Characterization of Juvenile Stages of Bursaphelenchus crenati Rühm, 1956 (Nematoda: Aphelenchoidoidea)

J2 differs from senior stages by the smallest number of cells in primordium as well as in the body size less than 200 µm vs body length of 300 µm or longer in juveniles of senior stages.

In the genital primordium, 3 to 5 germinal cells in the posterior part and 12 somatic nuclei in anterior part (Fig. 2G,H) are present. In addition, a single somatic apical nucleus at every extremity of the primordium is found. The primordium is situated in the middle of the intestine part of the body. Cloaca primordium is present as a dense agglomeration of somatic nuclei around the rectum (Fig. 2I,J).

In the genital primordium, 2 large germinal cells in the anterior part and 12 somatic nuclei in the posterior primordium part are present together with a single somatic apical nucleus on every extremity of the primordium (Fig. 2C,D). A circle of six somatic nuclei interrupt the ventral hypodermal cord chain of somatic nuclei. It is a primordium of vulva.

Differences of male juvenile of the third stage from female juvenile are manifested by the presence of cloaca primordium and the position of germinal cells in the posterior part of the primordium. In female juveniles, germinal cells are located in the anterior part of the primordium. Third stage juveniles differ from J2 in the number of cells of the primordium (more than 10 vs 4 in J2). From J4 juveniles, the third stage juveniles differ by the length of primordium occupying 6% of the body length or less vs 11% or more in J4 and adults.

Cloaca primordium is massive, with numerous somatic nuclei around rectum and a transparent cavity anterior to rectum with rudimentary spicules. The genital primordium is distinctly divided into an anterior germinal part of 30 to 60 large cells and a somatic part of two rows of somatic nuclei with 15 to 16 nuclei in every row. The somatic part is not divided into sections (Fig. 3G). Genital primordium occupies 11 to 25% of the body length (Fig. 4). Tail tip with narrowly conical 6 (4–9) µm hyaline zone is curved ventrally, corresponding to bursal flap of adult male.

Fourth stage female juvenile (Figs. 3, 5; Table 1). Genital primordium is divided into the anterior germinal part, consisting of 20 to 30 large cells and the posterior somatic part consisting of more than 60 somatic nuclei. On the ventral side of body wall in the middle of somatic primordium part, the lens-like invagination surrounded by massive structure attached to the ventral body wall, is distinct. It is the vulva primordium. Genital primordium occupies 16 to 33% of the body length (Fig. 5).

Differences between male juveniles of the fourth stage from female juveniles are manifested by the presence of cloaca primordium. In female juveniles, the lens-like transparent vulva primordium at the level of uterus primordium part is present. In J4 male juveniles, the vulva primordium is absent. The fourth stage juveniles differ from J3 in the larger genital primordium (11% of body length or more vs 6% or less in J3) and in numerous cells of the primordium: more than 50 in J4 vs 20 or less in J3. From adult nematodes, J4s differ in the absence of copulative organs vs spicules or vulva is present in adult nematodes.

Staining did not reveal any new morphological features in addition to detailed description (Gu et al., 2017).

In the center of primordium, 3 to 4 mitoses of somatic nuclei are distinct between germinal cells (Fig. 6A).

Figure 6

The genital primordium is initially straight, then transforming to loop-shaped (Fig. 6F,G), with the anterior apex and both somatic and germinal parts reversed posteriorly; the somatic part is set off by constriction from germinal part and its tip is curved hook-like (Fig. 6G). The cloaca primordium is enlarged with a transparent inner cavity anterior to the rectum (Fig. 6H). This molting phase is an indication of the orientation change of the somatic part, which in male juvenile J3 is located anteriorly and in J4 is shifted posteriorly, thus pulling a germinal part to the anterior direction (Fig. 6F,G).

The somatic part of the genital primordium in its posterior part possesses more than 20 somatic nuclei, the germinal part in the anterior part of the primordium with 5 to 10 large germinal cells.

The somatic part of the genital primordium reaches in some specimens the massive cloaca primordium, in which inner cavity transparent spicules outlines are distinct. The bursal flap is not formed, the tail is narrowly conical with 9 µm long hyaline zone.

Vulva primordium is a massive cellular agglomeration with an inner transversal slit, but without opening outside. All sections of the genital system are distinct: the ovary of 30 to 40 germinal cells, the oviduct, empty spermatheca, crustaformeria, and the uterus with inner empty cavity (Fig. 5).

Tabular key to juvenile stages of Bursaphelenchus crenati (Key is given in Table 3).

Figure 8

Figure 9

The dauer juveniles have the same genital primordium structure and size as J3. The differences are: the slender and straight body and straight long tail vs curved ventrally tail in the J3 of the propagative generation. Lateral field with two closely located bands of equal width (three incisures). Dauers differ in hemispherical continuous head with hyaline inner cap vs set off head and well developed cephalic circular framework. Stylet is very thin capillary tube, conus and basal thickenings not distinct. Median bulb elongated, its length twice as its width, central valve is weakly developed. Pharyngo-intestinal junction is one half of the median bulb length posterior to median bulb, surrounded by nerve ring with excretory pore at the posterior third of median bulb. Pharyngeal glands form very narrow band, gland nuclei indistinct.

In 3 hr after pouring beetle elytra with “nematangia” in water or 0.9% NaCl solution, the J3D juveniles started to molt to J4 juvenile with long genital primordium and well developed stylet and median bulb (Fig. 9).