Microscopic Observations on Form and Structure of the Worm Enchytraeus buchholzi (Clitellata: Enchytraeidae)

The worm Enchytraeus buchholzi is a new record species for Shaanxi, China, and a key pest on American ginseng Panax quinquefolium. To distinguish the species, the authors prepared its whole mounts and paraffin-embedded sections, and microscopically observed, photographed and measured. Besides, we conducted an experimental study on its DNA barcode. Cells, tissues and organs related to nervous, digestive, circulatory, excretory and reproductive systems were found, photomicrographed and described, including: prostomium, peristomium, segments, clitellum, pygidium, lateral and ventral chaetae; brain, cranial nerves, sensory papillae, ventral nerve cord; pharyngeal pad and glands, retractor muscles and muscular bundles, peptonephridia, esophagus, intestine; dorsal, lateral, ventral and intestinal parietal vessels, coelomocytes, coelomic cavity; nephridia, chloragogen cells; ovaries, groups of germ cells with developing oocytes, mature eggs, spermathecae; testes, seminal vesicles, sperm funnels, penial bulbs. Their shapes and sizes were given, and functions discussed briefly. The visual effect of staining specimens with hematoxylin plus eosin ranked the first, and that with acetocarmine the second. The supplementary and objective descriptions, with the microphotographs as forceful pieces of evidence, have expanded biological knowledge in aspects of the form, structure and function of the worm, which is helpful for professionals to recognize and understand this species and provide a solid basis for its integrated pest management.

The worm E. buchholzi has become a new record species for Shaanxi, China and a key pest on P. quinquefolium in Liuba County, Shaanxi Province. In order to distinguish this new pest, the authors prepared its whole mounts and paraffin-embedded sections, and microscopically observed, photographed and measured its sizes of forms and structures. These new observations would expand its biological knowledge, help professionals recognize the worm, and provide a basis for its integrated pest management (IPM).

Taxonomic Status
Three DNA sequences obtained in the formal experiment, with 676 base pairs each, were compared with those in BOLD Systems [17], which were matched to the species Enchytraeus buchholzi, with their similarities ranging from 98.89% to 100%. Thus, the DNA molecular analysis and identification suggested the specimens collected from Liuba be Enchytraeus buchholzi at species level. All the following descriptions are given under the new scientific name.

Body Form
Enchytraeus buchholzi is threadlike and monoecious or hermaphroditic. Living adults are 5 to 8 mm long; when fixed, length ca. 5 mm or slightly longer, width 180 ± 27 μm (sample size, n = 86) at segment V, and widest 285 ± 48 μm (n = 86) at clitellum. Its body consists of a prostomium, 27 -30 segments and a pygidium, with the number of segments changing slightly owing to individual variations (Fig. 1). Domelike projections, the sensory papillae, are distributed densely on the epidermis of the prostomium ( Fig. 2A, B); there is no chaeta on the epidermis of peristomium (segment I) ( Fig. 2A). Each segment from II to XXIX has a pair of lateral chaetal bundles with 2 or 3 chaetae, and a pair of ventral chaetal bundles with 3 chaetae except in XII and XIII (Fig. 11B). The pygidium has no chaeta but many sensory papillae on the epidermis ( Fig. 2C; Fig. 10B). Chaetal formula is 2-2,3: 3-3. Chaetae are straight with rarely weakly bent at the distal end, 35-44 μm long, only a few with nodules ( Fig. 2A, C; Fig. 11B) [18,19]. The clitellum covers XII and XIII, which can be distinguished by the two pairs of lateral chaetal bundles arranged longitudinally on each side though there is no intersegmental furrow outside and no septum inside ( Fig. 1A; Fig. 11B).

