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Abstract
© 2019. Published by The Company of Biologists Ltd | Biology Open (2019) 8, bio041616. doi:10.1242/bio.041616 RESEARCH ARTICLE Cold adaptive potential of pine wood nematodes overwintering in plant hosts 1,2 1,2, 1,2 1,2 1,2 Zhenkai Liu , Yongxia Li *, Long Pan , Fanli Meng and Xingyao Zhang ABSTRACT and Jiangsu Provinces, being the most seriously affected (Yu et al., 2011). By 2018, PWN infections had been found in 315 Chinese The pine wood nematode (PWN; Bursaphelenchus xylophilus) is the counties of 16 provinces, with the northeast regions in the mid- causal agent of pine wilt disease, which results in severe ecological temperate zone of China (N 41°, E 124°), including Liaoning and economic losses in coniferous forests. During overwintering, Provinces, being seriously affected (Provinces of Liaoning) PWNs undergo morphological and physiological changes to adapt to (State Forestry Administration of the People’s Republic of China, low temperature environments. Here, the physiological changes of 2018). As a potential habitat, the PWN may infest more areas the PWN populations sampled in the summer and winter were in Northeastern China and expand further into high-latitude compared to analyze the role of low temperatures in their response. cold regions. The PWN overwinters as third-stage dispersal juveniles, which The ability to survive cold is an important factor in determining showed significantly greater survival rates than summer populations the northern range limits of nematodes, but little is known about the (propagative forms) at sub-zero temperatures. The major PWN’s cold tolerance. To survive the cold season, many nematodes biochemical compounds in the populations were analyzed by gas display increasing levels of cold hardiness, which are regulated by chromatography. Eight dominant fatty acids, with stearic acid being antifreeze proteins and polyols as well as other low-molecular- the most important, were identified from PWN propagative stage and weight cryoprotectants, such as amino acids and sugars (Smith et al., third-stage dispersal stage. Compared with the propagative stage, the 2008; Wharton et al., 2003; Wu et al., 2018). Compared with other dispersal stage showed significant increases in the fatty acid content nematodes, the PWN has two developmental forms in its life cycle: and the proportion of unsaturated fatty acids. Three carbohydrates, propagative and dispersal. In the summer, individuals in the trehalose, glycerol and glucose, were detected in the PWN. propagative phase reproduce rapidly, developing from eggs through Compared with the summer population, the levels of trehalose and four larval stages (J –J ) to the reproductive adult, which increases 1 4 glycerol increased significantly, while glucose decreased, in the the number of nematodes in infected pines and causes severe winter population. The modifications in fatty acid composition and damage to healthy pines. In the autumn, with increasingly low cryoprotectant levels, as elements of its changing physiology, play temperature and food scarcity, the PWN molt from J into third- important roles in the overwintering success of the PWN. stage dispersal juveniles (J ). J molt to fourth-stage dispersal III III KEY WORDS: Pine wood nematode, Overwintering, Fatty acid juveniles (J ) when the juvenile receives a chemical signal released IV composition, Cryoprotectant by the last instar larvae and pupae of the Monochamus beetle (Zhao 2et al., 2007; Pereira et al., 2013). The J is considered III INTRODUCTION environmentally resistant to low temperatures and desiccation and is The pine wood nematode (PWN), Bursaphelenchus xylophilus the stage that survives and disperses in the winter (Kondo and (Nematode: Aphelenchoididae), is the causal agent of pine wilt Ishibashi, 1978; Zhao et al., 2007). disease (PWD), which causes severe ecological and economic Low temperatures bring several challenges to PWN survival. losses in coniferous forests (Mota and Vieira, 2008). The PWN is an Some challenges are associated with low temperature per se, which invasive species introduced from North America that is causing may cause changes in the viscosity, phase and organization of extensive damage to pine trees in Asia, especially in China and membranes, with a corresponding loss of function (Wharton and Japan. In 1982, a PWN infection was first reported in Nanjing Perry, 2011). Extreme sub-zero temperatures kill nematodes by (N 32°03′, E 118°50′), subtropical China (Yang et al., 2003). By freezing their cell contents and body water, and it is usually ice 2007, PWN infections had been found in 113 Chinese counties crystal formation inside nematodes that irrevocably damages its of 12 provinces, with the coastal regions in the subtropical and cells and body structures, eventually leading to death. To cope warm-temperate zones of China (N 29°–34°), including Zhejiang with these cold injuries, several physiological and biochemical mechanisms have evolved in overwintering nematodes. Physiological processes during this time are prioritized towards Laboratory of Forestry Pathogen Integrated Biology, Research Institute of Forestry surviving the extreme conditions and conserving resources for the New Technology, Chinese Academy of Forestry, Beijing, 100091, People’s Republic of China. Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing subsequent growing season, including the accumulation of lipid Forestry University, Nanjing Jiangsu, 210037, People’s Republic of China. droplets and sugars (Sinclair, 2015; Wharton et al., 2000; Jagdale and Gordon, 1997). *Author for correspondence ([email protected]) Lipids are important components of nematode overwintering Y.L., 0000-0002-1803-1214 energetics, and play important roles in structuring water and This is an Open Access article distributed under the terms of the Creative Commons Attribution ice during overwintering. Overwintering individuals, such as License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, Steinernema feltiae and other insects, have significantly greater distribution and reproduction in any medium provided that the original work is properly attributed. ratios of unsaturated to saturated fatty acids (Sinclair and Marshall, 2018; Jagdale and Gordon, 1997). Unsaturated fatty acids with Received 7 January 2019; Accepted 20 March 2019 Biology Open RESEARCH ARTICLE Biology Open (2019) 8, bio041616. doi:10.1242/bio.041616 low melting points probably allow lipid membranes to maintain Freezing temperature survival rate the sufficient fluidity required to maintain protein functions at No mortality was observed at temperatures greater than 0°C. The low ambient temperatures (Nieminen et al., 2013; Vukašinovic effects of sub-zero temperatures on PWN’s survival in the freezing et al., 2015). The accumulations of low-molecular-weight regime was significant. As the temperature decreased, the survival sugars and polyhydric alcohols (polyols) are well-known rate also decreased significantly (Fig. 3). Between 0°C and −15°C, responses to freezing, drought, salt and osmotic stresses in the the survival rate of J after the medium was frozen was notably III entomopathogenic nematodes S. feltiae and Antarctic nematodes greater than that of the propagative forms. Additionally, the S of Panagrolaimus davidi (Ali and Wharton, 2015; Wharton and the J was −8.2°C, which was lower than the −4.2°C of the III Perry, 2011). In S. feltiae and other insects, trehalose and glycerol propagative forms. When below −15°C, all the PWN died. The may accumulate as cryoprotectant agents that are correlated nematode suspensions could be kept in liquid phases at −5°C and with survival at low temperatures (Khani et al., 2007; Ali and −10°C when the suspensions were sufficiently clean and no ice Wharton, 2015). crystals were added. Under these conditions, there were no The aim of the present study was to describe the physiological significant differences between the survival rates of the and biochemical adaptations of the PWN to low temperature. The propagated PWN at −5°C and −10°C. The same was true for the winter populations, or overwinter states, were generally more dispersal forms. However, when the temperature was below −15°C, resistant to low temperature-associated stress than other populations. the nematode suspension froze. Compared with frozen nematode Therefore, the major physiological substances were measured by suspensions, the nematode survival rates were significantly greater gas chromatography in field-collected summer and winter when the nematode suspension did not freeze (Fig. 4). At −5°C and populations, to determine which factors are associated with the −10°C the survival rates of propagative forms increased 2.26 times winter survival of the PWN. and 40.1 times, and those of J increased 1.3 times and 2.94 times, III respectively. The lower the temperature, the greater the damage to RESULTS the PWN caused by freezing the suspensions. Morphological characteristics The nematodes isolated from the infected wood collected in August Fatty acid profile 2017 were propagative forms of the PWN, including larval stages Lipids comprised, on average 26.3% and 70.1% of the PWN dry (J –J ), and reproductive adults. The nematodes isolated in weights at the propagative stage and third-stage dispersal, 2 4 December 2017 were almost all J of the PWN (Fig. 1). respectively, and the average propagative PWN dry weight was III Morphologically, J is not clearly different from propagative 0.028 μg/ind, while that of the dispersal was 0.041 μg/ind. Eight III larvae; both having a stylet and median bulb; however, the former dominant fatty acids were identified from PWN propagative stage has a broadly rounded tail and a deep deposition of lipid droplets in and third-stage dispersal