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(ISSN
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2750-1396)
VOLUME
04
ISSUE
01
Pages:
19-29
SJIF
I
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FACTOR
(2021:
5.478
)
(2022:
5.636
)
(2023:
6.741
)
OCLC
–
1368736135
A
BSTRACT
Steinernema feltiae isolate Sf:1 changed into recovered from one soil pattern gathered from the
Surkhandarya region, Uzbekistan. The new isolate may be prominent from different isolates through
morphologic and morphometric data, and rRNA sequences. The first generation male is characterized
through spicule duration 79,5 (73±87) mm, SW ratio [(spicule length/anal div diameter)*100] 160 13
(137±177), GS ratio GS [(gubernaculum/ spicule length)*100] 79 8 (62±87), genital papillae association
and the second one technology male through a mucron 10 1 (9±11) mm long. For the infective juvenile, the
frame is 883 82 (754±975) mm long, lateral fields with 8 similarly evolved ridges, head smooth, barely
offset, massive gonad primordium cells, a protracted hyaline tail portion (identical to 1/2 of of tail
duration), and pore-like phasmid at 41% of the tail duration to the semicircle anus are the differentiation
factors.
K
EYWORDS
Journal
Website:
http://sciencebring.co
m/index.php/ijasr
Copyright:
Original
content from this work
may be used under the
terms of the creative
commons
attributes
4.0 licence.
Research Article
MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION
OF A BRAND NEW ISOLATE OF ENTOMOPATHOGENIC
NEMATODE, STEINERNEMA FELTIAE (FILIPJEV)
(RHABDITIDA: STEINERNEMATIDAE) FROM THE
UZBEKISTAN
Submission Date:
January 01,
2024,
Accepted Date:
January 03, 2024,
Published Date:
January 05, 2024
Crossref doi:
https://doi.org/10.37547/ijasr-04-01-04
Kurbonova Nafosat Sattor Qizi
Scientific Research Institute Of Plant Quarantine And Protection, Uzbekistan
Haytmuratov Arslоnbek Fayzull
aevich
Scientific Research Institute Of Plant Quarantine And Protection, Uzbekistan
Karimova Rixsiniso Miratxamovna
Scientific Research Institute Of Plant Quarantine And Protection, Uzbekistan
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(2021:
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)
(2023:
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)
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Uzbekistan, entomopathogenic nematode, Steinernema feltiae, morphology, morphometrics.
I
NTRODUCTION
Entomopathogenic nematodes (EPNs) infect
many one of a kind styles of bugs dwelling with
inside the soil inclusive of the larval sorts of
moths, butterflies, flies, and beetles in addition to
person sorts of grasshoppers and crickets. They
are soil-inhabiting organisms and may be used
efficiently to govern soil-borne insect pests,
however are typically now no longer powerful
while carried out to govern bugs with inside the
leaf canopy. The keys to achievement with EPNs
are expertise their existence cycles and functions,
matching the proper nematode species with the
pest species, making use of them in the course of
suitable environmental situations, and making
use of them handiest with like minded pesticides.
Because those dealers are dwelling organisms,
they require cautious coping with to live on cargo
and garage in addition to suitable environmental
situations to live on with inside the soil after
application (Lawrence and Georgis 2012).
Finding indigenous EPN populations and species
is an vital step to acquire powerful organic
manipulate towards pests because of their higher
model to nearby environment. Currently,
approximately sixty three species of the genus
Steinernema had been defined global and those
had been divided into 5 agencies consistent with
their morphology and molecular characteristics
(Nguyen and Hunt 2007). The maximum up to
date biogeographic account found out that those
nematodes had been remoted from all continents
(besides for Antarctica) and nearly all areas of the
world (Hominick 2002). S. feltiae isolate Sf:1 is
defined as a brand new isolate. The new isolate is
separated from different S. feltiae isolates with
the aid of using variations in a few morphological
and morphometric characters, the five,8S -r RNK
location, sequences.
