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33Russian Journal of Nematology, 2010, 18 (1), 49 - 57
Devibursaphelenchus wangi sp. n. (Nematoda: Ektaphelenchinae) feeding on Aphelenchoides sp.
Jianfeng Gu1' 2, Jiangling Wang2 and Jingwu Zheng1
'Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310029, China, e-mail: jwzheng@zju.edu.cn 2Technical Centre, Ningbo Entry-exit Inspection and Quarantine Bureau, 9 Mayuan Road, Ningbo 315012, Zhejiang, China e-mail: gujf@nbciq.gov.cn
Accepted for publication 12 April 2010
Summary. Devibursaphelenchus wangi sp. n. is described and figured. The new species was isolated from pine packaging wood from the USA and inspected in Ningbo harbour, China in 2009. The new species is characterised by relatively slender body (a = 36.5 and 37.1 for males and females, respectively); three lines in the lateral field; stylet with relatively wide lumen but lacking basal knobs; vulval flap absent, very short post-uterine sac; non-functional and indistinct female rectum and anus; spicules relatively small (14.2-15.6 |im) with a flattened cucullus; two pairs of caudal papillae. The new species is morphologically close to D. eproctatus and D. hunanensis and can be distinguished by shape and size of spicules and smaller stylet. The separate species status is supported by ITS-RFLP patterns and molecular phylogenetic analysis based on ITS1/2 and partial LSU sequences, which revealed that the new species is close to D. hunanensis. The feeding habit of the new species is also observed and discussed. Key words: Ektaphelenchidae, morphology, molecular taxonomy, new species.
In March 2009, during a routine inspection of imported packaging wood, a new species of Devibursaphelenchus was isolated together with an Aphelenchoides species from pine packaging wood coming from USA. It is described and figured herein as Devibursaphelenchus wangi sp. n.
MATERIAL AND METHODS
Sawn samples taken from packaging wood were cut into small pieces no more than 1 cm wide. Nematodes were extracted by the modified Baermann funnel technique for 24 h. The feeding habit of the new species was observed on a slide in water and recorded. Multiplication on agar-fungi plates (Botryotinia fuckeliana) failed. Measurements were made on permanent slides fixed in TAF and processed to glycerol following the method of Seinhorst (1959). The light micrographs were made using a Zeiss Imager Z1 microscope equipped with a Zeiss AxioCam MRm CCD camera.
DNA samples of Devibursaphelenchus wangi sp. n. were prepared according to Li et al. (2008). Two sets of primers (synthesised by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify the ITS1/2 region and the D2D3 LSU region of rDNA, respectively. Primers for amplification of ITS1/2 were forward primer F194 (5'- CGT AAC AAG GTA GCT
GTA G -3') (Ferris et al., 1993) and reverse primer 5368r (5'- TTT CAC TCG CCG TTA CTA AGG -3') (Vrain, 1993). Primers for amplification of D2/D3 LSU were forward primer D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and reverse primer D3Br (5'-TCG GAA GGA ACC AGC TAC TA-3') (De Ley et al., 1999). PCR conditions were as described by Li et al. (2008). PCR products were separated on 1% agarose gels and visualised by staining with ethidium bromide. PCR products of sufficiently high quality were purified for cloning and sequencing by Invitrogen, Shanghai, China.
For ITS-RFLP profiles, suitable aliquots of the amplified ITS rDNA were digested for at least 3 h at 37° using 10 U of each of the five restriction endonucleases (RsaI, HaeIII, MspI, HinfI and AluI) (Takara, Japan) following the manufacturer's instructions. Fragments were resolved by electrophoresis in a 2.5% agarose gel and stained with ethidium bromide.
The ITS1/2 and partial LSU sequences were analysed and aligned using the program ClustalW implemented in MEGA version 4.0 (Tamura et al., 2007). Phylogenetic trees were generated with the Neighbor Joining (NJ) method using the Tajima-Nei distance option. Bootstrapping analysis was performed with 1000 replicates.
Fig. 1. Devibursaphelenchus wangi sp. n. A: Female; B: Male; C: Anterior body; D: Lateral view of male tail; E: Spicules; F: Ventral view of male tail; G-I: Vulva region; J, K: Variation of female tail. (Scale bars=10 ^m).
DESCRIPTIONS
Devibursaphelenchus wangi sp. n.
(Figs. 1-3)
Measurements (Table 1).
