Description of Geocenamus vietnamensis sp. n. (Nematoda: Merliniidae) from Vietnam
Article Category: Arts & Humanities
Online veröffentlicht: 27. Mai 2019
Seitenbereich: 1 - 12
Eingereicht: 04. Dez. 2018
DOI: https://doi.org/10.21307/jofnem-2019-025
© 2019 Huu Tien Nguyen et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
The genus
Based on the observations from two studies, Sturhan (2011, 2012) concluded that the absence of longitudinal cuticular striae along the entire body appears to be the only essential character that distinguishes
Following the classification of Sturhan (2012), this study provides morphological and molecular characterizations of a new species of the genus
Soil and root samples were collected from the rhizosphere of
For morphometric and morphological examination, the extracted nematodes were killed by heat, fixed in a TAF solution, processed to anhydrous glycerol, following Seinhorst (1959), and mounted on permanent glass slides. Nematodes on permanent slides were observed through a Carl Zeiss Axio Lab. A1 light microscope. Measurements and pictures were taken using the ZEN lite software on ZEISS Axiocam ERc5s digital camera. Raw photographs were edited with Adobe Illustrator CS 3.
After examination and identification, good specimens were selected for SEM imaging, following the protocol of Abolafia (2015). The nematodes were hydrated in distilled water, dehydrated in a graded ethanol and acetone series, critical point dried, coated with gold, and observed with a Zeiss Merlin Scanning Electron Microscope.
DNA was extracted from a single individual nematode, following Waeyenberge et al. (2000). The nematode was transferred to a 0.5 ml Eppendorf tube, containing 18 µl of Worm Lysis Buffer (50 mM KCL, 10 mM Tris pH 8.3, 2.5 mMMgCl2, 0.45% NP 40, and 0.45% Tween 20) and 2 µl of proteinase K (600 mg ml−1) (Thermo Scientific). The tubes were incubated at 65oC (1 hr) and then at 95oC (15 min). PCR and sequencing protocols are described in detail by De Ley et al. (1999). Primers for D2–D3 of 28S rDNA amplification were D2A (59-ACAAGTACCGTGGGGAAAGTTG-39) and D3B (59-TCGGAAGGAACCAGCTACTA-39) (Subbotin et al., 2006). Primers for ITS rDNA amplification were modified from Vrain et al. (1992): VRAIN 2F (59-CTTTGTACACACCGCCCGTCGCT-39) and VRAIN 2R (59-TTTCACTCGCCGTTACTAAGGGAATC-39).
The BLAST homology search program was used to search for closely related species on GenBank. The sequence data set was aligned with the ClustalW software (Thompson et al., 1994). Sequence alignments were manually edited using ChromasPro software (ChromasPro 1.7.4; Technelysium Pty Ltd, Tewantin, QLD, Australia). The sequence data set was analyzed using the maximum likelihood (ML) method in the MEGA 6 program (Tamura et al., 2013). The best fit model for DNA evolution was obtained using the Modeltest in MEGA 6. The model TN93 + G was chosen for D2–D3 of 28S rDNA data set and model GTR + G was chosen for ITS rDNA data set; 1,000 bootstrap replications were executed. Outgroup taxa were chosen according to the results of previously published data (Subbotin et al., 2006; Ghaderi et al., 2014). The trees were visualized in FigTree v1.4.0.
