Differences of Pine Wood Nematode (Bursaphelenchus xylophilus) Developmental Stages under High-Osmotic-Pressure Stress

Abstract

Under ion imbalance, water deficiency, and salt stress, the osmotic pressure of the tree sap increases, and pine wood nematodes (Bursaphelenchus xylophilus, PWN) parasitizing in the trees may be subjected to high-osmotic-pressure stress. KCl, L-malic acid, sucrose, and glycerol solutions were used as osmolytes to explore the highest osmotic concentration that PWN can tolerate. Survival analysis showed that when the treatment concentration exceeded 90%, only a few nematodes in the glycerol group survived under 6 h treatment, and most of the survivors were third-stage dispersal juveniles (DJ3). Further examination revealed that under different concentrations of glycerol-induced high osmotic pressure, the survival rate and body length change rate were the highest in the DJ3 and the lowest in the second-stage propagative juveniles. In order to explore the molecular mechanism of resistance of DJ3 to high osmotic stress, transcriptome sequencing was performed at each developmental stage of PWN and differentially expressed genes that were up-regulated or down-regulated only in DJ3 were screened. The expression of genes related to CoA in DJ3, a key enzyme in metabolism, was significantly higher than the other developmental stages. In addition, the expression of the anti-reversal signal pathway-related gene AKT-1 in DJ3 was significantly lower than in the other development stages. Therefore, the specific expression of genes in DJ3 under high osmotic pressure may help them rapidly produce and accumulate energy-related compounds and activate the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathway to respond to damage caused by high-osmotic-pressure stress in time, thus promoting survival.

Keywords: Bursaphelenchus xylophilus; hypertonic osmotic stress; osmobiosis; pine wood nematode.

Figure 1

Phenotypic changes of male PWNs after high osmotic pressure treatment with different concentrations (10%, 30%, 40%, 50%, and 80%) of four types of osmolytes for 6 h. Scale bars: 100 μm.

Figure 2

Phenotype of male PWNs after 6 h of high osmotic pressure treatment and 12 h of rehydration. (A) Changes in lipid droplet occurrence and properties in males after 6 h of high osmotic pressure treatment with four types of osmolytes (treatment concentration of 20% for each osmolyte) and rehydration for 12 h. (B) Changes in lipid droplet occurrence and properties in males after 6 h of high osmotic pressure treatment with four types of osmolytes (treatment concentration of 40% for each osmolyte) and rehydration for 12 h. Scale bars: 100 μm.

Figure 3

Staining and survival rate of PWNs under high osmotic pressure treatment with different osmolytes for 6 h. (A) Staining of nematodes treated with a 20% saturated KCl solution for 6 h. The red line and the red box are circled for the enlarged view, i.e., Figure B. (B) Staining of nematodes treated with a 20% saturated KCl solution for 6 h. (C) Staining of nematodes treated with a 40% saturated KCl solution for 6 h. The red line and the red box are circled for the enlarged view, i.e., Figure D. (D) Staining of nematodes treated with a 40% saturated KCl solution for 6 h. (E) Staining of nematodes treated with a 20% saturated L-malic acid solution for 6 h. (F) Staining of nematodes treated with a 20% saturated sucrose solution for 6 h. (G) Staining of nematodes treated with a 20% saturated glycerol solution for 6 h. Scale bars: 100 μm.

Figure 4

The survival rate of PWNs treated with four types of osmolytes after 6 h dehydration and 12 h rehydration. (A) Survival rate of the glycerol treatment group. (B) The survival rate of the L-malic acid treatment group. (C) Survival rate of the KCl treatment group. (D) The survival rate of the sucrose treatment group. Lowercase alphabetical letters indicate a significant difference in the survival rate under different concentrations after dehydration treatment due to high osmotic pressure (n = 3, p < 0.05). Uppercase alphabetical letters indicate a significant difference in the survival rate after treatment with different osmolyte concentrations after rehydration (n = 3, p < 0.05). The error bars indicate a difference in the survival rate between the dehydration and the rehydration treatment same concentration (n = 3, * p < 0.05, ** p < 0.01). The color of the column differs based on the significance of survival rates.

Figure 5

Changes in PWN body length with glycerol-induced high-osmotic-pressure stress for 6 h and 12 h. (A) J2. (B) J3. (C) J4. (D) Male. (E) Female. (F) DJ3. The uppercase alphabetical letters indicate significant differences in PWN body lengths after 6 h of high osmotic pressure treatment (n = 3, p < 0.05). The lowercase alphabetical letters indicate significant differences in PWN body lengths after 12 h of high osmotic pressure treatment (n = 3, p < 0.05). The error bars indicate differences in the PWN body length at different treatment times at the same glycerol concentration (n = 3, * p < 0.05, ** p < 0.01). The color of the column differs with the significance of body length differences.

Figure 6

PWN body length change rate at each developmental stage after high osmotic pressure treatment. The change rate of body length was calculated based on the average body length (n = 30).

Figure 7

PWN survival rates after glycerol-induced high osmotic pressure treatment for 6 h and 12 h. (A) J2. (B) J3. (C) J4. (D) Male. (E) Female. (F) DJ3. The uppercase alphabetical letters indicate significant differences in the PWN survival rate after 6 h of high osmotic pressure treatment (n = 3, p < 0.05). The lowercase alphabetical letters indicate significant differences in PWN survival rates after 12 h of high osmotic pressure treatment (n = 3, p < 0.05). The error bars indicate differences in PWN survival rates at different treatment durations at the same concentration (n = 3, * p < 0.05, ** p < 0.01). The color of the column differs with the significance of the survival rate differences.

Figure 8

Gene expression analysis of PWN at different developmental stages. (A) Multi−group differential gene expression scatter plot at different developmental stages of PWN. (B) Statistics on the number of differentially expressed genes in DJ3. (C) Venn diagram of the up−regulated genes in DJ3. (D) Venn diagram of the down-regulated genes in DJ3.

Figure 9

Candidate gene screening and expression patterns. (A) Candidate gene expression analysis. The left side illustrates the candidate gene heat map, and the right side illustrates the pathways enriched with the candidate genes (detailed in Table S6). (B) Expression validation of the selected genes by RT−qPCR (detailed in Table S7).