MORPHOTAXOMETRIC AND MOLECULAR VALIDATION OF ...

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Bhat et al. World Journal of Pharmacy and Pharmaceutical Sciences

MORPHOTAXOMETRIC AND MOLECULAR VALIDATION OF

ENTOMOPATHOGENIC NEMATODE, STEINERNEMA ABBASI

(RHABDITIDA: STEINERNEMATIDAE) WITH MUCRONATE

PROCESSES IN ADULTS OF SECOND GENERATIONS OFF

SUBHUMID REGION, UTTAR PRADESH, INDIA

Aashaq Hussain Bhat1, Ashok K. Chaubey

1 and Sushil K. Upadhyay

2*

1Nematology Laboratory, Department of Zoology, Ch. Charan Singh University,

Meerut-250004, UP, India.

2Department of Zoology, Faculty of Science, Swami Vivekanand Subharti University,

Meerut-250005, UP, India.

ABSTRACT

The investigations were conducted on soil samples collected from the

sugarcane (Saccharum officinarum L.) agriculture fields of sub-humid

region, Uttar Pradesh. The worms were diagnosed as member of

bicornutum group due to the presence of two horns like cephalic

structures in third stage infective juveniles. The newly isolated worms

were not identical to pre-existing individuals of same group and can be

differentiated from the Steinernema abbasi (Rhabditida:

Steinernematidae) by the presence of mucronate processes in adults of

second generations and relative proportion of oesophagus with body

length in juveniles. In the present investigations the recovered

populations of entomopathogenic nematodes (EPN) were identified,

characterized and validated as an isolate of S. abbasi with the

application of advanced molecular tools. The phylogenetic analyses were based on the

efficacy of conserved genes (ITS, D2-D3 and coxI) sequences in taxa validations so that they

could later be used as biocontrol agents.

KEY WORDS: Steinernema abbasi, Morphotaxometry, Molecular phylogeny, Conserved

genes, Entomopathogenic nematode (EPN), Biocontrol agent.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.041

Volume 5, Issue 3, 1558-1579. Research Article ISSN 2278 – 4357

Article Received on

12 Jan 2016,

Revised on 03 Feb 2016,

Accepted on 24 Feb 2016

*Correspondence for

Author

Dr. Sushil K. Upadhyay

Department of Zoology,

Faculty of Science, Swami

Vivekanand Subharti

University, Meerut-

250005, UP, India


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INTRODUCTION

Entomopathogenic nematodes (EPN) of the genus Steinernema Travassos, 1927 are copious

and sundry group of soil-dwelling nematodes that parasitize the entomic fauna of valuable

agricultural crops. The 3rd

stage infective juveniles of this genus inhabited in symbiotic

association with bacterium Xenorhabdus (Thomas and Pionar, 1979) and it is the only stage

that enters into their host either via natural openings or topologically. Once they gain entry

into insect, they release their symbionts and ultimately kill the host within 24 to 48 hours

(Poinar, 1975; Griffin et al., 1991; Brown and Gaugler, 1997; Adams and Nguyen, 2002).

Because of these assets, they have been used as biological control agents (Bedding, 1990) and

exploration of new indigenous isolates of EPN is therefore getting attention around the world.

Discovery of new species and strains of EPN is a continuous process and it is assumed that

lots of species are waiting for their discovery. Identification of nematodes on the basis of

morphological features and their measurements along with molecular tools has proven

helpful in resolving ambiguities among the existing and newer species of EPN. The

ribosomal DNA consensus is an ideal choice for identification purpose as it contains highly

conserved regions and potentially highly variable regions. The most useful regions for EPN

identification are non-coding internal transcribed spacer regions (ITS1+5.8S+ITS2), D2-D3

and cytochrome oxidase subunit I (coxI). The aim of this study was to describe the existing

taxa of EPN as an isolate Steinernema abbasi based on the analyses of morphotaxometry and

afore mentioned conserved genes for molecular phylogeny so that they could later be used as

biocontrol agents.

MATERIALS AND METHODS

Isolation and storage of worms

The soil samples were collected from different sugarcane agricultural fields of Baghpat, Uttar

Pradesh, India using Wallace‟s sampling techniques (Wallace, 1971). The soil samples were

processed for EPN isolation by baiting technique after Bedding and Akhurst (1975).

