Trinh et al.

Egyptian Journal of
Egyptian Journal of Biological Pest Control (2021) 31:147
[Link] Biological Pest Control

RESEARCH Open Access

First report of entomopathogenic nematode

Steinernema surkhetense and its pathogenic
potential to larvae of the Greater Wax Moth
(Galleria mellonella L.) in Vietnam
Phap Quang Trinh1,2*, Duyen Thi Nguyen1,2*, Linh Thi Mai Le1,2 and Tien Huu Nguyen1,3

Abstract
Background: Entomopathogenic nematodes (EPNs) are evidently a useful nematode group for the biocontrol of
insect pests. It is well known that efficacy of different EPN strains, even belonging to the same species, can be signifi-
cantly varied in different localities. Therefore, exploring EPNs and testing their efficacy in various ecological regions
is of crucial importance to find out more efficient EPN strains. On the other hand, this practice is also needed to
enhance the knowledge on diversity and distribution model of EPNs over the world.
Results: In this study, a species belonging to the genus Steinernema, S. surkhetense, has been characterized for the
first time in Vietnam based on morphological and molecular characterizations. Morphological characterizations of
infective juveniles, the first and second-generation adults, and molecular characterization of D2-D3 expansion seg-
ment of 28S rRNA region were given. Molecular phylogeny of the genus Steinernema was also provided. In addition,
the study showed that the lethal efficacy of this local strain to larvae of Galleria mellonella L. was relatively higher than
other reported EPN strains in Vietnam.
Conclusions: The Vietnamese EPN population found in this study was determined to be conspecific with S. sur-
khetense, revealed its new distribution in Vietnam. Besides, detailed morphological and molecular characterizations of
it was provided with small variations compared to other populations in the world, and its relatively high lethal efficacy
on larvae of G. mellonella implied that this strain can be potentially a useful strain for biological control of insect pests.
Keywords: Entomopathogenic nematodes, Steinernema surkhetense, Lethal efficacy, Galleria mellonella, Central
Highlands, Vietnam, Biocontrol

Background animals (Nguyen and Hunt 2007). Although most EPN

Entomopathogenic nematodes (EPNs) are obligate par- species can control a wide range of insect pests, they are
asites and have significant potential in the biological also known that different EPN strains belonging to the
control of insects. They are capable of parasitizing and same species having different efficacy to control the same
causing death to hundreds of insect pests without having host insect (Laznik et al. 2010). Therefore, the works such
any clear adverse effect on humans and other vertebrate as isolating, identifying, and testing the efficacy of EPNs
strains from different localities are of crucial importance
in providing excellent EPN strains for producing bio-
*Correspondence: tqphap@[Link]; thuduyen0609@[Link] logical pesticides (Laznik and Trdan 2011). Besides, the
1
Institute of Ecology and Biological Resources, Vietnam Academy
of Sciences and Technology, 18 Hoang Quoc Viet, Cau Giay, 100000 Hanoi, above works also help to provide the information about
Vietnam diversity, distribution, and host range of EPNs over the
Full list of author information is available at the end of the article

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Trinh et al. Egyptian Journal of Biological Pest Control (2021) 31:147 Page 2 of 7

