Increasing the survival and efficacy of entomopathogenic nematodes on exposed surfaces by Pickering emulsion formulations offers new venue for foliar pest management.

Formulation technology has been the primordial focus to improve the low viability and erratic infectivity of entomopathogenic nematodes (EPNs) for foliar application. Adaptability to the fluctuating environment is a key trait in ensuring the survival and efficacy of EPNs. Hence, tailoring formulations towards EPNs foliar applications would effectively deliver consistent and reliable results for above-ground applications. EPNs survival and activity were characterized in novel Pickering emulsion post-application in planta cotton foliage. Two different types of novel formulations, Titanium Pickering emulsion (TPE) and Silica Pickering emulsion Gel (SPEG), were tailored for EPNs foliar applications. We report an extension of survival and infectivity to 96 hrs under controlled conditions by SPEG formulations for survival of IJ's on cotton foliage. In addition, survival of IJs (LT50) was extended from 14hrs in water to > 80 hrs and > 40 hrs by SPEG and TPE respectively. SPEG accounted for the slowest decrease of live IJs per surface area in comparison to TPE and control samples over time, exhibiting a 6-fold increase at 48 hrs. Under extreme conditions, survival and efficacy were extended for 8hrs in SPEG compared to merely 2hrs in control. Potential implications and possible mechanisms of protection are discussed.

Survival and efficacy of entomopathogenic nematodes on exposed surfaces.

Entomopathogenic nematodes (EPN) species differ in their capability to withstand rapid desiccation (RD). Infective juveniles of Steinernema carpocapsae are a better adaptable and tolerant than Steinernema feltiae or Heterorhabditis bacteriophora as, an optimal RH of > 90% is required by S. feltiae and H. bacteriophora while maintaining RH equivalent to 74% could sustain survival of S. carpocapsae under RD. Our findings from infectivity suggest that following application, shrunk IJs are acquired passively by the larvae, probably rehydrate and resume infection within the insect gut. Water loss rate is a key factor affecting survival of S. carpocapsae on exposed surfaces. The present study provides the foundation for characterizing mechanism of rapid rate of water loss in EPN. ATR-FTIR is a rapid and reliable method for analysis of water loss. Changes in peak intensity was observed at 3100-3600 cm-1 (OH bonds of water), 2854 cm-1 (CH stretching of symmetric CH2, acyl chains), 2924 cm-1 (CH stretching of anti-symmetric CH2, lipid packing heterogeneity), 1634 cm-1 (amide I bonds) indicate major regions for hydration dependent changes in all EPN species. FTIR data also indicates that, S. carpocapsae contains strong water interacting regions in their biochemical profile, which could be an influencing factor in their water holding capacity under RD. ATR-FTIR were correlated to water content determined gravimetrically by using Partial Least square -Regression and FTIR multivariate method, which could be used to screen a formulation's potential to maintain or delay the rate of water loss in a rapid and efficient manner.

Behavioral and molecular response of the insect parasitic nematode Steinernema carpocapsae to cues emitted by a host, the red palm weevil, Rhynchophorus ferrugineus.

As the larvae of the date palm pest, the red palm weevil, Rhynchophorus ferrugineus, feeds on the host tissue, they emit a distinctive sound which can be recorded outside of the infected tree. We evaluated the response of infective juveniles (IJs) of the entomopathogenic nematodes Steinernema carpocapsae to the R. ferrugineus larvae and it's sound source, separately. In the presence of the insect larvae, 50.2 % of total IJs moved toward those larvae. Recorded insect larvae sound emitted by the speaker resulted in 7% of total IJs near the sound source. RNA-Seq data indicated that more genes were downregulated in S. carpocapsae IJs exposed to insect and speaker compared to non-stimulated IJs. IJs exposed to insect exhibited more up-regulated genes than IJs exposed to speaker. Enriched pathways and biological processes in IJs were similar for both stimuli. The inhibition of locomotion, regulation of neurotransmitter secretion, response to biotic stimulus, and cellular response to chemical stimuli were enriched with unique GO terms for speaker treatment. The regulation of localization, sodium ion transmembrane transport, regulation of response to stress and response to organic substances were the GO categories enriched unique to insect. The host-parasitic interaction was regulated by the differential expression of Ras/MAP kinase, TGF-beta signaling, insulin signaling, AMPK signaling, PPAR signaling pathways and many developmental pathways. More prominent R. ferrugineus host localization by S. carpocapsae was primarily due to the differential transcriptional regulation of olfactory signal transduction, FOXO-family proteins, calcium signaling, WNT and mTOR signaling pathway. The neural basis for the nematode attraction to insect host is based on the chemosensation and the mechanosensation. Many neuropeptides and neuromodulators are involved in regulating the foraging behavior of S. carpocapsae. The results of this study provide new insights into the molecular mechanisms that allow these nematodes to seek insect hosts. Our finding, especially the molecular ones suggest that chemical cues emitted by the active insect host are stimulants of nematodes attraction. Whereas the sound emitted by the insect has minor effects on the nematode behavior.