Emerging 3rd

stage juveniles (IJ) were recovered from white trap (White, 1927) and stored in

culture flasks at 15±1°C in BOD incubator.

Light Microscopy

The 1st and 2

nd generation adults were obtained from cadavers after 2-3 days and 4-5 days of

post infection periods, respectively by dissecting them in Ringers solution (Woodring and

Kaya, 1988) while firstly emerged IJ were obtained from White traps. These were then killed


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with luke warm water, fixed in TAF (Courtney et al., 1955) and processed to glycerin after

Seinhorst (1959). Permanent slides were prepared using paraffin wax to avoid flattening of

specimens. Fifteen specimens of adults and twenty specimens of IJ were measured and

observed. Measurements are given in µm as mean ± standard deviation (SD) followed by

range in parentheses. All the measurements and photomicrographs were taken using Nikon

trinocular computerized unit with DLS1 software.

Scanning Electron Microscopy

Ultra topographic features of worm were investigated by the application of scanning electron

microscope. The infective juveniles (3rd

stage) and adults of 1st generation males were fixed

in 4% glutaraldehyde buffered with 0.1M phosphate buffer (pH 7.2). They were then washed

with phosphate buffer and post fixed with 2% osmium tetra-oxide followed washing with

0.1M phosphate buffer and dehydrated in graded ethanol series and dehydrated ethanol at

25ºC. They were dried at critical point with liquid CO2, mounted on SEM stubs with double-

sided carbon tape and coated with a 15nm layer of gold in a sputter coater (Nguyen and

Smart, 1995; 1997). The ultra-topographical microphotographs were captured with Neo

Scope JEOL 5000 FE scanning electron microscope (JEOL, Eching, Germany).

Cross-hybridization

Cross-breeding tests were performed between isolates of S. abbasi, CS17 and CS18 and two

other isolates present in lab viz. CS19 and CS20, using the modified hanging drop method

(Kaya and Stock, 1997). Twenty replicates were created for each treatment with control.

Sealed Petri dishes were incubated at 27-30°C and observations were recorded daily for

reproductive compatibilities. The closely related species Steinernema were not available to

carry out this test so far.

Molecular and phylogenetic analysis

The total genomic DNA was extracted from 3rd

stage of infective juveniles through Quigen

DNA Blood and Tissue Kit (Upadhyay, 2012) followed by Agarose Gel Electrophoresis

(AGE) for the detection of DNA in the collected elute. Molecular characterization were

based on internal transcribed spacer (ITS) regions of rDNA, partial sequence of 28S rDNA

(D2D3 domain) and mitochondrial gene encoding cytochrome C oxidase subunit 1 (coxI) for

validation of recovered worms as newer isolate of Steinernema. These regions were amplified

using the primers as suggested by Joyce et al. (1994). Amplified regions were sequenced,

annotated and submitted to National Center for Biotechnology Information (NCBI) under


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accession numbers KP219885, KU187260 and KU529463 for ITS, D2-D3 and coxI genes

respectively. The sequences of the present specimens were compared with available

sequences in GenBank using the BLASTN program (Altschul et al., 1990). The alignment

was worked out through BioEdit ver.7.0.5 (Hall, 1999) with Steinernema sp. sequences of

“bicornutum group”. Phylogenetic relationships among isolates were reconstructed by the

Maximum Parsimony (MP) method (Nei and Kumar, 2000) using Mega 6.0 program

(Tamura et al., 2007). Clades from trees of MP were supported by bootstrap analysis with

1000 replicates. The evolutionary distances were computed using LogDet method according

to Tamura and Kumar (2002) and are expressed as the units of the number of base

substitutions per site.

Abbreviations: n, characters; L, total body length; L‟, anterior end to anus; SW, stoma width;

EP, excretory pore; WEP, width at excretory pore; NR, nerve ring; ES, pharynx length;

ABW, anal body width; GBW, greatest body width; TR, testis reflection; SL, spicule length;

SW, spicule width; GL, gubernaculums length; GW, gubernaculums width; V, anterior end to

vulva length; V‟, posterior end to vulva length; WV, width at vulva; a, L/GBW; b, L/ES; c,

L/tail; c‟, tail/ABW; D%, EP/ES x 100; E%, EP/tail x 100; F%, GBW/tail x 100; V%, V

‟/L x

100; Muc, mucron.

