Bxy-glp-1 gene in the Notch pathway regulates the reproductive development of the pine wood nematode

Abstract

Studying the reproductive system of the pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer) Nickle, will identify its characteristics and life cycle. This is crucial for developing more targeted control strategies. In this study, the development of the gonads and reproductive organs were observed using microscopy, gonad dissection, and DAPI staining techniques. Second-stage juveniles (J2) had gonads composed of four primordial germ cells (Z1, Z2, Z3, and Z4) that form the adult gonads by proliferation within 72 h at 25 °C. There were subtle differences in somatic gonad cell morphology between males and females, which developed from Z1 and Z4 in third-stage juveniles (J3). These differences became more pronounced at fourth-stage juveniles (J4) and adult stages. Z2 and Z3 germ cells underwent mitosis and two rounds of meiosis, ultimately developing into male and female gametes. Female vulval precursor cells and male cloacal cells developed rapidly during the J4 stage. These results provide a basis for identifying the expression sites and biological functions of key genes regulating reproductive system development. Based on this, in situ hybridization and RNA interference (RNAi) were used to determine the function of the Bxy-glp-1 gene to show that it is involved in vulval formation and spermatogenesis. The results of this study will lay the foundation for disrupting critical stages in the reproduction of B. xylophilus.

Keywords

Bursaphelenchus xylophilus / Reproductive system / Gonad / Notch / Bxy-glp-1

References

[1] AgarwalP, ShemeshT, Zaidel-BarR. Directed cell invasion and asymmetric adhesion drive tissue elongation and turning in C. elegans gonad morphogenesis. Dev Cell, 2022, 57(17): 2111-2126

[2] AnanthaswamyD, CroftJC, WoozencroftN, LeeTW. C. elegans gonad dissection and freeze crack for immunofluorescence and DAPI staining. JoVE, 2022, 187: e64204

[3] AoyagiM, IshibashiN. Gametogenesis of the pine wood nematode, Bursaphelenchusxylophilus. Jpn J Nematol, 1983, 13: 20-25

[4] Artavanis-TsakonasS, MuskavitchMAT. Notch: the past, the present, and the future. Curr Top Dev Biol, 2010, 92: 1-29

[5] CrittendenSL, TroemelER, EvansTC, KimbleJ. GLP-1 is localized to the mitotic region of the C. elegans germ line. Development, 1994, 120(10): 2901-2911

[6] de la CovaCC, TownleyR, GreenwaldI. Negative feedback by conserved kinases patterns the degradation of Caenorhabditiselegans Raf in vulval fate patterning. Development, 2020, 147(24): dev195941

[7] FélixMA, BarkoulasM. Robustness and flexibility in nematode vulva development. Trends Genet, 2012, 28(4): 185-195

[8] GrayA, DullTJ, UllrichA. Nucleotide sequence of epidermal growth factor cDNA predicts a 128, 000-molecular weight protein precursor. Nature, 1983, 303(5919): 722-725

[9] HasegawaK, MotaMM, FutaiK, MiwaJ. Chromosome structure and behaviour in Bursaphelenchusxylophilus (Nematoda: Parasitaphelenchidae) germ cells and early embryo. Nematology, 2006, 8(3): 425-434

[10] HirschmannH, SasserJ. On the occurrence of an intersexual form in Ditylenchustriformis, n. sp.(Nematoda, Tylenchida). Proc Helminthol, 1955, 22(2): 115-123

[11] HirshD, OppenheimD, KlassM. Development of the reproductive system of Caenorhabditiselegans. Dev Biol, 1976, 49(1): 200-219

[12] IshibashiN, AoyagiM, KondoE. Comparison of the gonad development between the propagative and dispersal forms of pine wood nematode, Bursaphelenchuslignicolus (Aphelenchoididae). Jpn J Nematol, 1978, 8: 28-31

[13] KikuchiT, CottonJA, DalzellJJ, HasegawaK, KanzakiN, McVeighP, TakanashiT, TsaiIJ, AssefaSA, CockPJA, OttoTD, HuntM, ReidAJ, Sanchez-FloresA, TsuchiharaK, YokoiT, LarssonMC, MiwaJ, MauleAG, SahashiN, JonesJT, BerrimanM. Genomic insights into the origin of parasitism in the emerging plant pathogen Bursaphelenchusxylophilus. PLoS Pathog, 2011, 7(9): e1002219

[14] KimbleJ, CrittendenSL. Controls of germline stem cells, entry into meiosis, and the sperm/oocyte decision in Caenorhabditiselegans. Annu Rev Cell Dev Biol, 2007, 23: 405-433

[15] KimbleJ, HirshD. The postembryonic cell lineages of the hermaphrodite and male gonads in Caenorhabditiselegans. Dev Biol, 1979, 70(2): 396-417

