Virulence and Reproduction of Entomopathogenic Nematodes Isolated from a Single Mexican Locality¹

Entomopathogenic nematodes (EPNs) in the families Heterorhabditidae and Steinernematidae are effective biological control agents in the management of a variety of economically important insect pests (Agazadeh et al. 2010). In their invasion of healthy insects, these nematodes are symbiotically associated with insect-pathogenic bacteria in the Enterobacteriaceae: Photorhabdus is associated with Heterorhabditidae and Xenorhabdus with Steinernematidae (Campos-Herrera et al. 2015). The third-instar infective juvenile (IJ) is the only free-living stage that lives in soil in search of an insect host (Wang et al. 2014).

EPNs are ubiquitous in cultivated and noncultivated soils throughout the world (Stuart et al. 2006). The importance of interspecific and intraspecific variation has been recognized, and this variation accounts for differences in efficacy among species and strains/isolates of these entomopathogens.

There are different factors that regulate EPN effectiveness in biocontrol (e.g., the habits of the target insect) and environmental factors (e.g., temperature, humidity and soil texture, and pesticide use). The use of native strains or isolates of EPNs collected from, and adapted to, specific regions might contribute to the success of these agents against pests in those locales (Garcia-del-Pino et al. 2013, Parada Domínguez et al. 2019).

There are several reports on the presence of EPNs in central and southern Mexico (Morfín-Arriaga and Cortez-Madrigal 2018, Parada Domínguez et al. 2019) and northeastern Mexico (Treviño-Cueto et al. 2021). In the latter study, the larvicidal activity of local isolates of Heterorhabditis and Steinernema against larvae of the yellow fever mosquito, Aedes aegypti (L.), was determined in laboratory bioassays. General biological attributes of those isolates, however, were not the subject of that study. Several of those isolates, therefore, are the subject of the study reported herein. Attributes of newly isolated EPNs include virulence against selected target insects and production in the laboratory, both of which are essential for their utilization in biological control programs. In addition, production of IJs in susceptible hosts is a relevant consideration in that distribution of nematode-killed insect-containing IJs in soils is an effective method for deployment of these biocontrol agents (Shapiro-Ilan et al. 2006). Furthermore, collection, evaluation, and utilization of locally isolated biocontrol agents are relevant when considering restrictions to the importation and use of exotic biocontrol agents (Jaronski et al. 2013).

In order to contribute to the development of regional biological control programs utilizing local strains or isolates of beneficial organisms, our objective in this study was the general characterization of isolates of EPNs isolated from one locality in Saltillo, Mexico. Specifically, we determined (1) the mortality induced by each local isolate larvae of on yellow mealworm, Tenebrio molitor L. (Coleoptera: Tenebrionidae), following exposure to concentrations of IJs; and (2) the numbers of IJs produced per host larva by each isolate in the laboratory.

Our study was conducted starting in May 2017 at the experimental fields and the Biological Control Laboratory, Universidad Autónoma Agraria Antonio Narro (UAAAN), Saltillo, state of Coahuila, Mexico.

Twenty-eight soil samples were obtained at the Universidad Autónoma Agraria Antonio Narro, with differences in habitat and soil use as described by Treviño-Cueto et al. (2021). Each sample was a 1-liter volume of soil taken at a depth of 15 cm using hand shovels. The shovels were rinsed with 70% ethanol between samples. In the laboratory, nematodes were isolated from soil samples using the insect bait technique described by Kaya and Stock (1997) and Sanchez-Peña et al. (2011). Each sample was split among five 200-ml containers, and water was added to each so that the soil was slightly moistened but not saturated or muddy. Five last-instar T. molitor larvae obtained from a laboratory colony were added to each container. The containers were covered, placed upside down to bury the insect larvae, and incubated in bioclimatic chambers at 23 ± 2°C. After 7 to 9 d, parasitized larvae were recovered, identified by their typical symptoms of color change (e.g., reddish brown/purple for heterorhabditids and ochre/brown/black for steinernematids). Cadavers were placed in modified White traps (Kaya and Stock 1997) for emergence of IJs. The emerged IJs were recovered from the water suspension in the trap and washed by gentle decantation in several changes of purified water to remove insect remains. All isolates were stored in purified water in petri dishes at 15–20°C.

The nematodes were identified to family based on morphological characteristics and appearance of infected larvae (Kaya and Stock 1997, Poinar 1979). Further identification was by the methods described by Maafi et al. (2003) and Treviño-Cueto et al. (2021). From each tentative nematode family, DNA was extracted from several dozen individuals. Genetic regions amplified were: internal transcribed spacer (ITS)-rRNA gene, D2-D3 region of 28S rRNA gene. For steinernematids, the D2-D3 region of the 28S rRNA gene was sequenced. Voucher specimens were deposited at the Plant Pest Diagnostics Center, California Department of Food and Agriculture, Sacramento, CA.