RESULTS AND DISCUSSION

Type habitat: Soil around the roots of sugarcane plant (Saccharum officinarum L.)

Type locality: Baraut, Baghpat district, Uttar Pradesh, India.

Type specimen: Isolate CS17 and CS18 Steinernema abbasi (Rhabditida: Steinernematidae)

Infective juveniles (IJ, Fig. 1, 2)

The body of the IJ is thin, elongate and tapering smoothly at both extremities (from base of

esophagus to anterior end and from anus to terminus) and ensheathed within second-stage.

Body of heat relaxed specimens slender, almost straight or slightly curved with striated

cuticle and truncate cephalic region, continuous with body or offset. Stoma closed and cuticle

with faint transverse annulations. Exsheathed IJ with two horn like structures on the labial

region very distinct with light microscopy and SEM. Labial region smooth, usually

continuous with body. Oral aperture closed and phasmids inconspicuous. Pharynx long,

narrow, distinctly narrower at nerve ring level, terminating in a valvate basal bulb. Nerve ring

distinct and anterior to basal bulb. Excretory pore near the base of the metacorpus. Distance

from the anterior end to excretory pore always more than body width at excretory pore.


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Lateral field with eight ridges evenly spaced at the mid-body; beginning with a single line

becoming two ridges splitting into four, then six and eight at the mid-body ( Fig. 2). Towards

posterior region the number reduces to seven and then forming the last two ridges which

continue almost to the tail tip. Lateral field formula was: 2, 4, 6, 8, 7, 2. Cardia present,

rectum long and narrow, terminated with distinct anus. Tail was conical, tapering to a fine

pointed curved terminus.

A B

Fig. 1. Isolate CS17 of Steinernema abbasi (Infective juvenile): A- Pharyngeal region; B-

Tail region.

Fig. 2. Scanning electron microscopic images showing lateral line pattern in 3rd

stage

juveniles of Isolate CS17 Steinernema abbasi.

B

A A A A A

A

C

A A

D

E


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Male (1stgeneration, Fig. 3)

The 1st generation male of present worms were characterized by slender, ventrally curved, J-

shaped body with smooth cuticle, lateral fields and inconspicuous phasmids, collapsed stoma,

prominent cheilorhabdions beneath lips and separated by thick ring of sclerotized material.

Pharynx was muscular with cylindrical procorpus, slightly swollen metacorpus and narrow

isthmus with round basal bulb. Oesophagus was large extending near to mouth opening,

nerve ring distinct surrounding the middle of the isthmus, excretory pore at the level of nerve

ring and metacarpus. Distance from anterior end to excretory pore was always more than

body width at excretory pore, cardia well developed, gonads monarchic and testis reflexed

with varied length. Spicules with paired protuberant manubrium, thick lamina (blade) and

calomus with small rounded protrusions. A distinct velum extends almost to proximal end of

the lamina. Gubernaculum was boat-shaped, ventrally curved, slightly swollen in the middle

and gradually narrowing distally and bursa absent. Twenty-three genital papillae were visible

with light microscopy including 11 pairs and single mid-ventral precloacal papilla. Tail was

short, bluntly conical and no any terminal mucron.

A B C D

Fig: 3. Isolate CS17 of Steinernema abbasi (1st generation male): A, C- Pharyngeal

region; B, D- Anal region showing papillae.

Male (2nd

generation, Fig. 4)

The male worm of this generation was more or less similar to first generation, differing by

smaller body size, spicules and gubernaculums and a very short mucron in tail region.


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A B

Fig: 4. Isolate CS17 of Steinernema abbasi (2nd

generation male): A- Pharyngeal region;

B- Anal region.

