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Techniques in insect nematology
Abstract
Publisher Summary
This chapter focuses on the techniques used for identifying, isolating, propagating, assaying, and preserving nematodes that are parasitic in or pathogenic to insects. Nematodes are nonsegmented animals with excretory, nervous, digestive, reproductive, and muscular systems but lacking circulatory and respiratory systems. The stage of entomogenous and entomopathogenic nematodes that is infective varies depending on the group. A good stereomicroscope is essential for nematode identification and should have a range of magnification between 10 and 100X, a fairly fiat field, and good resolution. The gonads and other structures of fixed nematodes may be obscured by the granular appearance of the intestine. Specimens can be cleared by processing to lactophenol or glycerin. The cephalic structures and the number of longitudinal chords are diagnostic characters for genetic or specific determination of certain groups of nematodes. Extraction methods for insect nematodes are derived from techniques developed with plant-parasitic nematodes. It is found that the most common methods are the Baermann funnel, sieving, elutriation, and centrifugal flotation.
... In this study, mass production of G. mellonella larvae was ensured for the new generation nematode production in obtaining entomopathogenic nematodes from the soil and diagnosing the obtained entomopathogenic nematodes. For culturing G. mellonella, 2000 ml wheat bran, 250 ml glycerin, 200 ml honey, and 100 ml distilled water were mixed thoroughly in a container until homogenized, and then 200 ml of bee-wax was added to the mixture in small pieces (Kaya & Stock, 1997). Mass production of G. mellonella larvae was carried out at 25 °C in artificial diet prepared in this way. ...
... Dead larvae that were identified as infected were removed from the soil and placed in White's Trap (White, 1927), where entomopathogenic nematodes could be obtained after multiplying and leaving the host. After obtaining entomopathogenic nematodes, they were placed in 250 ml tissue culture flasks filled with distilled water and stored in climate cabinets at 15 °C (Kaya & Stock, 1997). In vivo production of entomopathogenic nematodes: To determine whether the isolates obtained from soil samples were entomopathogenic nematodes and to propagate those isolates with infectivity, inoculation was performed on G. mellonella larvae. ...
... Thus, in this environment, the next generation infective larvae were produced on G. mellonella larvae, which served as the host (Koppenhofer, 2000). The infective juveniles that moved into the water were transferred to flasks and stored in climate cabinets at 15 °C (Kaya & Stock, 1997). ...
With increasing concerns over the adverse environmental impacts of chemical pesticides, there has been a growing interest towards sustainable pest management strategies worldwide. In this context, entomopathogenic nematodes (EPNs) have emerged as promising biological control agents against insect pests in agricultural production. This study focused on the isolation, identification, and characterization of entomopathogenic nematodes (EPNs) in Şırnak Province, Türkiye. Over the period from July to September 2023, a comprehensive survey involving the collection of 256 soil samples led to the successful isolation, of 11 EPN isolates, which accounted for 4.3% of the samples. Morphological examinations and molecular diagnostics, including mitochondrial COI and 18S rDNA gene regions, facilitated the precise identification of these isolates. Notably, our findings revealed a diverse population of EPNs, predominantly Heterorhabditis bacteriophora, comprising five isolates, alongside four distinct species of Steinernema (one Steinernema carpocapsae, one Steinernema affine, two Steinernema feltiae and two Steinernema sp.). Phylogenetic analysis, utilizing the mitochondrial cytochrome oxidase subunit I (COXI) and the 18S rDNA gene regions, indicated low genetic variability within H. bacteriophora, whereas a higher level of diversity was observed among Steinernema species. Notably, the presence of diverse EPN species highlights their capacity as effective biocontrol agents against a range of insect pests. These findings provide valuable insights into the biodiversity of EPNs in Şırnak Province and underscore the significance of utilizing local isolates for sustainable agricultural practices. Further research is warranted to elucidate the efficacy of these isolates in integrated pest management strategies tailored to the region's specific agroecological conditions.
... molitor) late instar larvae were added as insect bait and incubated in the sealed ice-cream containers at ambient temperature for 1-2 weeks to assess for native EPN present in the soil samples, in an adaptation of the insect-baiting method of Bedding & Akhurst (1975). For the soil of each individual plot, mealworm cadavers potentially killed by EPN infection, and showing similar pathogenesis (based on colour), were rinsed in distilled water and placed on a modified White trap to collect emerging IJ (Kaya & Stock, 1997). ...
... Tenebrio molitor larvae/pupae cadavers from soil prebaiting in the semi-field trial showing signs of EPN infection were placed on modified White traps, and IJ collected upon emergence (Kaya & Stock, 1997). Phlyctinus larval cadavers from the semi-field trial showing signs of EPN infection were dissected to confirm mortality by infection. ...
... Phlyctinus larval cadavers from the semi-field trial showing signs of EPN infection were dissected to confirm mortality by infection. Phlyctinus larval cadavers with confirmed mortality by EPN infection that were showing different pathology than S. yirgalemense infection (typically dark yellow brown on Phlyctinus larvae) were kept on modified White traps in cases where dissection did not destroy the cadaver, and IJ collected upon emergence (Kaya & Stock, 1997). ...
Considerable progress has been made in the surveying, taxonomy, screening, mass production and formulation of entomopathogenic nematodes (EPN) and their associated symbiotic bacteria in South Africa. Steinernema yirgalemense isolate 157-C is one of the most promising native EPN candidates with regards to virulence, its broad insect-host spectrum, and can be readily mass-produced and formulated into a commercial product. The banded fruit weevils, Phlyctinus callosus sensu stricto and Phlyctinus xerophilus , previously grouped together under the Phlyctinus callosus sensu lato species concept, are native entimine weevils of economic importance to deciduous fruit, grapevine and berries in the Western Cape province of South Africa. This study investigated potential differences in baseline susceptibility of larvae and pupae of the two weevil species to S. yirgalemense in laboratory screenings. The test arena used was 24-well bioassay plates, with an inoculation concentration of 200 infective juveniles (IJ) insect ⁻¹ for larvae and 100 IJ insect ⁻¹ for pupae. Infection was determined 48 h and 96 h after inoculation. Field-efficacy of S. yirgalemense , applied at a concentration of 60 IJ cm ⁻² , against larvae of the two weevil species was determined in an ecologically relevant semi-field trial. In all cases in vitro mass-produced IJ of S. yirgalemense were used. No baseline differential susceptibility between P. callosus and P. xerophilus larvae was obtained in laboratory screenings. Phlyctinus pupae were approximately twice as susceptible compared to larvae, with significant differences between bioassay batches. Approximately 45% control of P. xerophilus larvae was obtained after 96 h of exposure to S. yirgalemense in the field, differing significantly from the control and P. callosus treatment. Low levels of Phlyctinus larval infection by native EPN (confirmed as Heterorhabditis bacteriophora from one P. xerophilus cadaver) occurred in both control and EPN treatment groups under field conditions.
... At each site, the soil sample (approximately 2000 g) was composed of five random sub-samples taken with a hand shovel to a depth of 30 cm over an area of 10 m 2 . The composite samples were placed in polyethylene bags to prevent water loss, and these were kept in coolers as recommended by Kaya and Stock (1997). The soil samples were transported to the Plant Protection Research Laboratory in the Faculty of Agriculture at Tishreen University for the isolation of EPNs. ...
... In each round, new G. mellonella larvae were added to the containers, and the soil moisture was adjusted as needed. The larvae in the containers were checked every two days (Kaya and Stock, 1997). Dead larvae that exhibited marks of infection with EPN were collected, washed twice in distilled water, and placed in a modified White trap (White, 1927). ...
... For the light microscope observations and assessment, 20 living specimens were examined from each life stage of males and IJ. Additionally, specimens were killed and fixed in TAF (7ml Formalin, 2ml Triethanolamine, 91 ml water) (Kaya and Stock, 1997). Morphological traits and morphometric measurements were observed under a light microscope equipped with software (Optika Proview, Optika, Ponteranica, Italy) using 4×, 10×, 40×, and 60× objectives. ...
The occurrence and distribution of entomopathogenic nematodes (EPNs) in the Syrian coast regions remain relatively uncharted. To address this gap in our knowledge, an extensive survey of these ecosystems was essential. This study aims to isolate and identify EPNs from diverse ecosystems within the coastal regions. The distribution of EPNs in cultivated and natural environments was analyzed according to habitat, altitude, and sampling season factors. Between 2017 and 2020, EPNs were recovered from 27 out of 821 soil samples (3.28%) and collected from 24 out of 375 sampling sites (6.4%). Based on morphological, morphometric, and molecular (ITS) characteristics, four EPN species were identified: Heterorhabditis indica (51.85%), representing the first report of its occurrence in the coastal regions, H. bacteriophora (33.33%), H. pakistanense (7.4%), which is also reported for the first time in Syria, and Steinernema affine (7.4%). There were statistical differences in the abundance and recovery frequency of EPNs in each type of habitat. Additionally, there were statistical differences in the altitude and sampling season recovery frequency.
Co-inertia analysis revealed correlation between the distribution and occurrence of EPNs in vegetation habitats, altitude, and sampling seasons, as well as some soil characteristics. H. indica and H. bacteriophora were associated with citrus orchards, low-altitude ranges, moderate organic matter, and acidic soil. More specifically, H. indica isolates were correlated with olive orchards, vegetable fields, autumn season, and clay, sandy, and sandy loam soils. Meanwhile, H. bacteriophora isolates were correlated with tobacco fields, grasslands, alkaline pH, spring season, silty loam, and clay loam soils. H. pakistanense was linked to pear orchards, vineyards, moderate pH, and low organic matter. S. affine occurred in walnut orchards, silty soil, higher altitudes, and winter season.
The virulence levels of three native EPN isolates ( S. affine , H. indica and H. bacteriophora ) were evaluated against 3 rd and 4 th instar larvae (outside and inside mines) and pupae of T. absoluta , a destructive pest in Syria. All three native EPN species exhibited ability to infect and kill the insect, with observed significant differences in their virulence. This study provides an understanding of EPN occurrence, distribution, and their potential for application in sustainable pest control strategies in Syria.
... A 100 g soil sample from each sampling site was placed into a glass container each with three last instar larvae of the wax Moth Galleria mellonella (L.) and covered with a lid. (Bedding and Akhurst, 1975;Kaya and Stock, 1997). The samples were then stored at room temperature. ...
... The samples were then stored at room temperature. After 10 days, dead larvae were collected and transferred to White traps to collect the emerging IJs (Kaya and Stock, 1997). ...
Nematode population densities were determined in 60 soil and root samples collected from 6 fruit orchards in the Bilecik province (western Turkey), between April 2022 and June 2022. The total number of identified nematodes have reached up to 2418 individuals (number of female: 1036; male: 154; and juvenile: 1228). They belong to 54 species, 54 genera, 33 families and 11 orders. Plant parasitic nematodes that were detected mostly are listed as follows: Helicotylenchus (6,12 %), Pratylenchus (5,74 %), Paratylenchus (4.83 %), Xiphinema (3,06 %), Tylenchorhynchus (2,19 %), Malenchus (1.94 %) and Tylenchus (1.19 %). According to the maturity index analysis, mean values showed the highest maturity level at peach trees (MI value: 3,52), followed by; walnut trees (MI value: 2.49), cherry trees (MI value: 2.15), nectarine trees (MI value: 1.86), plum trees (MI value: 1.57), and olive trees (MI value: 1.42). Mostly the diverse group in terms of species richness was within the order Dorylaimida. The nematodes associated with peach and walnut trees here showed the most stable environments in terms of soil nematode community structure.
... During 10 days as a holding period, the samples were checked for dead insects. Cadavers of insect larvae were transferred individually to the modified White traps (Kaya and Stock 1997). If the larvae were found still alive, the results were considered negative and the soil sample was discarded. ...