individuals, including four kinds of the intestine. Under an optical microscope, the PWN were dark but saturated fatty acids, lauric acid (C12:0), myristic acid (C14:0), the tails were bright (Fig. 2A). Internally, the large refringent bodies palmitic acid (C16:0) and stearic acid (C18:0), and four kinds of were observed in some samples (Fig. 2B). The lipid droplets within unsaturated fatty acids, myristoleic acid (C14:1), oleic acid (C18:1), the nematodes were stained red (Fig. S1). The intensity and linoleic acid (C18:2) and arachidonic acid (C20:4) (Fig. 5). The precision of the lipid staining was greater in the dispersal larvae than dispersal and propagative forms of PWN contained identical fatty the propagative larvae. acid components, but the relative quantities of the individual acids Fig. 1. Summer and winter pine wood nematode population structures. The summer population (August 2017) included 2nd instar juveniles (J ), 3rd instar juveniles (J ), 4th instar 2 3 juveniles (J ), females and males. The winter population (December 2017) included third-stage dispersal juveniles (J ), females and males. III Biology Open RESEARCH ARTICLE Biology Open (2019) 8, bio041616. doi:10.1242/bio.041616 Fig. 2. Morphological characteristics of the third-stage dispersal juveniles of the pine wood nematode. (A) Third-stage dispersal juveniles; (a) stylet and median bulb; (b) lipid droplets; (c) broadly rounded tail; (B) internal ‘gas bubbles’. often varied. The total fatty acid content of the dispersal forms 1.3: 1 to 1: 3.0: 3.8. The unsaturated fatty acids increased from was 3.8 times that of the propagative forms. The propagative 32.3% to 76.1%. forms contained mainly saturated fatty acids (68.7%), of which C18:0 (39.9%) and C16:0 (25.2%) were the two dominant fatty Sugars and polyols acids. However, the dispersal forms contained mainly unsaturated Three carbohydrates, trehalose, glycerol and glucose, were detected fatty acids (76.2%), with C18:2 (35.3%) and C18:1 (28.2%) being in the PWN propagative and dispersal stages (Fig. 7). The glycerol, the two dominant fatty acids. During the conversion of the PWN glucose and trehalose contents in the propagative stage were 0.81, from the propagative to dispersal stages, the unsaturated fatty acid 0.73 and 1.89 μg, respectively, and 2.59, 0.32 and 2.95 μg, content increased significantly more than the saturated fatty respectively, in the dispersal stage. The glycerol and trehalose acids, with oleic acid C18:1 increasing by 12.3 times, linoleic contents in the dispersal stage were greater than in the propagative acid C18:2 increasing by 19.7 times, palmitic acid C16:0 increasing type, and the glucose content was lower than in the reproductive by 2.2 times and stearic acid C18:0 increasing by 0.58 times. type. During the conversion of the PWN from the propagative to The accumulation of unsaturated fatty acids leads to an increased dispersal stage, the glycerol and trehalose concentration increased ratio of unsaturated fatty acids to saturated fatty acids during the significantly (P<0.05), with glycerol increasing by 220% and transition from the reproductive to dispersal type of PWN (Fig. 6). trehalose increasing by 56%. However, the glucose concentration The proportions of C18:0, C18:1 and C18:2 was changed from 3.5: significantly decreased (P<0.05) by 56.2%. Fig. 3. Effects of temperature on the survival of the pine wood nematode. Open circles, summer population; closed circles, winter population. Vertical bars represent standard errors. Biology Open RESEARCH ARTICLE Biology Open (2019) 8, bio041616. doi:10.1242/bio.041616 Fig. 4. Effects of supercooled water on the survival of the pine wood nematode. SP, summer population; WP, winter population. Vertical bars represent standard errors. DISCUSSION with other diapause or dauer nematodes, the J are considered to be III To overcome low temperature-associated stresses in winter, the more resistant to cold than other stages. In some J , the large III PWN molt to a particular developmental stage (J ), which refringent bodies became apparent, giving them a beaded III corresponds to the pre-dauer (J2) stages of C. elegans, but they appearance (Fig. 2B). In Ditylenchus dipsaci, the large refringent can sustain the diapause stage for a long time (Zhao et al., 2007). As bodies were formed by lipid droplets that coalesced in the Fig. 5. Fatty acid compositions of pine wood nematode populations. Using 2500 nematodes in a sample. Biology Open RESEARCH ARTICLE Biology Open (2019) 8, bio041616. doi:10.1242/bio.041616 Fig. 6. Fatty acid composition ratios of pine wood nematode populations. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. rehydration process (Wharton et al., 1985). In the PWN, the large (Raymond and Wharton, 2016). Survival rates for summer and refringent bodies may be formed by the encapsulation of many lipid winter populations indicated that the dispersal stage was more droplets in the rehydration process (Fig. 2B). resistant to low, and especially freezing, temperatures than the The low temperature survival rate was used to measure the cold propagative stage. Nematode suspensions cannot be kept unfrozen tolerance of nematode. Survival rates at −5°C and −10°C suggested at temperature less than −15°C (Huang et al., 2018). Therefore, the that PWN’s have a strong cold tolerance. In freezing water, the effects of whether the medium freezing on PWN survival rates were nematodes were seeded by exogenous ice nucleation and a not analyzed below −15°C. proportion were freeze tolerant. However, in supercool water the The J stage is considered environmentally resistant to freezing III nematode survival rate significantly increased. These results temperatures because it contains densely packed lipid droplets indicate that the PWN was freeze tolerant when exposed to frozen (Zhao et al., 2007). Like other nematodes and insects, the fatty acids surroundings, but cold intolerance was enhanced when the PWN of the PWN are mainly composed of palmitic acid, stearic acid, oleic was free of exogenous ice nucleation and able to supercool acid and linoleic acid, but palmitoleic acid (C16:1) was not detected Fig. 7. Types of carbohydrates in pine wood nematodes. Using 5000 nematodes in a sample. Biology Open RESEARCH ARTICLE Biology Open (2019) 8, bio041616. doi:10.1242/bio.041616 (Jagdale and Gordon, 1997; Vukašinovic et al., 2015). The PWN will contact free water, thereby avoiding the influence of difference in the fatty acid profiles may be related to species and inoculative freezing. Therefore, the cold tolerance of plant-parasitic environmental factors (Thompson, 1973). nematodes is a complex system, and the mechanisms behind the Compared with the propagative stage, the dispersal stage had overwintering of the PWN need to be further clarified. significant increases in the fatty acid content and the proportion of MATERIALS AND METHODS unsaturated fatty acids, which provide an energy base for PWNs and Nematode protect the fluidity of the cell lipid membrane to resist solidification In August 2017 and December 2017, trees infected by PWNs were sampled at low temperatures (Murray et al., 2007). Stearic acid was the most in the same Chinese red pine forest in Fushun (N 41°58′, E 124°24′), important fatty acid in PWN. In propagative forms, stearic acid Liaoning Province, northeastern China. Fushun belongs to the mid accounted for 40% of the fatty acid content. In dispersal forms, temperate zone, enjoying a continental monsoon climate. Summer is stearic acid was consumed to produce increased levels of oleic and warm and rainy, but winter is very cold. The average annual temperature is linoleic acids. This caused the proportions of C18:0, C18:1 and 6.3°C, and the average winter temperature is −12.1°C. In the Chinese red C18:2 to change from 3.5: 1.3: 1 to 1: 3.0: 3.8. If the fatty acids of pine forest, Pinus tabulaeformis can be classified into four categories based the PWN are self-synthesized, then there are a variety of desaturases. on the degree of PWN-associated damage. Seven Chinese red pines with Δ9-desaturas transforms saturated fatty acid (C18:0) into trunk diameters of ∼12 cm that were normal in appearance, or had a small monounsaturated fatty acid oleic acid (C18:1), and Δ12-desaturase number of needles turning yellow, were selected, and 5–10 cm thick discs were taken from the upper, middle and lower parts of each pine. Using the transforms 18:1 into 18:2 (Nieminen et al., 2013). Lipids, as an Baermann funnel method, the PWNs were immediately isolated from the energy store, are required for metabolic fuel over the winter and for wood samples. the energetic demands of development in the spring (Sinclair and Marshall, 2018). The modifications to the fatty acids can contribute Morphological characteristics to temperature adaptation by allowing lipid membranes to maintain The isolated nematodes were observed and photographed using optical their fluidity at low ambient temperatures (Lyons et al., 1975; microscopy and stained with Oil Red O. Oil Red O staining was performed Vukašinovic et al., 2015; Zhao et al., 2007). as outlined in Stamps’s method, to observe the nematode lipid droplet areas Cryoprotectants, such as sugars and polyhydric alcohols, protect (Stamps and Linit, 1995). organisms from chilling and freezing injury and thereby enhance their cold tolerance (Wharton et al., 2000). However, the cold Low temperature survival treatment conditions and seasonal changes determine the types of The nematodes isolated from the wood samples collected in winter were cryoprotectants in nematodes (Ali and Wharton, 2015; Grewal and placed in centrifuge tubes containing 1 ml of water to form a nematode Jagdale, 2002; Jagdale and Grewal, 2003). Trehalose, glycerol and suspension (5000 nematodes per ml). Then, 100 μl samples of the nematode