Selective breeding of entomopathogenic nematodes for enhanced attraction to a root signal did not reduce their establishment or persistence after field release

We recently showed that the efficacy of an entomopathogenic nematode (EPN) as a biological control agent against a root pest could be enhanced through artificial selection. The EPN Heterorhabditis bacteriophora was selected for higher responsiveness towards (E)-β-caryophyllene (EβC), a sesquiterpene that is emitted by maize roots in response to feeding damage by the western corn rootworm (WCR). EβC is normally only weakly attractive to H. bacteriophora, which is one of the most infectious nematodes against WCR. By selecting H. bacteriophora to move more readily along a EβC gradient we obtained a strain that was almost twice more efficient in controlling WCR population in fields planted with an EβC-producing maize variety. However, artificial selection for one trait may come at a cost for other important traits such as infectiousness, establishment and/or persistence in the field. Indeed, infectiousness was slightly but significantly reduced in the selected strain. Yet, this apparent cost was largely compensated for by the higher responsiveness to the root signal. Here we show that the selection process had no negative effect on establishment and persistence of field-released EPN. This knowledge, combined with the previously reported results, attest to the feasibility of manipulating key traits to improve the efficacy of beneficial organisms.Key words: entomopathogenic nematodes, tritrophic interactions, artificial selection, biological control, Diabrotica virgifera virgifera, western corn rootworm, persistence, establishmentDiabrotica virgifera virgifera LeConte (Chrysomelidae: Coleptera, western corn rootworm, WCR) is a major well established pest of maize in the American Corn Belt and more recently also in Europe.¹ The larval stages of this beetle can cause significant damages to maize roots, leading to reduction of plant growth, deficiencies in nutrient and water uptake, lodging, increased susceptibility to water stress and reduced grain yield.² This combination of factors result in an estimated loss of one billion US dollars per year in the USA.³ The pest has been introduced in Europe in the early ''90s,⁴ and it is expected that at full establishment the costs resulting from WCR damages will be half a billion Euros.⁵ Several strategies are available to control this soil-dwelling pest, including crop rotation, pesticides and transgenic Bt maize, but WCR can readily evolve resistance to each of these methods.^6–8 This is why efforts have been invested in biological control alternatives.Entomopathogenic nematodes (EPN) show great promise as biological agents against WCR.⁹ Root-produced volatiles appear to play an important role in the recruitment of EPN^10–13 and one such volatile, (E)-β-caryophyllene (EβC), has recently been identified for maize roots¹⁴ and was found to be an ideal below-ground alarm signal.¹⁵ EPN efficacy can be improved by exploiting the ability of WCR-damaged maize roots to emit the attractant.¹⁴ Further studies have shown the importance of choosing the right species of nematodes.¹⁶ Among the EPN species tested against WCR, Heterorhabditis bacteriophora has proven to be one of the most virulent nematodes,¹⁷ but it barely responds to EβC.¹⁶ We therefore recently selected H. bacteriophora for higher responsiveness to EβC.¹⁸ In the field, the selected strain exhibited better abilities to control WCR larvae, but logically only in maize plots with plants that emitted EβC. However, previous studies have shown that enhancing beneficial traits through selective breeding can incur costs and negatively alter other traits in the selected strain.¹⁹ For EPN such trade-offs after selective breeding have also been reported, for instance resulting in reduced storage stability²⁰ or a lower capacity to kill their hosts.²¹ After selection for enhanced responsiveness to EβC response, we observed a small, but significant negative effect on infectiousness of the selected strains. However, this drawback was readily outweighed by the improved ability to locate hosts in the field.¹⁸Not only infectiousness is a crucial trait for the successful use of EPN in biological control: establishment and persistence in the field are of decisive importance as well. These traits vary with EPN species and are determined by biotic factors such as pathogens and predators²² or abiotic factors such as soil type,²³ humidity,²⁴ temperature²⁵ or pH.²⁴ But the main factor that is thought to determine long-term persistence in the field is the presence of available host insects.²⁵ In field trials in Hungary, three EPN species, H. bacteriophora, H. megidis and Steinernema feltiae, were released to test their control potential against WCR. They all persisted at least as long WCR were present in soil, during the same year.²⁶ There was no significant difference between the three species in the establishment or persistence. Yet, independent of timing of application, EPN populations dramatically decreased within five months after application. The authors²⁶ propose that this short persistence is due to the absence of suitable alternative hosts in intensively cultivated crop fields in Europe.To determine if the selection for enhanced responsiveness to EβC went at a cost for establishment and persistence we compared these key traits for the original and the EβC-selected stains. Using a metal auger (2 cm diam.; 20 cm high), 310 soil samples were dug out either two days (establishment) or 28 days (persistence) after EPN application. The soil was placed in plastic boxes (4.5 cm diam.; 60 cm high) and as previously described²⁶ Tenebrio molitor (Coleoptera: Tenebrionidae) larva was placed as bait in the boxes. Presence/absence of EPN was evaluated by visually checking T. molitor larvae for EPN infection. Soil samples from areas where no EPN were applied served as controls. No significant differences were found between the original and selected strain of H. bacteriophora strain (factor “strain”), neither in establishment after two days nor in persistence after 28 days (factor “time”) (Fig. 1, two-way ANOVA, Ftime_1,35 = 2.937, p = 0.097; Fstrain_2,35 = 10.359, p < 0.001; Ftime × strain_2,35 = 1.202, p = 0.315, statistical differences within factors were calculated using a Bonferoni post-hoc test). Hence, the selection of H. bacteriophora for a better response to EβC had no consequence for how the nematodes settled in the experimental fields. Future efforts to improve the effectiveness H. bacteriophora against WCR might also include selection for increased persistence in soil. This would allow lower application rates and could provide growers with an affordable and efficient control strategy against this voracious pest.Open in a separate windowFigure 1Establishment and persistence of the original and a selected strain of H. bacteriophora. The selected strain (squares) established and persisted as well as the original strain (diamonds). The triangles represent control samples from plots where no nematodes were released. Establishment (after two days) and persistence (after 28 days) was equal for both strains. Moreover, the number of soil samples containing EPN after 28 days was not significantly lower than after 2 days, independently of treatment. A few nematodes were detected in the control samples but again no differences over time were detected. Error bars indicate the SEM. Different lower-case letters indicate statistical differences within establishment (after 2 days) or persistence (after 28 days) (p <0.05).So far, manipulation of tritrophic systems in order to improve biological control has been largely theoretical.^27–29 We show here that for EPN this approach is realistic and that their responsiveness to root-produced foraging signals can be enhanced without significant costs for other relevant traits. It has also been shown that the emissions of the signals by the plants can be enhanced.³⁰ Combining these strategies opens new perspectives for the development of ecologically sound strategies in pest management.     