Female (1st

generation, Fig. 5, 6)

Body became spirally coiled when heat killed. The body provided with smooth cuticle,

indistinct lateral lines and phasmids, truncated slightly rounded labial region. Oesophagus

was extending close to the oral opening, cheilorhabdions prominent located beneath the lips

composed of thick cuticularised ring, oesophagus muscular and cylindrical, metacorpus

slightly swollen, isthmus slightly narrowing terminating in a muscular basal bulb in the newly

recovered first generation female. Oesophagus set off from the intestine, posteriorly inserted

into the anterior portion of the intestine, cardia well developed, nerve ring usually

surrounding the anterior portion of basal bulb and excretory pore circular, located at the level

of metacorpus. Gonads amphidelphic, reflexed containing many eggs in random manner,

eggs commonly hatching inside female body and juveniles boring their way out. Vulva was

post-equatorial in position, slightly protruded and guarded with double flapped epiptygmata

and has transverse silt like opening. Rectum and anal opening was distinct, tail short, conical

with a pointed terminus without mucron. A ventral post anal swelling was always present in

the collected specimens.

A B C

Fig. 5. Isolate CS17 of Steinernema abbasi (1st generation female): A- Pharyngeal region;

B- Vulva region; C- Tail.


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A B C

Fig:6. Ultratopographic images of Isolate CS17 of Steinernema abbasi (1st generation

female): A- Pharyngeal region; B- Vulva region; C- Tail.

Female (2nd

generation, Fig. 7)

These were similar in general aspects to first generation female except smaller length and

width. Tail protruding longer, conoid and tapering evenly to salient posterior anal lip and

ending with a well-developed fine mucron.

A B C

Fig. 7. Isolate CS17 of Steinernema abbasi (2nd

generation female): A- Pharyngeal region;

B- Vulva region; C- Tail.

Diagnosis and relationships

Isolate CS17 of S. abbasi (Table 1) was characterized by noticeable facet i.e. presence of

mucron in 2nd

generation males (Figs. 4, 8) and females (Figs. 7, 8) and its absence in 1st

generation adults (Figs. 3, 5, 6, 8). Presence of two horn-like structures on the labial region of


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IJ (Figs. 1, 2, 8) places this in bicornutum group. Morphometrically (Table 1, 2), it can be

characterized by the IJ body length 549(504-599)µm, pharynx 101(91-106)µm, „a‟ratio

20(14-24), tail 53(44-66)µm, D% 50(43-57) and E% 96(67-122). The species is also

recognized by male (1st gen) characters of D% 54(36-66) and genital papillae. The 1

st

generation female with excretory pore 62(47-77)µm, pharynx 142(123-164)µm and tail

57(44-70)µm. The species is also characterized by sequence length of ITS1 (269bp), 5.8S

(157bp) and ITS2 (315bp) and by sequence composition (Table 3). The S. abbasi CS17 was

compared with eight species within bicornutum group which all are distinguished by the two

horn-like structures on their anterior end (Table 2). The present isolate showed similitude

with only one described species of Steinernema viz. S. abbasi while with others showed

incongruity in the following features with already species of bicornatum group. On

comparing males of the present specimen with S. abbasi (Elawad et al., 1997) males,

discrepancy was observed only by presence of well-developed mucron in second generation

males and females while only hardly visible in second females of S. abbasi. This might be

due to varied climatic conditions as well as different habitats. However, in all other

morphological characters much resemblance was noticed. Twenty-three genital papillae and

lateral field pattern of 2, 4, 6, 8, 7 and 2 was observed in the present and already described

species. As far as morphometry is concerned, little variation was observed in measurements

while most measurements were in close vicinity. The same was revealed by molecular

studies.


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Fig. 8. Microphotography of isolate CS17 of Steinernema abbasi (not to scale). Female 1st

generation: A- Pharyngeal region, B- Vulva region, C- Tail region; Male 1st

generation:

D- Anterior region, E- Tail region showing spicules; Female 2nd

generation: F- Anterior

region, G- Vulva region, H- tail region with mucron (arrowed); Male 2nd

generation: I-

Pharyngeal region, J- Tail region with spicules and gubernaculums; Infective juveniles:

K- Pharyngeal region, L- Tail region.


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S. abbasi CS17 can be segregated (Table 2) from S. bicornutum (Tallosi et al., 1995) by the

shorter length of IJ 549(504-599) vs 770(648-873)μm; EP, 50(43-61) vs 61(53-65)μm; NR,

73(52-88) vs 92(88-100)μm; ES, 101(91-100) vs 124(113-135)μm; ratio „a‟, 20(14-24) vs

27(23-29) and tail, 53(44-66) vs 72(63-78)μm. Males of the present species can be

distinguished (Table 2) from S. bicornutum by the number of genital papillae (23 vs 25),

presence of mucron only in the second generation males and females vs presence in all adults

generations except 1st generation males, by SL, 70(66-78) vs 65(53-70)μm; GL, 41(37-46) vs

48(38-50)μm; SW%, 191(145-231) vs 222(218-226) and GS%, 59(47-67) vs 72.

