Table 1
Measurements (in µm) and ratios of the development stages of Bursaphelenchus crenati.
| Stage/Character | J2 | J3 male | J3 female | J4 male | J4 female | Male | Female |
|---|---|---|---|---|---|---|---|
| n | 20 | 20 | 20 | 15 | 18 | 15 | 15 |
| L | 166 ± 22 | 367 ± 21 | 375 ± 26 | 455 ± 21 | 489 ± 14 | 698 ±69 | 843 ± 94 |
| (150–181) | (338–400) | (339–402) | (377–532) | (479–508) | (607–804) | (750–1082) | |
| Body width | 7 ± 1 | 10 ± 2 | 11 ± 2 | 14 ± 4 | 12 ± 0.2 | 17 ± 3 | 16 ± 3 |
| (6–8) | (6–12) | (8–13) | (12–17) | (10–15) | (14–216) | (13–20) | |
| Pharynx | 68 ± 4 | 111 ± 5 | 115 ± 6 | 106 ± 0.7 | 125 ± 12 | 122 ± 12 | 136 ± 22 |
| (65–70) | (104–118) | (109–125) | (105–106) | (111–140) | (105–144) | (108–178) | |
| Pharyngeal gland | 30 ± 5 | 50 ± 5 | 52 ± 5 | 49 ± 6 | 50 ± 6 | 50 ± 6 | 59 ± 14 |
| (27–34) | (44–58) | (45–60) | (45–53) | (42–55) | (41–64) | (40–85) | |
| Stylet | 7 ± 1 | 8 ± 1 | 8 ± 0.8 | 10 ± 1 | 10 ± 1 | 10 ± 0.1 | 11 ± 1 |
| (6–8) | (6–9) | (7–9) | (9–11) | (9–11) | (8–12) | (10–13) | |
| Lip region width | 4.0 ± 0.3 | 5 ± 0.2 | 5 ± 0.4 | 5 ± 0.2 | 5.8 ± 0.2 | 7.0 ± 0.6 | 7.0 ± 0.5 |
| (3.5–4.5) | (4–5) | (4–5) | (5–5.5) | (5.5–6.0) | (7–8) | (6.0–7.5) | |
| Lip region height | 2.3 ± 0.5 | 3 ± 0.3 | 2 ± 0.2 | 3 ± 0.2 | 2.5 ± 0.1 | 4 ± 0.5 | 3.6 ± 0.3 |
| (2.0–2.5) | (2–3) | (2–3) | (3–3.5) | (2.4–2.7)– | (3–5) | (3–4) | |
| Median bulb from | 30 ± 2 | 48 ± 2 | 48 ± 1.4 | 40 ± 3 | 56 ± 6 | 58 ± 5 | 60 ± 9 |
| anterior | (29–31) | (45–52) | (46–50) | (38–43) | (51–64) | (51–71) | (51–73) |
| Median bulb length | 9.4 ± 1 | 12 ± 1 | 12 ± 1.3 | 12 ± 0.3 | 12.7 ± 0.7 | 15 ± 2 | 18 ± 4 |
| (9–10) | (10–14) | (10–14) | (11.5–12) | (11.5–13.0) | (13–18) | (14–23) | |
| Median bulb width | 5.7 ± 0.3 | 9 ± 2 | 9 ± 1.2 | 9 ± 2 | 10.3 ± 1.3 | 12 ± 1 | 12 ± 1 |
| (5.5–6.0) | (6–11) | (7–10) | (7–10) | (10–12) | (11–14) | (10–13) | |
| Excretory pore | 35 ± 3 | 53 ± 2.1 | 54.0 ± 3.9 | 46 ± 4 | 61.3 ± 0.9 | 63 ± 2 | 70 ± 11 |
| from anterior | (33–37) | (50–57) | (49–61) | (44–49) | (60–62) | (60–77) | (59–90) |
| Tail | 9 ± 3 | 22 ± 3 | 25 ± 2 | 23 ± 3 | 27.5 ± 1.4 | 29 ± 3 | 31 ± 5 |
| (7–11) | (19–28) | (21–28) | (21–25) | (26–29) | (26–35) | (20–37) | |
| Anal body width | 4 ± 1 | 9 ± 1.1 | 9 ± 2 | 8 ± 1 | 10 ± 2 | 17 ± 2 | 11 ± 2 |
| (4–5) | (7–10) | (6–11) | (7–9) | (9–13) | (12–19) | (8–14) | |
| Genital primordium | 7 ± 1 | 20 ± 5 | 15 ± 2 | 78 ± 25 | 132 ± 40 | 472 ± 68 | 526 ± 90 |