... Infective juveniles (IJs) were transferred onto moist filter paper in Petri dishes where living G. mellonella larvae were added. The new generations of IJs were collected in a beaker and rinsed twice with sterile distilled water and stored at 13 °C as described by (Kaya and Stock 1997). ...
Background
Isolation of novel species of entomopathogenic nematodes (EPNs) with biocontrol potential against important insect pests is very important for the sustainable management of economic pests damaging food crops and providing protection to the agricultural environment. This study was aimed to new indigenous EPN isolates from Egyptian agricultural soils and studies its biocontrol potential for further use in the biological control programs. Five out of 15 soil samples obtained from a farm located at the Cairo–Alexandria desert highway was positive for the presence of EPN, using the greater wax moth baiting method.
Results
Sequencing of the internal transcribed spacer (ITS) region of 4 of the nematode isolates suggested that they belong to the species Heterorhabditis indica . However, one isolate does not show a high similarity to any of the H. indica previously recorded in the database of the Gen Bank and hence was identified as a new Heterorhabditis species and was deposited at the National Center for Biotechnology Information (NCBI) and registered under accession no. (OP555450) under the name of Heterorhabditis alii . This new species was also registered in the ZooBank under the registration link of: LSID urn: lsid: zoobank.org: act: 306F9D57-CC30-4B8E-8B19-4F0E42B08F34. No males were found in this species. Morphological characterization using the light microscope (LM) and scanning electron microscope (SEM) confirmed the identification of this nematode as a new species of the genus Heterorhabditis . Moreover, virulence of this new species against the fall armyworm (FAW), Spodoptera frugiperda (Smith 1797) (Lepidoptera: Noctuidae) was tested in comparison with the foreign EPN species, Heterorhabditis bacteriophora (HP88) and the local Heterorhabditis indica (Mango 2 isolate) and proved to be more effective against this devastative insect pest than the two compared species.
Conclusions
The present study found out a new species of the EPN genus, Heterorhabditis in Egypt. Our results were confirmed by both morphological and molecular analyses. The efficacy of this new species against the FAW proved to be a potent and safe biocontrol agent that can be used in biological control programs against this invasive insect pest of corn in Egypt and other global countries.
... Eleven treatments repetitions per treatment were evaluated with one insect per repetition. A techn selected from Insect Nematology [17] and the insects were observed every 24 h un mortality of the adult S. acupunctatus was obtained. The weevil cadavers were p white wet chambers and checked to determine whether there were any EPNs. ...
... Eleven treatments with 20 repetitions per treatment were evaluated with one insect per repetition. A technique was selected from Insect Nematology [17] and the insects were observed every 24 h until 100% mortality of the adult S. acupunctatus was obtained. The weevil cadavers were placed in white wet chambers and checked to determine whether there were any EPNs. ...
The weevil Scyphophorus acupunctatus Gyllenhal causes damage and losses in agave crops and has traditionally been controlled using contact and systemic agrochemicals. Implementing microbial control strategies is proposed as an alternative to mitigate the environmental impact associated with agrochemicals. The objective of this study was to determine the survival of entomopathogenic nematodes in oil emulsions for the control of adult S. acupunctatus. Three species of entomopathogenic nematodes were evaluated: Steinernema carpocapsae, S. glaseri, and Heterorhabditis bacteriophora. We used two concentrations (50 ± 5 and 100 ± 10 infectious juvenile nematodes), and oil emulsions derived from Salvia hispanica, Triticum vulgare, and Olea europea with oil purity of 20% and 40%. The effectiveness of these treatments was assessed by determining the mortality rate of S. acupunctatus. The results indicate that the combination of S. glaseri and H. bacteriophora, at concentrations of 50 ± 5 and 100 ± 10 nematodes, respectively, with T. vulgare and O. europea oils, achieved a mortality rate of 85.76% in S. acupunctatus adults at 24 h. At 120 h, a mortality rate of 100% was achieved with specific formulations, such as S. glaseri with 100 ± 10 nematodes + O. europea, and H. bacteriophora with 100 ± 10 nematodes + O. europea. Consequently, we conclude that oil formulations combined with nematodes show potential as an effective and environmentally friendly alternative for the control and management of S. acupunctatus.
... In turn, G. mellonella is also used to observe the development of the EPN, learn more about it, and obtain information including its life cycle duration and the number of generations it could produce within the host [13][14][15]. In terms of the abovementioned families, the storage conditions for steinernematids involve temperatures that range from 8 to 15 • C, and they survive for 6 to 9 months; on the other hand, heterorhabdithids, when kept under the same conditions, survive for 3 to 4 months [16]. Therefore, knowing the requirements of each species is important in this field of study because they can vary considerably; currently, it is believed that it is best to use native species of EPNs in geographical areas where biocontrol is applied [17][18][19]. ...
... Understanding EPNs in detail is essential, in addition to knowing whether or not the evaluated conditions affect their pathogenicity. According to Kaya and Stock [16], the optimum storage temperatures for steinernematids is 4 to 15 • C for 6 to 9 months, and the heterorhabdithids under similar conditions can only be kept for 3 to 4 months; they also mention that inocula between 1000 and 2000 are ideal for storage and that most species are active at a temperature of 25 ± 2 • C. ...
Entomopathogenic nematodes have been used in biological control for some time and are an alternative for the control of insect pests, but during their implementation, situations have arisen that can be improved. These vary with each species and include their production and storage. Oscheius myriophila, an entomopathogenic nematode (EPN), was monitored for its performance when produced in vivo, as well as its development using Galleria mellonella larvae, using the MC5-2014 strain isolated from soil samples in the municipality of Tepalcingo, Morelos, México. For a study with native strains of EPNs, a wide range of tests must be conducted because the required conditions can be very specific. In vivo production was quantified at initial infective juvenile (IJ) inocula of 50, 100 and 500, and we obtained the same production for the three inocula. The life cycle of the EPNs lasted 12 days, and two generations were observed in which adults were found at days 5 and 9. Both evaluations were performed at a temperature of 27 °C in G. mellonella larvae. In addition, the temperatures of 8, 12, 20 and 24 °C were evaluated for their storage, and we observed that the EPNs can be kept for at least 6 months, maintaining a survival rate of 58.67% and a good infective capacity at a temperature of 12 °C, remaining above 60%.
... Each nematode strain was multiplied separately on the last instar larvae of rice moth, Corcyra cephalonica (Stainton, 1866). Fifty larvae were kept in a 20 cm diameter petri dish lined with filter paper and inoculated with 1 × 10 3 IJs of a single EPN strain contained in 0.5 ml of sterilized distilled water (Kaya and Stock 1997). The same procedure was adopted for the other EPN strain used in the study. ...
Efficacy of two Indian isolates of entomopathogenic nematodes (EPNs) viz., Heterorhabditis bacteriophora MK256358 and Steinernema feltiae MK256355 were tested in laboratory against the larvae of cabbage butterfly, Pieris brassicae. Larval mortality was found directly proportional to initial inoculum level of infective juveniles (IJs). Susceptibility of larvae varied with respect to their variable size. H. bacteriophora MK256358 @ 25 IJs/larva caused 100% mortality to 3rd instar larvae at 72 h but @ 75 IJs/larva, the same mortality was achieved in 48 h. S. feltiae MK256355 @ 100 IJs/larva caused 100% mortality to 3rd instar larvae of P. brassicae at 48 h. H. bacteriophora MK256358 @ 25 and 100 IJs/larva resulted in 100% mortality to 4th and 5th instar larvae, respectively at 72 h, however S. feltiae MK256355 was unable to cause 100% mortality to either 4th or 5th instar larvae at any inoculum level or time period used in the study. LD50 and LT50 values of H. bacteriophora MK256358 were lower than S. feltiae MK256355 indicating that less nematode dose and time is required to kill 50% pest population. Reproduction capacity of nematode within the host was directly proportional to individual larval size and nematode inoculum level and for H. bacteriophora MK256358, it was higher and statistically significant (P ≤ 0.05) from S. feltiae MK256355. Our experimental findings open new avenues for utilization of EPNs against P. brassicae and set the basis for safe insect pest management programme.
... Following heat-killing, the nematodes were preserved in triethanolamine formalin (TAF) fixative before being processed using a gradual evaporation method with anhydrous glycerin for mounting (Poinar, 1976;Kaya and Stock, 1997). A drawing tube and differential interference components were added to an Olympus BX41 microscope for use in morphometric and morphology research. ...
Three species of nematodes within Acrobeloides (Cobb, 1924); Thorne, 1937 (Rhabditida, Cephalobidae) were collected and recorded for the first time in Iraq, based on morphometric and molecular data, these species, A. saeedi Siddiqi, Ley & Khan, 1992, A. apiculatus (Thorne, 1925), and A. bodenheimeri (Steiner, 1936), were molecularly characterized using the partial 28S rRNA gene sequences. The phylogenetic tree has been constructed to separate Acrobeloides species from closely related species.
... If no EPNs were detected initially, the assay was repeated. Dead larvae exhibiting signs of nematode infection were individually transferred onto modified White traps (Kaya and Stock 1997) and maintained at 23°C ± 1°C. The collected IJs underwent several cycles of exposure to fresh G. mellonella larvae to confirm pathogenicity and establish the new culture. ...
Entomopathogenic nematodes (EPNs) are closely associated with Popillia japonica and potentially used as their biological control agents, although field results proved inconsistent and evoked a continual pursuit of native EPNs more adapted to the environment. Therefore, we surveyed the Azorean Archipelago to isolate new strains of Heterorhabditis bacteriophora and to evaluate their virulence against the model organism Galleria mellonella under laboratory conditions. Six strains were obtained from pasture and coastal environments and both nematode and symbiont bacteria were molecularly identified. The bioassays revealed that Az172, Az186, and Az171 presented high virulence across the determination of a lethal dose (LD50) and short exposure time experiments with a comparable performance to Az29. After 72 hours, these virulent strains presented a mean determination of a lethal dose of 11 infective juveniles cm ⁻² , a lethal time (LT50) of 34 hours, and achieved 40% mortality after an initial exposure time of only 60 minutes. Az170 exhibited an intermediate performance, whereas Az179 and Az180 were classified as low virulent strains. However, both strains presented the highest reproductive potential with means of 1700 infective juveniles/mg of larvae. The bioassays of the native EPNs obtained revealed that these strains hold the potential to be used in biological control initiatives targeting P. japonica because of their high virulence and locally adapted to environmental conditions.
... To maintain the S. calcitrans colony, adult stable flies were confined within plastic containers (30 cm  35 cm  20 cm) and pro- in this study. These nematodes were cultured using the last larval stage of the greater wax moth (Galleria mellonella L.; Lepidoptera: Pyralidae) as a host, following the methods described by Kaya and Stock (1997). A suspension of EPN (200 IJs/700 μL of distilled water) was evenly applied to a 5.5 cm diameter Petri dish lined with two discs of Whatman ® No.1 filter paper. ...
The stable fly, Stomoxys calcitrans L. (Diptera: Muscidae), is a significant insect pest with global veterinary implications due to its capacity to both cause nuisance and transmit disease‐causing pathogens to livestock. This study aimed to determine the livestock bedding preferred for use as a development substrate by S. calcitrans larvae and field‐collected adults. The result showed that S. calcitrans larvae exhibited a preference (26.7%) for 7‐day‐old cow manure. Gravid females displayed a pronounced preference (55.0%) for fresh cow manure. As there were eight choices, indifference would result in 12.5% for each bedding substrate. Furthermore, the efficacy of four entomopathogenic nematodes (EPNs), namely Heterorhabditis bacteriophora (Poinar), Heterorhabditis indica Poinar, Karunakar & David (Rhabditida: Heterorhabditidae), Steinernema siamkayai Poinar, Karunakar & David and Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae), against S. calcitrans larvae and the persistence after application to livestock bedding substrates were evaluated under laboratory conditions. In filter paper bioassays, all four EPNs caused 76.7%–100.0% mortality in the second instar larvae of S. calcitrans when applied at 50 and 100 infective juveniles (IJs)/cm ² within 5 days after exposure. For the third instar larvae of S. calcitrans , only H. indica induced high mortalities of 86.6% when applied at 100 IJs/cm ² within 5 days after exposure, while the other EPNs resulted in mortalities of less than 70%. The data further demonstrated that H. bacteriophora , H. indica and S. siamkayai remained present in the substrates linked to S. calcitrans larvae for as long as 7 days after the application of EPNs. This study demonstrates the potential of EPNs as a biologically based control agent against larvae of S. calcitrans , a serious pest and significant vector for various livestock animals.