Table 1: Morphometrics of isolate CS17 of Steinernema abbasi. All measurements are in

μm and in the form: mean ± S.D. (range) except ratio and percentage.

Characters First Generation Second Generation Infective

Juvenile (IJ) Male Female Male Female

N 15 15 15 15 20

L 1335±80

(1244-1494)

5363±1361

(2977-7336)

941 ± 95

(800 - 1082)

1780 ± 516

(1046 - 2444)

549±29(504-

599)

A 12±1(10-14) 28±6(20 - 40) 16 ± 1(13 - 19) 16 ± 1(14 - 19) 20 ± 3(14 - 24)

B 9±0.9(7- 10) 30 ± 7(18 - 41) 8 ± 0.8(6 - 9) 12 ± 23(8 - 17) 5 ± 0.3(5 - 6)

C 47±5(40 -58) 125 ± 50(49 - 209) 39 ± 4(31 - 45) 31 ± 6(20 - 42) 10 ± 1(9 - 13)

c' 0.8±0.1(0.6 - 0.9) 0.8 ± 0.2(0.5 - 1.5) 0.8±0.1(0.7-1.0) 1.5±0.3(0.9-2.1) 3 ± 0.4(2 - 4)

V - 52 ± 6(33 - 56) - 54 ± 2(52 - 58) -

GBW 112±10(89 -132) 194±30(143 - 236) 58 ± 8(47 - 74) 108±29(72-142) 28 ± 5(23 - 44)

EP 80± 10(61 - 92) 77 ± 15(47 - 99) 71 ± 12(54 - 89) 62 ± 11(47 - 77) 50 ± 4(43 - 61)

NR 112±5(103 - 122) 125 ± 8(108 – 136 97 ± 6(87 - 114) 107±11(91-125) 73 ± 11(52 - 88)

ES 151±14(136 - 183) 177±11(158 - 202) 121 ±6(112 -

133)

142±12(123 -

164 101 ± 4(91-106)

TAIL (T) 29 ± 3(25 - 34) 47 ± 16(297 - 90) 24 ± 30(19 - 30) 57 ± 9(44 - 70) 53 ± 5(44 - 66)

ABW 37 ± 5(32 - 47) 59 ± 15(41 - 98) 30 ± 4(25 - 38) 39 ± 10(25 - 63) 16 ± 2(14 - 25)

SL 70 ± 3(66 - 78) - 56 ± 5(50 - 69) - -

SW 6 ± 1(4 - 8) - 4 ± 0.8(3 - 5) - -

GL 41 ± 3 (37 - 46) - 30 ± 3(25 - 36) - -

GW 6 ± 0.7(5 - 8) - 5 ± 0.3(4 - 5) - -

D% 54 ± 1(36 - 66) 44 ± 10(25 - 57) 59 ± 11(41 - 73) 43 ± 6(36 - 51) 50 ± 4(43 -57)

E% 279±29(221-308) 168 ± 53(92 - 230) 289±32(220-331) 110±10(93-127) 96 ± 13(67-122)

F% 390±44(327- 495) 453±158(228-735) 236±28(187-288) 187±31(140-

242) 53 ± 6(44 - 70)

SW% 191±23(145 - 231) - 190±24(131-218) - -

GS% 59 ± 5(47 - 67) - 54 ± 6(45 - 64) - -


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Table 2. Comparative morphometrics of infective juveniles and first generation males of S. abbasi isolates i.e. CS17 and CS18 and related

Steinernema sp. from the bicornutum-group. All measurements are in μm as mean followed by range in parentheses unless stated

otherwise.