Anterior Portion
The prostomium, the clitellum and segments between them constitute the anterior portion of the worm, ca. 1/3 of the full length, where the internal organs are relatively gathered.
Nervous System: There is the brain, long eggplantshaped in lateral view and inverted pear-shaped in dorsal or ventral view, 96 ± 14 μm long (n = 40), 19 ± 3 μm wide at the neck (n = 40), 48 ± 7 μm wide (n = 20) at the widest part, and 38 ± 6 μm thick (n = 20), located in I, II and partial III (Fig. 3A-C). The brain bifurcates in front part (Fig. 3B), and is rounded in posterior margin (Fig. 3C). It extends several cranial nerves, two thicker ones forward to the sensory papillae on the epidermis of the prostomium, two backward to the pharyngeal pad, and also stretches circumpharyngeal connectives downward to the subpharyngeal ganglia in II, linking the ventral nerve cord, 26 ± 9 μm wide and 18 ± 3 μm thick (n = 15), till the last segment before the pygidium (Fig. 10). The ventral nerve cord exposes only a little in I (Fig. 3D, E), and is relatively wider and thicker in II, III and IV than that in the rest segments ( Fig. 3A; Fig. 4D-G; Fig. 5D, G). Digestive System: The mouth opens on the ventral side of the peristomium (Fig. 3A, D), which is followed by the buccal cavity, pharyngeal pad, esophagus, intestine and anus (Figs. 3D,4,5,6,7,8,9 and 10). The dome-like pharyngeal pad, 92 ± 16 μm long (n = 32), 77 ± 11 μm wide (n = 11) and 40 ± 6 μm thick (n = 21), is rich in muscles and has, at the top, a muscular knot and ring ( Fig. 4A-E, G). The knot extends many retractor muscles to attach to the body wall on dorsal, lateral and ventral sides ( Fig. 4A-C, E, G), and the ring encircles the anterior part of the peptonephridia (esophageal appendages) and esophagus (Fig. 4C, F, G). The Fig. 2 Form of prostomium, segments I -III, XXVIII and XXIX, and pygidium. A. Prostomium, segments I (peristomium), II and III (mod-in-Hm), ventral view. B. Prostomium, transversal section (std-in-HE), anterior view. C. Segments XXVIII and XXIX, and pygidium (mod-in-Hm), ventral view. Labels abbreviated and their meanings (the same as below): lcb, lateral chaetal bundle; nod, nodule; pro, prostomium; py, pygidium; sp, sensory papillae; vcb, ventral chaetal bundle Fig. 3 Structure of prostomium, I, II and III (all std-in-HE). A. Prostomium, I, II and III, lateral view. B. Brain, showing bifurcation in its front part, dorsal view. C. Brain, showing round-shape in its posterior margin, dorsal view. D. Prostomium, I and II, longitudinal section, lateral view. E. Segment I, transversal section, anterior view. ac: anucleate coelomic corpuscle; ann, annulus; bc, buccal cavity; br, brain; bw, body wall; cpc, circumpharyngeal connective; crn, cranial nerve; dv, dorsal vessel; epc, epithelial cell; esc, epithelial sensory cell; isf, intersegmental furrow; lv, lateral vessel; mo, mouth; mu, muscle; per, peristomium; php, pharyngeal pad; pro, prostomium; rm, retractor muscle; spg, subpharyngeal ganglion; sub, sinus underneath the brain; vcs, ventral chaetal sac; vnc, ventral nerve cord peptonephridia, 48.6 μm long and 22.2 μm in diameter at its anterior part ( Fig. 4A-C), is attached to the back of the pharyngeal pad; the rest have two branches, coiled and hyaline, 96.4 μm long; the full length is 145 μm and the whole is confined in IV ( Fig. 4A-C, F, G). Each segment from IV to VI has a pair of pharyngeal (septal) glands, surrounding esophagus and squeezing the postseptum of the segment to the rear, especially in IV and V (Fig. 5A, B). The pharyngeal glands in IV and V are composed of elliptic dorsal and ventral lobes and thin strands, and the dorsal lobes are larger than the ventral ones (Fig. 5A, B; Table 1). The pharyngeal glands in VI are also made up of dorsal and ventral lobes and thin strands, but sometimes there appears a smaller middle lobe between them (Fig. 5B). The dorsal lobes in IV and V are separate (Fig. 5B), but the ones in VI connected dorsally (Fig. 5B, I). Their sizes are listed in Table 1.