... At this stage, nematodes are not fed, do not develop, and can survive for many months (Batalla-Carrera et al., 2010;Bathon, 1996;Grewal et al., 2003;Shapiro-Ilan et al., 2013). Insects have known about entomopathogenic nematodes since the 17th century (Nickel, 1984), but the use of nematodes to manage insect pests did not receive much attention until the 1930s Kaya & Patricia Stock, 1997;Smits, 1996). Before 1929, entomopathogenic nematode was not approved as an insect control agent, and Japanese beetles, Popillia japonica (Newman), were discovered in large numbers on the Tavistock Golf Course near Haddonfield, New Jersey, in 1929 by Glaser andFox (1930), infected with a nematode (Chandler et al. 1997;Gaugler et al. 1997;Kung et al. 1990;Rasmann & Turlings, 2008;Zhang et al. 2008, Ye et al. 2010. ...
An essential part of managing insect pests is the use of entomopathogenic nematodes and in preventing environmental contamination. Their use has been increasing in recent years. So far, about 30 to 40 nematode families are in contact with insects and other vertebrates. Among these families, the group widely studied as the so-called "entomopathogenic nematodes," also known as EPN, are Heterorhabditidae and Steinernematidae. Two species of Oscheius (Oscheius chongmingensis and Oscheius carolinensis) have been added in recent years to the EPNs group, and we expect that several species will be added to EPNs. ENP has a wide range of host insects found in a species of EPN that can attack over 250 different kinds of insects from several families. Suitable environments for EPNs include insect hemocoels, soil pores, or river bottoms that grow in contact with these environments. Occurrence, mobility, distribution, and stability of EPN under the influence of several factors, including intrinsic factors such as behavioral, physiological, and genetic characteristics. Biological nature included are hosted and non-host arthropods, predators, parasites, diseases, and aberrant environmental elements like temperature, moisture content, texture, pH, and UV radiation. Proper mass production and application are essential for the biological control effectiveness of entomopathogenic nematodes (EPN). In addition, there is no problem in applying EPNs because they are simple to spray with common equipment and are compatible with almost all chemical fertilizers, but the compatibility is different from chemical pesticides.
... The isolated nematodes were then processed into anhydrous glycerin (Seinhorst 1966), viewed under a microscope, and identified by species using a key. Isolated and identified species of entomopathogenic nematodes reared according to Kaya and Stock (1997). The newly emerged nematodes were kept at 10 °C in tissue culture flasks and used for further laboratory bioassays over a 2-4 week period to see how they were affected by the three biostimulants. ...
This study aimed to evaluate the effect of biostimulants derived from Streptomyces avermitilis metabolites on entomopathogenic nematodes: Steinernema carpocapsae , Steinernema feltiae , and Heterorhabditis bacteriophora , obtained from the soil of several Miscanthus × giganteus plantations in 2020–2023. The nematodes were isolated, identified, and cultured using live insects (the greater wax moth Galleria mellonella ). Three preparations containing biostimulants – Charkor, Stimpo, and Regoplant, were tested for compatibility with entomopathogenic and plant parasitic nematodes. Their effect on nematode survival was evaluated using the Petri dish test. The study showed that the effect of biostimulants on the survival of nematodes depended on the concentration of aversectin contained in the evaluated preparations. Stimpo and Regoplant had an adverse effect on plant parasitic nematodes. The highest G. mellonella mortality was observed at the higher dose of Charkor (0.4%), and the lowest at the lower dose of Regoplant (1.22%). The study showed that the virulence of the nematodes decreased after 48 hours of incubation in Regoplant and Stimpo solutions containing aversectin. The degree of interaction between nematodes depended on the nematode species, trophic preferences, and the concentration of active ingredients in the preparations. This conclusion is crucial for the selection of appropriate types of entomopathogenic nematodes and the application rate of plant parasitic nematodes in the assessment of their short- and long-term spread, persistence, and recycling in field conditions.
... The container then inversely placed and incubated at room temperature. After 3 to 4 days, the cadavers were rinsed with sterile water and the nematode was extracted according White trap method (Kaya and Stock, 1997). The suspension of nematode obtained from this trap was kept in the refrigerator at 10 0 C temperature for further study. ...
Bioefficacy of entomopathogenic nematode belongs to the genus Steinernema sp. were tested against mealworm Tenebrio mollitor with 50, 100, 150, 200 and 250 juveniles (IJs)/30 mealworm larvae. LC50 value was 105 IJs/ml for mealyworm. The invasion efficiency was lower as the density of nematodes increased. At highest density (250 IJs/ml), the efficiency of invasion of nematodes was 24.5 % whereas at lowest density (50 IJs/ml) the invasion efficiency reached 33%. For LT50 calculation, 30 larvae were put in contact with the nematode (density 250 IJs/ml) during 2, 4, 6, 8 and 10 hours. Mortality was recorded 48 hours after the contact periods, and LT50 value was obtained at 7.7 hours. After two weeks, nematode population could reach ca. 40,000 IJs/larva, 35,000 IJs/pupa and 29,000 IJs/imago. When applied using density 50 IJs/ml against Cylas formicarius, the nematode population grew up to ca. 4000 IJs/larva, 3600 IJs/pupa and 3400 IJs/imago with invasion efficiency of 10%, 8% and 7% respectively. A series of nematode density 1.5, 2.0, 2.5 and 3.0 x 104 IJs were poured on the soil surface in the plastic container where sweet potato tuber were buried. The tubers were previously and artificially infested by five pairs of weevil. Although in general the difference between treatments is not significant, the result showed that high mortality (>70%) of larva, pupa and adult of C. formicarius was observed in each tuber. It is concluded that the isolated nematode was not difficult to propagate in vivo, and their mobility to search and kill the weevil seemed promising as bioinsecticide.
... EPN were extracted from 160 soil samples obtained from 40 distinct locations in undisturbed areas such as conservation forests, waterfalls and caves across four provinces (Kanchanaburi, Ratchaburi, Phetchaburi and Prachuapkhirikhan) in western Thailand using the insect baiting technique (Kaya & Stock, 1997) during November to February 2022. Ten 6th instar larvae of G. mellonella were randomly distributed on top of 150 g of soil sample in a plastic container (7 × 10 × 5 cm) with four containers/sample. ...
The common cutworm, Spodoptera litura , is a serious insect pest of many vegetables and crops worldwide. Entomopathogenic nematodes (EPN) have been utilized as biological control agents for controlling various insect pests, including the larvae of S. litura . Many indigenous EPN have been recognized to be more effective in specific field applications. Among the 160 soil samples collected in undisturbed areas of western Thailand, three samples tested positive for EPN. Three indigenous EPN were identified as Steinernema siamkayai namely, EPNKU63, EPNKU70 and EPNKU85, based on ITS and D2/D3 expansion region analysis of the 28s rRNA genes. Additionally, genetic analysis of the symbiotic bacteria using recA rRNA sequences confirmed their identity as Xenorhabdus stockiae namely, PEPNKU63, PEPNKU70 and PEPNKU85. To evaluate their initial biocontrol potential against the 6th instar larvae of Galleria mellonella , virulence assays were conducted. The application of 100 infective juveniles (IJs)/insects resulted in the mortality of 80–100% of G. mellonella larvae after 72 h. When symbiotic bacteria were applied at 1 × 10 ⁶ cells/insect, they exhibited 63–93% mortality against G. mellonella larvae after 120 h. In further laboratory tests, three S. siamkayai isolates achieved 100% mortality of 3rd instar Spodoptera litura larvae within 72 h, with LC 50 values ranging from 29 to 30 IJs/insect. In screenhouse experiments, it was revealed that all S. siamkayai isolates displayed substantial virulence, ranging from 62% to 74%, against 3rd instar S. litura larvae within 72 h. This study demonstrates the biocontrol potential of S. siamkayai in controlling S. litura larvae.
... The nematode suspensions were topically applied on the larvae of T. molitor and S. exigua. Subsequent dead larvae were collected and placed onto a White trap that was kept at room temperature to facilitate the emergence of IJs [32]. The IJs were multiplied using S. exigua that were collected using the method described above. ...
An entomopathogenic nematode, Oscheius tipulae, was isolated from a soil sample. The identification of this species was supported by morphological and molecular markers. The nematode isolate exhibited pathogenicity against different target insects including lepidopteran, coleopteran, and dipteran insects. The virulence of this nematode was similar to that of a well-known entomopathogenic nematode, Steinernema carpocapsae, against the same insect targets. A comparative metagenomics analysis of these two nematode species predicted the existence of a combined total of 272 bacterial species in their intestines, of which 51 bacterial species were shared between the two nematode species. In particular, the common gut bacteria included several entomopathogenic bacteria including Xenorhabdus nematophila, which is known as a symbiotic bacterium to S. carpocapsae. The nematode virulence of O. tipulae to insects was enhanced by an addition of dexamethasone but suppressed by an addition of arachidonic acid, suggesting that the immune defenses of the target insects against the nematode infection is mediated by eicosanoids, which would be manipulated by the symbiotic bacteria of the nematode. Unlike S. carpocapsae, O. tipulae showed high virulence against dipteran insects including fruit flies, onion flies, and mosquitoes. O. tipulae showed particularly high control efficacies against the onion maggot, Delia platura, infesting the Welsh onion in the rhizosphere in both pot and field assays.
... Stock cultures of S. feltiae strain were obtained from David Shapiro-Ilan (USDA-ARS Southeastern Fruit and Tree-Nut Research Laboratory, Byron, Georgia, United States of America). Colonies were maintained on last-instar Galleria mellonella larvae according to standard protocols (Kaya and Stock, 1997). For all experiments described below, only freshly hatched IJs (maximum one week old) were used. ...
... All replicates were otherwise maintained the same way, with larvae placed individually on White traps throughout the infection and collection (White, 1927). The total number of emerging IJs was collected at the end of emergence (after 3 weeks) and counted to assess EPN progeny production (Kaya and Stock, 1997). Each treatment had three replicates with 10 larvae per replicate, and the entire experiment was repeated once. ...
... The pots were then inverted and placed in an incubator set at a temperature of 25 • C [54]. Dead larvae were identified after 7 days and individually placed in modified white traps [55]. Nematodes harbouring bacterial symbionts were first identified by morphological traits and placed in the intergeneric "glaseri group" [56]. ...
Entomopathogenic nematodes from the genus Steinernema (Nematoda: Steinernematidae) are capable of causing the rapid killing of insect hosts, facilitated by their association with symbiotic Gram-negative bacteria in the genus Xenorhabdus (Enterobacterales: Morganellaceae), positioning them as interesting candidate tools for the control of insect pests. In spite of this, only a limited number of species from this bacterial genus have been identified from their nematode hosts and their insecticidal properties documented. This study aimed to perform the genome sequence analysis of fourteen Xenorhabdus strains that were isolated from Steinernema nematodes in Argentina. All of the strains were found to be able of killing 7th instar larvae of Galleria mellonella (L.) (Lepidoptera: Pyralidae). Their sequenced genomes harbour 110 putative insecticidal proteins including Tc, Txp, Mcf, Pra/Prb and App homologs, plus other virulence factors such as putative nematocidal proteins, chitinases and secondary metabolite gene clusters for the synthesis of different bioactive compounds. Maximum-likelihood phylogenetic analysis plus average nucleotide identity calculations strongly suggested that three strains should be considered novel species. The species name for strains PSL and Reich (same species according to % ANI) is proposed as Xenorhabdus littoralis sp. nov., whereas strain 12 is proposed as Xenorhabdus santafensis sp. nov. In this work, we present a dual insight into the biocidal potential and diversity of the Xenorhabdus genus, demonstrated by different numbers of putative insecticidal genes and biosynthetic gene clusters, along with a fresh exploration of the species within this genus.