| length (GPL) | (6–8) | (14–28) | (12–17) | (60–95) | (77–160) | (355–581) | (413–630) |
| Genital primordium | 2.7 ± 0.1 | 5 ± 0.4 | 6 ± 1 | 5 ± 1 | 8 ± 2 | 14 ± 3 | 16 ± 3 |
| Width (GPW) | (2.5–3.0) | (4–5) | (4–7) | (4–6) | (6–10) | (11–14) | (12–21) |
| Spicule length | – | – | – | – | – | 17 ± 1 | – |
| along arc | (16–19) | ||||||
| a | 23 ± 2 | 40 ± 8 | 36 ± 4 | 31.6 ±1.2 | 40 ± 7 | 40 ± 5 | 53 ± 8 |
| (22–24) | (32–58) | (32–41) | (31–33) | (33–49) | (34–50) | (41–60) | |
| b | 4.5 ± 0.4 | 6.1 ± 0.3 | 6.0 ± 0.3 | 8.0–1.2 | 6.6 ± 0.6 | 9.7 ± 1.2 | 11.1–2.0 |
| (4.2–4.8) | (5.6–6.5) | (5.6–6.3) | (7.1–8.9) | (5.7–7.0) | (7.7–11.3) | (8.8–14.1) | |
| b’ | 2.5 ± 0.5 | 3.3 ± 0.2 | 3.3 ± 0.2 | 4.3 ± 1.1 | 3.9 ± 0.4 | 5.8 ±0.6 | 6.3 ± 1.0 |
| (2.1–2.8) | (3.1–3.6) | (3.0–3.6) | (3.6–5.1) | 3.4–4.3) | (4.9–6.6) | (4.6–7.6) | |
| c | 19.0 ± 4.3 | 16.6 ± 2.1 | 15.2 ± 0.5 | 20.0 ± 2.4 | 17.9 ±1.5 | 24.3 ± 2.9 | 27.7 ± 5.5 |
| (15.9–22.1) | (12.4–20.5) | (14.6–16.2) | (18.4–21.7) | (16.8–19.9) | (20.4–30.5) | (22–37) | |
| c’ | 2.1 ± 0.4 | 2.7 ± 0.4 | 2.8 ± 0.4 | 2.8 ± 0.1 | 2.8 ± 0.4 | 1.8 ±0.2 | 2.9 ± 0.3 |
| (1.8–2.4) | (2.3–3.4) | (2.4–3.4) | (2.7–2.9) | (2.3–3.1) | (1.4–2.2) | (2.4–3.4) | |
| V % or (V): genital primordium center from anterior, to body length, in % | 65 ± 2 | 61 ± 2 | 66 ± 2 | 58 ± 12 | 75 ± 2 | – | 75 ± 2 |
| (63–66) | (56–64) | (63–68) | (49–66) | (72–77) | (73–77) | ||
| GPL/GPW | 2.6 ± 0.4 | 4.3 ± 1.0 | 2.4 ± 0.2 | 17 ± 10 | 16.8 ± 3.0 | 34 ± 7 | 34 ± 8 |
| (2.4–2.9) | (3.0–5.7) | (2.2–2.8) | (10–24) | (13.6–20.6) | (26–49) | (24–46) | |
| GPL/L, % | 4.3 ± 0.04 | 5.4 ± 1.1 | 4 ± 0.3 | 18 ± 10 | 27 ± 8 | 67 ± 4 | 62 ± 6 |
| (4.26–4.32) | (4.2–7.1) | (3–4) | (11–25) | (16–33) | (58–72) | (55–71) | |
| Lip region length to its | 1.8 ± 0.3 | 1.9 ± 0.2 | 1.9 ± 0.1 | 1.7 ± 0.1 | 2.3 ± 0.1 | 2.0 ± 0.3 | 1.9 ± 0.2 |
| Width | (1.6–2.0) | (1.6–2.3) | (1.8–2.1) | (1.7–1.8) | (2.2–2.5) | (1.6–2.5) | (1.6–2.2) |
| Median bulb length to | 1.6 ± 0.1 | 1.4 ± 0.3 | 1.4 ± 0.1 | 1.4 ± 0.3 | 1.3 ± 0.2 | 1.3 ± 0.1 | 1.4 ± 0.2 |
| Its width | (1.6–1.7) | (1.0–1.9) | (1.2–1.6) | (1.2–1.6) | (1.0–1.5) | (1.1 ± 1.5) | (1.2–1.8) |
Figure 1
Bursaphelenchus crenati. Development in egg. A. Cleavage; B. Gastrula; C,D. First stage juvenile (J1) within egg shell, at different focal planes; E,F,G. Second stage juvenile (J2) molting with shed cuticle, at different focal planes; H. Hatching J2, preparing to leave the egg shell. Scale 20 µm.