Male. Body slender, cylindrical, posterior region sharply curved ventrally when heat-killed. Cuticle weakly annulated, lateral field with three incisures (i.e., two ridges). Lip region offset, about 3.5 ^m in
height and 7.1 ^m wide. Stylet short and relatively broad lumened, lacking basal swellings, conus forming ca. 38-40% of total length. Procorpus cylindrical. Median bulb strongly developed, elongate-oval, 15.7±0.8 ^m long, 9.7±0.8 ^m wide, with valve plates situated slightly posteriorly. Pharyngeal gland lobe slender and well developed, about six body diameters long, overlapping intestine dorsally. Nerve ring located at ca. 12-15 ^m posterior to median bulb. Excretory pore located at ca
Table 1. Measurements of Devibursaphelenchus wangi sp. n., all measurements in цт; mean ± s.d. (range).
Female Male
Holotype Paratypes Paratypes
- 15 6
655.0 704.3±62.0 614.3±22.9
(606.0-803.3) (578.0-644.0)
36.5 37.1±4.3 36.5±2.4
(26.4-45.1) (31.3-38.2)
7.7 8.5±0.8 7.2±0.4
(7.6-10.6) (6.8-8.1)
3.2 3.5±0.3 3.5±0.3
(3.0-4.0) (3.3-4.0)
- - 17.5±0.9
(16.2-18.8)
- - 2.8±0.1
(2.7-2.9)
78.6 78.2±1.7 29.1±0.6
(73.7-80.0) (28.0-29.7)
17.9 19.1±3.3 16.9±1.7
(15.0-29.5) (15.2-20.6)
7.0 7.7±0.8 7.1±0.7
(6.9-9.0) (6.9-8.8)
3.1 3.7±0.5 3.5±0.4
(3.1-4.5) (3.1-4.0)
16.8 17.1±0.3 14.8±1.4
(16.7-17.4) (12.4-16.6)
16.3 17.7±1.2 15.7±0.8
(16.3-19.8) (15.0-17.0)
10.3 11.0±1.8 9.7±0.8
(8.3-14.5) (8.3-11.1)
1.6 1.6±0.2 1.6±0.2
(1.3-2.0) (1.5-2.0)
95 105.4±8.2 98.2±3.2
(94.0-119.1) (93.0-104.0)
- - 18.3±0.6
(17.3-19.1)
- - 15.0±0.5
(14.2-15.6)
- - 15.9±0.3
(15.4-16.4)
350 298.6±34.6 180.7±4.7
(256.0-355.0) (160.0-199.0)
11.7 10.2±1.8 -
(7.6-13.2)
85.0 107.0±14.8 -
(79.0-128.0)
- - 35.2±2.2
(33.0-39.8)
n L a b b'
V or T Max body diam. Lip diam. Lip height Stylet length Median bulb length
Median bulb diam.
Median bulb length/diam.
Excretory pore position Spicule (dorsal limb)
Spicule (chord)
Spicule (curved median line from the middle of condylus to the end)
Ovary or testis length Post-uterine sac length Blind sac Tail length
c
30-35 ^m posterior to median bulb. Hemizonid located just posterior to excretory pore, but sometimes anterior to excretory pore. Testis single, about 180.7±4.7 ^m long, spermatocytes arranged in two rows. Cloacal opening lips slightly protruding. Spicules arcuate, condylus rounded, elongated, lamina smoothly and symmetrically curved, rostrum conical with bluntly pointed tip. Distal ends of spicules forming a flattened cucullus. Tail strongly recurved, terminus finely pointed, spade-shaped terminal bursa clearly visible
under light microscope. Two pairs of caudal papillae present: one pair located slightly precloacal and the second subventral pair located just anterior to the beginning of bursal flap.
Females. Body slightly ventrally arcuate when heat-relaxed. Cuticle and lip region similar to male. Ovary outstretched, developing oocytes in two rows. Vulva slightly inclined anteriorly, vulva lips not protruding, anterior vulva lip does not form a vulval flap. Vagina not sclerotized. Spermatheca elongate-
oval, sometimes containing round sperms. Post-uterine sac short, less than one body diameter long, rectum and anus indistinct. Intestine terminating as a blind sac. Tail conical, tapering to ventrally bent terminus, tail terminus finely rounded or sharply pointed.
Diagnosis and relationships. Devibursaphelenchus wangi sp. n. is characterised by relatively slender body (a = 36.5 and 37.1 for males and females, respectively); three lines in the lateral field; stylet with relatively wide lumen and lacking basal knobs; relatively high vulva position (average 78%); vulval flap absent, very short postuterine sac; nonfunctional and indistinct female rectum and anus; spicules relatively small (14.2-15.6 ^m) with a flattened cucullus; two pairs of caudal papillae; presence of a distinct bursal flap.