... Nine indigenous EPN isolates (Table 1) from the EPN collection at ARC-SG were used for this study. Isolation of the symbiotic bacteria was achieved by using a combination of the protocols of Kaya and Stock [42] as well as Muangpat et al. [43], with some modifications. Briefly, for each isolate, three last-instar Galleria mellonella (Linnaeus) (Lepidoptera: Pyralidae) larvae were infected and incubated for 48 h at 25 ± 1 • C in the dark. ...
Fungal diseases such as Fusarium head blight (FHB) are significant biotic stressors, negatively affecting wheat production and quality. This study explored the antifungal activity of the metabolites produced by the bacterial symbionts of entomopathogenic nematodes (EPNs) against FHB-causing Fusarium sp. Fusarium graminearum. To achieve this, the symbiotic bacteria of nine EPN isolates from the EPN collection at the Agricultural Research Council-Small Grains (ARC-SG) were isolated from the cadavers of Galleria mellonella (Lepidoptera: Pyralidae) larvae after infection with EPNs. Broth cultures (crude) and their supernatants (filtered and autoclaved) of each bacterial isolate were used as bacterial metabolite treatments to test their inhibitory effect on the mycelial growth and spore germination of F. graminearum. Mycelial growth inhibition rates varied among both bacterial isolates and treatments. Crude metabolite treatments proved to be more effective than filtered and autoclaved metabolite treatments, with an overall inhibition rate of 75.25% compared to 23.93% and 13.32%, respectively. From the crude metabolite treatments, the Xenorhabdus khoisanae SGI 197 bacterial isolate from Steinernema beitlechemi SGI 197 had the highest mean inhibition rate of 96.25%, followed by Photorhabdus luminescens SGI 170 bacteria isolated from Heterorhabditis bacteriophora SGI 170 with a 95.79% mean inhibition rate. The filtered metabolite treatments of all bacterial isolates were tested for their inhibitory activity against Fusarium graminearum spore germination. Mean spore germination inhibition rates from Xenorhabdus spp. bacterial isolates were higher (83.91 to 96.29%) than those from Photorhabdus spp. (6.05 to 14.74%). The results obtained from this study suggest that EPN symbiotic bacterial metabolites have potential use as biological control agents of FHB. Although field efficacy against FHB was not studied, the significant inhibition of mycelial growth and spore germination suggest that the application of these metabolites at the flowering stage may provide protection to plants against infection with or spread of F. graminearum. These metabolites have the potential to be employed as part of integrated pest management (IPM) to inhibit/delay conidia germination until the anthesis (flowering stage) of wheat seedlings has passed.
... After one hour, supernatant was discarded and the process of re-suspending IJs in sterilized distilled water and decanting was repeated three times till a clean nematode suspension is obtained. IJs were surface sterilized with 0.1% sodium hypochlorite [15] and washed with H2O. The resulting suspension was then re-suspended in distilled water at a concentration of approximately 1 x 10 3 IJs/ ml and stored in 250 ml tissue culture flasks in a BOD incubator maintained at 10 ± 1°C. ...
Efficacy of new and native species of entomopathogenic nematode (EPN), Heterorhabditis casmirica SKUAST-K 104 was evaluated against gram pod borer, Helicoverpa armigera in laboratory conditions. Larval mortality was directly proportional to time period as well as time period but inversely proportional to larval size. H. casmirica SKUAST-K 104 applied @ 50, 100, 150 and 200 IJs per 2nd instar larva resulted in pest mortality by 0.00, 8.33, 16.66, and 25.00 per cent, respectively at 24 hours and they were statistically significant (p ≤ 0.05) from each other. At 200 IJs inoculum level, 8.33, 16.66, 25.0, 41.66 and 50 per cent mortality of 5th instar larva was recorded at 24, 48, 72, 96 and 120 hours post inoculation, respectively. LC50 values was directly proportional to the size of larva but inversely proportional to size of nematode inoculum level. On the other hand, LT50 values was directly proportional to the size of larva but inversely proportional to size of nematode inoculum level. LC50 values calculated at 24 hours for 2nd, 3rd, 4th and 5th instar larvae was 256.88, 277.24, 326.25 and 384.25, respectively, whereas at 120 hours it was 126.11, 160.22, 184.36 and 219.14, respectively. Similarly, LT50 values calculated at inoculum level of 50 IJs per 2nd, 3rd, 4th and 5th instar larvae were 105.0, 113, 122 and 131 hours, respectively but at highest inoculum level of 200 IJs, it was 75, 89, 94 and 100 hours, respectively. Nematode multiplication rate within the host cadaver was directly proportional to the size of the host. Minimum and maximum number of IJs/ larva was 2.72 x 105 and 1.03 x 105, obtained from 2nd and 5th instar larva, respectively.
... Two isolates of Heterohabditis bacteriophora, the local and commercial isolates, were multiplied on last instar Galleria mellonella larvae under suitable conditions with a temperature ranging from 25 to 30 degrees Celsius and a relative humidity of up to 90 % according to methodology described by Hussein et. al [9]. After one week, the infective stages (IJs) were har- ...
The cucurbit fly is a pest with significant economic impact because it affects numerous agricultural crops and leads to fruit damage. This experiment was conducted to evaluate the efficacy of the local and commercial isolates of Heterohabditis bacteriophora nematodes on the larvae and pupae of the cucurbit fly insect Dacus ciliatus. Four concentrations were pre-pared for each isolate (10, 25, 50, and 100 infective juveniles/milliliter). The results showed clear variations in the isolates' ability to control the insects, and the effectiveness of the used concentrations varied in their impact on the mortality rate of the larvae and pupae. It was found that all concentrations could infect and control the cucurbit fly at both stages: larvae and pupae, but with varying percentages. Results showed that 100 IJs/mL scored the highest mortality rate compared to the other concentrations. The percentage of larvae and pupae mortality was higher when treated with the local isolate of nematodes compared to the commercial isolate at the same concentration.
... Biopreparations in the third stage of infective juveniles (IJ3) that were used for the experiment were purchased simultaneously with a guarantee of their highest quality. They were stored at 7 °C (Steinernema spp.) and 10 °C (Heterorhabditis spp.) before use (Kaya & Stock 1997). The nematodes were tested on the fifth-stage larvae, the greater wax moth (Galleria mellonella L.), a lepidopteran host highly susceptible to EPNs. ...
As trophic organisms, nematodes play an essential role in the soil environment: they mineralize nutrients into plant-available forms, are a food source for other soil organisms, and feed on pathogenic organisms and plant pests, therefore regulating populations of soil microorganisms. The plant-parasitic nematodes are important pests of crops. Nanoparticles (NP) are increasingly used in agriculture and other production sectors. They are present in the soil, not necessarily in trace amounts, and can affect various soil organisms, including nematodes. In this article, the effects of silver (Ag), gold (Au), and platinum (Pt) nanoparticles on the mortality and reproduction activity of selected nematode species from two trophic groups: entomopathogenic nematodes (EPN) ( Heterorhabditis bacteriophora and Steinernema feltiae ) and plant parasitic nematodes (PPN) ( Xiphinema diversicaudatum , Ditylenchus dipsaci , Heterodera schachtii ) were studied under laboratory conditions. All nanoparticles decreased the nematode population to an extent depending on the nematode species, nanoparticle type, and exposure time. AgNP had the greatest nematicidal effect, except for AuNP, which reduced the population of H. schachtii the most. The greatest sensitivity to AgNP was observed in X. diversicaudatum (100% mortality), D. dipsaci (90% mortality), and 37 to 13% mortality in other species. Effect of AuNP and PtNP on entomopathogenic nematodes was comparable to the control, not treated with nanoparticles. AuNP and PtNP similarly influenced nematode mortality. However, the effect of nanoparticles on new generations of entomopathogenic nematodes developing in host larvae Galleria melonella was inconclusive. All nanoparticles decreased the number of larvae of S. felitae and increased the number of H. bacteriophora migrating outside the cadavers compared to the control.
... Isolated from the soils of Kashmir valley, India, and identified according to specified methods, S. anantnagense was cultured using fully grown larvae of the Greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae). In 20 cm diameter petri dishes lined with filter paper, ten 5th instar larvae of G. mellonella were individually inoculated with approximately 1 x 10 3 infective juveniles (IJs) of the EPN strain in 0.5 ml of sterilized distilled water, following the protocol by Kaya and Stock (1997) [12] . The dishes were covered with an inverted petri bottom and stored in a BOD incubator (Dutky et al., 1964) [9] . ...
... An overview of all materials is provided in Table 1. WT strains were cultured in the greater wax moth Galleria mellonella following Kaya and Stock (1997). WT inbred lines were cultured on nematode growth gelrite (NGG) (3 g⋅l − 1 Gelrite, 2.5 g⋅l − 1 peptone from casein, 51 mM NaCl, 1 mM CaCl 2 ⋅2 H 2 O, 1 mM MgSO 4 ⋅7 H 2 O, 1 mM KH 2 PO 4 , 12 µM filter-sterilized cholesterol in 99 % ethanol) as described by Addis et al. (2014) and coated with P. laumondii strain DE2 bacteria. ...
... The EPN species used in the bioassay trials and field trials were all locally isolated EPN species obtained from the collection at the Department of Conservation Ecology and Entomology, Stellenbosch University (Table 1). The nematodes were cultured at 25°C according to the method adopted by Kaya and Stock (1997). The IJs were harvested from infected WML using White traps (Gaugler et al., 2000) and were then stored in 150 ml distilled water in vented 500-ml culture flasks (Nunc TM Cat. ...
The first recordings in South Africa of the highly invasive social wasps, Vespula germanica and Polistes dominula (Hymenoptera: Vespidae) was in 1974. These wasp species are known to represent a significant threat to the biodiversity of ecosystems in countries they invade. The susceptibility of V. germanica and P. dominula larvae to indigenous entomopathogenic nematodes (EPNs), namely Heterorhabditis bacteriophora, H. noenieputensis and Steinernema yirgalemense, and to the entomopathogenic fungus (EPF) Beauveria bassiana was tested both in laboratory bioassays and in situ wasp nests application in the Western Cape province. Bioassay results indicated that P. dominula and V. germanica larvae are susceptible to all biocontrol agents tested. Larvae of both wasp species were all dead and infected after exposure to 200 IJ/insect at 25°C four days after inoculation. Similar results were obtained seven days after inoculation (according to label) with B. bassiana. To test the in-field pathogenicity against P. dominula, wasp nests were treated with B. bassiana, H. bacteriophora, a mixture of the EPF and EPN species and a control of distilled water. The number of larvae and pupae infected 168 h after application were determined. In both cases, the mixture of EPF/EPN performed best, with a mean percentage of 31.39% ± 4.75% larvae infected, and with only 3.42% ± 0.68% of the pupae being infected. The results obtained suggest that higher concentrations of selected biologicals can be used as inundative biological control agents within an integrated management programme for the control of P. dominula and V. germanica in South Africa.
... The experiment was performed using a Petri dish assay, following Kaya and Stock (1997). IJs derived from the previous experiment were used to study the fitness potential of emerged IJs from the dead cadaver of PBW to re-infect fresh larvae. ...