Figure 2
Bursaphelenchus crenati. Methylene blue staining. Junior juveniles. A,B. Second stage juvenile (J2): A. Genital primordium; B. Tail; C,D. Genital primordium of J3 female juveniles (arrows: anterior germinal zone); E,F. Tails of female J3; G, H. Genital primordium of J3 male juveniles (arrows: posterior germinal zone); I,J. Tails of male J3 (arrows: cloaca primordium). Scale 20 µm.
Figure 3
Bursaphelenchus crenati. Methylene blue staining. Genital primordium and tails of the fourth stage juveniles and adults. A. Genital primordium of the fourth stage female juvenile (J4); B,C. Molting female juvenile J4-adult; B. Genital primordium; C. Tail; D. Genital system of adult female; E, F. Fourth stage male juvenile; E. Genital primordium; F. Tail and cloacal primordium; G. Genital system of adult male. Scale 50 µm.
Figure 4
Position of genital primordia in bodies of male juveniles and adults. Scale 50 µm.
Figure 5
Position of genital primordia in bodies of female juveniles and adults. Scale 50 µm.
Table 2
Measurements (in µm) and ratios of the development stages of Bursaphelenchus crenati.
| Stage/Character | J2→J3 male | J2→J3 female | J3→J4 male | J3→J4 female | J4→male | J4→female | Dauer J3D |
|---|---|---|---|---|---|---|---|
| n | 16 | 5 | 10 | 20 | 5 | 17 | 20 |
| L | 347 ± 7 | 323 ± 86 | 370 ± 3 | 377 ± 10 | 596 ± 91 | 544 ± 69 | 413 ± 20 |
| (338–354) | (181–410) | (366–374) | (364–398) | (532–660) | (433–614) | (392–463) | |
| Body width | 9 ± 1 | 10 ± 2 | 11 ± 2 | 12 ± 1 | 16 ± 0.3 | 13 ± 3 | 8 ± 1 |
| (8–10) | (8–14) | (9–13) | (11–13) | (14–17) | (9–17) | (6–9) | |
| Pharynx | 107 ± 4 | 103 ± 21 | 110 ± 5 | 110 ± 5 | 112 ± 9 | 117 ± 6 | 119 ± 9 |
| (104–112) | (65–116) | (105–115) | (102–117) | (105–118) | (109–123) | (101–128) | |
| Pharyngeal gland | 49 ± 5 | 51 ± 1 | 49 ± 4 | 48 ± 4 | 46 ± 1 | 48 ± 6 | 43 ± 7 |
| (44–56) | (49–53) | (44–55) | (41–54) | (45–47) | (40–55) | (35–55) | |
| Stylet | 7 ± 1.2 | 8 ± 1 | 7 ± 1 | 9 ± 0.7 | 9 ± 0.4 | 9 ± 1.4 | 9.4 ± 0.4 |
| (6–9) | (6–9) | (6–8) | (8–10) | (9–10) | (8–11) | (9–10) | |
| Lip region width | 5.0 ± 0.3 | 5 ± 0.6 | 5.6 ± 0.5 | 5 ± 0.6 | 5 ± 0.4 | 5.4 ± 0.4 | 4.8 ± 1.0 |
| (4–5) | (4–6) | (4.8–6.0) | (4–6) | (5–6) | (5–6) | (4–7) | |
| Lip region height | 3 ± 0.3 | 2.4 ± 0.2 | 2.4 ± 0.1 | 3 ± 0.3 | 3 ± 1 | 2.9 ± 0.4 | 2.9 ± 0.3 |
| (2–3) | (2–3) | (2.3–2.5) | (2–3) | (2–3)– | (2–3) | (2.5–4) | |
| Median bulb from | 46 ± 1 | 45 ± 8 | 47 ± 0.1 | 47 ± 3 | 46 ± 12 | 53 ± 3 | 49 ± 2 |
| Anterior | (45–47) | (31–50) | (46–48) | (40–50) | (38–54) | (51–58) | (45–51) |
| Median bulb length | 11 ± 1.2 | 11 ± 0.9 | 12 ± 1 | 12 ± 1 | 13 ± 1 | 13 ± 0.2 | 11 ± 2 |
| (10–13) | (10–12) | (11–13) | (10–13) | (12–14) | (12.5–13.5) | (9–15) | |
| Median bulb width | 9 ± 1.1 | 8 ± 1.5 | 10 ± 1 | 10 ± 1 | 9.9 ± 0.2 | 10 ± 1 | 8 ± 2 |
