Mating and reproductive characteristics of the pathogenic nematode Bursaphelenchus xylophilus

Yongxia Li · Mengge Gao · Baojun Liu ·Xuan Wang · Jiafu Hu ,2 · Zhenyu Liu ,3 ·Xingyao Zhang

Abstract Pine wood nematode ( Bursaphelenchus xylophilus), one of the most destructive invasive species, has caused extremely serious economic, ecological and social losses in many countries throughout the world. Since the high reproductive rate of B. xylophilus PWN is the main cause of rapid death of its pine hosts ( Pinus spp.), understanding the reproductive and population biology and the ecology of this nmatode are of great importance. This study mainly focused on analyzing the mating process and population structure under diff erent combinations of sex ratios for mating. Reproductive effi ciency of B. xylophilus peaked when the sex ratio (female to male) was 3.4:1. Phases of the mating process for the different sex-ratio combinations indicated that B. xylophilus had evolved alternative reproductive strategies to cope with complex copulating conditions to obtain a suitable population structure for further propagation. This research provides fundamental information on the mechanism that is responsible for the rapid population growth of B. xylophilus.

Keywords Reproductive strategy · Reproductive effi ciency · Sex ratio · Population structure · Pine wood nematode

Introduction

The pine wood nematode (Bursaphelenchus xylophilus), the pathogenic causal agent of pine wilt disease (PWD), is a native species to North America where it does not damage pine trees. This species was transported to Asia and Europe in the early 20th century and since then has caused enormous losses every year (Mamiya 1983; Williams 2006;Mamiya and Shoji 2009; Johnt et al. 2010). During the maturation of vector beetle (Monochamusspp.),B. xylophilusis transported and spread from dead pine trees to seemingly healthy trees via wounds as the beetle feeds on young branches and shoots (Katsuyama et al. 1989; Mota and Vieira 2008; Zhao et al. 2008). Generally, it takes 3 or 4 weeks for an infected pine tree to show wilting, then it dies within 40 days (Mamiya 1972; Zhao et al. 2008; Futai 2013; Zhao and Sun 2017). The high reproductive rate ofB. xylophilusis considered as the main reason for the rapid death of pine trees (Jung et al. 2010; Futai 2013; Shinya et al. 2013). Compared with the closely related native speciesBursaphelenchus mucnatus,B. xylophilushas a higher reproductive rate and stronger competitive ability, which are crucial to its ecological invasion mechanism (Vincent et al.2008; Cheng et al. 2009; Zhao et al. 2014). Elucidating its mating and reproductive strategies will help provide a fundamental basis for understandings mechanism of invasion and design Effective control measures.

Of the theories and models of mating and reproductive strategies developed over the past two centuries, the evolutionary stable strategy (ESS) theory states that individuals tend to choose the most stable strategy in the long term (Smith 1982). The status-dependent selection (SDS)assumes that individuals diff er in competitiveness and status and describes how a conditional ESS occurs under statusdependent decision rules. Diff erences such as body size or activity can inf luence the f itness and expression of the conditional strategy, in other words, selection between alternative tactics X and Y (Tomkins and Hazel 2007).

During preliminary research, we observed and described the behavior ofB. xylophilusat diff erent stages in the mating process. We found the mating success rate for the f irst contact was the highest when the population had three females for each male (Liu et al. 2014). Here, we continued this study of mating and reproductive characteristics ofB.xylophilusby analyzing the mating process and population structure under diff erent mating combinations, thus providing valuable information on the reproductive mechanism ofB. xylophilus.

Materials and methods

Source and culturing of nematodes

B. xylophilusNxy61 used in this study was originally isolated from a diseasedPinus massonianain Ningbo, Zhejiang province, People’s Republic of China. Nematodes were cultured onBotrytis cinereaon potato dextrose agar (PDA)plates at 25 °C in the dark.

Acquisition of virgin adult nematodes

Nematodes were isolated from the fungal cultures using the Baermann funnel method (Kiyohara and Suzuki 2010). Eggs were collected and incubated in phosphate-buff ered saline(PBS) at 25 °C without food for approximately 2 days until they all hatched as second-stage juveniles (J2). Then, the J 2 population was collected and synchronously cultured on PDA withBotrytis cinereaat 25 °C for approximately 52 h to obtain fourth-stage juveniles (J4). At this time, most J4nematodes can be clearly distinguished as male or female according to genital morphology. Virgin J4adults were obtained by culturing J4males and females separately on a lawn ofB. cinereafor 24 h. Only virgin adults were used in this study unless specif ically mentioned.

Microscopic observation

Thirty pairs of individuals were used to observe the entire mating process ofB. xylophilusas previously described(Liu et al. 2014). Before females and males were placed together for mating observations, virgin adults were kept separately in sterile water for 1 h to adapt to the new environment. Then a male and female were selected and placed into the well (15 mm diameter × 1 mm depth) of concave microscope slides with 200 μL sterile water for mating observation with a light microscope at 25 ± 1 °C. Each treatment was done 30 times.