world, which is necessary to better understand the evolu- 3.2.6 Add-in in Geneious R11 under GTR + G + I model
tion of life. (Markov chains were set with 1 × 106 generations, four
Currently, more than 121 valid EPNs species have been runs, 20% burn-in, and subsampling frequency of 500
described in the world (Didiza et al. 2021). Among them, generations) following Nguyen et al. (2020).
Steinernema surkhetense Khatri-Chhetri, Waeyenberge,
Spiridonov, Manadhar and Moens 2011 belongs to car- Virulence of S. surkhetens from Vietnam
pocapsae-group (presence of short juveniles). This species In this study, an experiment on the lethal efficacy of S.
has been reported from several regions of Nepal, India, surkhetense to larvae of G. mellonella was done, following
and China (Bhat et al. 2020). In Vietnam, 13 EPN species Nguyen (2008). There were 10 treatments with the con-
belonging to the genera Steinernema and Heterorhabdi- centration of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 IJs
tis have been found (Phan et al. 2014). The present study per larva of G. mellonella. Each treatment was repeated 3
provides the first morphological and molecular charac- times, 15 larvae were used for a replication resulted in a
terizations of the EPN S. surkhetense from Vietnam, and total of 45 larvae per treatment. In the control treatment,
molecular phylogeny of the genus Steinernema. Besides, IJs were replaced by distilled-water. The experiment was
the pathogenic potential of this strain to the larvae of checked after 48 h. at 28 °C. Cadavers of larvae were used
great wax moth, G. mellonella, was also tested. for a white trap to confirm the infection of IJs (Nguyen
2008).
Methods
Nematode isolation and rearing Statistical analysis
Fifty soil samples were collected randomly from Lam Results were analyzed using SPSS version 25 to deter-
Dong and Ninh Thuan, Vietnam. Nematodes were recov- mine ­LC50, to detect significant differences and correla-
ered from soil samples using the modified Galleria bait tion (IBM Corp 2017).
method (Bedding and Akhurst 1975). Positive samples
were found at the following coordinates: 11°45′ 37″ N, Results
109°35′ 47″ E and 11°45′ 55″ N, 109°11′ 01″ E. Subse- Measurements
quently, they were cultured on the larvae of G. mellonella Data of different measurements are presented in Table 1.
using the method described by Nguyen (2008). The first
and second-generation adults were obtained by dissect- Morphological characterization
ing infected cadavers after 3 and 5 days, respectively. Female (first generation)
Infective juveniles (IJs) were obtained when they emerged Body was spiral-shaped. Cuticle was smooth under LM
from the cadavers after 8–10 days. (Fig. 1A). Lip region was rounded and slightly offset to
body contour. Mouth was funnel-shaped. Cheilorhab-
Morphological characterization dions and cardia were prominent. Secretory-excretory
Heat-killed nematodes were fixed using TAF (Formalin pores were prominent and located anterior to nerve ring
40%: 7 ml, Triethanolamine: 2 ml, distilled-water: 91 ml) (Fig. 1B). Vulva was slit-like with protruding lips, located
for 3–4 days and subsequently transferred to glycerin at mid-body with short epiptygmata (Fig. 1C). Genital
to make permanent slides, following Seinhorst (1959). tract was amphidelphic-didelphic with reflexed ovaries.
Measurements and pictures of all nematode stages were Tail was conical with short mucron (Fig. 1D).
taken from permanent slides using Carl Zeiss Axio Lab.
A1 light microscope equipped with a Zeiss Axiocam Female (second generation)
ERc5s digital camera. Similar to the first-generation females. Body size was
smaller and slightly curved ventrally. Tail was elongate
Molecular characterization and tapering to a pointed tail end. (Fig. 1E and F).
The D2-D3 region of 28S rRNA was amplified using D2A
(5′-ACA​AGT​ACC​GTG​AGG​GAA​AGTTG-3′) and D3B Male (first generation)
(3′-TCC​TCG​GAA​GGA​ACC​AGC​TACTA-5′) primers Body was J-shaped. Head region was similar to female.
(De Ley et al. 1999). The obtained sequence was analyzed Testis was reflexed ventrally (Fig. 1H). Spicules were
following Nguyen et al. (2020). Blast search was used paired, symmetrical, and moderately curved. Gubernacu-
to search for closely related species on GenBank (Alts- lum was boat-shaped (Fig. 1I). A single precloacal papilla
chul et al. 1997). Forward and reverse sequences were and 11 pairs of genital papillae were arranged in normal
assembled and analysed using Geneious R11. The best fit position for Steinernema (i.e. 6 or 7 pairs precloacal sub-
model was chosen using Mega 7, following Nguyen et al. ventral, one pair adcloacal, one pair lateral, 2 pairs sub-
(2020). Phylogenetic trees were created using MrBayes terminal, and one pair subdorsal).
Table 1 Morphometric data of Steinernema surkhetense from different localities. All measurements are in μm (except for ratio)
Characters S. surkhetense from Vietnam S. surkhetense from Nepal (Khatri-Chhetri et al., 2011)

Females Males Infective Females Males Infective

juveniles juveniles
1st generation 2nd generation 1st generation 2nd 1st generation 2nd generation 1st generation 2nd
generation generation

n 10 10 10 10 10 20 20 20 20 20
L 7405 ± 1103 1632 ± 108 1664 ± 91 879 ± 65 474 ± 21 2970 ± 922 1072 ± 101 1224 ± 194 824 ± 89 415 ± 15
(5962–9990) (1400–1768) (1549–1828) (802–993) (437–500) (1894–5692) (875–1290) (887–1576) (621–945) (393–450)
a 28 ± 3 (24–34) 13 ± 0.7 (12–14) 10.6 ± 0.8 (9–11) 15 ± 1 (13–16) 20 ± 1 (19–22) 19 ± 2.7 (13–25) 13 ± 1.4 (11–16) 12 ± 1.3 (10–14) 12 ± 1.9 (8.8–15) 19 ± 1.8 (16–24)
b 33 ± 4 (26–41) 10.5 ± 1 (9.0–12) 11.2 ± 1 7.2 ± 0.2 5 ± 0.2 (4.7–5.2) 24 ± 8.8 (14–49) 5.6 ± 0.5 11 ± 1.6 (8.0–15) 6.8 ± 0.8 5 ± 0.2 (4.3–4.8)
(10–12.7) (6.9–7.5) (4.6–6.7) (5.2–8.0)
c
Trinh et al. Egyptian Journal of Biological Pest Control