suspensions, each containing approximately 500 PWNs were placed in glucose were the principal carbohydrates detected in the PWN. Eppendorf tubes (four replicates) and transferred to the Low-temperature Compared with the propagative type, the glycerin and trehalose Reaction Bath. content was greater in the dispersal stage of PWN, while the glucose The nematodes isolated from the wood samples collected in summer were content was lower. placed in centrifuge tubes containing 1 ml of water to form a nematode In the summer and winter populations of the PWN, the suspension (5000 nematodes per ml). Then, 100 μl samples of the nematode principal carbohydrate that had the highest content was glycerol, suspensions, each containing approximately 500 PWNs, were placed in which is a penetrating cryoprotectant and readily permeates across Eppendorf tubes (four replicates) and transferred to the Low-temperature membranes, playing an important role in freezing-avoiding insects Reaction Bath. (Qiu and Bedding, 2002). Therefore, its role during freezing could be more important than that of trehalose. Among non- Frozen survival penetrating cryoprotectants, trehalose had the greatest content in The samples were controlled from 1°C to various temperatures (T :5, 0, min −1 −5, −10, −15°C) at 1°C min and frozen by adding ice crystals. They were the PWN. Trehalose stabilizes membranes and protects against the −1 held at T for 12 h and then rewarmed to 1°C at 1°C min . After thawing, dehydrating effects of osmotic stresses under low temperature stress min the samples were placed at room temperature for 24 h. (Wharton et al., 2000). The decrease in glucose may result from its use as a fuel for the basal metabolism during the overwintering Unfrozen survival period (Kimur et al., 1992). The samples were cooled from 1°C to two minimum temperatures (T : −5, min Under laboratory conditions, the mortality rate of the PWN at −1 −10) at 1°C min . They remained unfrozen at T for 12 h. They were then min −15°C was 100%. However, it can overwinter in northern China, −1 rewarmed to 1°C at 1°C min and placed at room temperature for 24 h. where the lowest winter temperature can reach −30°C, the monthly Ultra-pure water was used to prepare the nematode suspensions to ensure minimum temperature is −21.6°C, and the monthly average they remained unfrozen. Survival was determined by counting the temperature is −14.3°C (January 2018, Fushun). This illustrates proportion of moving nematodes after a mechanical stimulus. Control that the PWN is able to survive low temperatures in the field, where samples consisted of 100 μl nematode suspension in Eppendorf tubes held at the hosts play important roles in the overwintering of the PWN. The room temperature for the duration of the experiment. PWN spends the winter months in the host, which have minimum Gas chromatography for lipid temperatures that are higher than the air temperature. According to The nematodes isolated from the wood samples collected in winter and the model established by Vermunt et al. (2012), the mean daily summer were independently added to centrifuge tubes containing 1 ml of under-bark minimal temperature was, on average, ∼2.1°C higher water to form two nematode suspensions (25,000 nematodes per ml). Then, than the minimum air temperature (Vermunt et al., 2012). The 100 μl samples of the nematode suspensions containing 2500 winter or injuries to PWNs caused by low temperature were mainly the result summer forms of the PWN were independently placed in Eppendorf tubes. of inoculative freezing. In winter, PWNs are distributed in the resin The samples were centrifuged, the dH O supernatant removed and 20 μl canals. The free water content in the host is reduced and xylem C19:0 added as an internal standard. The mixtures were homogenized on parenchyma cells adapt to sub-freezing temperatures by deep ice. The homogenates were transferred to glass tubes and then incubated with 1 ml of methanol +2% H SO for 1 h at 80°C. Once the samples supercooling (Kasuga et al., 2006). This reduces the chance that the 2 4 Biology Open RESEARCH ARTICLE Biology Open (2019) 8, bio041616. doi:10.1242/bio.041616 Jagdale, G. B. and Gordon, R. (1997). Effect of temperature on the composition of cooled, 1 ml of hexane was added to each. Samples were oscillated, fatty acids in total lipids and phospholipids of entomopathogenic nematodes. centrifuged at 1500× g for 3 min and the upper organic layers were extracted J. Therm. Biol. 22, 245-251. doi:10.1016/S0306-4565(97)00019-3 for GC analysis. Jagdale, G. B. and Grewal, P. S. (2003). Acclimation of entomopathogenic Samples were then analyzed on an Agilent 7890A GC (Agilent, USA) nematodes to novel temperatures: trehalose accumulation and the acquisition of equipped with an AB FFAP column (30 m×250 μm×0.25 μm). A constant thermotolerance. Int. J. 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Published: May 15, 2019