Background
The emergence of pink bollworm (PBW), Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae), in cotton due to Bt resistance and concealed feeding habit has created a need for alternative, eco-friendly, and cost-effective control methods. This study aimed to evaluate the bio-efficacy and reproductive potential of two native strains of entomopathogenic nematodes (EPNs), Heterorhabditis indica , namely CICR-HI-CL and CICR-HI-MN, against PBW larvae and pupae under in-vitro conditions.
Results
The larval assay revealed that strain CICR-HI-CL exhibited higher potency than strain CICR-HI-MN against 2 nd , 3 rd , and 4 th instar larvae, with median lethal dose ( LD 50 ) values of 5.45, 4.45, and 4.60 infective juveniles (IJs) per larva, respectively. In case of pupal bioassay, both EPN strains demonstrated greater virulence when applied directly ( LD 50 values: 29.65 and 73.88 IJs per pupa for strains CICR-HI-CL and CICR-HI-MN, respectively) compared to soil application (147.84 and 272.38 IJs per pupa). Both EPN strains successfully penetrated and reproduced on 4 th instar larvae, resulting in maximum production of 19.28 and 20.85 lakh IJs per larva in the next generation when inoculated at 30 IJs per larva.
Conclusion
The present study has generated useful information on the virulence and reproductive potential of two strains of EPN H. indica (CICR-HI-CL and CICR-HI-MN) against PBW, a dreaded pest of cotton. Higher virulence and reproductive potential of EPN strains demonstrated their ability to multiply, sustain and perpetuate on larval and pupal stages of PBW. The knowledge generated will help formulate effective management strategies for PBW with the inclusion of EPN as a potential biological control candidate. The soil-dwelling life stages viz., last instar hibernating larvae and pupae of PBW can be the ideal weak links to make a successful use of H. indica for sustainable management of PBW in the cotton ecosystem. However, before taking these EPN strains to field for managing PBW, detailed studies investigating their biocontrol potential against PBW under field conditions are needed.
... Inoculum of H. indica isolate SGS (GenBank: KU945293) were produced in vivo through recycling using mealworm, Tenebrio molitor Linnaeus, 1758 (Coleoptera: Tenebrionidae) and collected in filtered water in a modified white trap (Kaya & Stock, 1997). ...
Phlyctinus callosus and P. xerophilus (Coleoptera: Curculionidae) are two cryptic species of native entimine weevils, previously grouped together under the P. callosus sensu lato concept, that are pests of economic importance to the deciduous fruit and vine industry in the Western Cape province of South Africa. Laboratory bioassays were conducted using entomopathogenic fungi (EPF) isolates of Beauveria and entomopathogenic nematodes (EPNs), Heterorhabditis indica and Steinernema yirgalemense , to determine differences in susceptibility of adult P. callosus and P. xerophilus to potential biological control agents. The test arena used was 24‐well bioassay plates with an inoculation rate of 200 infective juveniles (IJs)/insect for EPNs and 5 × 10 ⁵ conidia/insect for EPF. Insects were inoculated using a 12.7 mm filter paper impregnated with 50 μL of entomopathogen suspension. Infection was determined after 96 h incubation for EPNs through dissection of cadavers. Insects inoculated with EPF were incubated in the wells for 18 days and mortality recorded daily. Cadavers were surface sterilized and observed for overt mycosis. Differential susceptibility between P. callosus and P. xerophilus was observed in EPF but not in EPN bioassays. Differential susceptibility to EPF could be due to methodology. Low adult weevil pathogenicity was found for all entomopathogens, with variable results obtained from different bioassay batches.
... The nematodes H. bacteriophora HP88 were provided by the Laboratory for Microbial Control of Arthropods of Federal Rural University of Rio de Janeiro (UFRRJ). For multiplication of EPNs, last-instar caterpillars of Tenebrio molitor were used, provided by the Center for Scientific and Technological Development in Phytosanitary Management of Pests and Diseases (NUDEMAFI UFES), following the method proposed by Kaya & Stock (1997). ...
Many studies about fasciolosis control have been carried out, whether acting on the adult parasite or in Pseudosuccinea columella, compromising the development of the larval stages. The present study aimed to evaluate, under laboratory conditions, the susceptibility of P. columella to Heterorhabditis bacteriophora HP88, during for 24 and 48 hours of exposure. The snails were evaluated for 21 days for accumulated mortality; number of eggs laid; hatchability rate; biochemical changes; and histopathological analysis. We found that exposure induced a reduction in glucose and glycogen levels, characterizing a negative energy balance, due to the depletion of energy reserves as a result of the direct competition established by the nematode/endosymbiont bacteria complex in such substrates. A mortality rate of 48.25% and 65.52% was observed in the group exposed for 24 h and 48 h, respectively, along with significant impairment of reproductive biology in both exposed groups in relation to the respective controls. The results presented here show that P. columella is susceptible to the nematode H. bacteriophora, with the potential to be used as an alternative bioagent in the control of this mollusk, especially in areas considered endemic for fascioliasis, in line with the position expressed by the World Health Organization Health.
... Standard procedures for the rearing and storage of EPNs were followed. In brief, each of the five EPN species was separately inoculated in a 9 cm diameter Petri dish lined with filter paper at a concentration of 1000 IJs in 800 μl (equivalent to 200 IJs/larva), followed by the introduction of 10 wax moth larvae, Galleria mellonella Linnaeus, (Kaya and Stock, 1997). After 24 h, the dead wax moth larvae were removed from the Petri dish, rinsed with water, and transferred into a new Petri dish lined with filter paper that was incubated for 48 h. ...
... The flasks were incubated in an incubating growth chamber (IncoShake, Labotec) at 140 rpm at 28°C for 48 h. After sterile glycerol was added to each Erlenmeyer as a cryopreservation agent (15% glycerol (v/v); 4.5 ml), the culture was shaken vigorously and pipetted into 1.5 ml Eppendorf tubes and stored in a -80°C freezer (Kaya & Stock, 1997). The bacteria in the stock culture, which was used throughout the study, were replaced with fresh bacteria every few months, or when the supply was running low. ...
Optimising the in vitro liquid mass production process for entomopathogenic nematodes (EPN) is a critical step in the development of a cost-effective EPN biopesticide product. Recording the nematode population and growth dynamics in an Erlenmeyer shake flask environment is essential to gaining a better understanding of which factors may influence the final yield. Although infective juvenile (IJ) inoculum concentration has previously been studied, no consensus yet exists as to whether it influences the final yield. This study sought to determine the impact of IJ inoculum concentration and timing on the recovery, growth and yield of the South African EPN isolate of Steinernema yirgalemense in shake flasks. The results indicated that the IJ inoculum concentration positively affects the final IJ yield of 2.88 × 15 ⁵ IJ ml ⁻¹ . Moreover, at higher IJ inoculum concentrations, a higher number of reproductive females and males are produced, when recovery percentages were similar. The lowest IJ inoculum concentration treatment was also the only treatment to show signs of an unwanted second generation in the final yield. The IJ inoculation timing trials confirmed that once the bacterium, Xenorhabdus indica , has ended the exponential growth phase and entered the stationary phase, it is ideal to then inoculate the IJ. However, it was found that the S. yirgalemense can be inoculated into the X. indica bacteria solution after 36 h of growth, instead of after 44-48 h. These trials demonstrated the need for further optimisation to produce consistent yields of the highly pathogenic South African EPN isolate of S. yirgalemense .
... After 48 h, sterile glycerol was added to the Erlenmeyer flasks as a cryopreserving agent (15% glycerol v/v; 4.5 ml) and stirred vigorously to ensure homogeneity. A 1-ml sample was then pipetted into a 1.5-ml Eppendorf tube and stored in a -80 °C freezer (Kaya and Stock 1997). The bacteria were verified and confirmed as being P. thracensis (Booysen et al. 2022). ...
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.
... Tenebrio molitor Linnaeus, 1758 (Coleoptera: Tenebrionidae) larvae were used to determine the pathogenicity after in vitro production. Galleria mellonella larvae were reared in the laboratory (Kaya and Stock, 1997), whereas T. molitor larvae were purchased from a local distributor. ...
... The EPN, Heterorhabditis bacteriophora HP88 (Nemaplus® Provided by e-nema, Schwentinental, Germany) strain was used in all experiments. EPN were produced in vivo using last instar of greater wax moth Galleria mellonella L. larvae (Lepidoptera: Pyralidae) as hosts (Kaya and Stock, 1997). Each larva was infected with 50 IJs of the HP88 strain in silver sand with 10% moisture and incubated at 25 • C. Four days after incubation, dead larvae were transferred to White traps. ...
... Galleria larval mortality was recorded on a daily basis. Dead larvae were placed into White traps (White 1927), and infective juveniles were collected and used to infect live G. mellonella larvae to confirm Koch's postulates (Kaya & Stock 1997). For taxonomic studies, 30 G. mellonella were exposed to infective juveniles (IJ) (200 IJ per G. mellonella) of nematodes in a 9.0 cm diameter Petri dish lined with a moistened filter paper and kept in the dark at 28 ± 2°C. ...
In this study, morphological and molecular features were used to identify a new Steinernema
sp. from Chhattisgarh, India. Morphological and molecular features provide evidence
for placing the new species into the “bicornutum” clade. The new species is characterized by
the following morphological features: infective juveniles with a body length of 587 (494–
671) μm; a distance from the anterior end to excretory pore of 46 (43–50) μm; a distance
from anterior end to nerve ring of 72 μm (61–85 μm); and E% of 88 (77–97). The firstgeneration
males are characterised by 27 genital papillae and very short spicules, with a
length of 61 μm (53–67) μm. The SW% and GS% ratio of S. shori n. sp. are 139 (107–190)
and 75 (62–90), respectively. The new species is further characterized by sequences of the
internal transcribed spacer and partial 28S regions of the ribosomal DNA. Phylogenetic
analyses show that S. shori n. sp. is most closely related to S. abbasi, S. kandii, and
S. yirgalemense.
In the southern United States, corn earworm, Helicoverpa zea (Boddie), and soybean looper, Chrysodeixis includens (Walker) are economically important crop pests. Although Bt crops initially provided effective control of target pests such as H. zea , many insect pests have developed resistance to these Bt crops. Alternative approaches are needed, including biological control agents such as entomopathogenic nematodes (EPNs). However, the effectiveness of EPNs for aboveground applications can be limited due to issues such as desiccation and ultraviolet radiation. Effective adjuvants are needed to overcome these problems. Ten strains of EPNs were tested for virulence against eggs, first to fourth instars, fifth instars, and pupae of H. zea and C. includens in the laboratory. These 10 EPN strains were Heterorhabditis bacteriophora (HP88 and VS strains), H. floridensis (K22 strain), Hgkesha (Kesha strain), Steinernema carpocapsae (All and Cxrd strains), S. feltiae (SN strain), S. rarum (17c+e strain), and S. riobrave (355 and 7–12 strains). EPNs could infect eggs of H. zea or C. includens in the laboratory, but the infection was low. The mortality caused by 10 EPN strains in seven days was significantly higher for the first to fourth instars of H. zea compared to the control, as was the fifth instars of H. zea . Similarly, for the first to fourth and fifth instars of C. includens , the mortality was significantly higher compared to the controls, respectively. However, only S. riobrave (355) had significantly higher mortality than the control for the pupae of H. zea . For the pupae of C. includens , except for H. bacteriophora (HP88) , S. rarum (17c+e) , and H. floridensis (K22), the mortality of the other seven strains was significantly higher than the control. Subsequently, S. carpocapsae (All) and S. riobrave (7–12) were chosen for efficacy testing in the field with an adjuvant 0.066% Southern Ag Surfactant (SAg Surfactant).
In field experiments, the SAg Surfactant treatment significantly increased the mortality and EPN infection for S. carpocapsae (All) on first instars of H. zea in corn plant whorls. On soybean plants, with the SAg Surfactant, S. carpocapsae (All) was more effective than S. riobrave (7–12) on fifth instars of C. includens . This study indicates that EPNs can control H. zea and C. includens , and SAg Surfactant can enhance EPN efficacy.