201 ± 37 40 ± 4 (36–48) 58 ± 6 (49–67) 44 ± 4.8 (35–50) 10.5 ± 0.3 161 ± 54 11 ± 1.6 (8.7–15) 65 ± 12 (43–85) 44 ± 6.9 (31–59) 9 ± 0.5 (8.3–10)
(152–283) (10–10.8) (91–309)
c’ 0.4 ± 0.1 (0.3–0.6) 1 ± 0.1 (0.9–1.1) 0.7 ± 0.0 0.8 ± 0.1 3.9 ± 0.1 – – – – –
(0.6–0.8) (0.7–0.9) (3.8–4.1)
V 53 ± 2.3 (48–54) 57 ± 2.6 (53–62) – – – 54 ± 3.4 45–59 50 ± 2.7 (42–54) – – –
W
(2021) 31:147

268 ± 28 123 ± 11 163 ± 15 60 ± 5.2 (51–69) 23 ± 0.8 (22–25) 154 ± 34 80 ± 10 (64–97) 105 ± 19 73 ± 13 (56–96) 21 ± 1.8 (18–25)
(225–318) (103–142) (145–186) (102–236) (75–132)
EP 92 ± 16 (56–107) 76 ± 5 (69–83) 73 ± 6 (62–84) 61 ± 4 (55–66) 34 ± 1 (32–35) 53 ± 13 (33–86) 137 ± 21 55 ± 10 (43–78) 44 ± 4 (35–50) 32 ± 1.5 (28–34)
(96–195)
NR 154 ± 14 108 ± 6 (98–118) 106 ± 11 90 ± 5.4 67 ± 3.4 (61–70) 93 ± 17 (65–114) 159 ± 13 92 ± 19 (59–140) 90 ± 10 63 ± 3.1 (57–70)
(134–179) (82–117) (82–100) (142–198) (73–109)
ES 226 ± 12 157 ± 11 149 ± 10 122 ± 7 95 ± 3 (90–99) 132 ± 31 193 ± 16 115 ± 15 122 ± 11 92 ± 4.1 (84–101)
(207–244) (139–180) (124–160) (114–133) (52–190) (173–236) (86–146) (106–141)
ABD 92 ± 8.8 (80–110) 41 ± 3.8 (33–46) 41 ± 1.4 (40–44) 26 ± 1.4 (24–28) 11.6 ± 0.5 47 ± 10 (25–68) 28 ± 3.2 (21–34) 31 ± 3.8 (23–38) 27 ± 2.3 (23–32) 12 ± 0.8 (11–14)
(10.8–12)
T 37 ± 4.4 (31–46) 41 ± 5 (31–47) 29 ± 1.7 (25–32) 20 ± 1.4 (18–23) 45 ± 1.5 (43–47) 19 ± 5.0 (12–30) 103 ± 15 19 ± 2.1 (16–23) 19 ± 3.6 (15–27) 45 ± 3.2 (38–53)
(65–135)
HT – – – – 19.6 ± 1 – – – – 26 ± 3.9 (20–32)
(18.4–22)
SPL – – 74 ± 6 (62–81) 60 ± 3.6 (57–68) – – – 70 ± 5.2 (58–78) 64 ± 5.4 (55–76) –
SPW – – 11.6 ± 1 10 ± 0.7 – – – – – –
(10.2–13) (8.6–10.8)
GUL – – 53 ± 4 (48–58) 43 ± 4.2 (38–50) – – – 52 ± 5.1 (42–63) 44 ± 5.6 (35–53) –
GUW​ – – 7 ± 0.6 (6.3–7.8) 6 ± 0.8 (4.7–7.6) – – – – – –
H% – – – – 44 ± 2.5 (40–47) – – – – 57 ± 7.4 (44–73)
D% 41 ± 7 (24–46) 49 ± 3.7 (43–56) 49 ± 4.8 (40–55) 50 ± 2.5 (46–55) 35 ± 1 (34.7–37) 44 ± 20 (28–117) 71 ± 10 (50–87) 48 ± 8.7 (37–64) 36 ± 3.3 (31–42) 35 ± 2.2 (31–40)
E% 247 ± 42 193 ± 26 256 ± 29 301 ± 34 75 ± 2 (72–78) 292 ± 84 137 ± 28(85– 295 ± 66 237 ± 45 72 ± 6.4 (54–84)
(160–302) (162–250) (203–296) (239–340) (156–453) 189) (205–429) (167–313)
Page 3 of 7
Trinh et al. Egyptian Journal of Biological Pest Control (2021) 31:147 Page 4 of 7

