The nervous system of plant-parasitic nematodes and their behaviors

Nematodes are the most abundant animals on earth. The majority of nematodes are free-living which feed on bacteria and fungi in the soil. However, some nematodes are parasitic to other animals and plants and have a huge impact on human health and agriculture worldwide. The nervous system of the non-parasitic nematode Caenorhabditis elegans has been studied extensively. Based on C. elegans and a few other species, nematode nervous systems were thought to be highly conserved. However, using a comparative neuroanatomy approach, I found unexpected variation in the number and structural properties of neurons among ten species across four clades. To further study the nervous system of plant-parasitic nematodes, I investigated how the neurotransmitter serotonin regulates their behaviors. Neurotransmitters are endogenous molecules used by neurons for signal transmission. Serotonin regulates feeding and reproductive behaviors in C. elegans, but its role in plant-parasitic nematodes remains unknown. In the root-lesion nematode Pratylenchus penetrans, I detected serotonergic neurons in cells adjacent to feeding and reproductive structures. I observed that exogenous serotonin induced P. penetrans feeding and reproductive behaviors. Also, using pharmaceutical compounds that disrupt serotonin signaling, my data suggest that these neurons regulate feeding and reproductive behaviors through endogenous serotonin. The soybean cyst nematode Heterodera glycines has a distinct life cycle: both juvenile males and females lose their mobility when feeding is initiated; however, the adult males regain their mobility while females never regain mobility. As a part of a collaborative study with other lab members, I have studied the change in the mobility of H. glycines from a neuronal aspect. GABA is the most prominent inhibitory neurotransmitter in nematodes. I have constructed a map of GABAergic neurons and cloned the gene encoding the key enzyme in GABA synthesis (hg-unc-25) in H. glycines. I have used heterologous rescue in a Caenorhabditis elegans mutant and validated that HG-UNC-25 is indeed the GABA synthesizing enzyme in H. glycines. Also, I have found the sedentary stages of H. glycines is associated with a reduction of GABAergic neurons in the ventral nerve cord. Together, my research has provided evidence that the nervous systems of nematodes are not as conserved as we thought and there is a need to further investigate the nervous system. A better understanding of the nervous system of plant-parasitic nematodes may be important to understand the evolution of these parasitic nematodes. More importantly, the knowledge of the how the nervous system regulates the specific behaviors of plant-parasitic nematodes may provide insights into new control strategies

Assessing the damage potential of Pratylenchus penetrans on red raspberry cultivars

Washington State is the nation's largest producer of red raspberries for processing. The root lesion nematode, Pratylenchus penetrans, is a major constraint to the industry, shortening the productive lifespan of many plantings. Current management of P. penetrans relies upon preplant soil fumigation, but federal regulations of soil fumigants have made this practice increasingly difficult and expensive for growers. In addition, few post-plant nematode management options exist. A better understanding of the damage potential of this nematode to raspberry is necessary to guide future management recommendations. The objective of this project was to determine whether several popular raspberry cultivars in Washington State, Cascade Bounty, Chemainus, Meeker and Saanich, differ in susceptibility to P. penetrans. Field trials in existing plantings of these cultivars were established in northwest Washington. Treated plots were sprayed with the nematicides oxamyl and fosthiazate in the spring and fall of 2011 and 2012, while non-treated plots received no nematicides. Pratylenchus penetrans population densities in soil and root samples were assessed before and after each nematicide application. Pratylenchus penetrans root population densities in nematicide-treated plots were consistently v lower than those in non-treated plots at all the samplings. However, P. penetrans soil population densities in non-treated plots varied during the two year study. In summer 2011, yield of raspberry fruit in nematicide-treated plots was not significantly greater than in non-treated plots in any of the trials. In summer 2012, treated plots in the 'Chemainus' and 'Meeker' 1 trials had reduced yields along with increased fine root biomass compared to non-treated plots. In addition, in a microplot study evaluating the damage potential for P. penetrans to newly established 'Meeker' plants in a local silt loam soil and a sandy loam soil, P. penetrans had more severe damage potential to raspberry plant growth in the sandy loam soil than in the silt loam soil, but populations of P. penetrans increased faster in the silt loam soil than the sandy loam soil

GABA Immunoreactivity and Pharmacological Effects vary Among Stylet-Bearing Nematodes

Plant-parasitic nematodes conduct a series of sophisticated behaviors to complete their life cycles. Among these, locomotion behaviors, including finding the host and migrating to the feeding site, directly affect the success of parasitism. Thus, disrupting locomotion behaviors has the potential to control these parasites. γ-Aminobutyric acid (GABA) is the prominent inhibitory neurotransmitter in nematodes. GABA-immunoreactive neurons are mostly found in motor neurons, where they regulate behaviors in the model nematode C. elegans. However, the GABA system in most stylet-bearing nematodes has received little attention. Using immunohistochemistry, we found variation in the pattern of GABA-immunoreactivity among two major plant-parasites and a fungal feeder. Some of these GABA-immunoreactive neurons lack clear homologs to C. elegans. Pharmaceutical assays showed that applying GABA, its agonist, and its antagonist, can disrupt the locomotion behaviors of these nematodes, although sensitivity to a given compound varied between species. Our data suggest that the GABA system is a potential target for the control of plant-parasitic nematodes

Microbiota succession influences nematode physiology in a beetle microcosm ecosystem

