A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.
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.
The pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, is one of the most destructive invasive species, causing the wilting and eventual death of pine trees. This pathogenic nematode is believed to be native to the USA, but it was transported to several countries, including China, Japan, South Korea, and Portugal. Recently, it has also been reported in Spain, killing millions of pine trees at huge economic losses and threatening forest production and ecological stability1-5.
Once a host pine tree is infected by pinewood nematodes, thousands of millions of offspring are propagated rapidly within the trunk. This causes xylem dysfunction, resulting in wilting and eventually in the death of the host tree6. However, there is currently no efficient way to control this disease. Mating behavior might play an important role in the high fecundity of this nematode7. We therefore investigated the mating behavior of B. xylophilus in the laboratory, trying to find an effective way of disturbing its mating and decreasing its fecundity.
This protocol is intended to introduce the detailed methods of how to obtain the virgin adults of B. xylophilus and how to observe and analyze the mating behavior using a video recorder and a stereomicroscope. This protocol can also be used as a reference for behavioral studies of other nematodes.
1. Acquisition of Virgin Adult Nematodes
2. Observation and Video Recording of Mating Behavior
3. Data Collection
The mating success rate (R) of virgin adults was 86.67% on average, which was significantly higher than that of the randomly selected adults: 13.33 ± 4.65% (F = 1301.71, df = 1, P = 0.0001) (Figure 2). This finding suggests that it is easier to observe the mating behavior of B. xylophilus with virgin adults than with randomly selected adults.
When one male was paired with one female, the entire mating process lasted 82.8 min on average. The whole process could be described by four different phases: searching, contacting, copulating, and lingering. The duration for these four phases were 21.8 ± 2.0 min, 28.0 ± 1.9 min, 25.8 ± 0.7 min, and 7.2 ± 0.5 min, respectively (Figure 3). In a recent publication, we described the 11 sub-behaviors as cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming9.
When one male met two or more females simultaneously, the male seemed to mate randomly with one of them, without any selection. However, obvious mating choice was observed for female B. xylophilus. When a female contacted a male, the female tended to touch the male's head, body, tail, and genital region gently with her head and appeared to be evaluating her potential partner. If the female did not accept the male, the female left him quickly and swam to another male for the next evaluation. One of the males was then selected by the female for mating. Mating success rates after the first contact (P) of the 1♀ + 1♂, 3♀ + 1♂, and 1♀ + 3♂ combinations were 46.7 ± 9.3%, 80.0 ± 7.4%, and 23.4 ± 7.4%, respectively, and the differences between them were significant (F = 8.21, df = 2, P = 0.0124) (Figure 4).
Intrasexual competition was observed in both males and females. When one virgin female was mixed with 3 virgin males, the males tried to approach and contact the female as much as possible to get the chance of mating with her. When the vulva was occupied by one male, the other males tried to disturb the mating male by using violent body swings or dragging to separate them. Female intrasexual competition was seen to be more or less similar to male intrasexual competition.
Mating success mainly depended on the female. When a female accepted a male, the male moved more slowly and curled or even formed a hoop, and the female moved forward actively to make it easier for the male to locate her vulva and to avoid interference from other males. If a female did not accept a male, the female tried to shake him off, even if the male had mounted and positioned himself by her vulva. Because of the intrasexual competition, copulation frequencies of the 1♀ + 1♂, 3♀ + 1♂, and 1♀ + 3♂ combinations were 1.43 ± 0.091, 1.17 ± 0.074 and 1.83 ± 0.074, respectively, and the differences between them were significant (F = 14.50, df = 2, P = 0.0001) (Figure 5).
In our investigation, the copulation duration varied greatly, with a minimum of 20 s and a maximum of 42 min. According to our data, the duration of copulation could be obviously differentiated into two groups: short-term copulations, which lasted less than 5 min, and long-term copulations, which lasted longer than 10 min. Obvious differences were observed between the different groups for the average duration of short-term copulation (F = 3.81, df = 2, P = 0.0296), long-term copulation (F = 179.52, df = 2, P = 0.0001), and all copulations (F = 2.93, df = 2, P = 0.0367) (Figure 6).
Figure 1: Genitalia of Male and Female B. Xylophilus. (a) spicule of male (blue arrow). (b) vulva of female (red arrow). Please click here to view a larger version of this figure.