M
ATERIALS AND METHODS
The nematode populace become recovered with
the aid of using the Galleria-entice approach from
one soil pattern amassed in the course of a survey
carried out with inside the Surkhandarya
location, a potato fields, Shurchi in April - May
2022. The nematodes had been sooner or later
reared on G. mellonella larvae and installed as a
laboratory tradition on the Nematology Lab,
institute of Plant quarantine and protection,
Tashkent and named because the Sf:1 isolate.
Light microscopy
First generation females and men had been
amassed from 4
–
five days post inoculated
Galleria cadavers (dissected out in distilled
water). Infective juveniles and 2d technology
adults had been amassed in the course of the
week after their first emergence from Galleria
cadavers and had been killed the use of hot (50
–
60°C) Ringer`s solution (Nguyen and Smart
1994). Dead nematodes had been constant in
triethanolamine
formalin,
processed
to
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anhydrous glycerin with the aid of using a
sluggish evaporation approach (Woodring and
Kaya 1988), and set up on microscopic slides. In
lady specimens, cowl slips had been supported
the use of portions of hair to keep away from
knocking down of nematodes. Morphological
research and morphometric measurements had
been made the use of an Olympus BX41
microscope (Olympus Corporation, Rochester,
NY, USA) geared up with interference contrast,
thru a virtual DP50 camera (Olympus, Shibuya-
ku, Tokyo, Japan) and the use of UTHSCSA Image
device software, model 3.0 (Department of Dental
Diagnostic Science on the University of Texas, San
Antonio, TX, USA) (Vilcox et al 2002).
Molecular characterization
Total genomic extraction of small subunit
ribosomal RNA gene, partial sequence; inner
transcribed spacer 1, five.8S ribosomal RNA gene,
and inner transcribed spacer 2, entire sequence;
and massive subunit ribosomal RNA gene, partial
sequence. of different S. feltiae isolates received
from GenBank.
Sequences for isolate Sf:1 had been deposited in
GenBank beneathneath the accession numbers
OQ189902 respectively.
R
ESULTS
Morphological characteristics
Infective juvenile
Body narrow, habitus fairly curved ventrally
upon heat-killed, on occasion enclosed in cuticle
of 2d-degree juvenile, tapering often from base of
pharynx to anterior give up and from anus to tail
terminus. Mouth and anus closed. Head broad,
offset from frame, labial papillae now no longer
observed,
transverse
slit-like
amphidial
apertures posterior to labial disc however at the
extent of 4 awesome cephalic papillae. Pharynx
long, narrow, isthmus awesome, surrounded with
the aid of using nerve ring (Figure 1E), basal bulb
elongate and valvate. Cardia distinguished.
Secretory
–
excretory pore at mid-pharynx level.
Hemizonid awesome, anterior to the bottom of
terminal bulb (Figure 1E). Bacterial vesicle
elongate (
43μm long).
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Figure 1. Steinernema feltiae isolate Sf:1. A
–
C,
first generation lady: A, anterior location,
pharynx, nerve ring, and secretory-excretory
pore; B, vulva with double epiptygma and vulval
lips; C, tail in lateral view, anus, and terminal peg;
D, tail of 2d technology lady; E, anterior location
of 0.33-degree infective juvenile; F, posterior
location of 0.33-degree infective juvenile. G
–
L,
first generation male; G, anterior location; H and
I, posterior location, spicules, gubernaculum, and
mucron; J and K, gubernaculum; L, posterior
location of male displaying range and association
papillae and mucron. Scale-bars: A = ninety two
μm, B = 22 μm, C = sixty two μm, D = 32 μm, E =
forty two μm, F = 35 μm, G = fifty nine μm, H,
I = fifty t
wo μm, J, K = forty two μm, L = seventy
seven μm.
Female, first generation
Body robust, habitus C-shaped. Cuticle acting
clean beneathneath mild microscopy, lateral
fields now no longer observed. Amphids
inconspicuous. Buccal hollow space funnel- or
cup-shaped, stoma shallow. Pharynx with
cylindrical procorpus, metacorpus barely swollen
and nonvalvate, isthmus awesome, basal bulb
pyriform and valvate.