| (8–10) | (6–10) | (9–11) | (7–10) | (9.5–10.0) | (9–11) | (6–10) | |
| Excretory pore | 51 ± 1 | 50 ± 8 | 53 ± 2 | 54 ± 4 | 52 ± 12 | 58 ± 3 | 40 ± 3 |
| from anterior | (50–52) | (37–56) | (51–54) | (45–59) | (44–61) | (54–62) | (37–44) |
| Tail | 22 ± 4 | 21 ± 6 | 22 ± 1 | 26 ± 1.5 | 28 ± 5 | 28 ± 3 | 37 ± 3 |
| (20–28) | (11–27) | (21–24) | (24–28) | (25–32) | (23–31) | (30–41) | |
| Anal body width | 8 ± 0.4 | 8 ± 3 | 11 ± 2 | 11 ± 1.1 | 11 ±3 | 10.1 ±0.9 | 8 ± 1 |
| (7–8) | (5–12) | (8–13) | (10–13) | (9–13) | (9–11) | (5–9) | |
| Genital primordium | 18 ± 4 | 10 ± 3 | 20 ± 2 | 18 ± 5 | 158 ± 138 | 197 ± 105 | 14.8 ± 2.1 |
| Length (GPL) | (14–23) | (6–14) | (17–22) | (13–28) | (60–255) | (122–369) | (11–18) |
| Genital primordium | 5 ± 1 | 5 ± 1 | 4.8 ± 05 | 5 ± 1 | 10 ±5 | 9 ± 3 | 3.9 ± 0.4 |
| Width (GPW) | (4–5) | (3–7) | (4.0–5.3) | (3–6) | (6–13) | (7–14) | (3–5) |
| Spicule length | – | – | – | – | – | – | – |
| Along arc | |||||||
| a | 40 ± 5 | 34 ± 8 | 34.8 ± 5.2 | 32 ± 2 | 39 ± 12 | 41.5 ± 4.6 | 54 ± 8.3 |
| (34–46) | (22–41) | (28.4–41.8) | (29–35) | (31–48) | (36.8–48.9) | (44.6–69.6) | |
| b | 6.0 ± 0.3 | 5.6 ± 0.6 | 6.1 ± 0.2 | 6.1–0.2 | 9.1 ± 0.3 | 7.8 ± 0.6 | 5.5 ± 0.4 |
| (5.7–6.4) | (4.8–6.4) | (5.9–6.3) | (5.8–6.5) | (8.9–9.2) | (6.7–8.3) | (5.1–6.4) | |
| b’ | 3.2 ± 0.1 | 3.1 ± 0.3 | 3.4 ± 0.2 | 3.4 ± 0.2 | 5.3 ± 0.4 | 4.6 ±0.5 | 3.5 ±0.3 |
| (3.1–3.3) | (2.8–3.6) | (3.2–3.6) | (3.1–3.7) | (5.1–5.6) | (4.0–5.2) | (3.0–4.1) | |
| c | 16.0 ± 2.5 | 15.6 ± 0.6 | 16.6 ± 0.9 | 14.8 ± 0.8 | 21.1 ± 0.9 | 19.7 ± 1.7 | 11.2 ± 0.9 |
| (12.4–17.9) | (14.7–16.2) | (15.1–17.3) | (13.8–1.58) | (20.5–21.7) | (18.0–22.3) | (9.9–13.2) | |
| c’ | 2.8 ± 0.4 | 2.8 ± 0.5 | 2.1 ± 0.3 | 2.3 ± 0.2 | 2.6 ±0.1 | 2.8 ± 0.3 | 4.7 ± 0.5 |
| (2.4–3.4) | (2.2–3.4) | (1.7–2.5) | (2.0–2.8) | (2.6–2.7) | (2.3–3.2) | (4.3–5.5) | |
| V % or (V): genital primordium center from anterior, to body length, in % | 61 ± 2 | 64 ± 3 | 62 ± 1 | 66 ± 2 | 52 ± 3 | 73.2 ± 1.6 | 62 ± 5 |
| (59–64) | (59–68) | (60–64) | (63–68) | (49–54) | (70.5–74.3) | (56–72) | |
| GPL/GPW | 4.0 ± 1.1 | 2.3 ± 0.6 | 4.2 ± 0.2 | 3.9 ±1 | 15 ± 6 | 20 ± 4 | 3.8 ± 0.5 |
| (3.0–5.4) | (1.5–2.9) | (3.9–4.5) | (2.7–6.1) | (10–19) | (16–27) | (3–4.5) | |
| GPL/L, % | 5.2 ± 1.2 | 3.3 ± 1.0 | 5.5 ± 0.6 | 5 ± 1 | 25 ± 19 | 35.3 ±15.2 | 4 ± 1 |
| (4.2–6.6) | (1.8–4.3) | (4.5–5.9) | (4–7) | (11–39) | (24.3–60.0) | (3–5) | |
| Lip region length to its | 1.8 ± 0.3 | 2.0 ± 0.3 | 2.3 ± 0.2 | 1.9 ± 0.2 | 2.1 ± 0.6 | 1.9 ± 0.1 | 1.6 ± 0.3 |
| Width | (1.6–2.3) | (1.6–2.4) | (2.0–2.6) | (1.6–2.4) | (1.7–2.5) | (1.8–2.0) | (1.4–2.4) |
| Median bulb length to | 1.4 ± 0.2 | 1.4 ± 0.2 | 1.2 ± 0.1 | 1.2 ± 0.1 | 1.3 ± 0.2 | 1.3 ± 0.1 | 1.4 ± 0.2 |
| Its width | (1.1–1.6) | (1.3–1.7) | (1.2–1.3) | (1.0–1.4) | (1.2–1.4) | (1.2 ± 1.4) | (1.1–1.7) |