Figure 2: Mating Success Rate of Virgin Adult Couples and Randomly Selected Adult Couples of B. xylophilus. The calculated mean values are from 30 replicates. The vertical bar shows the standard error. The different lowercase letters indicate significant differences at P <0.05 when compared by Tukey's test. Please click here to view a larger version of this figure.
Figure 3: Duration of the 4 Phases in the Mating Process of B. xylophilus. The calculated mean values are from 30 replicates. The vertical bar shows the standard error. The different lowercase letters indicate significant differences at P <0.05 when compared by Tukey's test. The average duration of the copulating phase is 25.80 ± 0.71 min, which is 31.17% of the whole mating process. Please click here to view a larger version of this figure.
Figure 4: Mating Success for the First Contact of B. xylophilus. The calculated mean values are from 30 replicates ±SE. Means with the same lowercase letter within each column are not significantly different at P <0.05 when compared by Tukey's Honest Significant Difference test. Please click here to view a larger version of this figure.
Figure 5: Copulation Frequency of B. xylophilus in Different Groups. The calculated mean values are from 30 replicates ±SE. Means with the same lowercase letter are not significantly different at P <0.05 when compared by Tukey's Honest Significant Difference test. Please click here to view a larger version of this figure.
Figure 6: Copulation Duration of B. xylophilus. Means with the same lowercase letter within each set of columns are not significantly different at P <0.05 when compared by Tukey's Honest Significant Difference test. Please click here to view a larger version of this figure.
Behavioral tests are a fundamental aspect of chemical ecology, neurobiology, molecular biology, and genetics. Nematodes, and particularly C. elegans, are extensively used for studies in these areas. The mating behavior of C. elegans has been investigated previously10-11. However, B. xylophilus is gonochoristic and different from the hermaphroditic C. elegans, and its mating process has different behavioral features9. Recently, the mating behavior of Bursaphelenchus okinawaensis, a hermaphroditic sister species of B. xylophilus, was reported12. Though different experimental methods were used, this hermaphroditic species showed similar sex attraction to B. xylophilus, and inter-species attraction between them was observed. This hermaphroditic sister species might be used as a model to conduct further investigation on the biology of Bursaphelenchus.
This protocol provides a guideline on how to observe and investigate the behavioral features of B. xylophilus during mating. Our findings showed that using virgin adult nematodes is critical for increasing the chance of successfully observing the mating process (Figure 1). Obtaining J4 larvae and precisely distinguishing their sexes are two important fundamental steps for the acquisition of virgin adults. Under our experimental conditions (i.e., at 25 °C in the dark), the period of 52 h is the proper time to obtain J4 from J2 larvae. However, distinguishing sex at the early J4 stage mostly depends on personal experience. To ensure accuracy, it is necessary for different people to double-check the sex allocation.
The behavioral features of B. xylophilus during the mating process can be described qualitatively and analyzed quantitatively by recording the durations of the four different phases, the percentage of mating success after first contact, the copulation frequency, and the copulation duration of different combinations. Interestingly, for the first time, we observed mating choice by females and strong intra-sexual competition in both males and females. No promiscuity was observed; however, this could be due to the limited number of nematodes used in our investigation. This protocol can not only be used for investigating the mating behavior of B. xylophilus, but it can also be useful and valuable as a reference for other nematodes.
The authors have nothing to disclose.
Najie Zhu and Liqun Bai contributed equally to this work. This work was financially supported by the Special Fund for Forestry Scientific Research in the Public Welfare (201204501) and the National Natural Science Foundation of China (31170604, 31270688, and 31570638). We thank Dr. Holighaus Gerrit and Dr. Danielle Hickford for their helpful suggestions on English writing.
B. xylophilus isolate Nxy61 | Extracted from Pinus massoniana in Ningbo area of China | ||
Grey mold strain | Obtained from Forestry Academy of China | ||
Baermann funnels | Sengong | ||
falcon tubes | Sengong | ||
centrifuge | Sengong | ||
pipette | Sengong | ||
sterile dd H2O | Sterilized by high pressure of steam at 121°C for 30 min. | ||
Petri dishes | Sengong | ||
PDA medium | Huankai Bio. | 021050 | |
incubator | Sengong | ||
Laminar flow | Sengong | ||
glass capillary | Sengong | ||
Stereoscope | Leica | LED5000 RL | |
inverted stereo microscope | Zeiss | A1 | |
concave slides | Sengong |