Nematodes are among the most diverse but least studied organisms. The classic morphology-based identification has proved insufficient to the study of nematode identification and diversity, mainly for lack of sufficient morphological variations among closely related taxa. Different molecular methods have been used to supplement morphology-based methods and/or circumvent these problems with various degrees of success. These methods range from fingerprint to sequence analyses of DNA- and/or protein-based information. Image analyses techniques have also contributed towards this success. In this review, we highlight what each of these methods entail and provide examples where more recent advances of these techniques have been employed in nematode identification. Wherever possible, emphasis has been given to nematodes of agricultural significance. We show that these alternative methods have aided nematode identification and raised our understanding of nematode diversity and phylogeny. We discuss the pros and cons of these methods and conclude that no one method by itself provides all the answers; the choice of method depends on the question at hand, the nature of the samples, and the availability of resources.

Citrus root weevil (Diaprepes abbreviates) causes significant yield loss in citrus, especially in Florida. A promising source of control for this pest are biological control agents, namely, native entomopathogenic nematodes (EPN) within the genus Steinernema. Two species of endemic EPN in Florida are S. diaparepesi, abundant within the central ridge, and S. khuongi, dominating the flatwood regions of the state. These citrus-growing regions differ significantly in their soil habitats, which impacts the potential success of biological control measures. While the genome sequence of S. diaprepesi is currently available, the genome sequence of S. khuongi and identity of the symbiotic bacteria is still unknown. Understanding the genomic differences between these two nematodes and their favored habitats can inform successful biological control practices. Here, MiSeq libraries were used to simultaneously sequence and assemble the draft genome of S. khuongi and its associated symbionts. The final draft genome for S. khuongi has 8,794 contigs with a total length of ∼82 Mb, a largest contig of 428,226 bp, and N50 of 46 kb; its BUSCO scores indicate that it is > 86% complete. An associated bacterial genome was assembled with a total length of ∼3.5 Mb, a largest contig at 116,532 bp, and N50 of 17,487 bp. The bacterial genome encoded 3,721 genes, similar to other Xenorhabdus genomes. Comparative genomics identified the symbiotic bacteria of S. khuongi as Xenorhabdus poinarii. These new draft genomes of a host and symbiont can be used as a valuable tool for comparative genomics with other entomopathogenic nematodes and its symbionts to understand host range and habitat suitability.

Wireworms are immature stages of click beetles (Coleoptera: Elateridae) and are considered a serious threat to sweet potato production in the southern United States. The major wireworm species collected in North Florida sweet potato fields in 2017 and 2018 were Conoderus scissus, C. rudis, C. amplicollis, and C. falli. These species vary in their behavior and biology. During a 2-yr study period, we conducted two insecticide field trials using eleven insecticides belonging to organophosphates, neonicotinoids, pyrethroids, and botanical classes, and three field trials using entomopathogenic nematode (EPN) species to control wireworms. In 2017, all insecticide treatments significantly reduced new feeding holes and total holes (old + new + other) as compared to the untreated control. In 2018, the result was similar with a few variations. In both years, all insecticides showed a percentage reduction in wireworm damage holes (2017: 34.88–96.19%; 2018: 12.38–97.02%) with the highest by Regent. In the EPN field study, one application of EPN near planting significantly reduced soil insects. In a laboratory study conducted at the Tropical Research and Education Center, UF-IFAS, chlorpyrifos caused higher percentage mortality of C. rudis (55.5%) than C. scissus (22.2%). At the present experiment rates, none of the insecticides caused the mortality of C. amplicollis. Heterorhabditids strain ‘FL-2122’ was more susceptible to chlorpyrifos than other strains of EPN.

Entomopathogenic nematodes within the genus Steinernema are used as biological control agents against significant agricultural pests. Steinernema diaprepesi is native to Florida and very effective in controlling citrus root weevil, a devastating pest of citrus, ornamental plants, and vegetables. Here, we present the draft genome of Steinernema diaprepesi, which is a valuable tool for understanding the efficacy of this nematode as a biological control agent.

Sex determination is a key developmental event in all organisms. The pathway that regulates sexual fate has been well characterized at the molecular level in the model free-living nematode Caenorhabditis elegans. This study aims to gain a preliminary understanding of sex-determining pathways in a plant-parasitic nematode Meloidogyne incognita, and the extent to which the roles of the sex determination genes are conserved in a hermaphrodite species, C. elegans, and plant-parasitic nematode species, M. incognita. In this study, we targeted two sex-determining orthologues, sdc-1 and tra-1 from M. incognita using RNA interference (RNAi). RNAi was performed by soaking second-stage juveniles of M. incognita in a solution containing dsRNA of either Mi-tra-1or Mi-sdc-1 or both. To determine the effect of RNAi of the target genes, the juveniles treated with the dsRNA were inoculated onto a susceptible cultivar of cowpea grown in a nutrient pouch at 28 °C for 5 weeks. The development of the nematodes was analyzed at different time points during the growth period and compared to untreated controls. Our results showed that neither Mi-sdc-1 nor Mi-tra-1 have a significant role in regulating sexual fate in M. incognita. However, the silencing of Mi-sdc-1 significantly delayed maturity to adult females but did not affect egg production in mature females. In contrast, the downregulation of Mi-tra-1 transcript resulted in a significant reduction in egg production in both single and combinatorial RNAi-treated nematodes. Our results indicate that M. incognita may have adopted a divergent function for Mi-sdc-1 and Mi-tra-1distinct from Caenorhabditis spp. However, Mi-tra-1 might have an essential role in female fecundity in M. incognita and is a promising dsRNA target for root-knot nematode (RKN) management using host-delivered RNAi.