Abstract Unravelling the multifaceted and bidirectional interactions between microbiota and host physiology represents a major scientific challenge. Here, we utilise the nematode model, Pristionchus pacificus, coupled to a laboratory-simulated decay process of its insect host, to mimic natural microbiota succession and investigate associated tripartite interactions. Metagenomics reveal that during initial decay stages, the population of vitamin B-producing bacteria diminishes, potentially due to a preferential selection by nematodes. As decay progresses to nutrient-depleted stages, bacteria with smaller genomes producing less nutrients become more prevalent. Lipid utilisation and dauer formation, representing key nematode survival strategies, are influenced by microbiota changes. Additionally, horizontally acquired cellulases extend the nematodes’ reproductive phase due to more efficient foraging. Lastly, the expressions of Pristionchus species-specific genes are more responsive to natural microbiota compared to conserved genes, suggesting their importance in the organisms’ adaptation to its ecological niche. In summary, we show the importance of microbial successions and their reciprocal interaction with nematodes for insect decay in semi-artificial ecosystems

Complete genome sequencing of nematode Aphelenchoides besseyi, an economically important pest causing rice white-tip disease

Abstract Aphelenchoides besseyi is a seed-borne plant-parasitic nematode that causes severe rice yield losses worldwide. In the present study, the A. besseyi Anhui-1 strain isolated from rice in China was sequenced with a hybrid method combining PacBio long reads and Illumina short reads, and subsequently annotated using available transcriptome references. The genome assembly consists of 166 scaffolds totaling 50.3 Mb, with an N50 of 1.262 Mb and a maximum scaffold length of 9.17 Mb. A total of 16,343 genes were annotated in the genome, with 94 gene families expanded while 70 families contracted specifically in A. besseyi. Furthermore, gene function analysis demonstrated that the genes related to drought tolerance were enriched, and cellulase genes were horizontally acquired from eukaryotic origin. Our findings provide resources to interpret the biology, evolution, ecology, and functional diversities of Aphelenchoides spp. in the light of genomics

Improving Transgenesis Efficiency and CRISPR-Associated Tools Through Codon Optimization and Native Intron Addition in Pristionchus Nematodes

A lack of appropriate molecular tools is one obstacle that prevents in-depth mechanistic studies in many organisms. Transgenesis, clustered regularly interspaced short palindromic repeats (CRISPR)-associated engineering, and related tools are fundamental in the modern life sciences, but their applications are still limited to a few model organisms. In the phylum Nematoda, transgenesis can only be performed in a handful of species other than Caenorhabditis elegans, and additionally, other species suffer from significantly lower transgenesis efficiencies. We hypothesized that this may in part be due to incompatibilities of transgenes in the recipient organisms. Therefore, we investigated the genomic features of 10 nematode species from three of the major clades representing all different lifestyles. We found that these species show drastically different codon usage bias and intron composition. With these findings, we used the species Pristionchus pacificus as a proof of concept for codon optimization and native intron addition. Indeed, we were able to significantly improve transgenesis efficiency, a principle that may be usable in other nematode species. In addition, with the improved transgenes, we developed a fluorescent co-injection marker in P. pacificus for the detection of CRISPR-edited individuals, which helps considerably to reduce associated time and costs

Immobility in the sedentary plant-parasitic nematode H. glycines is associated with remodeling of neuromuscular tissue.

The sedentary plant-parasitic nematodes are considered among the most economically damaging pathogens of plants. Following infection and the establishment of a feeding site, sedentary nematodes become immobile. Loss of mobility is reversed in adult males while females never regain mobility. The structural basis for this change in mobility is unknown. We used a combination of light and transmission electron microscopy to demonstrate cell-specific muscle atrophy and sex-specific renewal of neuromuscular tissue in the sedentary nematode Heterodera glycines. We found that both females and males undergo body wall muscle atrophy and loss of attachment to the underlying cuticle during immobile developmental stages. Male H. glycines undergo somatic muscle renewal prior to molting into a mobile adult. In addition, we found developmental changes to the organization and number of motor neurons in the ventral nerve cord correlated with changes in mobility. To further examine neuronal changes associated with immobility, we used a combination of immunohistochemistry and molecular biology to characterize the GABAergic nervous system of H. glycines during mobile and immobile stages. We cloned and confirmed the function of the putative H. glycines GABA synthesis-encoding gene hg-unc-25 using heterologous rescue in C. elegans. We found a reduction in gene expression of hg-unc-25 as well as a reduction in the number of GABA-immunoreactive neurons during immobile developmental stages. Finally, we found evidence of similar muscle atrophy in the phylogenetically diverged plant-parasitic nematode Meloidogyne incognita. Together, our data demonstrate remodeling of neuromuscular structure and function during sedentary plant-parasitic nematode development

Crowdsourcing and the feasibility of manual gene annotation: A pilot study in the nematode Pristionchus pacificus

Nematodes such as Caenorhabditis elegans are powerful systems to study basically all aspects of biology. Their species richness together with tremendous genetic knowledge from C. elegans facilitate the evolutionary study of biological functions using reverse genetics. However, the ability to identify orthologs of candidate genes in other species can be hampered by erroneous gene annotations. To improve gene annotation in the nematode model organism Pristionchus pacificus, we performed a genome-wide screen for C. elegans genes with potentially incorrectly annotated P. pacificus orthologs. We initiated a community-based project to manually inspect more than two thousand candidate loci and to propose new gene models based on recently generated Iso-seq and RNA-seq data. In most cases, misannotation of C. elegans orthologs was due to artificially fused gene predictions and completely missing gene models. The community-based curation raised the gene count from 25,517 to 28,036 and increased the single copy ortholog completeness level from 86% to 97%. This pilot study demonstrates how even small-scale crowdsourcing can drastically improve gene annotations. In future, similar approaches can be used for other species, gene sets, and even larger communities thus making manual annotation of large parts of the genome feasible