Morphometric data of
| Paratypes | ||||
|---|---|---|---|---|
|
|
Holotype female | Allotype male | Female ( |
Male ( |
| Body length (L) | 877 | 712 | 875 ± 64 (776–979) | 706 ± 35 (631–754) |
| Lip region height | 3.1 | 3.6 | 3.6 ± 0.5 (3.1–4.2) | 3.4 ± 0.3 (3.1–3.6) |
| Lip region width | 6.2 | 5.7 | 6.5 ± 0.7 (5.2–7.3) | 6.1 ± 0.5 (5.2–6.8) |
| Stylet cone | 18.7 | 15.6 | 15.7 ± 1.4 (13.5–18.7) | 15.1 ± 1.4 (13–17.7) |
| Stylet shaft | 9.4 | 8.3 | 10.4 ± 0.5 (9.4–11.4) | 8.8 ± 1.6 (5.7–10.9) |
| Stylet length | 28 | 24 | 26 ± 1 (24–28) | 24 ± 1 (23–26) |
| Stylet knob height | 2.1 | 1.6 | 2.0 ± 0.4 (1.6–2.6) | 1.9 ± 0.4 (1.6–2.6) |
| Stylet knob width | 4.2 | 3.1 | 3.5 ± 0.8 (1.6–4.2) | 3.6 ± 0.2 (3.12–3.6) |
| Stylet base to dorsal gland orifice (DGO) | 3.1 | 3.12 | 2.9 ± 0.3 (2.6–3.1) | 3.0 ± 0.3 (2.6–3.6) |
| Body width at stylet base | 16.6 | 11.9 | 16.7 ± 2.8 (13.0–20.8) | 13.7 ± 1.0 (11.9–15.6) |
| Maximum body diameter (MBD) | 31 | 22 | 29 ± 3 (25–34) | 22 ± 1 (22–25) |
| Body width at vulva | 22 | 27 ± 3 (22–34) | ||
| Body width at anus | 18.7 | 13 | 19.1 ± 2.4 (15.6–23.9) | 16.1 ± 2.6 (13–21.8) |
| Anterior end to the end of pharynx | 152 | 125 | 138 ± 9 (124–152) | 122 ± 7 (114–135) |
| Anterior end to the end of median bulb | 89 | 73 | 81 ± 8 (74–99) | 72 ± 5 (67–81) |
| Anterior end to median valve | 78 | 65 | 72 ± 7 (66–88) | 63 ± 5 (57–71) |
| Median bulb width | 10.9 | 9.8 | 11.0 ± 0.7 (10.4–12.5) | 9.6 ± 1.2 (6.8–10.4) |
| Median bulb length | 15.6 | 15.6 | 17.0 ± 1.6 (14.6–18.7) | 15.5 ± 1.2 (13.5–17.7) |
| Anterior end to nerve ring | 86 | 86 | 90 ± 6 (83–104) | 83 ± 8 (74–99) |
| Anterior end to secretory–excretory pore | 94 | 89 | 94 ± 5 (86–102) | 86 ± 6 (76–97) |
| Isthmus length | 39 | 31 | 32 ± 4 (26–39) | 30 ± 2 (26–31) |
| Basal bulb length | 27 | 21 | 25 ± 2 (23–28) | 22 ± 2 (19–25) |
| Tail length | 86 | 75 | 104 ± 14 (85–125) | 84 ± 9 (68–96) |
| Spicule length | 21 | 20 ± 1 (19–22) | ||
| Gubernaculum length | 5.2 | 6.5 ± 1.0 (5.2–7.8) | ||
| Anus to phasmid | 23 | 17.7 | 23 ± 3 (18.7–26) | 18.6 ± 2.6 (13.5–23) |
| Tail annuli (ventral) | 61 | 48 | 64 ± 5 (58–73) | 59 ± 6 (48–65) |
| a = L/MBD | 28 | 33 | 30 ± 3 (25–34) | 32 ± 1 (29–33) |
| b′ = L/distance from anterior end to pharyngo–intestinal valve | 5.8 | 5.7 | 6.4 ± 0.5 (5.8–7.2) | 5.8 ± 0.2 (5.5–6.2) |
| c = L/Tail length | 10.2 | 9.5 | 8.5 ± 0.8 (7.3–10.2) | 8.5 ± 0.7 (9.3–9.5) |
| c′ = Tail length/ABD | 4.6 | 5.8 | 5.5 ± 0.7 (4.3–6.5) | 5.3 ± 0.7 (4.3–6.3) |
| V = Anterior end to vulva/L ×100 | 50 | 49 ± 2 (47–52) | ||
| M = stylet conus length/stylet length ×100 | 67 | 65 | 60 ± 3 (56–67) | 62 ± 4 (57–69) |
| MB = % distance from anterior end to median bulb in relation to the length of pharynx | 51 | 52 | 52 ± 3 (49–59) | 51 ± 1 (50–53) |
| O = % distance of dorsal pharyngeal gland opening from stylet knobs in relation to the stylet length | 11.1 | 13.0 | 11.0 ± 1.3 (9.6–13.0) | 12.5 ± 1.3 (10.2–14.3) |
| Phasmid % tail | 27 | 24 | 22 ± 2 (18.3–27) | 22 ± 3 (17–28) |
Figure 1:
Figure 2:
Figure 3:
Figure 4:
Figure 5:
Phylogenetic tree generated from D2–D3 of 28S rDNA sequences based on ML method (TN93 + G model) with 1,000 replications.
