Entomopathogenic nematodes are effective biocontrol agents against arthropod pests. However, their efficacy is limited due to sensitivity to environmental extremes. The objective of the present study was to establish a foundation of genetic-based selection tools for beneficial traits of heat and desiccation tolerance in entomopathogenic nematodes. Screening of natural populations enabled us to create a diverse genetic and phenotypic pool. Gene expression patterns and genomic variation were studied in natural isolates. Heterorhabditis isolates were phenotyped by heat-and desiccation-stress bioassays to determine their survival rates compared to a commercial line. Transcriptomic study was carried out for the commercial line, a high heat-tolerant strain, and for the natural, low heat-tolerant isolate. The results revealed a higher number of upregulated vs. downregulated transcripts in both isolates vs. their respective controls. Functional annotation of the differentially expressed transcripts revealed several known stress-related genes and pathways uniquely expressed. Genome sequencing of isolates with varied degrees of stress tolerance indicated variation among the isolates regardless of their phenotypic characterization. The obtained data lays the groundwork for future studies aimed at identifying genes and molecular markers as genetic selection tools for enhancement of entomopathogenic nematodes ability to withstand environmental stress conditions. Domestication and improvement of crop plants and animals have been part of agriculture for thousands of years. Genetic manipulation of beneficial arthropods, such as silkworms and honeybees, has been conducted for hundreds of years 1,2 and genetic improvement programs have also provided innovative methods for controlling insect pests 3,4. Beneficial arthropods have been selected for climate tolerance 5,6 host-finding ability, host preference 7,8 , improved sex ratio 9,10 , increased fecundity 10,11 , and resistance to insecticides 12,13. Unlike the long history and vast research on the use of beneficial insects for biological control, the use of entomopathogenic nematodes (EPNs) and the genetic improvement of EPNs is in its infancy. As the use of EPNs for biological control of insect pests becomes practical and commercial due to improvements in production methods 14,15 , the use of powerful genetic tools to improve their performance has been strongly advocated (see reviews 16,17). The only free-living stage of the nematode is the third stage infective juvenile (IJ), a non-feeding larva that lives in the soil, and seeks out and penetrates its host through natural openings 18,19. The IJ is exposed to changing environmental conditions and its lack of tolerance to extreme environmental conditions directly influences the shelf life, quality and field performance. Survival, persistence and shelf-life are critical limiting factors for the commercial use of nematodes as biological control agents 20. These difficulties stem mainly from EPNs' sensitivity to heat and desiccation...

Direct contact between the conidia of entomopathogenic fungi (EPF) and their host is a prerequisite to successful infection; the host can, therefore, be infected by both direct treatment and by transmission of fungal inoculum from infested surfaces. This unique characteristic makes EPF especially relevant for the control of cryptic insects. In the case of the red palm weevil (RPW) Rhynchophorus ferrugineus, the eggs and larvae are almost inaccessible to direct-contact treatment. The objective of the present study was to investigate the mechanism of conidia transmission from a treated surface to host eggs and larvae. Foam pieces infested with Metarhizium brunneum conidial powder, conidial suspension, or distilled water were used as a laying surface for RPW females. The number of eggs laid was not affected by the EPF treatments and ranged from 2 to 14 eggs per female. However, hatching rate and larval survival were significantly reduced in the conidial powder treatment, resulted in 1.5% hatching and no live larvae. In the conidial suspension treatment, 21% of laid eggs hatched, compared to 72% in the control treatment. In both M. brunneum treatments, females’ proboscis, front legs and ovipositor were covered with conidia. The females transferred conidia in both treatments to the laying holes, reaching up to 15 mm in depth. This resulted in reduced egg-hatching rate and significant larval mortality due to fungal infection. The stronger effect on egg and larval survival using dry conidia seemed to result from better conidial adhesion to the female weevil in this formulation. In future studies, this dissemination mechanism will be examined as a prevention strategy in date plantations.

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