Braasch (2009) re-established the genus Devibursaphelenchus Kakuliya, 1967 belonging to Ektaphelenchinae, which contains five species: D. typographi Kakuliya, 1967; D. eproctatus (Sriwati, Kanzaki, Phan & Futai, 2008) Braasch, 2009; D. hunanensis (Yin, Fang & Tarjan, 1988) Braasch, 2009; D. lini (Braasch, 2004) Braasch, 2009 and D. teratospicularis (Kakuliya & Devdariani, 1965) Braasch, 2009.
Devibursaphelenchus wangi sp. n. is particularly close to D. eproctatus and D. hunanensis in the shape of spicules and female tail. Devibursaphelenchus wangi sp. n. is distinguished from D. eproctatus by the shape and size of spicules (15.4-16.4 pm vs 18.8-20.8 pm long measured along curved median line, condylus not recurved dorsally vs sometimes recurved dorsally ); different size of stylet (12.4-16.6 pm and 16.7-17.4 pm for males and females, respectively vs 15-20 pm and 19-22 pm); different ovary length (256-355 pm vs 152-243 pm) and testis length (160-199 pm vs 129-143 pm); different c ratio of males (c = 16.2-18.8 vs c = 13.315.0).
Devibursaphelenchus wangi sp. n. is distinguished from D. hunanensis by the presence of three vs four lateral lines, absence vs presence of a functional rectum and anus, shorter stylet (12.4-16.6 pm and 16.7-17.4 pm for males and females, respectively, vs 19-21 pm and 20-26 pm); the shape of spicules (distal end of spicules with a distinct cucullus vs distal end of spicules obtuse, without cucullus).
Devibursaphelenchus wangi sp. n. is distinguished from D. typographi by the body shape (a = 31.338.2 and 26.4-45.1 for males and females, respectively vs a = 21.2-22.5 and 20.2-20.9); shorter stylet (averaging 17 and 15 pm for females and males, respectively, vs 21-22 pm); the shape of
female tail terminus (finely rounded or sharply pointed vs broadly rounded), the V value (V = 73.780.0 vs V = 85.3-85.8), and the size of spicules (14.2-15.6 pm vs 12 pm long measured in chord).
Devibursaphelenchus wangi sp. n. is distinguished from D. lini by the shape and size of spicules (14.215.6 pm long vs 16-21 pm long measured in chord, rostrum bluntly pointed vs sharply pointed); different length of stylet (12.4-16.6 pm and 16.7-17.4 pm for males and females, respectively vs 17-21 pm and 1823 pm); and the vulva structure (no sclerotization in the vulva region vs a strong half ring-like sclerotization in the anterior vulva part).
Devibursaphelenchus wangi sp. n. is distinguished from D. teratospicularis by stylet length (averaging 17 and 15 pm for females and males, respectively, vs 1822 pm); lack of basal swellings at the stylet vs presence of minute swellings in D. teratospicularis; finely rounded or pointed vs blunt tip of female tail; and by shape of spicules (rostrum position in the anterior part of spicules vs rostrum in the middle part of spicules due to very high condylus of D. teratospicularis, smoothly ventrally curved dorsal limb vs sunken distal part of dorsal limb).
Molecular profiles and phylogenetic status. The rDNA based sequences of ITS1/2 and D2D3 LSU are deposited in the GenBank database with the accession numbers GQ894739 and GQ903770, respectively. The molecular phylogenetic status of the new species is shown in Figures 4 and 5, and the ITS-RFLP profiles of rDNA are shown in Figure 6 and Table 2. The ITS-RFLP pattern of D. wangi sp. n. is different from the patterns of D. hunanensis and D. lini (Burgermeister et al., 2009).
Table 2. Sizes of PCR products and DNA restriction fragments obtained in ITS-RFLP analysis and calculated on sequencing results of the ITS1/2 regions
Species PCR product (bp) Restriction fragments (bp)1
Rsa I Hae III Msp I Hinf I Alu I
D. wangi sp. n. 966 403 335 155 73 717 249 769 197 488 433 45 798 73 65 15 15
D. hunanensis2 947 374 580 765 497 641
305 367 182 181 293
196 164 13
72 63
42
1 Fragment sizes (bp) were calculated with a computer program DNASTAR MapDraw 5.01.
2 According to Burgermeister et al., 2009.
Fig. 2. Light photomicrographs of Devibursaphelenchus wangi sp. n. (female) A: Whole body; B: Vulva region; C, D: Feeding on Aphelenchoides sp.; E, F: Tail; G, H: Vulva region (lateral view); I: Vulva region (ventral view). (Scale bars=10 дт).
