Currently, most nematode identification methods focus on morphological characteristics. These techniques are only helpful when a trained taxonomist is available.
However, precise categorization based on morphological features is often complex due to specific morphological characteristics and limited samples. Molecular techniques are promising in overcoming these problems.
Molecular methods utilize nucleic acid-based techniques to identify nematode species. Species identification is crucial to nematological research, quarantine enforcement for regulatory purposes, and nematode management that does not rely on nematicides. Accurate species identification increases samples’ detection sensitivity and the speed of quantifying nematode species in research or management.
The simplest molecular method uses restriction enzymes to digest DNA and generate banding patterns that differentiate nematode species based on the degree of sequence divergence. PCR-based methods utilizing sequence-specific primers for the nuclear ribosomal DNA (rDNA) or mitochondrial cytochrome C oxidase subunit I (COX1) are also standard and practical tools for nematode identification.
Protein-based identification methods use unique protein composition and structures to delineate nematode species. They utilize the 20-plus alphabet of amino acids compared to four DNA bases, allowing for structural variations such as post-translational modifications and conformational changes. However, protein-based identification methods are less sensitive than DNA-based approaches.
With the shift from traditional morphological identification techniques, various modern-day and molecular methods have been used to classify nematodes. These methodologies range from DNA- and protein-based characterization/identification to high-throughput approaches like next-generation sequencing.
These approaches allow researchers to study how nematodes respond to various environmental and genetic stimuli. They also allow scientists to manipulate nematodes and investigate their phenotypic responses to host-seeking behaviors.
For example, scientists have generated stable transgenic nematode lines of Strongyloides stercoralis resistant to the toxic effects of certain antibiotics. These nematodes are amenable to transgenesis by intragonadal microinjection, where exogenous plasmid or linear DNA is injected directly into the syncytial testes of adult hermaphrodites and then transferred to their progeny. Scientists have understood how these nematodes’ sensory nervous systems respond to host-emitted sensory cues using this method. This mechanistic understanding may help develop novel methods for nematode control.
Nematodes, the most common animals on Earth, are critical to soil ecology. Global-scale ecological studies are required to understand better nematode abundance and community composition and the environmental forces that impact them.
The resulting global dataset provides a unique opportunity to identify environmental factors that drive nematode community structure.
A curated reference database specific to nematodes is also critical for molecular tools like metabarcoding. Currently, most metabarcoding analyses use a general 18S rRNA nematode primer set (NF1_F and 18Sr2b_R).
Integrated Nucleic Acid Detection System
Molecular identification and characterization of nematodes is essential for understanding nematode biodiversity and developing important management practices. Traditional morphological methods need more observable characteristics and taxonomic classification due to the many strongly linked nematode species.
PCR-based molecular methods compensate for the shortcomings of morphological analysis and provide advantages such as sensitivity, correctness, and time savings. In addition to PCR-based identification, molecular approaches such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA) have been developed to improve the detection of pathogenic nematodes.
Commercial kits have also been developed to extract nematode DNA from soil samples directly, which can significantly reduce the labor and cost of amplification. In addition, protein-based detection and identification methods such as MALDI-ToF mass spectrometry can classify nematodes by analyzing their proteomes. This will further enhance nematode identification and characterization. These advances are essential to shift the paradigm of nematode research from classical morphological characterization to modern-day molecular identification and characterization.