Excretory System: The nephridia are spindle-shaped, 126 ± 28 μm long and 31 ± 5 μm wide (n = 16) excluding tubules; there is a pair of them in each of the segments from VII to X, in septa 6/7, 7/8, 8/9 and 9/10, lying on both sides of the ventral nerve cord ( Fig. 5H; Fig. 6A, B). In addition, there are similar structures in each of the segments between the clitellum and the pygidium (Fig. 9B, C, E; Fig. 10A).
Reproductive System: Male genital organs: There is a pair of elliptic testes, 14.0 ± 0.9 μm long and 9.7 ± 0.0 μm wide (n = 5), lying both sides of the ventral vessel and attaching to the anteseptum of XI closely (Fig. 7C). Nearby is a pair of seminal vesicles in different shape and size, 122 ± 34 μm long (n = 81), 102 ± 34 μm wide (n = 36) in lateral view and 74 ± 25 μm wide (n = 45) in dorsal or ventral view, with many spots on their surfaces ( Female genital organs: Attaching to both sides of the ventral nerve cord and near anterior margin of XII, there appears a pair of grape-like ovaries in which the immature germ cells, oogonia, are located (Fig. 8A); on the same site the figure also shows that XII is filled with groups of germ cells with developing oocytes that are globular, oval, or in other shape, with only a few cut open but the majority covered with many nurse cells stained blue except their nucleus stained slightly red. These groups of germ cells are aggregated into clusters in different shape, and the sizes of the clusters are: 181 ± 55 μm long and 172 ± 56 μm wide (n = 28) in lateral view; 149 ± 45 μm long and 93 ± 24 μm wide (n = 46) in dorsal or ventral view (Fig. 8A-D). They are also found in XI (Fig. 7A, C, D), and spirally arranged around two centers in dorsal or ventral view (Fig. 7C). The distance is 90 ± 14 μm (n = 5) between the two centers.  (Fig. 8C, F-J). When there are more mature eggs, they may extend backward to other segments after the clitellum in vivo.
There is a pair of spermathecae connecting with their short ectal and long ental ducts, located on both sides of the esophagus and above the ventral lobes of pharyngeal glands in V (Fig. 5A, B, E-G), which open on the ventral body wall, with glandular tissues surrounding their ectal orifice inside (Fig. 5A) and muscular epidermis (cushion) outside (Fig. 5E). The two pieces of muscular epidermis are located along the two longitudinal lines of ventral chaetal bundles, and close to the anteseptum of V ( Fig. 4A; Fig. 5E). Their ental ducts stretch diagonally forward to and attach to lateral body wall (Fig. 5B). The ampullae of spermathecae are elliptical or spherical, 42 ± 8 μm long and 32 ± 7 μm wide (n = 26), full of cilialike spermatozoa inside (Fig. 5F, G).
Other Systems: The body wall consists of thin cuticle, epidermal and muscular layers (circular and longitudinal) ( Fig. 3A; Fig. 5F; Fig. 9D, E). Both the muscular layers and the coelomic fluids, through the former acting against the latter, form a hydrostatic skeleton, which maintains the shape and toughness of the organism under living conditions. Exchange between oxygen and carbon dioxide is performed through the moist body wall.