Fig. 1 LM pics of Steinernema surkhetense from Vietnam. A–D first-generation female. A entire body; B pharyngeal region; C vulva region; D tail
region. E–F second-generation female. E entire body; F tail region. G–I first-generation male. G pharyngeal region; H entire body; I tail region. J
entire body of second-generation male. K–N juvenile. K entire body; L pharyngeal region; M lateral field; N tail region
Trinh et al. Egyptian Journal of Biological Pest Control (2021) 31:147 Page 5 of 7

Fig. 2 BI Phylogenetic tree generated from D2-D3 of 28S rRNA sequences under GTR + G + I. Bayesian posterior probabilities (in percentage) are
given next to each node. Sequence of Steinernema surkhetense from Vietnam is in red and bold
Trinh et al. Egyptian Journal of Biological Pest Control (2021) 31:147 Page 6 of 7

Table 2 Lethal efficacy of Steinernema surkhetense to larvae of a highly significant correlation between nematode inocu-
Galleria mellonella lation concentration and the mortality rate of larvae was
Treatments No. of innoculation Average found based on the Pearson correlation test (R = 0.752,
concentration (IJs/larva) mortality p < 0.01). The One-way ANOVA analysis showed that the
rate (%) lethal efficacies of all treatments are significantly different
Control 0 0a than the control (Table 2).
1 10 60b
2 20 73.33c Discussion
3 30 86.67de Generally, the morphology of S. surkhetense from Viet-
4 40 93.33ef nam is highly in agreement with the description of
5 50 100f Khatri-Chhetri et al. (2011). Variations in some meas-
6 60 100f urements between these populations were found,
7 70 100f including larger L, EP, ES, body diam., T, a, b, c, E% in
8 80 100f the first-generation females; longer body length L, EP,
9 90 100f ES, T, smaller c and E values in the first-generation
10 100 100f males; longer body length and smaller H% value in juve-
*
Note: different superscript letters in the third column indicate significant niles (Table 1). However, according to Hunt and Nguyen
difference (2016), large variations in measurements of adult Stein-
ernema spp. are known to be present since their body
Male (second generation) sizes are much larger than juveniles. Besides, other pop-
Similar to first-generation of males, but body size was ulations of S. surkhetense from different localities in the
smaller (Fig. 1J). world also showed variations in measurements.
Khatri-Chhetri et al. (2011) differentiated S. sur-
khetense from S. nepalense using a combination of mor-
Infective juvenile phological and molecular characterizations. Especially,
Body was short, slender, slightly curved ventrally, and ITS rDNA sequence of S. surkhetense was significantly
tapering to both two ends (Fig. 1K). Lip region was different from that of S. nepalense. However, obtained
rounded and continuous to body contour (Fig. 1L). result showed that D2-D3 expansion segment of 28S
Lateral field was with 9 lines at mid-body (Fig. 1M). rRNA sequence of S. surkhetense in this study was very
Amphids was slit-like. Hemizonid was distinct and similar (only 4 nucleotides difference) to that of S. nepa-
located at beginning of basal bulb. Secretory-excretory lense (accession number: HQ190045). On the other hand,
pore was prominent and located at anterior third of phar- Nguyen and Hunt (2007) reported that cross-hybridiza-
yngeal region. Hyaline region was occupying half of tail tion study can be applied to separate closely related spe-
length. Tail was tapering to pointed tail end (Fig. 1N). cies of Steinernema spp., therefore, such studies can be
Phasmid was pore-like and located at mid-tail. done in the laboratories where populations of both S.
surkhetense and S. nepalense present to support species
Molecular characterization status of these species in future.
Obtained D2-D3 region of 28S rRNA sequence of S. sur- The study of De Doucet et al. (1999) suggested that
khetense from Vietnam was 695 bp long (assession num- larvae of G. mellonella can be used as a standard host
ber: MW703809). It was 100% similar to other sequences for testing and comparing virulence (based on ­ LC50)
of S. surkhetense from GenBank (accession number: of EPNs. Their experiments on larvae of G. mellonella
MF621001, HQ190043). The phylogenetic tree showed showed that ­ LC50 of Steinernema rarum = 6 IJs/larva,
that the D2-D3 region of 28S rRNA sequence of S. sur- Steinernema feltiae = 9 IJs/larva, and Heterorhabditis
khetense from Vietnam had a sister relationship to the bacteriophora = 3 IJs/larva. In Vietnam, Nguyen (2008)
sequences of S. surkhetense, S. nepalense Khatri-Chhetri reported that ­LC50 of 2 potential strains of EPNs (Stein-
et al. (2011), S. siamkayai, S. huense, and S. carpocapsae ernema S-TK10 and Heterorhabditis H-TN3) on lar-
with a maximal support (1 PP). (Fig. 2). vae of Spodoptera litura (Fab.), Omiodes indicate (Fab.),
Agrotis ipsilon (Huf.), Spodoptera exigua (Hub.), Pieris
rapae (L.), and Plutella xylostella (Linn.) were 13–95 IJs/
Virulence of S. surkhetense from Vietnam larva. In this study, ­LC50 of S. surkhetense from Vietnam
Probit analysis in SPSS version 25 showed that L ­C50 was 14 IJs/larva, indicating that this is a relatively poten-
(nematode inoculation concentration kill 50% larvae of tial strain of EPNs to be used in the biocontrol of insect
G. mellonella) of S. surkhetense was 14 IJs/larva. Besides, pests. However, it is also important to test the virulence
Trinh et al. Egyptian Journal of Biological Pest Control (2021) 31:147 Page 7 of 7