The entire mating process refers to the period from introduction of the nematodes into the water environment through completion of copulating when they swim away from each other. Duration of searching refers to the period from nematode introduction into the water environment until a male and female encounter and contact each other.Duration of contact refers to the period when the male and female start touching each other until the male precisely locates the female’s vulva. Duration of mating choice includes the search phase and the contact phase. Duration of copulating includes the period from male attachment and insertion of its spicule into the female’s vulva until they separate from each other. Mating success rate for the f irst contact means the rate of successful mating immediately after the f irst contact. Contact frequency means the average number of times that a female and male encounter and contact with each other (not including copulation).

Reproductive effi ciency and off spring population structure for various mating combinations

Monogamous and polygamous mating combinations were used to investigate the reproductive effi ciency and off -spring structure ofB. xylophilus, as follows: (1) one male with one female (1♀ + 1♂); (2) multiple males with one female (1♀ + 3♂, 1♀ + 2♂), (3) multiple females with one male (3♀ + 1♂, 2♀ + 1♂, 4♀ + 1♂, 5♀ + 1♂); and (4) multiple females with multiple males (3♀ + 3♂). Nematodes in the four combinations were cultured on 60 mm PDA petri dishes withB. cinereafor 4 days (about one generation) to obtain a trend line of reproductive effi ciency and on 90 mm PDA petri dishes withB. cinereafor 12 days(about three generations). After the incubation, nematodes for each combination were collected using the Baermann funnel method. Then the offspring were counted, and reproductive effi ciency (total off spring of per female and per male), ratio of larvae to adults and sex ratio of the off spring (female to male) from each combination were calculated. Each combination had 30 replicates.

Statistical analyses

Data were analyzed by one-way ANOVA with the post hoc Tukey HSD test using SPSS 22.0 (IBM, Armonk, NY, USA)to compare diff erences in mean values between diff erent treatments (Brown 2005).

Results

Optimal sex ratio and population structure of B.xylophilus

Among the various male-female combinations tested,reproductive effi ciency after one generation (approximately 4 days) was highest for 3♀ + 1♂ (198.26 ± 2.03,P< 0.05)(Fig. 1 a).

Using the reproductive effi ciencies of the diff erent mating combinations, we obtained a reproductive effi ciency curve and calculated a mono basic quadratic equation,y= -9.4167x2 + 64.264x+ 90.214. Obviously, the reproductive effi ciency curve reached its highest point for the ratio 3.4 females to 1 male (Fig. 1 b).

To determine whether the results of population structure were consistent with the sex ratio, we analyzed the population structure of the four main combinations (1♀ + 1♂,1♀ + 3♂, 3♀ + 1♂, 3♀ + 3♂) after culturing for 4 days(approximately one generation) and 12 days (approximately three generations) (Table 1). The off spring number increased by 85.65% when the male number was f ixed and the female number was three times greater, i.e., comparing the combination of 1♀ + 1♂ with 3♀ + 1♂. However, the off spring number only slightly increased when comparing 1♀ + 3♂with 1♀ + 1♂. This result suggests that off spring number depends mainly on the number of females.

Accordingly, the sex ratio of the off spring for the combination of 3♀ + 1♂ was prominently higher and closer to 3.4:1 than were the other three combinations after 4 days(3.56 ± 0.06,P< 0.05). Based on the reproductive effi ciency curve above, the off spring sex ratio under this combination is more conducive to further population growth. Conversely,the sex ratio of the off spring from the other three combinations appeared to deviate from 3.4:1, illustrating that the reproductive effi ciency for these combinations would be lower.

The ratio of larvae to adults was much higher for the combination of 3♀ + 1♂ compared with the other three combinations (0.53 ± 0.01,P< 0.5). The ratio of larvae to adults determines the long-term reproductive trend for the population. Thus, the population growth would be the fastest with the combination 3♀ + 1♂ (Table 1).

Fig. 1 Mean (± SEM) reproductive effi ciency and reproductive effi ciency curve of B. xylophilus: a reproductive effi ciency of different male-female combinations after 4 days (approximately one generation). b Reproductive effi ciency for diff erent sex ratios. c Reproductive effi ciency of the four mating combinations after 12 days (approximately three generations). N = 30 independent replications. Means with the same lowercase letter within each column are not signif icantly diff erent at P < 0.05 based on one-way ANOVA with the post hoc Tukey HSD test. Reproductive effi ciency isbased on totalnumberof off spring per male and female parents:Reprod uctive efficiency?

Moreover, the reproductive effi ciency for the four combinations after 12 days was consistent with the results after 4 days (Fig. 1 c). The population structure on the 12th day for the four combinations revealed a similar tendency(Table 1). These f indings all conf irmed that 3♀ + 1♂ was the optimal mating combination forB. xylophilus.

