papillate bursa and dauer stages capable of entering the haemocoel of healthy insects. Rhabditis hambletoni Pereira, 1973 is transferred to Heterorhabditis. Aside from Neoaplectana spp., H. bacteriophora is the only nematode known as a vector for a bacterial disease of insects. The infective stage juveniles of this nematode carry a specific bacterium in their intestines which is released after the parasites enter the body-cavity of a healthy insect. The bacteria kill the insect in 48 hrs., and the juveniles develop into hermaphroditic females that produce young which develop into males and females. The latter mate and produce juveniles that develop into infective stages, which leave the cadaver and search for a new host. The infective stage of H. bacteriophora can invade and kill larvae of Galleria mellonella in 48 hours and it can also destroy larvae of Culex pipiens.
- . The normal habitat of these bacteria is the intestinal lumen of nematodes or the body cavity of host insects mately associated with entomogenous nematodes. The normal habitat of these bacteria is the intestinal lumen of nematodes or the body cavity of host insects into which they have been introduced by the nematodes. The genus is placed in bacteria is the intestinal lumen of nematodes or the body cavity of host insects into which they have been introduced by the nematodes. The genus is placed in the family Enterobacteriaceae since the bacteria possess
- Abstract Rats inoculated either per os or intraperitoneally with infective-stage juveniles of Neoaplectana carpocapsae showed no signs or symptoms of pathogenicity, toxicity, evidence of infection, or nematode-related histopathology. Blood and urine samples did not reveal nematode presence. No significant differences were noted in weight gain between treated and control animals after 36 days. Nematodes recovered in fecal samples from per os inoculated rats were virtually all dead. Those recovered from intraperitoneally-inoculated rats were usually encapsulated.
- Materials and methods Nematode - Neoaplectana (Steinernema) carpocapsae Host – Greater wax moth (Galleria mellonella L. Soil type (i) pure silica sand (ii) coarse sandy loam (iii) silty clay loam (iv) clay
- Abstract Rats inoculated either per os or intraperitoneally with infective-stage juveniles of Neoaplectana carpocapsae showed no signs or symptoms of pathogenicity, toxicity, evidence of infection, or nematode-related histopathology. Blood and urine samples did not reveal nematode presence. No significant differences were noted in weight gain between treated and control animals after 36 days. Nematodes recovered in fecal samples from per os inoculated rats were virtually all dead. Those recovered from intraperitoneally-inoculated rats were usually encapsulated.
- Objective Select Steinernema feltiae strain for enhanced host finding ability Materials and method Placed the nematode at a point away from host larva Select the nematode migrating towards (positive) or away (negative) from the host Repeated the assay 13X for the positive nematodes
- Findings: 26% of sample 8 out of 10 survey regions No nematode in desert and redwood regions Steinernematids were predominantly found Steinernematids were found in coniferous forests, oak woodlands and grasslands Heterorhabditids were isolated from coastal marshes.
- Compatibility of the insecticides with EPNs was evaluated by observing mortality and infectivity of infecting juveniles (IJs) 48 h after immersion in solution of the insecticide formulations. Among all insecticides tested, LorsbanÔ (chlorpyrifos), DecisÔ (deltamethrin), MatchÔ (lufenuron), DeltaphosÔ (deltramethrin þ triazophos), DimilinÔ (diflubenzuron), StallionÔ (gamacyha- lothrin), Karate ZeonÔ (lambdacyhalothrin) TracerÔ (spinosad), VexterÔ (chlorpyrifos), GalgotrinÔ (cypermethrin), CerteroÔ (triflumuron), and TalcordÔ (permethrin) were compatible (class 1) with the three nematode species tested under laboratory conditions
- Materials and method Nematode- Steinernema carpocapsae and S. feltiae Hosts- Galleria mellonella, Tenebrio molitor L, Curculio caryae, and Hermetica illucens
- Materials and method Nematode- Steinernema carpocapsae and S. feltiae Hosts- Galleria mellonella, Tenebrio molitor L, Curculio caryae, and Hermetica illucens