Female, second generation
Similar to first generation however smaller,
frame period about 1/2 of that of the primary
technology and one-0.33 of its frame diameter.
Eggs are organized in a single row. Tail conoid
with out mucron. Postanal location barely
swollen.
Male, first generation
Body ventrally curved, habitus C- or J-shaped,
tons smaller and extra narrow than lady. Cuticle
clean beneathneath mild microscope, lateral
fields now no longer observed. Head rounded,
barely depressed from frame. Six pointed labial
papillae and 4 cephalic papillae seen
beneathneath SEM. Amphids inconspicuous.
Buccal hollow space funnel- or cup-shaped, stoma
shallow.
Pharynx
muscular,
procorpus
cylindrical,
metacorpus
barely
swollen,
nonvalvate, isthmus awesome, basal bulb
pyriform and valvate. Nerve ring simply anterior
to the basal bulb. Cardia distinguished sticking
out into intestinal lumen, deirids now no longer
seen. Secretory-excretory pore at center of
pharynx, excretory duct cuticularized. Testis
monorchic, reflexed, with brief reflection.
Spicules paired, barely brownish in color,
strongly curved, period/width 6.2, head
(manubrium) width is about identical to period,
blade arcuate with directly tip, dorsal lobe nicely
curved, terminating at spicule tip, lateral lobe
distinguished, terminating at spicule tip, ventral
lobe enlarged anteriorly at dorsal and Ventral
facet to shape outstanding apex and rostrum,
terminating posterior to rounded spicule tip,
velum massive, now no longer masking spicule
tip.
Gubernaculum about 75% of spicule period, boat-
formed in lateral view, swollen at middle, with
barely ventrally curved knob at proximal end, in
ventral view, cuneus short, pointed posteriorly,
wing of corpus increasing laterally. There are 23
genital papillae comprising eleven pairs of
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papillae and a unmarried ventral papilla
positioned anterior to cloacal opening. Of these,
seven pairs are positioned precloacal (three pairs
lateral and four pairs subventral), one pair
adcloacal and 3 pairs postcloacal (1 pair
subdorsal and a couple of pairs subterminal
almost subventral). Tail conoid with eight μm
lengthy mucron that continually present.
Phasmids inconspicuous. Figure 2. Light
microscope pics of first era men of Steinernema
feltiae isolate Sf:1 : A
–
F, version in spicule and
gubernaculum shape. Scale bars: A = 22 μm, B =
18,7 μm, C = 35 μm, D = 31 μm, E and F = 17,7 μm.
Male, 2nd era Similar to first era male however
greater slender, frame diameter about 1/2 of that
of the primary era male. Testis flexure extending
greater posteriorly. Mucron greater wonderful,
about 12 ± 1 μm lengthy.
Figure 2. Light microscope photographs of first generation males of Steinernema feltiae isolate
Sf:1 : A
–F, variation in spicule and gubernaculum shape. Scale bars: A = 22 μm, B = 18,7 μm, C = 35
μm, D = 31 μm,
Male, second generation
Similar to first generation male but more slender,
div diameter approximately half that of the first
generation male. Testis flexure extending more
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posteriorly. Mucron more distinct, approximately
12 ± 1 μm long.
Discussion
S. feltiae isolate Sf:1 is morphologically
characterised via way of means of a mixture of the
capabilities of diverse developmental levels of the
nematode (Table 1). Infective juveniles are
prominent via way of means of their frame period
of 882 ± 80 (760
–984) μm, clean head and barely
offset, lateral fields with 8 similarly advanced
ridges (nine traces or incisures) at mid-frame
area, hyaline tail element lengthy (identical to 1/2
of of tail period) and massive gonad primordium
cells.