Character abbasi bicornutum ceratophorum pakistanense riobrave yirgalemense bifurcatum papillatum CS17 CS18 goweni

IJ

L 541

(496-579)

770

(648-873)

706

(591-800)

683

(649-716)

622

(361-701)

635

(548-693)

521

(460-590)

652

(572-720)

549

(504 - 599)

530

(494- 578)

640

(531-719)

EP 48(46-51) 61(53-65) 55(47-70) 54(49-58) 56(51-64) 51(45-59) 45 (40-49) 50(44-58) 50(43 - 61) 51(40 - 65) 51(32-58)

NR 68(64-72) 92(88-100) 92(79-103) 80(76-83) 87(84-89) 88(82-93) 68(64-72) 88(81-96) 73(52 - 88) 77(69 - 87) 81(69-94)

ES 89(85-92) 124(113-135) 123(108-144) 113

(108-122)

114

(109-116)

121

(115-128) 89(85-92) 110(103-121)

101

(91 - 106) 98(85 -111)

119

(109-126)

T 56(52-61) 72(63-78) 66(56-74) 58(53-62) 53.5(46-54) 62(57-67) 53.6(51-59) 54(40-78) 53(44 - 66) 54(46 -61) 67(59-89)

A 18(17-20) 27(23-29) 26(24-28) 24(21-27) 23(20-24) 21(20-25) 18(17-20) 27(22-30) 20(14 - 24) 20(17 - 22) 25(22-29)

B 6(5.5-6.6) 6.2(5.6-6.9) – 6.0(5.0-6.0) 5.4(4.9-6-0) 5.2(4.8-5.9) 6(5.5-6.6) 5.9(5.0-6.4) 5(5 - 6) 5(5 - 6) 5.4(4-6)

C 9.8(8.1-

0.8) 10.7(9.7-12.0) 10.6(8.8-12.9)

11

(10.0-12.0)

11.6

(10.1-2.4)

10.3

(9.2-11.2) 9.8(8.1-10.8) 12.1(8.3-15.0) 10(9 - 13) 10(8 -12) 9(6-11)

D% 53(51-58) 50(40-60) 45(40-56) 47(42-53) 49 (45-55) 42 (38-48) 39.7 (33-47) 46(40-53) 50(43 -57) 50(41 - 64) 43(27-49)

E% 86(79-94) 84(80-100) 84(74-96) 91(87-102) 105 (93-111) 83(67-98) 84.7 (77-94) 93(66-121) 96(67 -

122) 94(70 - 123) 77(48-94)

Male (1st)

EP 80(68-89) 82(67-98) 85(50-104) 81(72-92) 103(94-111) 86(74-107) 70(58-86) 73(54-96) 80(61 - 92) 86(71 - 99) 61(40-860

NR 103

(99-123) 123(108-137) 123(90-147) 99(88-107)

103

(106-134) 108(98-136) 117(100-130) 104(74-125)

112

(103- 122)

111

(101- 128)

114

(101-134)

ES 133

(121-144) 156(138-167) 165(149-190)

132

(126-146)

144

(128-154)

148

(132-165) 145(130-158) 136(94-163)

151

(136- 183)

138

(120- 165)

142

(131-150)

T 26(20-31) 32(25-35) 30(23-38) 25(24-27) 31(29-35) 20(17-27) 30(22-34) 25(16-35) 29(25 - 34) 30(24 - 37) 28(23-32)

SL 61(51-69) 65(53-70) 71(54-90) 68(62-73) 67(63-75) 64(51-77) 68.8(60-85) 52(42-62) 70(66 - 78) 73(62 - 96) 55(50-57)

GL 43(35-48) 47(38-50) 40(25-45) 41(36-45) 51(48-56) 48(42-54) 38.9(30-49) 31(23-36) 41(37 - 46) 43(32 - 48) 35(30-40)

D% 56(50-70) 52(50-60) 51(33-65) 60(50-60) 71(60-80) 58(50-66) 48(42-58) 54 (43-65) 54(36 - 66) 62(53 - 71) 42(28-59)


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The present isolate showed divergence from S. pakistanense (Shahina et al., 2001) by the presence of mucron in 2nd

generation adults while in

case of S. pakistanense indistinct in 1st generation males and visible only in 2

nd generation females and in morphometry, the males of the two

differed by SL, 70(66-78) vs 62(62-73)μm. IJs are shorter than S. pakistanense 549(504-599) vs 683(649-716)μm (Table 2), an anal swelling in

the 2nd

generation female present vs absent. When compared with S. ceratophorum (Jian et al., 1997), again the IJ of the present specimen were

shorter than S. ceratophorum and differed in most morphometrical measures (Table 2) which was also noticed in their first generation males.