Table 1 Population structure of four mating combinations after 4 days (approximately one generation) and 12 days (approximately three generations)

Mating and reproductive strategies of B. xylophilus

When mating behavior of females and males in the diff erent combinations (3♀ + 1♂, 2♀ + 1♂, 1♀ + 1♂, 3♀ + 3♂,1♀ + 2♂, 1♀ + 3♂) was observed and analyzed, the duration of the mating choice (searching and contact) under the six mating combinations differed (31.4 ± 0.5 min,39.0 ± 0.7 min, 80.6 ± 3.9 min, 42.6 ± 0.7 min,52.6 ± 0.6 min and 65.9 ± 1.3 min, respectively; Fig. 2 a);the combination of 3♀ + 1♂ (T = 31.4 ± 0.5 min,P< 0.05)required the least time to finish the choice process.Statistical analysis indicated that combinations with more females (3♀ + 1♂, 2♀ + 1♂) required less time for mating choice and yielded a higher mating success rate for the f irst contact (Fig. 2 a, b). For combinations with more females, females attempted to mate with the male as early as possible by shortening the selection time and increasing the mating success rate for the f irst contact. The male also tended to decrease its contact frequency and shortened the mating time with the female, likely to reserve and obtain energy to mate as soon as possible with other females. Consequently, the males were able to produce more off spring. This strategy is called the Y mating strategy or early mating strategy.

Fig. 2 Mean (± SEM) duration of mating phases and success rate for mating combinations with diff erent female to male ratios. a Duration of mating choice. b Mating success rate for f irst contact. c Contact frequency of females and males. d Duration of copulating. N = 30 independent replicates. Means with the same lowercase letter over columns are not signif icantly diff erent at P < 0.05 based on one-way ANOVA with the post hoc Tukey HSD test

On the other hand, combinations with more males, e.g.,1♀ + 3♂ and 1♀ + 2♂, always needed a longer choice process since females usually required several contacts to choose suitable mates. This behavior often led to the contact frequency increasing and yielded a low mating success rate for the f irst contact. Once paired, they also took more time to copulate (Fig. 2 b-d). For combinations with more males,the female tended to increase the selection time to f ind a better mating partner, for example, the strongest one, and thus reduced the mating success rate of the f irst contact. Males also increased their contact frequency and copulating duration to ensure mating success. This “X mating strategy” (or“preferential mating strategy”) allows the selection of the best mate for copulating to ensure higher quality off spring.

The female f inally mated with the male after contacting it several times in the monogamous combination (1♀ + 1♂);this strategy is def ined as the limited mating strategy. For combinations with multiple females and multiple males, for example, 3♀ + 3♂, females and males both chose the one that interested them, def ined as the ‘self-choose strategy’.

Discussion

Reproductive strategy is a key feature in determining the evolutionary performance of an organism (Álvarez et al.2005; Shinya et al. 2015 ). Conditional strategies are superior to f ixed strategies (West-Eberhard 1989; Scheiner 1993;Plaistow 2004; Schlichting and Pigliucci 2010). Individuals always evolve conditional strategies in response to various environment conditions; each strategy is assumed to be based on a unique physiological mechanism that aff ects lifehistory traits (Smith 1978). So far, most published reports on the reproduction ofB. xylophilusfocused on the Effects of pine volatiles, associated fungi and other environment factors on reproductive ability (Maehara and Futai 1997;Niu et al. 2012).

In the controlled laboratory study, we systematically monitored the mating process of diff erent sex-ratio mating combinations and analyzed subsequent mating and reproductive characteristics ofB. xylophilus. The data indicated that the reproductive strategy ofB. xylophilusconforms to the SDS theory: nematodes tended to use the Y mating strategy when more females were present to obtain more off spring and the X mating strategy when more males were present,which should result in fewer but higher quality off spring.These strategies may further inf luence the population structure ofB. xylophilus.

Sex ratio is important for population propagation and will ultimately inf luence the evolution of the species by aff ecting population structure, mating effi ciency and the diff erentiation of sexual dimorphism (Pröhl 2002; Cui et al.2020). An optimal sex ratio will result in greater fecundity and rapid population increase (Triantaphyllou 1973). Results of this study showed the sex ratio of off spring under the four sex-ratio mating combinations were all near the optimal sex ratio of 3.4:1 after one generation and three generations.Furthermore, the ratio of larvae to adults for each combination also increased from the f irst to the third generation.These results suggest that the population structure ofB. xylophilustends to be optimized under stable conditions. The off spring sex ratio for 3♀ + 1♂ was closest to 3.4:1, and the ratio of larvae to adults was also the highest among all mating combinations, suggesting to some extent that sex ratio signif icantly inf luences population dynamics and that the population structure will maintain a trend that is conducive to the reproduction ofB. xylophilusfor some period in the future.

In summary, the optimal mating sex ratio was 3 females to 1 male, and the reproductive strategy ofB. xylophilusconformed to the SDS theory: when more females were present, the Y mating strategy led to more off spring, when more males were present, the X mating strategy led to fewer but higher quality off spring. Because rapid population growth ofB. xylophilusis a key factor in the severity of pine wilt,understanding the reproductive biology characteristics ofB. xylophilusshould help to further clarify its reproduction mechanism at the molecular level. Meanwhile, Effective measures can be taken through controlling the biological parameters such as sex ratio and mating effi ciency ofB. xylophilus, to manage the reproductive rate of f ield populations.

Author’s contributionsXZ, ZL, JH designed the study; YL, MG,BL, XW performed the experiments and analyzed the results; YL, MG wrote the manuscript. All authors read and approved the manuscript.

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