Fig. 3. Light photomicrographs of Devibursaphelenchus wangi sp. n. (male) A: Whole body; B, C: Anterior body; D: Lateral field; E, F: Spicules; G & I: Tail (ventral view, showing papillae and bursa); H: Tail. (Scale bars=10 ^m).
48
36
54
89
100
100
99
-Bursaphelenchus xylophilus AM179515
— B. macromucronatus EU256381
-B. burgermeisteri EU034530
Ektaphelenchoides compasai DQ257622
-Aphelenchoides subtenuis EF312109
Devibursaphelenchus lini EU559192
E. pini DQ257620 — D. hunanensis EU400449 D. wangi n. sp. GQ894739
Aphelenchus avenae AB368919
Fig. 4. Molecular phyl&genetic status of Devibursaphelenchus wangi sp. n. based on ITS1/2 sequences. Aphelenchus avenae served as the outgroup species. Numbers at branching points are bootstrap values obtained using 1000 repetitions. Scale bar: substitutions/site.
67
100
99
97
60
-Bursaphelenchus obeche EU159108
■ B. burgermeisteri EU159109 - B. africanus AM397024
100
■ B. luxuriosae AM396571
- B. xylophilus AM396580
- B. arthuri AM396564
99 56
99 32
96
98
73
64
79
77
83
93
96
72
88
■ B. thailandae AM396577
-Cryptaphelenchus sp. EU287596
-Ektaphelenchus obtusus AB368533
-Ektaphelenchoides compasai DQ257625
100
- Devibursaphelenchus wangi n. sp. ■ D. hunanensis GQ337012 -D. lini AM396570
Ektaphelenchoides pini DQ257623 - Ruehmaphelenchus asiaticus AM269475
- Aphelenchoides besseyi AY508109 -Laimaphelenchus preissii EU287598
— L. australis EU287600 -A. fragariae AB368540
■ Schistonchus guangzhouensis DQ912927
— A. xylocopae AB434933 -L. heidelbergi EU287595
— Schistonchus aureus DQ912925 -S. centerae DQ912928
Aphelenchus avenae AB368536
0.05
Fig. 5. Molecular phylogenetic status of Devibursaphelenchus wangi sp. n. based on partial LSU sequences. Aphelenchus avenae served as the outgroup species. Numbers at branching points are bootstrap values obtained using 1000 repetitions. Scale bar: substitutions/site.
The two molecular trees show that the new species is close to D. hunanensis (Figs 4 and 5), and that the genus Devibursaphelenchus is close to other genera of Ektaphelenchinae such as Ektaphelenchoides, Ektaphelenchus and Cryptaphelenchus (Fig. 5).
Type habitat and locality. Packaging wood of Pinus sp. from USA, inspected in Ningbo Entry-exit Inspection and Quarantine Bureau, China, in 2009.
Feeding habitat. Seven specimens of Devibursaphelenchus wangi sp. n., including males, females and juveniles were found feeding on Aphelenchoides sp. The bodies of Aphelenchoides sp. were penetrated by the stylets of D. wangi sp. n., which could not easily be separated from their food (Fig. 2).
Types. Holotype male, ten female and five male paratypes (slide numbers 848-1 to 848-11) deposited in the nematode collection of Ningbo Entry-exit Inspection and Quarantine Bureau, China. One paratype male and four paratype females (slide numbers 9319 and 9320) deposited in the Canadian National Collection of Nematodes, Ottawa, Ontario, Canada. One paratype male and four paratype females (slide numbers 848-12 and 848-13) deposited in the Institute of Biotechnology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China.
Etymology. D. wangi sp. n. is named after Wang Songqing, the deputy director of Ningbo Entry-exit Inspection and Quarantine Bureau, who had supported the authors' research.
Fig. 6. ITS-RFLP pattern of Devibursaphelenchus wangi sp. n. M = Molecular size marker (100 bp ladder); Lane 1: rDNA amplification product; Lanes 2-6: Digestion products obtained with Rsal, HaeIII, MspI, Hinfl and AluI. Sizes of PCR product and its restriction fragments are shown in Table 2.