Figure 6:
Phylogenetic tree generated from ITS rDNA sequences based on ML method (GTR + G model) with 1,000 replications.
Their body is ventrally curved after fixation, tapered at both ends. The lateral field is present with 6 to 8 lines without areolation at mid-body, about 6.0 µm wide (Figs. 1F, 2K, 3D). Body annuli are distinct, 1.2 ± 0.1 µm wide, and they are divided into blocks. The longitudinal striation is conspicuous with 18 to 20 lines at the ventral side near vulva (Fig. 3D). The labial region bears 6 to 7 annuli without longitudinal striae, not offset or very slightly offset by a shallow depression. A projected, round-to-hexagonal oral disc is present. First labial annulus is divided into six sectors; the lateral sectors are much smaller than the other four. Lateral sectors of first annulus are present with oval amphidial apertures (Fig. 3C). The head framework is not sclerotized. A well-developed stylet is present with elongated conical part and sloping and rounded knobs (Figs. 1C,E, 2B,E). The dorsal gland orifice is located 2.6 to 3.1 µm posterior to stylet knobs. An oval median bulb and a saccate terminal bulb, not overlapping intestine, are present. Secretory–excretory pore is located at the level of the nerve ring, 86 to 102 µm from the anterior end; hemizonid is located anterior to the secretory–excretory pore; deirid is absent. Vulva is a transverse slit, sunken or flush to body surface; epyptigma is double; vaginal length is 30% of the body diameter, moderately sclerotized; spermatheca is rounded, with rounded sperm cells; ovaries are outstretched with a single row of oocytes. The tail is conical, annulated with hyaline, and the tail tip is pointed; 58 to 73 tail annuli are present on the ventral side (Figs. 1H, 2L, 3I,J). Phasmid is slightly enlarged, at about one-fourth of the tail length. The lateral field ends before the hyaline part of the tail tip.
Males are similar to females but they are more ventrally arcuate (Figs. 1B, 2A). The male reproductive apparatus is present with slightly bent spicules and a curved gubernaculum; cloacal lips are protuberant. Two posterior hypoptygmata are well developed, with equal lengths (Fig. 4I). Bursa is absent. Phasmid is located 22 to 28 µm posterior to the cloacal aperture. The tail is conical, annulated with hyaline, and the tail tip is pointed (Figs. 1I, 2I, 4F,G,H).
The alignment of D2–D3 of 28S rDNA sequences contained 31 sequences including the sequences from two outgroup taxa (
The phylogenetic relationships between
The ITS rDNA sequence alignment contained 27 sequences including the sequences from
The phylogenetic relationships between the ITS rDNA sequence of
Among
One holotype female, one allotype male and 33 paratypes (18 females and 15 males) from a population were extracted from the rhizosphere of
The specific epithet is derived from Vietnam, the country where the species was found.
Holotype and paratypes were deposited in the Nematode Collection of the Institute of Ecology and Biological Resources (IEBR), Vietnamese Academy of Science and Technology, 18 Hoang Quoc Viet Road, Hanoi, Vietnam, and 10 female paratypes were deposited in the nematode collection of the Zoology Museum, Ghent University, K. L. Ledeganckstraat 35, Ghent, Belgium. The D2–D3 and ITS sequences were deposited in GenBank with accession numbers MH191361 and MH191362, respectively.
In terms of morphology, this new species possesses a relatively long stylet compared to other plant-parasitic nematode groups, a slightly offset labial region bearing 6 to 7 annuli without striation, a non-sclerotized head framework, a body cuticle with 26 to 28 longitudinal striae (excluding lateral lines), 6 to 8 lateral lines at lateral field, a conical and pointed tail with annulated tip, and the absence of a bursa in the male. These characteristics clearly indicate that this species belongs to the subfamily Merliniinae. According to Siddiqi (2000) and Sturhan (2011), this species should belong to the genus
In both D2–D3 of 28S and ITS rDNA trees, our new species always shows a very close relationship with species of