Nematode IJs were harvested from the White traps and resuspended in 10 ml of purified water. The number of IJs per milliliter was determined and adjusted using the Neubauer chamber and microscopic observation. Six isolates included in the bioasay were three Steinernema carpocapsae (Weiser) isolates designated as M5, M6, and B7, and three Heterorhabditis bacteriophora Poinar isolates designated M18, MZ9, and MZ6. Last-instar T. molitor larva were exposed to nematodes.

The bioassay was performed in experimental units consisting of petri dishes (100 × 15 mm) each lined on the bottom with a circle of rinsed, moistened brown paper. Suspensions of either 0, 10, 25, 50, 100, or 200 IJs/larva were evenly distributed on the moistened paper, and 10 last-instar T. molitor larvae were placed in each dish. The IJs used were less than a month old. There was a total of six isolates with six concentrations of IJs for each isolate. Each isolate/concentration was replicated 10 times. Larval mortality was recorded every 24 h up to 72 h, verifying that larvae did not show mobility when poked with a probe.

Nematode development and IJ production in T. molitor larvae were quantified for each EPN isolate using the bioassay testing method but only with one concentration of IJs (e.g., 100 IJs/larva) and five EPN isolates (the M6 isolate of S. carpocapsae was not available for this test). After 7–9 d, dead larvae were randomly selected from each EPN isolate treatment, identified by the color change described (reddish brown/purple for heterorhabditids and ochre/brown/black for steinernematids), and placed in modified White traps (Kaya and Stock 1997) for emergence of IJs. The IJs in the White trap were transferred individually to petri dishes (10-cm diameter) containing 20 ml of sterile distilled water. Total IJ production was determined from dilutions using the Neubauer chamber.

Mortality data were not corrected since mortality in the control was zero. An analysis of variance was performed for mortality, and Tukey’s test was applied to separate the means in the R Studio program. Probit analysis (Finney and Tattersfield 1952) and estimation of the median lethal concentration (LC₅₀) were performed with the StatPlus (2022) statistical program. The production of IJs was compared by analysis of variance followed by Bonferroni’s test for mean separation in the R Studio program.

Six EPN isolates were obtained from the 120 samples collected from University soils. The six were determined to belong to the genera Heterorhabditis and Steinernema (Table 1) based on the characteristic coloration of the T. molitor cadavers (Woodring and Kaya 1988). A reddish-dark wine color of larvae killed by EPNs is caused by the bacterium Photorhabdus associated with Heterorhabditis; whereas Xenorhabdus, associated with Steinernema spp., induces a brownish-dark color in EPN-killed larvae. We did not observe the reported fluorescence in our putative Heterorhabditis isolates (Woodring and Kaya 1988).

Sequences of the ITS-rRNA and D2-D3 of 28S rRNA genomic regions of the Heterorhabditis isolates showed 100% identity to many isolates of H. bacteriophora from Mexico (e.g., MK421440.1), Argentina, the Middle East, and India (Machado et al. 2019), underscoring the cosmopolitan distribution of this species (Campos-Herrera et al. 2012). Sequences of H. bacteriophora obtained herein are deposited in GenBank with the 28S D2-D3 rRNA region as accession MN191509 and the ITS-rRNA region as MN191510. The D2-D3 region of 28S rRNA gene sequence of the Steinernema isolates matched with 100% identity that of isolates of S. carpocapsae from different areas of the world (e.g., Colombia, Iran, Switzerland, and Mexico) (Noujeim et al. 2011). It also matched strain DD-136 of S. carpocapsae cited in earlier studies (Welch and Bronskill 1962). The sequence of the 28S D2-D3 rRNA region of S. carpocapsae derived from this work is deposited in GenBank (accession MN503268).

The pathogenicity and virulence of entomopathogenic nematodes isolated from Saltillo, Coahuila, Mexico, on T. molitor larvae were determined in laboratory bioassays. Control mortality was very low (0–1%) in these tests; therefore, mortality correction was not required (Abbott 1925). When insects were exposed to each isolate at varying concentrations ranging from 25 to 200 IJs/larva, the evaluated isolates induced mortality ranging from 15.16% to 100% in a lapse of 24 to 72 h (Tables 1–6). In general, within isolates, mortality did not increase markedly when larvae were exposed to increasing EPN concentrations. The lowest percent mortality values were observed in isolates such as Heterorhabditis MZ9 with 15.16% T. molitor larval mortality at a concentration of 50 IJs/larva after 24 h, or the Heterorhabditis MZ6 isolate with <63% larval mortality with 200 IJs/larva after 72 h (Tables 4 and 6).

