Multi-locus phylogenetic analyses uncover species boundaries and reveal the occurrence of two new entomopathogenic nematode species, Heterorhabditis ruandica n. sp. and Heterorhabditis zacatecana n. sp.

Abstract

Species of the nematode genus Heterorhabditis are important biological control agents against agricultural pests. The taxonomy of this group is still unclear as it currently relies on phylogenetic reconstructions based on a few genetic markers with little resolutive power, specially of closely related species. To fill this knowledge gap, we sequenced several phylogenetically relevant genetic loci and used them to reconstruct phylogenetic trees, to calculate sequence similarity scores, and to determine signatures of species- and population-specific genetic polymorphism. In addition, we revisited the current literature related to the description, synonymisation, and declaration as species inquirendae of Heterorhabditis species to compile taxonomically relevant morphological and morphometric characters, characterized new nematode isolates at the morphological and morphometrical level, and conducted self-crossing and cross-hybridization experiments. The results of this study show that the sequences of the mitochondrial cytochrome C oxidase subunit I (COI) gene provide better phylogenetic resolutive power than the sequences of nuclear rRNA genes and that this gene marker can phylogenetically resolve closely related species and even populations of the same species with high precision. Using this gene marker, we found two new species, Heterorhabditis ruandica n. sp. and Heterorhabditis zacatecana n. sp. A detailed characterization of these species at the morphological and morphometric levels and nematode reproduction assays revealed that the threshold for species delimitation in this genus, using COI sequences, is 97% to 98%. Our study illustrates the importance of rigorous morphological and morphometric characterization and multi-locus sequencing for the description of new species within the genus Heterorhabditis, serves to clarify the phylogenetic relationships of this important group of biological control agents, and can inform future species descriptions to advance our efforts towards developing more tools for sustainable and environmentally friendly agriculture.

Comparative Screening of Mexican, Rwandan and Commercial Entomopathogenic Nematodes to Be Used against Invasive Fall Armyworm, Spodoptera frugiperda

“…We intend to use locally isolated EPNs to avoid introducing foreign organisms. This is important, as it is increasingly evident that EPN species are not always correctly identified [24,25]. Misdiagnosing closely related EPN species increases the risk of accidently introducing non-native EPNs in a targeted region, as has been reported previously [26].…”

“…The virulence and killing speed of the 29 Mexican EPN strains (five species) were evaluated on third-instar FAW caterpillars. One strain of Heterorhabditis atacamensis, twentyone of Heterorhabditis bacteriophora, three of Heterorhabditis zacatecana (newly described by [25]), three of Heterorhabditis mexicana and one of Steinernema riobrave were used (Table 1). The virulence of each Mexican EPN strain was compared to the virulence of the commercial EPN Steinernema carpocapsae strain All, which was used as the positive control.…”

“…Five of the most promising EPN strains were selected, from the preceding two steps, with at least one strain from each origin (Mexican, Rwandan or commercial). The Rwandan EPN were S. carpocapsae RW14-G-R3a-2 and H. ruandica Rw18_M-Hr1a (formerly considered to be H. bacteriophora [25,30]). The Mexican EPN was H. zacatecana MEX-41.…”

“…The five most virulent Mexican strains (four species) were selected for the second step, in which they were compared with five Rwandan (three species) and six commercial strains (six species). In the third step, the five most promising EPN from the second step (one Mexican, two Rwandan and two commercial EPN) were tested at three concentrations (5,25,125 EPN per FAW) against second-, third-and sixth-instar caterpillars as well as against pupae of FAW. In addition, we evaluated reproductive output of each of these EPN strains in FAW caterpillars.…”

Laboratory and field trials reveal the potential of a gel formulation of entomopathogenic nematodes as biocontrol against the fall armyworm (Spodoptera frugiperda)

“…Two entomopathogenic nematode species were used in this study. These were Steinernema carpocapsae (strain RW14-G-R3a-2) and Heterorhabditis ruandica (strain Rw18_M-Hr1a;), both being among the strains isolated from soil samples in Rwanda in 2014 and 2018, respectively (Fallet et al, 2020; Machado et al, 2021; Yan et al, 2016). They were among the most effective strains in killing FAW caterpillars in a screening test where we compared 40 EPN strains, representing twelve species, originating from Rwanda, Mexico and from a few commercial sources (Fallet et al, in press).…”