The effects of plant cysteine proteinases on the nematode cuticle

Background Plant-derived cysteine proteinases of the papain family (CPs) attack nematodes by digesting the cuticle, leading to rupture and death of the worm. The nematode cuticle is composed of collagens and cuticlins, but the specific molecular target(s) for the proteinases have yet to be identified. Methods This study followed the course of nematode cuticle disruption using immunohistochemistry, scanning electron microscopy and proteomics, using a free-living nematode, Caenorhabditis elegans and the murine GI nematode Heligmosomoides bakeri (H. polygyrus) as target organisms. Results Immunohistochemistry indicated that DPY-7 collagen is a target for CPs on the cuticle of C. elegans. The time course of loss of DPY-7 from the cuticle allowed us to use it to visualise the process of cuticle disruption. There was a marked difference in the time course of damage to the cuticles of the two species of nematode, with H. bakeri being more rapidly hydrolysed. In general, the CPs’ mode of attack on the nematode cuticle was by degrading the structural proteins, leading to loss of integrity of the cuticle, and finally death of the nematode. Proteomic analysis failed conclusively to identify structural targets for CPs, but preliminary data suggested that COL-87 and CUT-19 may be important targets for the CPs, the digestion of which may contribute to cuticle disruption and death of the worm. Cuticle globin was also identified as a cuticular target. The presence of more than one target protein may slow the development of resistance against this new class of anthelmintic. Conclusions Scanning electron microscopy and immunohistochemistry allowed the process of disruption of the cuticle to be followed with time. Cuticle collagens and cuticlins are molecular targets for plant cysteine proteinases. However, the presence of tyrosine cross-links in nematode cuticle proteins seriously impeded protein identification by proteomic analyses. Multiple cuticle targets exist, probably making resistance to this new anthelmintic slow to develop. Graphic Abstract

Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes

Background The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. Results 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45–11.5 ng/μL, and on Qubit™ ranged between undetectable – 0.962 ng/μL. Median A260/280 ranged between 0.505–3.925, and median A260/230 ranged − 0.005 – 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. Conclusions A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

Identification and Nematicidal Characterization of Proteases Secreted by Endophytic Bacteria Bacillus cereus BCM2

The endophytic bacterium Bacillus cereus BCM2 has shown great potential as a biocontrol organism against Meloidogyne incognita, which causes severe root-knot diseases in crops. In our previous study, the metabolite of BCM2 showed high nematicidal activity against the M. incognita second-stage juveniles. However, the mechanism employed by endophytic bacteria to infect and kill nematodes is still unclear. Here, we investigate both the endophytic bacterial extracellular proteins with nematicidal activity and their mechanism of killing nematodes. The first step was detecting the nematicidal activities of crude proteins. The results show that the nematode mortality rate reached 100% within 72 h, and the crude proteins damaged both the cuticle and eggshell, before finally destroying the targets. This suggests possible proteinaceous pathogeny in BCM2. Throughout the process, the fine-detail changes in the nematode cuticle and the intestinal structure were observed using scanning electron microscopy and transmission electron microscopy. These images show that BCM2 extracellular proteins did not damage the internal organization of the nematode but did severely damage its cuticle, which led to content leakage. From the crude proteins, chitosanase, alkaline serine protease, and neutral protease were purified and identified. The M. incognita–B. cereus BCM2 microenvironment simulation demonstrates that BCM2 adheres to the surface of nematodes and helps the metabolites that were produced by BCM2 to rapidly recognize and kill M. incognita. This relationship between plants, endophytic bacteria, and nematodes offers insight into the biological mechanisms that can be utilized for of nematode management.

Fabrication of sharp silicon arrays to wound Caenorhabditis elegans

ABSTRACTUnderstanding how animals respond to injury and how wounds heal remains a challenge. These questions can be addressed using genetically tractable animals, including the nematode Caenorhabditis elegans. Given its small size, the current methods for inflicting wounds in a controlled manner are demanding. To facilitate and accelerate the procedure, we fabricated regular arrays of pyramidal features (“pins”) sharp enough to pierce the tough nematode cuticle. The pyramids were made from monocrystalline silicon wafers that were micro-structured using optical lithography and alkaline wet etching. The fabrication protocol and the geometry of the pins, determined by electron microscopy, are described in detail. Upon wounding, C. elegans expresses genes encoding antimicrobial peptides. A comparison of the induction of antimicrobial peptide gene expression using traditional needles and the pin arrays demonstrates the utility of this new method.

