Observation and Quantification of Mating Behavior in the Pinewood Nematode, <em>Bursaphelenchus xylophilus</em>

Abstract

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 °C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 ±3.91 min (mean ±SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 ± 2.0, 28.0 ± 1.9, 25.8 ± 0.7 and 7.2 ± 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Bxy‐fuca encoding α‐L‐fucosidase plays crucial roles in development and reproduction of the pathogenic pinewood nematode, Bursaphelenchus xylophilus

“…The synchronized J2 were collected and transferred onto a lawn of B. cinerea on a PDA plate. Worms were collected after 24, 48 and 72 h for J3, J4 and adult animals respectively …”

An oleaginous yeast platform for renewable 1-butanol synthesis based on a heterologous CoA-dependent pathway and an endogenous pathway

“…As a result, we successfully deleted ten genes in parallel (except for YALI0A16379g) individually by using the disruption cassettes with long homologous sequences in the Y. lipolytica strain Po1g KU70 Δ. Details of gene deletion experiments and results have already been described in our previous paper [ 20 ]. After confirming that the targeted gene had been deleted, the knockout mutant strains were then tested for 1-butanol production by using GC/MS.…”

“…5 ). A detailed procedure for the gene deletion in Y. lipolytica was already described in our previous paper [ 20 ]. In order to clone those endogenous genes into Y. lipolytica expression vector pYLEX1, another pYLEX1 derivative pYLEX1-NEW was created by adding Xba I, Avr II, Mfe I and Mlu I sites into the Bam HI site of pYLEX1 (Additional file 1 : Figure S6).…”

“…For the heterologous CoA-dependent pathway, the following engineered Y. lipolytica strains were generated: (a) Po1g::pYLEX1 (used as a negative control); (b) Po1g::pYLEX1-EuET; (c) Po1g::pYLEX1-All; (d) Po1g::pYLEX1-All-GPD; and (e) Po1g::pYLEX1-All-MDH. For the endogenous pathway, ten Y. lipolytica single-gene deletion mutants were generated [ 20 ]. Sixteen genes in the twelve steps of the proposed endogenous 1-butanol pathway were selected for overexpression and thus sixteen gene-overexpression strains were generated accordingly.…”

Annulohypoxylon sp. FPYF3050 produces volatile organic compounds against the pine wood nematode, Bursaphelenchus xylophilus

“…After carefully removing the water and worms, the eggs were rinsed repeatedly with sterile water and transferred to a centrifuge tube to obtain the pure eggs. To obtain synchronous development of J2, the collected eggs were kept in sterile water in an incubator at 25°C for 24 h (Shinya et al, 2009;Zhu et al, 2016).…”