Entomopathogenic nematodes (EPNs) of the genera Heterorhabditis and Steinernema are insecticidal agents used against diverse groups of key insect pests. Yet, their application cost and reliability frequently relegate them as niche products in the biopesticide market. A full spectrum of developments is being addressed to solve the two issues. Among them, molecular and non-molecular methods play a significant role in improving their biocontrol traits. Relevant programs usually comprise selection, hybridization, and/or mutagenesis/genetic engineering techniques. Progress to boost EPN-biocontrol traits like infectivity, virulence, survival/persistence, and co-optimization of EPNs and their symbionts is discussed herein, showing possible molecular processes as bases for further optimization. Trait improvement may also impact other parts of the cascade that lead to positive outputs such as increasing EPN efficiency or negative trade-offs like decreased baseline fitness. Thus, understanding related molecular processes would help to exploit/strengthen positive ones and fix weaknesses. The purposes of advanced molecular techniques like RNA interference, qPCR, CRISPR-Cas9, and RNA sequencing and their contribution in EPN improvement, as well as their main limitation and validation, are presented. Also, ligand-target interactions of relevant symbionts are amplified to be scalable for efficient exploitation of their secondary metabolites, which are toxic to key hosts. Using reverse transcription quantitative PCR (RT-qPCR) gave molecular cues for understanding how adding dimethyl sulfoxide to the culture medium can boost commercial EPN production, a novel technology for increasing such production. Other molecular cues of certain plant volatiles demonstrated increased EPN efficacy by directing EPN behavior and improving infectivity. The pros and cons of traditional molecular techniques applied on EPNs are presented for their optimal usage. Yet, advanced molecular tools like qPCR analysis combined with traditional ones can offer novel insights to elucidate complicated soil biotic interactions and detect abiotic elements that modify EPN effectiveness. Modern techniques like sequencing of the whole nuclear and mitochondrial genome have rightly resolved Heterorhabditis spp. phylogenetic relationships, avoiding former inconsistent tree topologies mostly due to using different gene markers. Other knowledge gaps along with a forward-looking roadmap to catalysts for future studies over the next years are highlighted. Expanding advanced molecular techniques such as metabarcoding can offer perfect EPN uses and secure the best EPN-host matching for economical and sustainable agricultural practices.