La inducción del estado anhidrobiótico de los nematodos entomopatógenos en agregados granulares (AG) mejora la estabilidad de almacenamiento y es función de su tasa de perdida de humedad. El objetivo fue investigar el comportamiento del contenido de humedad en AG y su efecto sobre el tiempo de supervivencia y la infectividad de Steinernema glaseri (NJ-43). El material desecante utilizado fue tierra diatomea y la encapsulación se llevó a cabo usando un mezclador de vórtice. Los AG se almacenaron a temperatura de 25±2 °C y humedad relativa de 96±2%, se midió el peso de los AG y se realizó el conteo de los EPN vivos diariamente. La infectividad se probó mediante la exposición 10±1 nematodos contra larvas de Galleria mellonella. Cada observación se repitió 5 veces y el experimento se llevó a cabo por duplicado. El análisis de la supervivencia se realizó con gráficas de Kaplan-Meier. El contenido de humedad de S. glaseri fue 73% y el contenido de humedad inicial del AG fue 40%. El contenido dehumedad de equilibrio del AG fue 6% y el tiempo requerido para llegar a este valor fue de ocho días. El tiempo de supervivencia fue 8.839 días y la infectividad de 50%. Se concluye que la tasa de pérdida de humedad de 4.24 % por día afecta negativamente la supervivencia e infectividad de S. glaseri.
Rhabditis (Rhabditella) axei is a free-living, pseudoparasitic, necromenic, and parasitic nematode, depending on the host. This species feeds mainly on bacteria present in decaying organic matter, soil, and other substrates; however, in its parasitic form, it can colonize some species of snails. Moreover, the presence of R. axei has also been detected in birds and mammals, including humans.
In 2021–2023, during monitoring of the palm borer Paysandisia archon in Central Italy, R. axei emerged from dead larvae of this alien invasive moth and was extracted from palm fibres of Trachycarpus fortunei in three independent sites. The nematode was identified by morphological and morphometric analyses. Molecular analyses using SSU and LSU gene fragments were used to confirm the identification and to perform Bayesian reconstruction of the phylogeny. Each sampling site showed a unique haplotype.
Concerning the pathogenicity of this nematode against insects, the test performed on Galleria mellonella larvae did not show any entomopathogenic effect. This is the first time that R. axei was found associated with P. archon, and this recurrent association was discussed.
The growing interest in the use of entomopathogenic nematodes and their symbiotic bacteria as promising biocontrol agents of many arthropod pests and pathogens has created running technologies to expand their use globally. The related laboratory procedures and tests on these nematodes such as their isolation, count, culture, identification, pathogenicity, virulence, and environmental tolerance should form the solid basis for such an expansion with reliable uses. Extensive practical details of such procedures and tests as well as how to identify and overcome the problems associated with these aspects are addressed in this chapter.
A new species of entomopathogenic nematode, Steinernema adamsi n. sp., was recovered from the soil of a longan tree (Dimocarpus sp.) in Mueang Lamphun District, Thailand, using baiting techniques. Upon analysis of the nematode's morphological traits, we found it to be a new species of Steinernema and a member of the Longicaudatum clade. Molecular analyses of the ITS rDNA and D2D3 of 28S rDNA sequences further confirmed that S. adamsi n. sp. is a new species of the Longicaudatum clade, which is closely related to Steinernema guangdongense and Steinernema longicaudam. Using morphometric analysis, the infective juveniles measure between 774.69 and 956.96 μm, males have a size range of 905.44 to 1,281.98 μm, and females are within the range of 1,628.21 to 2,803.64 μm. We also identified the symbiotic bacteria associated with the nematode based on 16S sequences as Xenorhabdus spp. closely related toXenorhabdus griffiniae. Furthermore, we have successfully assessed a cryopreservation method for the long-term preservation of S. adamsi n. sp. Successful cryopreservation of this new species will allow for the longer preservation of its traits and will be valuable for its future use. The discovery of this new species has significant implications for the development of effective biological control agents in Thailand, and our work contributes to our understanding of the diversity and evolution of entomopathogenic nematodes.
Entomopathogenic nematodes (EPNs) are successfully used in the biological control of agricultural insect pests. This study aims to determine the body length of hermaphrodite individuals, egg diameter and reproductive capacity obtained from Infective Juveniles (IJs) stored at different temperatures and durations. Heterorhabditis bacteriophora Poinar, 1976 (Rhabditida: Heterorhabditidae)’s Hybrid Strain HBH was used in the study. IJs stored at 15, 25 and 35°C for 7, 14 and 21 days were inoculated onto Galleria mellonella L., 1758 (Lepidoptera: Pyralidae) last instar larvae at a dose of 100 IJs. On the 2nd day of infection, hermaphrodite individuals and eggs were obtained by dissecting the larvae. The reproductive capacity was determined 10-12 days after infection. The study was conducted in Bursa Uludağ University, Faculty of Agriculture, Plant Protection Department, Nematology Laboratory in 2023. In conclusion, the longest hermaphrodite individuals and egg diameter were obtained as 6207.22 µm and 55.65 µm, respectively from the IJs stored for 7 days at 15°C. The highest reproductive capacity was also observed as 167.500 IJs per G. mellonella larva in IJs stored under the same conditions with respect to temperature and time. This study is important for assessing the morphological effects of different temperature values and storage durations on EPNs.
The tomato leafminer, Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae) is an important pest of tomato crops in South America and it has recently been introduced to the Mediterranean area including Egypt. This devastative insect pest affecting tomato plantations. The susceptibility of T. absoluta larvae and pupae to the entomopathogenic nematode species, [Steinernema monticolum and Heterorhabditis bacteriophora (HP88)] was determined in both the laboratory and the field. Leaf bioassays were conducted to evaluate the nematode’s efficiency in the laboratory to kill the insect larvae. The nematode, Heterorhabditis bacteriophora (HP88) has killed 80% of the 4th instar larvae at 600 nematode infective juveniles (IJs)/ml. and 100% at 1200 IJs/ml. 72 hours post treatment. At the same time, when Steinernema monticolum infective juveniles were used in the laboratory at 600 nematode infective juveniles (IJs)/ml., 80-87% mortality was recorded on the 3rd and 4th instar larvae respectively. While mortality percent was 84 and 89% when 1200 IJs/ml. of Steinernema monticolum were used against 3rd and 4th instar insect larvae respectively. Field efficacy of the two tested nematode species after foliar application to tomato plants was evaluated in 42 field plots of 33.3 m2 each. Total recorded mortality when H. bacteriophora was used has varied along 3 consecutive years from around 60% during the years 2011 and 2012 to 80% mortality in the year 2013. When Steinernema monticolum was applied in the field, mortality percent varied from 58 to 61% in the years 2011 and 2012 respectively and reached a maximum of 67% in the year 2013. These results indicate that the nematode H. bacteriophora (HP88) was more virulent than the S. monticolum against the tomato borer, Tuta absoluta in both the laboratory and the field.
key words: Entomopathogenic nematodes, field tests, Heterorhabditis bacteriophora (HP88), laboratory tests, Steinernema monticolum, tomato borer, Tuta absoluta.
Entomopathogenic Nematodes (EPNs) found in a variety of soil types, geographical regions, and hosts, which belong to the genera Steinernematidae and Heterorhabditidae, have the potential to act as biocontrol agents. In comparison to chemical and microbial pesticides, they performed better. A total of 87 soil samples were taken from regions where maize was grown in Tuticorin district, Tamil Nadu, India and they were examined for the presence of EPN in 2022–2023. By using the Corcyra baiting approach, a total of 9 samples (10.33%) showed EPN-positive sites. A total of 8 Steinernema sp. (13.33%) and 1 Heterorhabditis sp. (5.00%) were isolated from that population. EPN is identified at a generic level using the cadaver’s colour. Heterorhabditis displays brick red, while Steinernema exhibits creamy white. At a dose of 100–600 IJs/larva for the third and fifth instar, the isolated efficient native EPN strain (Kayathar strain) demonstrated mortality of 95.00–100.00% and 94.50–99.80%. According to the study, EPN showed considerable potent against Spodoptera frugiperda. So, EPNs may be used as a promising bio-control agent to battle pests of the maize crop.
Aim of study: Pine wilt disease (PWD) is a serious threat to the susceptible pine forests. It is caused by Bursaphelenchus xylophilus (Nematoda: Parasitaphelenchidae) (Steiner and Buhrer 1934), Nickle 1970 and transmitted by Monochamus Dejean beetles. In the recent study, we assessed the effects of entomopathogenic nematode, Steinernema carpocapsae (Nematoda: Steinernematidae) against Monochamus galloprovincialis larvae in Anatolian black pine and Scots pine logs. Area of the study: The experiments were conducted in Duzce University, Faculty of Forestry and in a pine forest at Duzce University campus area. Material and methods: The mean number of eggs per pine logs, and the productivity of S. carpocapsae in M. galloprovincialis larvae were compared under laboratory conditions. The nematode experiments were conducted using oviposited pine logs in the field. Main results: The females of M. galloprovincialis oviposited more eggs on Scots pine compared to black pine logs. Both in black pine and in Scots pine, the survival rates of M. galloprovincialis after nematode application was significantly lower than control. Highlihts: As a result of the study, S. carpocapsae can be an efficient biological control agent of this wood-boring insect.
Achromobacter nematophilus sp. nov., isolated from the intestinal lumen of a species of Neoaplectana (Steinernematidae: Nematoda), is described. It can be distinguished from other species of Achromobacter by its larger cell size and action on gelatin and litmus milk.
The effect of entomopathogenic nematodes on nontarget arthropods in the laboratory, field soils, and a stream were assessed. In the laboratory, adult predators were less susceptible to the nematodes Steinernema carpocapsae (Weiser) (Rhabditida: Steinernematidae) and Heterorhabditis bacteriophora Poinar (Rhabditida: Heterorhabditidae) than the immature stages. In field tests, entomopathogenic nematodes that had significantly suppressed pest populations (Popillia japonica Newman, Japanese beetle, Scapteriscus vicinus Scudder, tawny mole cricket, Otiorhynchus sulcatus (F.), black vine weevil, Delia radicum (L.), cabbage maggot, and Diabrotica virgifera virgifera LeConte, western corn rootworm) did not adversely affect the numbers of nontarget soil arthropods in comparison with the untreated control. In contrast, broad-spectrum chemical insecticides (isofenphos, ethoprop, or chlorpyrifos used as chemical checks) significantly reduced or showed a tendency to reduce nontarget arthropod populations. In a stream trial, S. carpocapsae significantly reduced black fly larval populations, but the nontarget insects often increased in the treatment sites. Decreases in nontarget populations were matched by approximately equal or greater reductions in the upstream controls. We conclude that entomopathogenic nematodes do not adversely affect nontarget arthropods when used for short-term control of insect pests.
Queens of Bombus terrestris, B. hypnorum, B. lucorum, B. hortorum, B. lapidarius and Psithyrus sp., infected with the parasitic nematode, Sphaerularia bombi, were collected and studied in the Netherlands from April to September, 1971. In June, the infected queens gathered in a special wooded area which partially overlapped with a normal hibernation site. These queens were observed digging in the soil and depositing third-stage juvenile nematodes through their anus. The nematodes entered the soil and molted to the adult stage in approximately 2 months. Mating occurred and the infective females were ready to penetrate a new host. Two molts occurred in the egg of S. bombi and the nematodes matured to third-stage juveniles in the host. The free-living stages of S. bombi are re-described with special emphasis on the pharyngeal region.
