The pine wood nematode Bursaphelenchus xylophilus is the causal agent of pine wilt disease, one of the most devastating forest diseases in East Asian and West European countries. The lifecycle of B. xylophilus includes four propagative larval stages and gonochoristic adults which are involved in the pathogenicity, and two stages of dispersal larvae involved in the spread of the disease. To elucidate the ecological roles of each developmental stage in the pathogenic life cycle, we performed a comprehensive transcriptome analysis using RNA-seq generated from all developmental stages of B. xylophilus and compared transcriptomes between stages. We found more than 9000 genes are differentially expressed in at least one stage of the life cycle including genes involved in general nematode biology such as reproduction and moulting but also effector genes likely to be involved in parasitism. The dispersal-stage transcriptome revealed its analogy to C. elegans dauer and the distinct roles of the two larval stages from each other regarding survival and transmission. This study provides important insights and resources to understand B. xylophilus parasitic biology.

The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer, J. Agric Res. 48, 1934, 949), Nickle (J. Nematol. 2, 1970, 375), is the causative agent of the pine wilt disease and causes serious damage to pine forests around the world. During a survey for the pinewood nematode, four other Bursaphelenchus species (Bursaphelenchus mucronatus, B. sexdentati, B. anamurius and B. vallesianus) were isolated from wilted pine trees in Turkey. To understand the effects of these Bursaphelenchus species on wilting of pine trees, a study was conducted under greenhouse conditions. Two-year-old seedlings of three pine species (Pinus nigra, P. brutia and P. pinea) and one cedar species (Cedrus libani) were used. Fifteen seedlings of each species were inoculated with nematodes and 10 seedlings of each species served as controls. The inoculum densities used for each seedling contained approximately 1000 (±100) nematodes of all life stages in 0.25 ml of distilled water. The first wilting symptoms were observed in the fifth week in all pine species but not in the cedar seedlings. All seeding mortality occurred between the 5th and 13th weeks of the study; no mortality was observed outside of this period. The most pathogenic nematode species was B. mucronatus, closely followed by the other species. The most susceptible seedling species was P. nigra, and C. libani was the most resistant species.

The small RNA (sRNA) pathways identified in the model organism Caenorhabditis elegans are not widely conserved across nematodes. For example, the PIWI pathway and PIWI-interacting RNAs (piRNAs) are involved in regulating and silencing transposable elements (TE) in most animals but have been lost in nematodes outside of the C. elegans group (Clade V), and little is known about how nematodes regulate TEs in the absence of the PIWI pathway. Here, we investigated the role of sRNAs in the Clade IV parasitic nematode Strongyloides ratti by comparing two genetically identical adult stages (the parasitic female and free-living female). We identified putative small-interfering RNAs, microRNAs and tRNA-derived sRNA fragments that are differentially expressed between the two adult stages. Two classes of sRNAs were predicted to regulate TE activity including (i) a parasite-associated class of 21–22 nt long sRNAs with a 5′ uridine (21-22Us) and a 5′ monophosphate, and (ii) 27 nt long sRNAs with a 5′ guanine/adenine (27GAs) and a 5′ modification. The 21-22Us show striking resemblance to the 21U PIWI-interacting RNAs found in C. elegans, including an AT rich upstream sequence, overlapping loci and physical clustering in the genome. Overall, we have shown that an alternative class of sRNAs compensate for the loss of piRNAs and regulate TE activity in nematodes outside of Clade V.