Characterization of the phenotypic and genotypic tolerance to abiotic stresses of natural populations of Heterorhabditis bacteriophora.

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.

Can an entomopathogenic nematode serve, as proxy for strongyles, in assessing the anthelmintic effects of phenolic compounds?

As gastro-intestinal nematodes (GINs) become increasingly resistant to chemical anthelmintics, and because consumers scrutinize chemical residues in animal products, the use of herbal anthelmintics and in particular, phenolic compounds, has become attractive. Most life stages of GINs cannot be grown in the lab as they are obligatory parasites, which limits our understanding of the effects of phenolic compounds on their parasitic stages of life. We hypothesized that a species phylogenetically close to GINs and grown in vitro, the insect-parasitic nematode Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade), when fed with Photorhabdus luminescens exposed to plant phenolics, can serve, as proxy for strongyles, in assessing the anthelmintic effects of phenolic compounds. We compared the development of H. bacteriophora infective juveniles (IJ) and the exsheathment rate of L3 larvae of the strongyle Teladorsagia circumcincta and Trichostrongylus colubriformis when exposed to catechin, rutin, chlorogenic and gallic acids, and myricetin. Gallic acid had the highest impact in terms of IJ mortality but the highest impairment of IJ development to adulthood was imposed by myricetin. The studied compounds were not lethal to GINs stricto sensu but we consider that the practical implications of total exsheathment inhibition and mortality on GIN populations are similar. Catechin and rutin had similar effects on rhabditid and strongyles: they imposed ca. 90% lethality of IJs at concentrations higher than 1200 ppm and the remaining live IJs did not develop further, and they also totally inhibited strongyle L3 exsheathment in a dose-response fashion. Gallic acid was 100% lethal to IJs exposed above 300 ppm and chlorogenic acid caused 87% mortality above 1200 ppm, with no development for the surviving IJs but for all lower concentrations, all the IJs developed to adult stages. Likewise, gallic and chlorogenic acids did not affect the exsheatment of GIN L3 larvae. Therefore, a discrepancy between the effects of gallic and chlorogenic acids on the development of rhabditid IJs and exsheathment of GIN L3 larvae was found only when they were exposed to high concentrations. A dose-response of IJ lethality to myricetin was found, with no IJ development between 150 and 2400 ppm; but contrary to the other compounds, myricetin also impaired IJ development of IJs above 10 ppm in a dose-response manner and showed dose-responses in the L3 exsheathment. Apart for the high rates of lethality imposed on IJs by gallic and chlorogenic acids at high concentration, these results suggest that H. bacteriophora fed P. luminescens exposed to phenolics shows potential to serve as model in studies of the anthelmintic effects of phenolics in GIN.

Toxicity of phenolic compounds to entomopathogenic nematodes: A case study with Heterorhabditis bacteriophora exposed to lentisk (Pistacia lentiscus) extracts and their chemical components.

Insects show adaptive plasticity by ingesting plant secondary compounds, such as phenolic compounds, that are noxious to parasites. This work examined whether exposure to phenolic compounds affects the development of insect parasitic nematodes. As a model system for parasitic life cycle, we used Heterorhabditis bacteriophora (Rhabditida; Heterorhabditiade) grown with Photorhabdita luminescens supplemented with different concentrations of plant phenolic extracts (0, 600, 1200, 2400 ppm): a crude ethanol extract of lentisk (Pistacia lentiscus) or lentisk extract fractionated along a scale of hydrophobicity with hexane, chloroform and ethyl acetate; and flavonoids (myricetin, catechin), flavanol-glycoside (rutin) or phenolic acids (chlorogenic and gallic acids). Resilience of the nematode to phenolic compounds was stage-dependent, with younger growth stages exhibiting less resilience than older growth stages (i.e., eggs < young juveniles < young hermaphrodites < infective juveniles < mature hermaphrodites). At high concentrations, all of the phenolic compounds studied were lethal to eggs and young juveniles. The nematodes were able to survive in the presence of medium and low concentrations of all studied compounds, but very few of those treatments allowed for reproduction beyond the infective juvenile stage and, at low concentrations, the crude 70% ethanol extract, chloroform and hexane extracts, and myricetin were associated with some impaired reproduction. The ethyl-acetate fraction and gallic acid were extremely lethal to the young stages and allowed almost no development beyond the infective juvenile stage. We conclude that exposure of infective juveniles to phenolics before they infect insects and post-infection exposure of other nematode developmental stages may affect the initiation of the infection, suggesting that the chemistry of dietary phenolics may limit H. bacteriophora's infection of insects.

Ethanolic extracts of Inula viscosa, Salix alba and Quercus calliprinos, negatively affect the development of the entomopathogenic nematode, Heterorhabditis bacteriophora - A model to compare gastro-intestinal nematodes developmental effect.