Nematodes as evolutionary commuters between marine, freshwater and terrestrial habitats

Nematodes are the only major metazoan group which is persistently abundant and diverse across marine, freshwater and terrestrial ecosystems. This could be the result of a few major habitat transitions followed by extensive diversification, or numerous habitat transitions followed by moderate diversification. To pinpoint habitat transitions, we superposed nematode habitat associations on an available phylum-wide phylogenetic tree based on small subunit ribosomal DNA sequences (≈2730 SSU rDNA sequences covering ≈1750 nematode taxa). Our analysis revealed at least 30 major habitat transitions within the phylum Nematoda. These transitions as well as their directionality were unevenly spread over the 12 major clades. Most transitions reside in Clades 1–6, and these transitions are bidirectional. Members of Clades 8–12 showed five full transitions, and these took place exclusively from terrestrial to marine systems. We relate our results to the distinct secretory–excretory systems in Clades 1–6 and Clades 8–12, as well as to differences in water permeability of the nematode cuticle. Hence, the phylum Nematoda is characterized by a relatively large number of habitat transitions followed by moderate diversification. The identification of multiple habitat transitions at a low taxonomic level will facilitate future investigations into the mechanisms underlying this unusual ecological flexibility.

Sexual dimorphism of the cuticle and body-wall muscle in free-living mycophagous nematodes

Sexual dimorphism in motility-related traits is widespread among animals, including several species of Nematoda. However, no study has examined motility-related structural components and compared them between sexes. We examined the motility-related components in four species: Bursaphelenchus conicaudatus Kanzaki, Tsuda and Futai, 2000; Bursaphelenchus rainulfi Braasch and Burgermeister, 2002; Bursaphelenchus doui Braasch, Gu, Burgermeister and Zhang, 2005; Parasitaphelenchus costati Kanzaki, Ekino, Ide, Masuya and Degawa, 2018. We measured the structure and amount of cuticle and body-wall muscles and estimated their relationship to body diameter or total cross-sectional area. Although no structural differences were observed in muscle, the relevant muscle area of B. doui and P. costati was significantly smaller in females than in males. This difference was greatest in P. costati. In all but B. doui, the relative cuticle thickness was significantly smaller in females than in males. Furthermore, only P. costati females had no striated basal zones in their cuticles; these are thought to be cross-linked proteins that provide strength to nematode cuticle during body movement. These results indicate that sexual dimorphism in motility-related structural components is present in P. costati and that females invest less energy in the components than do males.

Signal pathways involved in microbe–nematode interactions provide new insights into the biocontrol of plant-parasitic nematodes

Plant-parasitic nematodes (PPNs) cause severe damage to agricultural crops worldwide. As most chemical nematicides have negative environmental side effects, there is a pressing need for developing efficient biocontrol methods. Nematophagous microbes, the natural enemies of nematodes, are potential biocontrol agents against PPNs. These natural enemies include both bacteria and fungi and they use diverse methods to infect and kill nematodes. For instance, nematode-trapping fungi can sense host signals and produce special trapping devices to capture nematodes, whereas endo-parasitic fungi can kill nematodes by spore adhesion and invasive growth to break the nematode cuticle. By contrast, nematophagous bacteria can secrete virulence factors to kill nematodes. In addition, some bacteria can mobilize nematode-trapping fungi to kill nematodes. In response, nematodes can also sense and defend against the microbial pathogens using strategies such as producing anti-microbial peptides regulated by the innate immunity system. Recent progresses in our understanding of the signal pathways involved in microbe–nematode interactions are providing new insights in developing efficient biological control strategies against PPNs. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management'.

Substrate-induced changes in dynamics and molecular motions of cuticle-degrading serine protease PL646: a molecular dynamics study

Cuticle-degrading serine proteases secreted by nematophagous fungi can degrade the nematode cuticle during the infection processes.

Development of a transformation system for Hirsutella spp. and visualization of the mode of nematode infection by GFP-labeled H. minnesotensis

Hirsutella rhossiliensis and H. minnesotensis are endoparasitic fungi of the second-stage juvenile (J2) of the soybean cyst nematode (Heterodera glycines) in nature. They also parasitize both H. glycines J2 and Caenorhabditis elegans on agar plates. Agrobacterium tumefaciens-mediated transformation conditions were established for these Hirsutella spp. The resulting transformants were similar to the corresponding wild-type strains. The infection processes of H. glycines J2 and C. elegans second larval stage (L2) by H. minnesotensis expressing ZsGreen were microscopically analyzed. Conidia of H. minnesotensis adhered to passing nematodes within 8 h post-inoculation (hpi), formed an infection peg between 8 and 12 hpi and penetrated the nematode cuticle between 12 and 24 hpi for C. elegans L2 and between 12 and 32 hpi for H. glycines J2. Hyphal proliferation inside of the nematode coelom was observed at approximately 32 hpi for C. elegans L2 and at approximately 40 hpi for H. glycines J2. The fungus consumed the whole body and grew out to produce conidia at approximately 156 and 204 hpi for C. elegans L2 and H. glycines J2, respectively. The efficient transformation protocol and a better understanding of infection process provide a solid foundation for studying the molecular and cellular mechanisms underlying fungal parasitism of nematodes.