Soils from 351 sites representing ecologically diverse habitats from six Hawaiian Islands (sea level to 4,200 m) were assessed for entomopathogenic nematodes using the Galleria baiting technique. Twenty-four sites (6.8%)were positive for entomopathogenic nematodes. Twenty-two sites (6.3%) were positive for a Heterorhabditis sp. from the islands of Kauai (6), Oahu (5), Maui (4), Molokai (1), and Hawaii (6), and two sites were positive for a Steinernema sp. from Maui. No entomopathogenic nematodes were recovered from soils on the island of Lanai. Heterorhabditids were highly correlated with ocean beaches within 100 m of seashore (0 m elevation). These positive sites had soils containing sand grains from coral and shells with moderately alkaline pH (8.0) and low organic content (12%). The steinernematid isolates came from inland areas in silty clay and silt loam soils with higher organic content (15-35%).
Pheromermis pachysoma (von Linstow) n. gen., n. comb., a parasite of the yellowjacket, Vespula pensylvanica (Saussure), is described from California. The genus, Pheromermis, is characterized by the presence of four submedian cephalic papillae; large anteriorly placed cup-shaped amphids; an S-shaped vagina not bent in a transverse plane to the body; six hypodermal cords; paired, short, separate spicules; cuticle with cross fibers; and eggs lacking processes. The development of P. pachysoma is unique because a paratenic or transport host is required for completion of the life cycle. The adult nematodes occur in water or saturated soil and the eggs are fully embryonated at oviposition. The eggs hatch in the gut of various insects and infective stage juveniles penetrate the gut wall and enter a quiescent state in the tissues of these paratenic hosts. Wasp larvae are probably infected when they are fed paratenic hosts captured by worker yellowjackets. Postparasitic juveniles of P. pachysoma emerge from adult wasps when the latter visit wet sites after their fall emergence from the nest. The ant parasite, Mermis myrmecophila Baylis, is transferred to the genus Pheromermis.
Simple new methods are described for rearing species of Neoaplectana and Heterorhabditis monoxenically and cheaply in large quantities for use in biological control. Yields of more than half a million nematodes/g of an homogenate of pig kidney/fat on crumbed polyether polyurethane sponge in conical flasks were obtained consistently for some species. The method lends itself to industrial development.
SUMMARYA method for producing up to 2000 million infectives of Neoaplectana bibionis per container is described and is applicable to other species of Neoaplectana and Heterorhabditis spp. The nematodes were cultured within autoclavable plastic bags, on crumbed polyether polyurethane sponge coated with chicken offal homogenate that had been sterilised and inoculated with the primary form of the appropriate symbiotic bacterium (Xenorhabdus spp.). Procedures for extracting and cleaning the nematodes on a large scale are described. Nematodes were stored and transported on clean sponge in aerated polyethylene tubes. The techniques are suitable for industrial use with little further development.
Survival and pathogenicity of two entomopathogenic nematodes, Steinernema carpocapsae (=Neoaplectana) and Steinernema glaseri, were tested in four types of soil: sand, sandy loam, clay loam, and clay over a period of 16 weeks. Significant differences in the survival and pathogenicity of both nematodes occurred in all soil types. S. carpocapsae survival was 44.9, 38.8, 32.9, and 26.7% in sandy loam, sand, clay loam, and clay, respectively, whereas S. glaseri survival was 30.1, 25.9, 22.5, and 19.3% in sand, sandy loam, clay loam, and clay, respectively, at the end of the test. S. glaseri survival was significantly lower than S. carpocapsae in all soil types. Survival for S. carpocapsae was greatest in sandy loam, while survival for S. glaseri was best in sand. Pathogenicity assays with recovered nematodes supported the survival data. Persistence of both species decreased as the proportion of clay increased.
There are over 3000 reported parasitic and phoretic associations between nematodes and insects, with less than ten involving phytoparasitic nematodes (Poinar, 1975). Nematodes are aquatic metazoans with very limited powers of dispersion. They are greatly benefited by a synchronized association with an insect host for increased mobility and protection during travel to an insect’s breeding or feeding sites.
A new subspecies, Xenorhabdus nematophilus subsp. beddingii, is described to accommodate the bacterial symbionts of two undescribed species of Steinernema, which are entomopathogenic nematodes. Strains of this subspecies are gram-negative, facultatively anaerobic, peritrichously flagellated rods. They occur in two forms; one of these forms is positive for pigmentation (brown), adsorption of bromothymol blue, and production of antimicrobial compounds, whereas the other form is negative for these characteristics. They are pathogenic when injected into insects. Like other X. nematophilus strains, they are negative for catalase and nitrate reductase. All strains produced acid from glucose (no gas), dextrin, fructose, maltose, mannose, and trehalose, and some produced acid from glycerol, ribose, or salicin. The new subspecies is distinguished from the other three subspecies of X. nematophilus by being positive for phosphatase and esculin hydrolysis. The type strain is strain Q58/1 and has been deposited in the University of Queensland Microbiology Collection as strain UQB 2871.
A new species, Xenorhabdus japonicus, is proposed as the bacterial symbiont of Steinernema kushidai isolated from field soil in Shizuoka Prefecture, Japan. Xenorhabdus japonicus could be distinguished phenotypically and genetically from other Xenorhabdus spp. The type strain of the species, SK-1, a Gram-negative, facultative anaerobe and peritrichously flagellated rod, has colonies with primary and secondary forms. The strain can be differentiated from the type strain of Xenorhabdus nematophilus by several characters, including the formation of arginine dehydrolase, phenylalanine deaminase and lysine decarboxylase, the assimilation of inosine and L-proline and acid production from inositol. The major cellular fatty acids are 16:0, cyclo 17:0 and 18:1. The ubiquinone system is Q-8. The G+C content of DNA is 45.9 mol%. The DNA of strain SK-1 has 20 to 58% homology with that of the type strains of other Xenorhabdus spp.
It is well known that some insect-pathogenic nematodes always live in
symbiosis with Gram negative bacteria belonging to the genus Xenorhabdus
(earlier referred to as Achromobacter). We have here used diapausing
pupae of Hyalophora cecropia as a model system and investigated the
Mexican strain of Neoaplectana carpocapsae and its bacterium Xenorhabdus
nematophilus for their ability to withstand insect immunity separately
and in the symbiotic unit. We have prepared a reconstituted nematode
containing a streptomycin-resistant mutant of X. nematophilus. In normal
pupae LD50 (the injected dose that causes death of 50% of the
population) of the bacteria was about 500 cells; for immunized pupae it
was 5 × 105 cells. Cecropia immunity did not affect the
nematodes. Fewer than ten nematodes with bacteria were lethal, while
LD50 of axenic nematodes was about 500. Immune haemolymph
caused lysis of X. nematophilus. Immune proteins P9A and P9B were
identified as the active components in Cecropia immunity against X.
nematophilus. It was found that the nematodes help the bacteria by
excreting an immune inhibitor that selectively destroys both forms of P9
as well as immune protein P5. These results contribute to our
understanding of the symbiotic relationship between nematodes and their
bacteria and its survival value against induced insect immunity.
Bacterial (Enterobacteriaceae) symbionts isolated from newly collected Steinernema glaseri (insect pathogenic nematodes) were found to differ from those previously isolated in being able to produce antibacterial compounds. They were identified as Xenorhabdus nematophilus subsp. poinarii. Study of the new strains showed that the dimorphism common to Xenorhabdus spp. is expressed differently in X. nematophilus subsp. poinarii and that this subspecies differs from other X. nematophilus in several other respects. X. nematophilus subsp. poinarii produced colony variants that did not differ from the parent strain in antibacterial activity whereas some strains lost antimicrobial activity without changing colony morphology; no variant of any strain absorbed bromothymol blue from agar media. There was also considerable variation in colony morphology between different isolates. Moreover, although the nematode/bacterium complex was an efficient pathogen, X. nematophilus subsp. poinarii strains were unlike other X. nematophilus in not being highly pathogenic when injected intrahaemocoelically into insect larvae (Galleria).
The mermithid parasite, Agamermis unka, is the most important natural enemy of the brown planthopper (BPH), Nilaparvata lugens, in Korean rice fields. Very little is known about many aspects of the mermithid's life cycle and behavior, and a study was undertaken to close the data gap. The sex ratio of A. unka isolated from field-collected BPH showed a strong female bias. Even when several A. unka occurred within a BPH, the majority were females. Similarly, the sex ratio of field-collected A. unka adults that were in the soil was strongly biased towards females and, in many instances, the females were found in the absence of males. Females collected from the field from January to May and maintained in water at 25 C had a mean pre-oviposition period of 17-28 days and a mean oviposition period of 17-37 days, and averaged 543-1851 eggs/female. The eggs averaged 20, 17 and 36 days to hatch at 30, 25 and 20 C respectively, but none hatched at 15 C. Most of the eggs (96%) hatched at 25 and 20 C, but only 64% hatched at 30 C. Agamermis pre-parasites could be found on rice stems in the field and laboratory. In the field, BPH-susceptible and BPH-resistant rice cultivars showed no significant difference in the numbers of pre-parasites on the stem. In the laboratory, the number of pre-parasites recovered/rice stem was significantly higher in two out of three trials when BPH nymphs and adults were present. When BPH nymphs were exposed to the pre-parasites in the laboratory, 39% of the brachypterous females and 4.5% of the brachypterous males were parasitized, whereas 0.3% of the macropterous females and 0% of the macropterous males were parasitized. The parasitism data obtained under field conditions showed similar trends. The reason(s) for these differences in parasitism between the BPH sex and wing types that have been observed both in the laboratory and field is unknown. Because brachypterous males and macropterous females and males occur in lesser numbers than the brachypterous females, this may, in part, account for the differences in parasitism observed. However, BPH behavior cannot be discounted as a factor in the differential parasitism.
A simple filter paper method for estimating a wide range of moisture potentials has been tested for fifteen soils ranging in texture from sands to heavy clays. The method has given estimates of moisture potential from -0.1 to -900 bars with an accuracy that should be acceptable for many types of field experimentation. Relationships between 15-bar percentages determined by both the filter paper and pressure membrane methods, and biologically determined permanent wilting percentages for wheat, are discussed.
A survey of adult sciarid root gnats in greenhouses at the University of California, Riverside, demonstrated that 9.0% were infected with Tetradonema plicans. The mean number of gravid T. plicans females per infected sciarid adult was 5.1 with a range of 1 to 48 parasites per host fly. Only 1.4% of the maggots collected from pots in the greenhouses were infected. To study the bionomics of T. plicans a simple system for culturing sciarid flies and T. plicans and extraction of T. plicans eggs was developed. Fermented sphagnum moss and cellulose (4:1) with rabbit chow was the basic culture medium. Up to 3 × 109T. plicans eggs were produced from an inoculum of 6 × 104 eggs per culture container. Eggs were collected by seiving or sucrose flotation. Infections in 1-liter containers showed that T. plicans (10 T. plicans eggs per sciarid larva) reduced sciarid fly populations by 74 to 80% for 4 months. Steinernema carpocapsae had no effect on sciarid flies, but in contrast, Heterorhabditis bacteriophora (10 juvenile nematodes per host larva) decreased sciarid populations by 60%. At 100-fold higher infection level all sciarid larvae were killed, but H. bacteriophora was unable to reproduce in the sciarid larvae.
When Beauveria bassiana conidia were mixed in nonsterile Yolo fine sandy loam (YFSL) or Staten peaty muck (peat) soil at water potentials ranging from 0.0 bars (saturation) to −1500 bars, conidia half-lives were longest at −15 bars and decreased as the water potential approached either 0.0 or −200 bars. Conidia half-lives increased as the water potential decreased from −200 to −1500 bars. Conidia half-lives were longest at a soil temperature of 10°C and decreased both at 2°C and as the temperature approached 50°C where no conidia were recovered after 2 weeks. The longest mean half-life value was 44.4 weeks for conidia in YFSL at −10 bars and 10°C; the shortest half-life value was 0.3 weeks in peat soil at 0 bars and 28°C. Clay-coating lengthened conidia survival in all treatments in a factorial experiment involving the two soil types and several combinations of soil temperature and water potential. When two strains of B. bassiana were compared, colony counts of strain ABG-6178 always decreased relative to an initial baseline count taken soon after mixing conidia and soil; colony counts of strain IL-116 routinely increased after the baseline count was taken before decreasing later. Conidia survival was often significantly longer in the low organic YFSL than in the high organic peat. The results suggest that conidia survival is influenced by the direct effect of physical factors and soil microbial populations. Since B. bassiana conidia survival is highly variable, its potential as a microbial insecticide is much greater in some soil environments than in others.