Double flapped epiptygmata present in both generation females while these characters are not scrutinized in S. ceratophorum. Mucron was

observed in 2nd

generation male and female only while in S. ceratophorum it was reported only in 1st and 2

nd generation females.

SW% 159

(128-180)

222

(218-226)

140

(100-200)

180

(100-220) 114

171

(121-213) 138(120-170) 156(125-194)

191

(145 231)

171

(135- 218)

146

(105-208)

GS% 70(58-85) 72 60 (40-80) 60(50-60) 76 74(65-85) 59(51-79) 59(48-70) 59(47 - 67) 60(46 - 72) 64(49-790

GBW 87(82-98) 109(80-127) 146(104-185) 102(80-128) 133

(116-160) 112(97-138) 108(85-117) 69(54-87)

112 (89 -132)

144(96-171) 100

(85-115)

MUC A A A P A A A A A A A


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The males (1st generation) of the present specimen could be separated from males of S.

riobrave (Cabanillas et al., 1994) by EP, 80(61-92) vs 103(94-111)μm; SL, 70(66-78) vs

67(63-75)μm; GL, 41(37-46) vs 51(48-56)μm; GBW, 112(89-132) vs 133(116-160)μm; D%,

54(36-66) vs 71(60-80); SW%, 191(145-231) vs 114 and GS%, 59(47-67) vs 76. The

recovered IJs were shorter than S. riobrave viz. 549(504-599) vs 622(361-701)μm. The

number of genital papillae in male generation also varies which is 23 vs 24. In present isolate

double-flapped epiptygmata were present in both generations of females vs no epiptygma in

the second generation female of S. riobrave. In addition mucron was present in 2nd

generation

males and females vs absence in all generations of S. riobrave. CS17 isolate of S. abbasi might

be differentiated from S. yirgalemense (Nguyen et al., 2004) by shorter IJ 549(504-599) vs

longer IJ 635(548-693). Males of present specimen could be separated from males of S.

yirgalemense by tail length 29(25-34) vs 20(17-27)μm; SL, 70(66-78) vs 64(51-77)μm; GL,

41(37-46) vs 48(42-54)μm; SW%, 191(145-231) vs 171(121-213); GS%, 59(47-67) vs

74(65-85), genital papillae, 23 vs 25 and to be likely presence of a mucron in the second

generation males of S. yirgalemense.

CS17 isolate of S. abbasi can be distinguished from S. bifurcatum (Fayyaz et al., 2014) by

longer body length and nerve ring comparatively posterior in position (Table 2). However,

males can be differentiated by SW%, 191(145-231) vs 138(120-170). The proximal end of

gubernaculum in the present specimen was not bifurcated while in case of S. bifurcatum male

gubernaculum was bifurcate at both proximal and distal ends, a key diagnostic feature. The

2nd generation female was comparatively longer than S. bifurcatum (Table 2). S. abbasi CS17

can be separated from S. papillatum (Ernesto et al., 2015) by shorter body length of IJs, NR

and „a‟ ratio (Table 2). Male of the new species can be distinguished from S. papillatum

males by genital papillae (23 vs 25); SL, 70(66-78) vs 52(42-62)μm; GL, 41(37-46) vs 31(23-

36)μm; SW%, 191(145-231) vs 156(125-194)μm and GBW, 112(89-132) vs 69(54-87)μm.

Presence of mucron in only 2nd

generation adult vs absence in all adult generations also

segregates two. The 2nd

generation females were shorter in total body length than S.

papillatum. Males of S. abbasi CS17 can be also differentiated from S. goweni (Ernesto et al.,

2016) males by the higher values of SL, GL, D% and lower value of GS% (Table 2). IJs of S.

abbasi CS17 are shorter than S. goweni 549(504 - 599) vs. 640(531–719) µm. The presence of

mucron in 2nd

generation male and female vs only in females also distinguished two species.


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Cross hybridization

The positive results were observed between the cross of CS17 and CS18 but the progeny were

not observed from cross-breeding treatments with CS19 and CS20 although they were

produced in the self-cross controls.