Posterior Portion
Segment XIV, the pygidium and segments between them constitute the posterior portion; all the segments in this portion take on homonomous metamerism except the ipv, intestinal parietal vessel; mu, muscle; meg, mature egg; miv, microvillus; ne, nephridium; n, nucleus; nc, nurse cell; ov, ovary; pb, penial bulb; pm, posterior margin of XIII; spf, sperm funnel; ucg, unicellular gland; vnc, ventral nerve cord; vv, ventral vessel; yog, yolk granule pygidium. Expressed from outside to inside sequentially, the structure of each layer in these segments is: body wall, ventral nerve cord, nephridia, coelomic cavity, coelomocytes, ventral vessel, chloragogen cells, intestinal parietal vessel, muscular wall of intestine, intestinal parietal cells, and intestinal lumen (Fig. 9B-F). In addition, segments from VII to X in the anterior portion have similar structures (Fig. 5H; Fig. 6A, B), with the intestinal parietal vessel substituted by the dorsal vessel. The segment before the pygidium contains many intestinal parietal cells, and the ventral nerve cord becomes relatively thicker there (Fig. 10A, B).

Eggs and Their Development
Under living conditions, E. buchholzi is pale to milky white ( Fig. 1A; Fig. 11D), and reproduces asexually by parthenogenesis; cross fertilization may occur at lower temperatures (12-18 °C). Several mature eggs are wrapped within a cocoon (Fig. 11A-C), which is deposited singly or collectively, and hidden with dusts and debris by parent adults. An adult produces several tens of cocoons in its life time when feeding on American ginseng root powders in a wet-sandy dish indoors. The number of mature eggs is 1-2 per cocoon in early stage, and rises quickly to 3-10 per cocoon with time elapsing, sometimes even up to 15 in a cocoon. In the first 50 days of adults' reproductive period, the mean number was ever maintained at ca. 8 eggs per cocoon as shown in Fig. 11C, possibly due to the rich nutrition of American ginseng root powders. The outline of mature eggs in a cocoon is clear when the latter is just laid (Fig. 11C); as the embryonic development proceeds, the outline of the eggs inside becomes blurred in vivo. A newly hatched young worm is about 1 mm long; after continuous feeding and direct developing, the worm grows up to an adult when there are about two mature eggs appearing in its clitellum and its color turns from pale to milky white. After that, the adult continues injuring American ginseng severely within roots (Fig. 11D) as well as reproducing more offspring.

Functional Discrimination and Analysis
Internal organs of the worm are relatively concentrated in its anterior portion, especially in both sections from the prostomium to VI and from XI to XIII. The worm moves freely and quickly inside the host plant and between particles of wet-sandy soils, which is closely related to its developed brain, ventral nerve cord, densely sensory papillae on the prostomium and pygidium, muscular tissues in the body and intestinal walls, and lateral and ventral chaetae.
The pharyngeal pad proper is rich in retractor muscles, and with the help of the muscular knot and ring that extends several muscular bundles out to attach to the body wall, it thus strengthens the effect of grinding food. The anterior part of the peptonephridium opens to basal pharyngeal pad; three pairs of pharyngeal glands connect the pharyngeal pad through tubules or strands [20,21]. Their functions should be secreting digestive enzymes and enhancing digestive powers by means of extracellular digestion, an important link in its life process.
Seen in I to XI, the dorsal, lateral and ventral vessels were stained red with the HE dyeing way. The ventral and intestinal parietal vessels can also be seen in segments in the posterior portion (Fig. 9B-F), which shows: 1) the ventral vessel extends between the ventral nerve cord and the layer of chloragogen cells, transporting metabolic wastes to the rear; and 2) the intestinal parietal vessels form a network that encircles the digestive tract, absorbing nutritional fluids from intestinal parietal cells, turning them into fresh colorless blood, and carrying it forward. The inlet of dorsal vessel acts as a valve that prevents blood from returning to the rear. The color of the nephridia in the ventral side of VII to X and other segments in posterior portion is pale, showing the excretory function may reduce staining extent in corresponding organs. Nucleate coelomocytes may be the weaver of septa and anucleate coelomocytes may function as lymphatic bodies.
Although the size of the ovaries is small, they reproduce oogonia continuously. Generally, after numerous growing and mitotic divisions, some oogonia develop and enlarge into primary oocytes; undergoing a full meiosis, each oocyte forms three polar bodies and a large haploid ootid; the polar bodies disintegrate and the ootid matures into the egg [22]. The germ-line cysts in Enchytraeus albidus ovaries consist of 16 cells; during oogenesis, the fate of interconnected germ cells differentiates and only one cell develops as the future egg, while the other 15 become nurse cells [23]. Recalling the photomicrographs in Figs. 7 and 8, almost all the features of oogenesis in the target worm are similar to those in E. albidus. The nurse cells should have produced all the yolk granules, with their chemical properties changing from basophilic to acidophilic, thus stained red. Being bathed in nutrientrich coelomic fluids in the clitellum, each oocyte and accompanied nurse cells are able to develop rapidly.