of this strain on other insect pests to evaluate its poten- database search programs. Nucleic Acids Res 25:3389–3402. [Link]
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EPNs: Entomopathogenic nematodes. Armonk, NY
Khatri-Chhetri HB, Waeyenberge L, Spiridonov S, Manandhar HK, Moens M
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Not applicable. tive juveniles from Nepal. Russ J Nematol 19:53–74
Laznik Ž, Tóth T, Lakatos T, Vidrih M, Trdan S (2010) Control of the Colorado
Authors’ contributions potato beetle (Leptinotarsa decemlineata [Say]) on potato under field con-
PQT was the supervisor of the project. DTN prepared the data. LTML was ditions: a comparison of the efficacy of foliar application of two strains of
responsible for molecular data. THN prepared the manuscript. All authors con- Steinernema feltiae (Filipjev) and spraying with thiametoxam. J Plant Dis
tributed to writing and editing the manuscript. All authors read and approved Prot 117:129–135
the final manuscript. Laznik Z, Trdan S (2011) Entomopathogenic nematodes (Nematoda: Rhab-
ditida) in Slovenia: from tabula rasa to implementation into crop produc-
Funding tion systems. In: Perveen F (ed) Insecticides - advances in integrated pest
This research was funded by the Institute of Ecology and Biological management. InTech, Rijeka, pp 627–656
Resources, Vietnam Academy of Sciences and Technology (project code: Nguyen HT, Nguyen TD, Le TML, Trinh QP (2020) First report of Xiphinema
VAST04.04/21-22). hunaniense Wang & Wu, 1992 (Nematoda: Longidoridae) in Vietnam. J
Nematol 52:e2020–e2078. [Link]
Nguyen KB, Hunt DJ (2007) Entomopathogenic nematodes: systematics, phy-
Declarations logeny and bacterial symbionts. Brill, Leiden, The Netherlands
Nguyen NC (2008) Entomopathogenic nematodes in Vietnam (in Vietnamese).
Ethics approval and consent to participate Publishing House for Science and Technology, Hanoi, Vietnam
Not applicable. Phan KL, Mráček Z, Půža V, Nermut J, Jarošová A (2014) Steinernema huense sp.
n., a new entomopathogenic nematode (Nematoda: Steinernematidae)
Consent for publication from Vietnam. Nematology 16:761–775
Not applicable. Seinhorst JW (1959) A rapid method for the transfer of nematodes from fixa-
tive to anhydrous glycerin. Nematologica 4:67–69. [Link]
Availability of data and materials 1163/​18752​9259x​00381
All data generated or analysed during this study are included in this published
article.
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Competing interests Springer Nature remains neutral with regard to jurisdictional claims in pub-
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Author details
1
Institute of Ecology and Biological Resources, Vietnam Academy of Sciences
and Technology, 18 Hoang Quoc Viet, Cau Giay, 100000 Hanoi, Vietnam.
2
Graduate University of Science and Technology, Vietnam Academy of Sci-
ences and Technology, 18 Hoang Quoc Viet, Cau Giay, 100000 Hanoi, Vietnam.
3
Nematology Research Unit, Department of Biology, Ghent University, K.L.
Ledeganckstraat 35, 9000 Ghent, Belgium.

Received: 24 September 2021 Accepted: 23 November 2021

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