A comparison of mortality levels induced by the six isolates observed at 72 h postexposure is included in Table 7. Again, mortality induced by some isolates (e.g., M6, B7, MZ6) exhibited a weak response to increasing IJ concentrations, for example, mortality levels consistent and at levels similar to those observed after exposure to the lowest concentrations tested (Table 7). Comparisons of mortality levels induced at the same concentration among the six isolates indicated that, in general, the Steinernema isolates induced significantly higher mortality levels than the Heterorhabditis isolates.

The mortality response observed at 72 h postexposure was used for estimating the LC₅₀ of each isolate (Table 8). The lowest LC₅₀ among the six isolates was with the S. carpocapsae M6 isolate (4.99 IJs/larva). The least virulent isolate (i.e., exhibiting the highest LC₅₀) was the H. bacteriophora MZ6 isolate with an LC₅₀ of 45.91 IJs/larva. Although the confidence limits of intervals were broadly overlapping, the analysis indicated important differences in the LC₅₀s among the isolates. These values ranged from 4.99 to 45.91 IJs/larva, which is almost a 10-fold difference among the isolates. We saw no evidence of statistically significant higher virulence for one nematode genus over the other in these assays, although there was a very general trend for Steinernema isolates being more pathogenic (Tables 7 and 8).

The IJ production by the different EPN isolates in T. molitor ranged from 2,227 to 17,320 IJs/larva (Table 9). The isolate exhibiting the greatest IJ production was the S. carpocapsae M5 isolate, which also was the isolate with the lowest LC₅₀.

Labaude and Griffin (2018) indicated that laboratory screening is essential to detect EPN strains of low virulence, while noting that the factors controlling virulence have not been identified with complete certainty. Virulence and reproductive capabilities are essential traits in the measurement of the fitness of EPNs. Rolston et al. (2009) found a wide range of virulence among isolates of Steinernema feltiae (Filipjev) collected from a small Irish island (Bull Island). Likewise, we found a range of lethal responses of T. molitor larvae to the isolates we tested. The lowest LC₅₀ values we observed were with the M5 (4.99 IJs/larva) and M6 (6.23 IJs/larva) isolates of S. carpocapsae, and the MZ9 isolate of H. bacteriophora yielded an LC₅₀ of 11.18. These values are similar to those reported by Doucet et al. (1992) with LC₅₀ values of 4 to 9 IJs/larva for H. bacteriophora tested against Galleria mellonella L. larvae.

Bedding et al. (1983) evaluated several isolates of Heterorhabditis and Steinernema against several species of soil insects and G. mellonella larvae. They found no trend indicating higher virulence of one genus over the other, and the virulence of nematode species and strains towards insect species was variable. In their study, no one nematode taxon or isolate was the most infective for all insect targets. On the other hand, several studies indicate higher activity of Steinernema than Heterorhabditis against specific insect species (Molyneux 1986, Tarasco et al. 2015). Our comparison of mean mortality values indicated a similar trend (Table 7). These and other results emphasize the importance of evaluating several nematode species or isolates against specific insect targets before development and deployment as biological control agents.

Regarding IJ production of IJs in T. molitor larvae, the S. carpocapsae M5 isolate produced greater numbers of IJs per larva than the other isolates tested. The M5 isolate was also the most active against T. molitor larvae in our study (Tables 1–8). Treviño-Cueto et al. (2021) also found that M5 was the most pathogenic among the same isolates against A. aegypti larvae, and it ranked second among those isolates in IJ production in A. aegypti larvae.

Prabowo et al. (2019) cultivated in vivo S. carpocapsae in T. molitor larvae, obtaining 3,620, 6,290, 11,300, and 15,400 IJs/larva using EPN concentrations of 100, 200, 300, and 400 IJs/larva. Our results were comparable for insect larvae exposed to 100 IJs with mean production within a range of 1,650 to 17,320 IJs/larva across isolates from both species tested. For S. carpocapsae, the values obtained herein were 1,650 and 17,320 IJs/larva.

Shapiro-Ilan et al. (2008) indicated that a key factor for the in vivo reproduction of EPNs is the nutritional content of the host’s diet in determining the reproductive capacity and quality of the IJs produced. The numbers they reported ranged from 100,000 to 200,000 IJs per G. mellonella larva. It must be mentioned that last-instar larvae of this insect are considerably larger and heavier than last-instar T. molitor larvae, thus explaining in part the higher numbers of IJs obtained by Shapiro-Ilan et al. (2008).

In conclusion, our results exhibit considerable variability in virulence and reproductive potential among the isolates we evaluated. These observations indicate that there are large differences, likely caused by only partially known underlying mechanisms, among coexisting, sympatric EPN isolates and that these differences must be considered when choosing local EPN isolates for development in biological control programs.

We thank Consejo Nacional de Ciencia y Tecnología (CONACYT) for providing scholarship support to D.T.C. (CVU 503782). Project 389721, Dirección de Investigación, UAAAN. The editors’ comments greatly improved this manuscript.