Comparisons with the Type Species and Early Descriptions
The worm Enchytraeus buchholzi was first described as a widespread species by Vejdovský in German in 1878 [4]; characters such as body length, segment number, chaetae per bundle, salivary glands (peptonephridia, esophageal appendages), seminal vesicles, sperm funnels, and vas deferens or sperm duct were introduced (Table 2), with other phenomena (intestinal canal covered with glands, segmental organs flowing to outside, ovaries maturing earlier, mature eggs falling into the body cavity, etc.) recorded. A monograph on the class Oligochaeta was compiled by Michaelsen in German in 1900 [24], with the species E. buchholzi included and corresponding characters cited especially from the one published by Vejdovský in 1879 [25]. Morphotaxonomic studies and geographical distributions of the worm have been continuing in different parts of the world [26][27][28][29][30], and some early literature is cited to make comparisons with the specimens from Liuba (Table 2).
Comparisons in Table 2 show the specimens from Liuba are very similar to the type species on many traits, especially on the first three: body length, segment number, and chaetae per bundle. In conjunction with the result of the DNA molecular analysis, the targets are identified into Enchytraeus buchholzi, and thus recovered their original identity. There are, of course, some differences among those descriptions, probably owing to distinct habitats and individual variations ( Table 2). Based on the traits that seminal vesicles are larger than half diameter of segment V and the ental duct of the spermatheca attaches to lateral body wall ( Table 2; Fig. 5B and the text written above), the specimens may be identified into a subspecies, namely Enchytraeus buchholzi ssp. liubaensis.
The number of chaetae and their shape also contribute significantly to the identification of species in the family Enchytraeidae [4]; according largely to the trait, Vejdovský ever distinguished and named seven new species and redescribed two in the genus Enchytraeus [4]. The first two characters, segment number and chaetae per bundle, are the most important traits to distinguish enchytraeid species; the third one, body length, should be less important because of its varying with segment number. Largely relying on the trait, 2-3 chaetae per bundle, with supports of some other traits, several enchytraeids were identified new at species level after 1959; for example, the potworm Enchytraeus bulbosus was new to science because its penial bulb is larger than half diameter of the clitellum [9], and the terrestrial species Enchytraeus luxuriosus was new because of its segment number around 45 [30].

Making Slide Specimens
Among the five methods used to make slide specimens, four led to whole mounts and one to paraffin sections, each of which has its own advantage though they are different in fixation, staining and mounting process. The HE dyeing way is recommended since different structures can be colored properly and thus easily recognized; staining in acetocarmine liquid is convenient for fast dyeing, with bright color and good effect. Compared with the slide specimens sectioned, the whole mounts are relatively thicker, and some parts are seen blurred because of beyond the depth of field under higher objective lens and of more tissue layers for light to penetrate. Specimens sectioned can be cut thinner, but some fine structures may be easy to wash away, resulting in image distortion.

Conclusions
All descriptions and microphotographs of the worm E. buchholzi, verified, revised and newly supplemented here, express essential characteristics of the species. Simultaneously, the new results expanded text descriptions, which would be helpful for professionals to recognize this small worm, understand its morphology, structure and physiological function. As forceful pieces of evidence, these microphotographs may support corresponding free-hand drawings published previously and correct something wrong, if any. Concerning IPM practice, the studies would help ginseng farmers aim at the target pest, manage it scientifically, and promote a vigorous development of the American ginseng planting industry.