[16] KimbleJE, WhiteJG. On the control of germ cell development in Caenorhabditiselegans. Dev Biol, 1981, 81(2): 208-219

[17] KodoyianniV, MaineEM, KimbleJ. Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditiselegans. Mol Biol Cell, 1992, 3(11): 1199-1213

[18] Kramer-DraubergM, LiuJL, DesjardinsD, WangY, BranickyR, HekimiS. ROS regulation of RAS and vulva development in Caenorhabditiselegans. PLoS Genet, 2020, 16(6): e1008838

[19] LehmannR, JiménezF, DietrichU, Campos-OrtegaJA. On the phenotype and development of mutants of early neurogenesis in DrosophilaMelanogaster. Wilhelm Roux’s Arch Dev Biol, 1983, 192(2): 62-74

[20] LivakKJ, SchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) method. Methods, 2001, 25(4): 402-408

[21] MahadikSS, BurtEK, LundquistEA. SRC-1 controls growth cone polarity and protrusion with the UNC-6/Netrin receptor UNC-5 in Caenorhabditiselegans. PLoS ONE, 2024, 19(5): e0295701

[22] MamiyaY. The life history of the pine wood nematode, Bursaphelenchuslignicolus. Jpn J Nematol, 1975, 5: 16-25

[23] MichaelyP, BennettV. The ANK repeat: a ubiquitous motif involved in macromolecular recognition. Trends Cell Biol, 1992, 2(5): 127-129

[24] PimentelCS, FirminoPN, AyresMP. Comparison of methods to obtain and maintain cultures of the pinewood nematode Bursaphelenchusxylophilus. J Res, 2020, 25(2): 101-107

[25] QiuXW, YangLL, YeJR, WangW, ZhaoTT, HuH, ZhouGX. Silencing of cyp-33C9 gene affects the reproduction and pathogenicity of the pine wood nematode Bursaphelenchusxylophilus. Int J Mol Sci, 2019, 20(18): 4520

[26] RigsbyRE, ParkerAB. Using the PyMOL application to reinforce visual understanding of protein structure. Biochem Mol Biol Educ, 2016, 44(5): 433-437

[27] RudelD, KimbleJ. Conservation of glp-1 regulation and function in nematodes. Genetics, 2001, 157(2): 639-654

[28] SommerRJ, BumbargerDJ. Nematode model systems in evolution and development. Wires Dev Biol, 2012, 1(3): 389-400

[29] SulstonJE, AlbertsonDG, ThomsonJN. The Caenorhabditiselegans male: postembryonic development of nongonadal structures. Dev Biol, 1980, 78(2): 542-576

[30] SulstonJE, HorvitzHR. Post-embryonic cell lineages of the nematode. Caenorhabditis Elegans Dev Biol, 1977, 56(1): 110-156

[31] ViglierchioDR, SchmittRV. On the methodology of nematode extraction from field samples: comparison of methods for soil extraction. J Nematol, 1983, 15(3): 450-454

[32] VlachakisD, PapageorgiouL, PapadakiA, GeorgaM, KossidaS, EliopoulosE. An updated evolutionary study of the Notch family reveals a new ancient origin and novel invariable motifs as potential pharmacological targets. PeerJ, 2020, 8: e10334

[33] WangY, SuzukiK, SakaueD, YamadaT. Variations in life history parameters and their influence on rate of population increase of different pathogenic isolates of the pine wood nematode Bursaphelenchusxylophilus. Nematology, 2005, 7(3): 459-467

[34] YushinV, RyssA. Sperm development and structure in Bursaphelenchusmucronatus (Nematoda: Aphelenchoidea: Aphelenchoididae). Nematology, 2011, 13(4): 395-407

[35] ZhaoLL, ZhangXX, WeiYN, ZhouJ, ZhangW, QinPJ, ChintaS, KongXB, LiuYP, YuHY, HuSN, ZouZ, ButcherRA, SunJH. Ascarosides coordinate the dispersal of a plant-parasitic nematode with the metamorphosis of its vector beetle. Nat Commun, 2016, 7: 12341

[36] ZhouLF, ChenFM, YeJR, PanHY. Selection of reliable reference genes for RT-qPCR analysis of Bursaphelenchusmucronatus gene expression from different habitats and developmental stages. Front Genet, 2018, 9: 269

[37] ZhouLF, LiuWY, BaiLQ, LiuHL, WangJH, MaXX, HuangLJ, GuoK, YuHS, HuJF. Female’s war: a story of the invasion and competitive displacement between two xylophilus group nematode species. J Pest Sci, 2023, 96(3): 1301-1311

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Northeast Forestry University