2D proteome analysis initiates new Insights on the Salmonella Typhimurium LuxS protein

Background  

Quorum sensing is a term describing a bacterial communication system mediated by the production and recognition of small signaling molecules. The LuxS enzyme, catalyzing the synthesis of AI-2, is conserved in a wide diversity of bacteria. AI-2 has therefore been suggested as an interspecies quorum sensing signal. To investigate the role of endogenous AI-2 in protein expression of the Gram-negative pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), we performed a 2D-DIGE proteomics experiment comparing total protein extract of wildtype S. Typhimurium with that of a luxS mutant, unable to produce AI-2.     

A tritrophic signal that attracts parasitoids to host-damaged plants withstands disruption by non-host herbivores

Background  

Volatiles emitted by herbivore-infested plants are highly attractive to parasitoids and therefore have been proposed to be part of an indirect plant defense strategy. However, this proposed function of the plant-provided signals remains controversial, and it is unclear how specific and reliable the signals are under natural conditions with simultaneous feeding by multiple herbivores. Phloem feeders in particular are assumed to interfere with plant defense responses. Therefore, we investigated how attack by the piercing-sucking cicadellid Euscelidius variegatus influences signaling by maize plants in response to the chewing herbivore Spodoptera littoralis.     

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.     

Indigo: an annotated bibliography

This bibliography lists and comments on publications describing the history, preparation, chemistry and some applications of indigo and related dyes. Note that the commentary by the author is in italics after the bibliographic information.     

PCR gut analysis reveals that Tenuiphantes tenuis (Araneae: Linyphiidae) is a potentially significant predator of Argentine stem weevil,Listronotus bonariensis (Coleoptera: Curculionidae), in New Zealand pastures

Polymerase chain reaction (PCR) gut analysis was conducted on specimens of the introduced spider Tenuiphantes tenuis collected from dairy pasture in Canterbury, New Zealand. PCR primers were specifically designed to amplify a fragment of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) from Listronotus bonariensis and revealed that this major pasture pest species is consumed in the field by T. tenuis. The field predation rate of L. bonariensis by T. tenuis was estimated from our PCR results together with published data on the degradation of DNA and the density of T. tenuis in Canterbury pastures. We found that T. tenuis is a potentially significant predator of L. bonariensis in New Zealand pastures.     