The first era men are characterised via way of
means of lengthy, head rounded, barely brownish
spicules with 81 ± 5 (73
–87) μm lengthy,
advanced velum, spicule period/width 6.four ±
0.eight (5.2
–
6.nine), ratio SW (%) 162 ± 13 (137
–
177), GS ratio (%) 81 ± eleven (65
–
90), posterior
location with 23 genital papillae (eleven pairs and
a unmarried ventral preanal).
The second generation have an extended mucron,
10 ± 1 (nine
–eleven) μm lengthy. The infective
juvenile of the brand new isolate is prominent
from all different isolates via way of means of a
clean genital primordium, lateral discipline
morphology at mid frame, with 8 similarly
advanced ridges as opposed to 8 ridges with
submarginal pair vague in different isolates, and
a wonderful hyaline tail element approximately
1/2 of of the tail period.
Males of isolate Sf:1 are separated from kind
species and different Uzbekistan isolates via way
of means of an extended spicule, 81 (73
–
87) as
opposed to 72 (65
–
77); SW, 162 (137
–
177) as
opposed to 115 (99
–
130); GS, 81 (65
–
90) as
opposed to 59 (52
–
61) (Table 2), advanced
velum, and spherical head spicule. In addition, the
gubernaculum withinside the new isolate is
longer than that of different isolates of S. feltiae
(sixty two vs. fifty one μm). The referred to
variations may be taken into consideration as
intraspecific versions and discriminate this
isolate from others. Furthermore, infective
juveniles of isolate Sf:1 are absolutely coiled
below four
–
7°C temperatures, while none of the
opposite Uzbekistan species (belonging to the
`feltiae-kraussei-oregonensis` institution species)
display comparable behavior.
Table 1. Morphometrics of Steinernema feltiae isolate Sf:1. Measurements are in μm and in the
form: mean ± standard deviation (range).
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Character
First generation
Second generation
Infective
juvenile
Male
Female
Male
Female
Number of
specimens
examined
20
20
20
20
20
Body length (L) 1361 ± 99
(1226
–
1520)
5022 ± 950
(4392
–
7200)
871 ± 81
(677
–
983)
1852 ± 216 (1810
–
1921)
885 ± 82
(754
–
975)
a (L/W)
9,2 ± 1
(7
–
10)
25 ± 2 (22
–
25)
17 ± 2 (12
–
19)
17 ± 1 (14
–
17)
25 ± 4
(18
–
28)
b (L/ES)
10 ± 1 (7
–
11)
27 ± 1 (25
–
27)
6,2 ± 1 (5
–
7)
13 ± 1 (10
–
13)
7,2 ± 0.7
(5
–
7.5)
c (L/T)
37 ± 3
(30
–
39)
230 ± 14
(220
–
242)
33 ± 5 (25
–
38)
45 ± 2 (38
–
46)
12,2 ± 1
(9
–
13)
Vulva %
–
56 ± 3 (49
–
61)
–
54 ± 2 (48
–
55)
–
Body diameter
(W)
157 ± 18
(123
–
182)
232 ± 44
(185
–
260)
57 ± 5 (49
–
63)
128 ± 36 (108
–
217)
40 ± 3
(34
–
44)
Excretory pore
(EP)
88 ± 7
(75
–
94)
97 ± 12 (88
–
120)
67 ± 8 (50
–
78)
66 ± 8 (54
–
82)
62 ± 5
(54
–
66)
Nerve ring
105 ± 11
(95
–
116)
155 ± 17
(140
–
172)
–
96 ± 12 (73
–
111)
113 ± 6
(89
–