DISCUSSION
The genus Devibursaphelenchus clearly belongs to Ektaphelenchinae with the following typical characters: stylet with relatively wide lumen, rectum and anus obscure in females, intestine ending in a blind sac. The molecular phylogenetic analysis also showed that Devibursaphelenchus species cluster together with those of the genera of Ektaphelenchinae (Fig. 5). Additionally, the predatory behaviour of Devibursaphelenchus species supports their placement within Ektaphelenchinae. Among the genera of Ektaphelenchinae, only Devibursaphelenchus has a bursa, which is otherwise typical for Parasitaphelenchinae.
The genus Devibursaphelenchus differs from Ektaphelenchus by having a male tail with a terminal bursa, but if the male is absent, the females of these two genera are similar especially for those Ektaphelenchus species without distinct basal swellings (all the stylets of Devibursaphelenchus species are without basal swellings).
According to Braasch (2009), the genus Devibursaphelenchus is characterised by a slender body; strong stylet (20-26 pm long) with relatively wide lumen and usually without basal thickenings; metacorpus elongate-oval, with valve plates located posterior to the middle of bulb; excretory pore posterior to metacorpus; vulva relatively posterior (V = 76-80), not protruding and without flap; vagina sclerotized; post-uterine branch short (up to 1.5 times the corresponding body diameter long); rectum and anus obscure in females; male tail strongly recurved, with distinct terminal bursa seen in dorso-ventral position; two pairs of male caudal papillae; spicules strong, almost straight and arcuate
in their distal part, with prominent rostrum, relatively high condylus, without cucullus, and in some species with a hook-like appendix at the distal end, with appearance of a cucullus.
The re-establishment of Devibursaphelenchus is supported by this new species description. However, the new species lacks a highly sclerotized vagina shown in other species of the genus (Braasch, 2009) and has a cucullus. Therefore, these features may not be used for genus characterisation.
Devibursaphelenchus hunanensis has several features in common with the other Devibursaphelenchus species (Braasch, 2009), but it was described as having four lateral lines (two refractive inner lines), and distinct rectum and anus of females (Yin et al, 1988). Gu et al. (2006) reported that D. hunanensis was detected in Ningbo, China, but the rectum and anus of the reported species were not clearly seen. The identity of this species with the originally described D. (B.) hunanensis remains questionable.
Braasch (2004) observed that a specimen of D. lini was feeding on a Bursaphelenchus xylophilus juvenile. D. hunanensis has been observed feeding on B. mucronatus and B. rainulfi (unpubl. observations). Devibursaphelenchus wangi sp. n. has been observed feeding on Aphelenchoides sp. Therefore, we assume that Devibursaphelenchus species are predatory on other nematodes and cannot be multiplied on fungi. It will be very interesting and of practical interest to investigate the potential efficiency of Devibursaphelenchus species for a possible future control strategy of B. xylophilus.
Because packaging wood is a circulating product and there is no phytosanitary treatment mark, the exact geographic origin of the new species remains unclear. The vector of the new species is also unknown.
ACKNOWLEDGEMENT
The research presented here was supported by General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China (2010IK269). The authors thank Dr. Helen Braasch, Potsdam, Germany and Dr. Wolfgang Burgermeister, Julius Kühn Institute, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany, for reading the manuscripts.
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Jianfeng Gu, Jiangling Wang, Jingwu Zheng. Devibursaphelenchus wangi sp. n. (Nematoda: Ektaphelenchinae), питающийся на Aphelenchoides sp.
Резюме. Приводится описание и иллюстрации для Devibursaphelenchus wangi sp. n. Новый вид выделен из прибывшей из США упаковки, изготовленной из древесины сосны. Древесина упаковки была обследована в гавани Нингбо в Китае в 2009 году. Новые вид характеризуется сравнительно тонким телом (a = 36.5 и 37.1 для самцов и самок, соответственно); тремя линиями латерального поля, стилетом со сравнительно широким просветом и без базальных утолщений, отсутствием вульварной складки, не функционирующим и не различимым ректумом и анальным отверстием у самок; сравнительно небольшими (14.2-15.6 дт) спикулами с уплощенным кукулюсом, двумя парами хвостовых папилл. Новый вид морфологически близок к D. eproctatus и D. hunanensis, но может быть дифференцирован от них по размеру и форме спикул и меньшей длите стилета. Независимый статус этого нового вида подтвержден спектрами ITS-RFLP, а также результатами молекулярно-филогенетического анализа, основанного на сравнении полной ITS последовательности и частичной LSU последовательности. Показано, что новый вид близок к D. hunanensis. Исследован характер питания нематод нового вида.