Heterorhabditis bacteriophora can represent a model system for herbal medication against gastro-intestinal strongylid parasites in determining the recovery and development due to their unique parasitic infectious cycle. The fact that plant extracts impair nematode development is known but their differential impact on stages of the life cycle of H. bacteriophora has never been investigated. We examined the developmental stages resumed from eggs, young juveniles (J1-3), infective juveniles (IJs), young and adult hermaphrodites of H. bacteriophora upon exposure to crude ethanolic extracts of Inula viscosa, Salix alba, and Quercus calliprinos at concentrations of 600, 1200, and 2400ppm. Our results showed that plant extracts were highly toxic to the survival of the eggs and young juveniles J1 to J3 at all concentrations. The plant extracts inhibited their development and were associated with low reproduction parameters (i.e. fecundity and viability of eggs). The IJs, J4, young and developed hermaphrodites displayed concentration-dependent negative effect on development with less egg count, poor vulval muscle development, loss of egg laying capacity and progeny development by matricidal hatching. Plant extract of I. viscosa at low (600ppm) concentration did not impair vulval development. These results suggest that these plant extracts show potential for the control of parasitic rhabditids.

Transcriptome analysis of stress tolerance in entomopathogenic nematodes of the genus Steinernema.

Entomopathogenic nematodes of the genus Steinernema are effective biological control agents. The infective stage of these parasites can withstand environmental stresses such as desiccation and heat, but the molecular and physiological mechanisms involved in this tolerance are poorly understood. We used 454 pyrosequencing to analyse transcriptome expression in Steinernema spp. that differ in their tolerance to stress. We compared these species, following heat and desiccation treatments, with their non-stressed counterparts. More than 98% of the transcripts found matched homologous sequences in the UniRef90 database, mostly nematode genes (85%). Among those, 60.8% aligned to the vertebrate parasites including Ascaris suum, Loa loa, and Brugia malayi, 23.3% aligned to bacteriovores, mostly from the genus Caenorhabditis, and 1% aligned to EPNs. Analysing gene expression patterns of the stress response showed a large fraction of down-regulated genes in the desiccation-tolerant nematode Steinernema riobrave, whereas a larger fraction of the genes in the susceptible Steinernema feltiae Carmiel and Gvulot strains were up-regulated. We further compared metabolic pathways and the expression of specific stress-related genes. In the more tolerant nematode, more genes were down-regulated whereas in the less tolerant strains, more genes were up-regulated. This phenomenon warrants further exploration of the mechanism governing induction of the down-regulation process. The present study revealed many genes and metabolic cycles that are differentially expressed in the stressed nematodes. Some of those are well known in other nematodes or anhydrobiotic organisms, but several are new and should be further investigated for their involvement in desiccation and heat tolerance. Our data establish a foundation for further exploration of stress tolerance in entomopathogenic nematodes and, in the long term, for improving their ability to withstand suboptimal environmental conditions.

Effects of tannin-rich host plants on the infection and establishment of the entomopathogenic nematode Heterorhabditis bacteriophora.

Parasitized animals can self-medicate. As ingested plant phenolics, mainly tannins, reduce strongyle nematode infections in mammalian herbivores. We investigated the effect of plant extracts known to be anthelmintic in vertebrate herbivores on the recovery of the parasitic entomopathogenic nematode Heterorhabditis bacteriophora infecting African cotton leafworm (Spodoptera littoralis). Nematode infective juveniles (IJs) were exposed to 0, 300, 900, 1200, 2400 ppm of Pistacia lentiscus L. (lentisk), Inula viscosa L. (strong-smelling inula), Quercus calliprinos Decne. (common oak) and Ceratonia siliqua L. (carob) extracts on growth medium (in vitro assay). In control treatments, 50-80% of IJs resumed development to J4, young and developed adult hermaphrodites, whereas all extracts, except for C. siliqua at 300 ppm, impaired IJ exsheathment and development. The highest concentration of I. viscosa extract (2400 ppm) had the strongest effect, killing 95% of exposed nematodes. Surviving nematodes did not recover, remaining at the IJ stage. Over the whole cycle, I. viscosa extract inhibited recovery to 25% or less, and did not allow full development to adulthood, whereas 65% of IJs in the control treatment recovered and resumed development, 12% reaching complete maturation within 72 h of incubation. When herbivorous S. littoralis larvae were fed with different plant extracts in vivo, I. viscosa had the strongest effect at concentrations above 300 ppm, with 90% of insect-invading IJs not developing to parasitic stages, whereas in the control treatment, 85% of IJs resumed development. Exposure to C. siliqua extract also inhibited exsheathment and development of 75% of the IJs. Half of those that resumed development reached full maturation. P. lentiscus and Q. calliprinos extracts also inhibited development of 50% IJs. Our results suggest that H. bacteriophora can be used to study herbal medication against parasites in animals.