Antagonistic factors, broadly identified as antibiosis, competition and natural enemies, impact on entomopathogenic nematodes. Antibiosis can occur through the release of plant chemicals from the roots into the soil, which may adversely affect the host-finding behavior of the infective stage nematode, or the presence of these chemicals in the host insect may negatively affect nematode reproduction. In laboratory studies, intra-specific and inter-specific competition reduces nematode fitness, and inter-specific competition can cause local extinction of a nematode species. For example, after concomitant infection of a host, a steinernematid species usually excludes a heterorhabditid species. The mechanism for the steinernematid superiority has been postulated to be a bacteriocin(s) produced by Xenorhabdus, the symbiotic bacterium of the steinernematid, which prevents Photorhabdus, the symbiotic bacterium of the heterorhabditid, from multiplying. Inter-specific competition between two steinernematid species shows that both can co-exist in a host, but one species will eventually prevail in the environment. By having different foraging strategies, however, both steinermatid species may co-exist in the same habitat. An important issue is whether the introduction of an exotic entomopathogenic nematode species will competitively displace an indigenous nematode species. Although the environmental risks are small, the recommended policy is that the introduction of exotic nematodes be regulated. With other pathogens, entomopathogenic nematodes can out-compete entomopathogenic fungi, but not Bacillus thuringiensis, for the same host individual when both the nematode and entomopathogen are applied simultaneously. The best studied natural enemy is the nematophagous fungus, Hirsutella rhossiliensis, which causes higher mortality in Steinernema glaseri compared with Heterorhabditis bacteriorphora. Differential susceptibility to the fungus may be associated with the retention of the second-stage cuticle by H. bacteriophora. Invertebrate predators including mites and collembolans feed on entomopathogenic nematodes. Although a number of studies have been conducted with antagonists, there is a dearth of field data. We suggest that long-term research plots be established where natural populations of entomopathogenic nematodes occur and include antagonists as a component of such studies.
Understanding the ability of natural populations of predators, parasitoids, and pathogens to suppress prey populations is important in determining their potential as biological control agents. We measured the seasonal population dynamics of endemic entomopathogenic nematodes (Steinernema carpocapsae and Heterorhabditis bacteriphora) in turfgrass and their effect on populations of Japanese beetle, Popillia japonica, larvae and mobile arthropods associated with the soil surface and thatch layer. Both nematode species were recovered in central New Jersey in samples collected from April to December. S. carpocapsae tended to be more prevalent in the plots (32.6 and 37.1% of sections) than H. bacteriophora (8.3 and 0.3% of sections). However, when only the positive sections were analyzed H. bacteriophora had a higher density (63.6 ± 49.3 nematodes/cm2) than S. carpocapsae (17.0 ± 4.9 nematodes/cm2). Sections with H. bacteriophora had significantly lower P. japonica densities compared to sections without nematodes (58.5 ± 20.2 compared to 112.7 ± 9.2 larvae/m2). S. carpocapsae did not have a measurable impact on P. japonica populations. Mobile surface arthropods, as measured by pitfall catches, were higher in H. bacteriophora positive sections than in sections without nematodes. This difference was due to higher catches of Araneae and Lithobiomorpha. Mobile surface arthropod populations did not differ between sections with and without S. carpocapsae. The scope of H. bacteriophora′s impact on host populations is likely to be limited because of the nematodes patchy distribution.
Eine Methode wird beschrieben bei der Nematoden innerhalb 24 Stunden von der Fixierungsflüssigkeit in wasserfreies Glyzerin überführt werden. Die Nematoden werden nach Fixierung in ein Gemisch von 20 Teilen Aethanol 96%, I Teil Glyzerin und 79 Teilen Wasser gebracht. Das Aethanol in diesem Gemisch wird durch Wasserentzug im Exsiccator mit Aethanol 96% bei 35°-40°C in nicht weniger als 12 Stunden bis zu etwa 95% konzentriert. Danach wird ein Gemisch von 5 Teilen Glyzerin in 95 Teilen Aethanol 96% hinzugefügt. Dieser wird bei 40°C in wenigstens 3 Stunden zu reines Glyzerin konzentriert.
Heterotyplenchus autumnalis Nickle, a parasite of the face fly, Musca autumnalis Dc Geer, was found in face flies in Nebraska late in 1965. Surveys in 1966 showed that the parasite was present when the flies first appeared in the field in June. The nematode was active through- out the season and was present in flies entering hibernation in the fall of 1966. About 30% of face flies produced on 2 farms during the season were infected. H. autumnalis was not found in at least 10 other species of flies collected from cattle manure.
S. kushidai was xenically propagated on dog food/agar and pig offal/agar media supplemented with peptone (1.2%). It propagated to a lesser extent on Wouts' lipid agar medium. The nematode was also xenically propagated on a medium of soluble starch, D-glucose, lard, peptone, yeast extract, agar and distilled water. pH and media composition required for optimal production of the nematode were examined. Maximum yield of infective juveniles was on soluble starch (0.6%), D-glucose (1.0%), lard (3.0%), peptone (1.5%), yeast extract (1.5%), agar (0.3 to 1.0%) and 33 mM Bis-Tris buffer (pH 6.7).
A nematode new to science, S. monticolum sp. n. (Rhabditida: Steinernematidae) found during a survey of entomopathogenic nematodes conducted throughout the nine provinces of Korea, is described. It can be separated from other members of the genus by the length of the tail mucro in adults of both generations, the shape and length of the male spicules and gubernaculum, and the arrangement of the genital papillae. The infective juvenile can be distinguished from other Steinernema species by the body width and the value of ratio a (total length divided by greatest width). An amendment of the specific name of several Steinernema species is presented. A key to the new and other Steinernema species is provided.
Insect parasitic nematodes were surveyed in the Republic of Ireland between October 1986 and October 1987. A total of 551 soil samples was tested for the presence of nematodes by baiting with Galleria mellonella larvae. Steinernema feltiae (= S. bibionis) and S. affinis were recovered from 7.1 and 3.3% of samples, respectively. Heterorhabditis sp. was found in one sample. There was a significant association between locality (county) and frequency of nematode recovery. Nematodes were more frequently recovered from sandy and peaty soils than from clays and clay loams, but differences between soil types were not significant. Nematodes were present in tilled fields (41/365 samples), grassland (13/143) and woodland (4/43). There was a significant association between sampling time and frequency of recovery. Nematodes were less likely to be recovered in May-June than at other times of the year.
Face flies, Musca autumnalis (Diptera: Muscidae), of both sexes infected with the nematode, Heterotylenchus autumnalis (Tylenchida: Allantonematidae), visited the faces of cattle and fresh cattle dung. However, female flies greatly outnumbered males at both sources. Generally. infected male flies found on cattle and dung contained young nematodes. In contrast, infected females on cattle contained nematodes of all ages, and most infected females from dung contained older nematodes. The propensity of healthy female flies to visit faces of cattle and dung depended on their gonadotrophic age. The majority of flies with immature eggs were found on cattle while the majority with mature eggs were found on dung. Infected flies containing early nematode stages appeared to behave like healthy ones with immature eggs. However, once the nematodes invaded the flies' ovaries, the flies' behavior changed. These infected flies were found primarily on dung and apparently became “terminal” dung feeders.
Three techniques were compared for efficiency at extracting entomophilic rhabditoid nematodes from soil samples. The Baermann funnel extraction was superior to centrifugation flotation, and flotation sieving methods in recovering Neoaplectana carpocapsae Weiser dauerlarvae from sand with 71.2, 45.8, and 7.6% recovery rates, respectively. In a petri dish bioassay, larvae of the greater wax moth, Galleria mellonella (L.), were exposed to filtered Baermann funnel extracts of nematode-spiked field soil. This method was effective in detecting populations of entomophilic rhabditoids.
Soil from 403 hedgerow, roadside verge, woodland, heathland or field sites was assessed for presence of entomopathogenic rhabditid nematodes with aGalleria larva baiting technique. Steinernematids were recovered from 48 % of the sites but only one site yielded Heterorhabditis sp. The species recovered most frequently was Steinernema bibionis (Bovien). Another unidentified Steinernema sp. was also isolated. Prevalence of steinernematids in the different types of habitat formed continuum so that roadside verges harboured them most often while heathland sites yielded them least often. Mean soil temperatures at 5 cm were about 15 °C in fields and verges, 13 °C in hedgerows and heathland and 12 °C in woodland. Prevalence of the nematodes varied in different parts of Britain and was influenced by soil type. They were associated with calcareous soils, especially those with a calcareous subsoil horizon. Soils which are periodically or seasonally water-logged were suitable. Soils enriched by humus and hence high in organic matter frequently harboured the nematodes and some suitable soils had significant clay contents or subsoils high in clay. It appears that the two steinernematids are relatively unspecialized species, occurring in diverse habitats and soil types and are adapted to cooler temperatures.
The presence of entomopathogenic nematodes in soil from 15 sites on 10 sampling occasions over a period of 28 months was documented. Sites were chosen to represent habitats classified as heathland, hedgerow, roadside verge, pasture or woodland. Bioassays utilized Galleria larvae and most of the nematodes obtained were identified asSteinernema (= Neoaplectana) bibionis (Bovien). A sibling species which fails to interbreed with S. bibionis was also found at 2 of the sites. Bioassays were at 15 and 20°C and there was no significant difference in the results at these temperatures. There was no evidence for seasonality in the presence of the nematodes and at 2 sites they were found on every sampling occasion. Otherwise, sites converted unpredictably from positive to negative or negative to positive over the 28-month sampling period.
In a number of surveys using baiting techniques, entomogenous nematodes of the families Heterorhabditidae and Steinernematidae were detected in all states and territories of Australia. At least 4 Heterorhabditis species (3 previously unknown), 6 Steinernema species (4 previously unknown) and one species of an undescribed genus of Steinernematidae were found. In Tasmania, where collections were made over 10 years, the nematodes were detected more commonly in winter-spring than in summer-autumn; there was no significant difference in their occurrence in forest from that in grassland.
A more general use of the filter-paper method for measuring soil-water potential over a very wide range of values is advocated, both for in situ and laboratory situations. Using Whatman® No. 42, both HgCl2-treated and untreated, filter papers, the calibration curves measured on two different batches two years apart were almost identical and were in good agreement with a curve published previously by other authors. Routine use of the same papers could therefore be made without recalibrating. Examples using the method for constructing water-release curves are given, and other uses are suggested.
Samples of soil (25 g) were treated with 1 or 2 ml of propylene oxide, 400 or 800 parts/106 of sodium azide, or autoclaved for 1.5 or 3.0 h. Soil sterilization was achieved by the propylene oxide and autoclaving treatments. Sodium azide inhibited the bacteria and actinomycetes and drastically reduced the fungal population. The autoclaving treatment decreased the soil pH 0.2 unit, while propylene oxide and sodium azide treatments increased it 0.5–1.1 units. Extractable manganous—Mn was increased 2- to 3-fold by all treatments except for a 90- to 120-fold increase in an autoclaved soil; extractable Ca was not affected; and the extractable K changes were slight. Total extractable N was increased 10–20 parts/106, and available P was generally increased by the treatments. Propylene oxide induced the least chemical alterations upon sterilization and is considered an appropriate sterilant to study chemical transformations in soils; but, germination and growth of wheat and alfalfa were retarded in propylene oxide treated soil.