115)
Pharynx length
(ES)
157 ± 10
(140
–
168)
212 ± 11
(195
–
236)
140 ± 9
(128
–
157)
168 ± 11 (142
–
179)
131 ± 7
(122
–
142)
Tail length (T) 39 ± 3
(34
–
42)
22 ± 5 (18
–
31)
29 ± 5 (22
–
36)
43 ± 4 (38
–
47)
78 ± 10
(65
–
93)
Anal div
diameter (ABD)
52 ± 3
(44
–
54)
61 ± 6 (45
–
64)
36 ± 3 (30
–
38)
25,2 ± 2 (23
–
27)
20 ± 2
(15
–
21)
Mucron length 8 ± 0.5
(7
–
8.5)
–
10 ± 1 (9
–
11)
–
–
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Character
First generation
Second generation
Infective
juvenile
Male
Female
Male
Female
Spicule length
(SL)
79 ± 5
(73
–
87)
–
58 ± 4 (55
–
64)
–
–
Spicule width
(SW)
13 ± 2
(11
–
15)
–
–
–
–
Gubernaculum
length (GL)
62 ± 4
(56
–
64)
–
42 ± 3 (37
–
46)
–
–
D%
[(EP/ES) × 100]
57 ± 7
(48
–
65)
47 ± 6 (42
–
52)
47 ± 4 (39
–
50)
40 ± 4 (36
–
43)
47 ± 3
(42
–
48)
E%
[(EP/T) × 100]
223 ± 25
(187
–
255)
415 ± 35
(382
–
451)
221 ± 35
(182
–
254)
152 ± 21 (129
–
156)
79 ± 4
(73
–
81)
SW
(SL/ABD) × 100
164 ± 13
(137
–
177)
–
176 ± 14
(153
–
190)
–
–
GS
(GL/SL) × 100
83 ± 11
(65
–
90)
–
73 ± 5 (56
–
76)
–
–
Table 2. Comparative morphometrics of Steinernema feltiae with its Uzbekistan isolates (mean
and range in μm).
Stage/character
S. feltiae
(Type)
S. feltiae
Sf:1
S. feltiae
Sf:17
S. feltiae
Sf:28
Infective juvenile
L
849 (736
–
950)
883 ± 82 (754
–
975)
678 ± 70 (546
–
784)
655 ± 80 (533
–
810)
EP
62 (53
–
67)
60 ± 5 (54
–
66) 57 ± 3 (53
–
62)
49 ± 5 (42
–
60)
ES
136 (115
–
150)
131 ± 7 (122
–
142)
107 ± 4 (101
–
115)
113 ± 11 (96
–
134)
T
81 (70
–
92)
78 ± 10 (65
–
93)
74 ± 7 (63
–
85)
59 ± 8 n/a
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Stage/character
S. feltiae
(Type)
S. feltiae
Sf:1
S. feltiae
Sf:17
S. feltiae
Sf:28
D%
[(EP/ES) × 100]
45 (42
–
51)
45 ± 3 (42
–
48) 51 ± 2 (48
–
54)
44 ± 3 (39
–
51)
E%
[(EP/T) × 100]
78 (69
–
86)
77 ± 4 (73
–
81) 77 ± 7 (67
–
87)
83 ± 8 (59
–
96)
Ridges
8
8
8
8
1st generation male
D%
[(EP/ES) × 100]
60 (56
–
62)
55 ± 7 (48
–
65) 49 ± 6 (36
–
56)
58 ± 6 (52
–
68)
SL
70 (65
–
77)
79 ± 5 (73
–
87) 65 ± 3 (62
–
72)
72 ± 3 (70
–
75)
SW
113 (99
–
130)
160 ± 13 (137
–
177)
151 ± 7 (141
–
161)
152 ± 14 (137
–
172)
GS (GL/SL) × 100 59 (52
–
61)
79 ± 11 (65
–
90)
64 ± 5 (56
–
69)
71 ± 6 (64
–
75)
Mucron
P
P
P
P
EP = excretory pore; ES = pharynx length; GL = gubernaculum length; L = length; n/a = no data
available; P = present; SL = spicule length; SW = spicule width; T = tail length.
A
CKNOWLEDGMENTS
The authors thank Dr Khodjaeva Sevarakhan,
Norov Tokhirbek, Yunusov Alisher, Botirova
Nasiba (Agency of plant protection and
quarantine of the Republic of Uzbekistan) for the
sequencing and providing helpful comments on
disacussion.
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)
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