Figure 6
Bursaphelenchus crenati. Molting juveniles (shedding cuticle in tail). A,B. Molting male juvenile from J2 to J3; A. Genital primordium (gp); B. Tail and cloacal primordium (cp); C-H. Molting male juvenile from J3 to J4; C. Genital primordium of molting J3-J4 (aac – anterior apical cell, sz – somatic zone, gerz -germinal zone); D, E, H. Tail and cloacal primordium (cp) of J3-J4 male juvenile; F. Genital primordium of molting male J3-J4 juvenile at “loop stage”; G. Drawing of F with explanation of the reversion of the somatic zone (sz) with anterior apical cell (aac) flexed to the germinal zone (gerz); gc – germinal cells; pac – posterior apical cell, sn – somatic nuclei, vc – ventral cord nuclei; I,J – molting female J3-J4 juvenile; I. Genital primordium with anterior germinal zone (gerz); J. Tail. Scale 50 µm.
Table 3
Tabular key to juvenile stages of Bursaphelenchus crenati.
| C1 | C2 | C3 | C4 | C5 | C6 | |
|---|---|---|---|---|---|---|
| Stage/Character | Copulative structures | Average body size, in µm | Ratio: genital primordium to body length, in % | Genital primordium cellular structure | Cloaca primordium or cloaca around rectum | Genital primordium somatic part: a division to sections |
| J2 | Absent | 170 (150–180) | 4 | Small with two large germinal cells in center | Absent | Absent |
| J3 male | Absent | 370 (340–400) | 5 (4–7) | 2–5 large germinal cells in the posterior part of primordium, 12 small somatic nuclei in anterior part | Present | Absent |
| J3 female | Absent | 380 (340–400) | 4 (3–4) | 2–5 large germinal cells in the anterior part of primordium, 12 small somatic nuclei in posterior part | Absent | Absent |
| J4 male | Absent | 460 (380–530) | 18 (11–25) | 20–60 large germinal cells in anterior part of primordium and 30–60 somatic cells in posterior part | Present | Absent |
| J4 female | Absent | 490 (480–510) | 27 (16–33) | 20–60 large germinal cells in anterior part of primordium and 30–60 somatic cells in posterior part | Absent | Absent |
| Male, adult | Paired spicules in cloaca, at tail end | 700 (610–800) | 67 (58–72) | More than 60 germinal cells in anterior part | Present | Absent |
| Female, adult | Vulva at 75% body length from anterior end | 840 (750–1100) | 62 (55–71) | More than 60 germinal cells in anterior part | Absent | Present |
Figure 7
Diagrams of juvenile growth. A. Body length; B. Ratio of genital primordium length to body length (GP/L, %). Standard deviation (STD) levels are marked for mean values.
Figure 8
Dauer juveniles. Methylene blue staining. A. Male juvenile J3D; B. Female juvenile J3D; C. Genital primordium of male dauer juvenile; D. Tail of male dauer juvenile. Scale 50 µm for A,B and 20 µm for C,D.
Figure 9
Dauer juveniles molted to J4D after 3 hr in 0.9% NaCl solution. A. Male juvenile; B. Female juvenile.