Empirical and deterministic accuracies of across-population genomic prediction

Background

Differences in linkage disequilibrium and in allele substitution effects of QTL (quantitative trait loci) may hinder genomic prediction across populations. Our objective was to develop a deterministic formula to estimate the accuracy of across-population genomic prediction, for which reference individuals and selection candidates are from different populations, and to investigate the impact of differences in allele substitution effects across populations and of the number of QTL underlying a trait on the accuracy.

Methods

A deterministic formula to estimate the accuracy of across-population genomic prediction was derived based on selection index theory. Moreover, accuracies were deterministically predicted using a formula based on population parameters and empirically calculated using simulated phenotypes and a GBLUP (genomic best linear unbiased prediction) model. Phenotypes of 1033 Holstein-Friesian, 105 Groninger White Headed and 147 Meuse-Rhine-Yssel cows were simulated by sampling 3000, 300, 30 or 3 QTL from the available high-density SNP (single nucleotide polymorphism) information of three chromosomes, assuming a correlation of 1.0, 0.8, 0.6, 0.4, or 0.2 between allele substitution effects across breeds. The simulated heritability was set to 0.95 to resemble the heritability of deregressed proofs of bulls.

Results

Accuracies estimated with the deterministic formula based on selection index theory were similar to empirical accuracies for all scenarios, while accuracies predicted with the formula based on population parameters overestimated empirical accuracies by ~25 to 30%. When the between-breed genetic correlation differed from 1, i.e. allele substitution effects differed across breeds, empirical and deterministic accuracies decreased in proportion to the genetic correlation. Using a multi-trait model, it was possible to accurately estimate the genetic correlation between the breeds based on phenotypes and high-density genotypes. The number of QTL underlying the simulated trait did not affect the accuracy.

Conclusions

The deterministic formula based on selection index theory estimated the accuracy of across-population genomic predictions well. The deterministic formula using population parameters overestimated the across-population genomic accuracy, but may still be useful because of its simplicity. Both formulas could accommodate for genetic correlations between populations lower than 1. The number of QTL underlying a trait did not affect the accuracy of across-population genomic prediction using a GBLUP method.     

Compaction and particle segregation in myelin membrane arrays

Compacted membrane arrays are formed in the nerve myelin sheath by lowering the water activity (through evaporation or immersion in hypertonic solutions of nonelectrolytes or monovalent salts) or by binding specific cations (Ca(++), La(+++), and tetracaine at concentrations above 5-10 mM). X-ray diffraction observations on intact, hydrated nerves treated to induce compaction provide a control to assess the significance of structural changes seen by freeze-fracture electron microscopy. Compaction inevitably leads to lateral segregation of particles away from the closely packed membrane arrays into contiguous normal, or slightly expanded, period arrays. In the particle-enriched layers, the E fracture face is more particle-dense than the P face, whereas no particles are found on either face in the compacted layers. Morphologically, compaction induced by the all-or-nothing, relatively irreversible action of specific cations cannot be distinguished from compaction to the same extent induced by the graded, reversible effects of nonelectrolytes. Compaction by sodium chloride resembles that by specific- cation binding in that the repeat period is independent of reagent concentration; but, like dehydration by nonelectrolytes, the extent of compaction is reversibly related to reagent concentration. Sodium chloride-compacted myelin can be distinguished morphologically by a lack of the elongated border particles at the boundary between smooth and particle-enriched membrane observed for other compacting treatments. Fracture faces in compacted arrays are not always smooth, but the unusual appearances can be duplicated in purified myelin lipid multilayers subjected to similar treatments, which indicates that the particle-free membrane fracture faces are uninterrupted lipid hydrocarbon layers. Correlation of x-ray diffraction and electron microscopy observations provides a direct basis for identifying the intramembrane particles with transmembrane protein. The transmembrane protein appears to play a significant role in maintaining the normal membrane separation; swelling of the particle-enriched arrays in myelin compacted by tetracaine at low ionic strength provides information about the charge distribution on the transmembrane protein. Swelling of the compacted arrays following irreversible particle segregation shows that the interaction properties of the particle-free membranes are similar to those of pure lipid multilayers. Compaction and the consequent particle segregation in lyelin results from conditions stabilizing close apposition of the lipid bilayers. Particle segregation in areas of close contact between other cell membranes may also be driven by interbilayer attractive forces.