Molecular analysis

CS17 isolate of S. abbasi was characterized by sequences of non-transcribed spacer region i.e.

ITS (KP219885), D2-D3 region (KU187260) and coxI gene (KU529463). The pairwise

distances of the ITS and D2D3 regions between species of the “bicornutum” group were

calculated by modern biostatistical tools and summarized in Table 3. The length of the ITS

+5.8S+ITS2 sequence was 739bp, including ITS1, 268bp; 5.8S, 157bp and ITS2, 314bp and

its composition was: G+C, 0.3694; A+T, 0.6306; A, 0.2368; C, 0.1502; G, 0.2192 and T,

0.3938 (Table 4). S. abbasi CS17 thus has same nucleotide sequences as S. abbasi (ITS1 and

ITS2) and it was the closest taxon. The interspecific relationships were worked out by

distance matrix comparison and found too significant. The sequence of the D2D3 region of

isolate of S. abbasi CS17 was 922bp long and its composition was; G+C, 0.452; A+T, 0.547;

A, 0.271; C, 0.162; G, 0.289 and T, 0.276 (Table. 4). The coxI sequence was 634bp and its

composition was: A, 0.463; C, 0.208; G, 0.132 and T, 0.195. Sequence length and

composition of other earlier species of belonging groups were summarized in Table 4.

Phylogeny

The maximum parsimony phylogenetic analysis by the application of ITS region revealed

that the alignment covered 1229 characters among which 262 characters conserved, 321

variables parsimony uninformative while 646 were parsimony informative in nature. The

phylogenetic relationships among 22 nucleotide sequences were carried out and were found

to be remarkable (Fig. 9). The MP tree length was 919; consistency index 0.676259; retention

index 0.778978 and composite index 0.588260 (0.526791). The MP tree was obtained using

the SPR (Subtree-Pruning-Regrafting) algorithm with search level 3 in which the initial trees

were obtained by the random addition of sequences (10 replicates). All positions containing

gaps and missing data were eliminated and there were a total of 618 positions in the final

dataset. In this consensus tree, the present two isolates of S. abbasi viz. CS17 and CS18 form a

monophyletic group with already described isolates of S. abbasi (Fig. 9). The phylogenetic

analysis of D2-D3 region by maximum parsimony (MP) revealed that the alignment results in

935 characters, out of that 643 were constant, 116 variable characters with parsimony


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uninformative and 176 characters were parsimony informative. The phylogenetic

relationships of currently recovered worms (CS17 and CS18) with 13 nucleotide sequences

were carried out and found to be significantly different from the other existing members of

same group (Fig. 10). The MP tree length was 201; consistency index 0.746988; retention

index 0.851064 and composite index 0.673230 (0.635734). All positions containing gaps and

missing data were eliminated and there were a total of 585 positions in the final dataset. In

this consensus tree, the present two isolates of S. abbasi i.e. CS17 and CS18 form a

monophyletic group (single clade) with earlier described species, S. abbasi and S. bifurcatum

with 100% bootstrap values (Fig. 10). The maximum parsimony analysis of coxI region,

refelected that the alignment resulted in 844 characters, of which 255 characters were

conserved, 195 variable characters were parsimony uninformative and 394 characters were

parsimony informative (Fig. 11).

Table: 3. Pairwise distances of the ITS and D2D3 regions between species of the

“bicornutum group” (percentage similarity). Data for Steinernema abbasi CS17 and

CS18 in bold.

ITS region CS17 CS18 abb bif bic cer pak rio yir Pap gow

KP219885 S. abbasi CS17

KR029843 S. abbasi CS18 100

EF469773 S. abbasi 100 100

JX989267 S. bifurcatum 73 73 73

AY171279 S. bicornutum 74 74 74 68 86

KF312236 S. ceratophorum 79 79 79 73 68

AY748449 S. pakistanense 73 73 73 99 74 73

DQ835613 S. riobrave 73 72 73 80 70 78 74

AY748450 S. yirgalemense 82 81 82 70 75 75 70 72

KJ913707 S. papillatum 76 76 76 75 75 79 74 87 72

KR781038 S. goweni 75 75 75 74 75 79 73 81 74 83

D2D3 region CS17 CS18 abb bic cer rio yir bif pap gow

KU187260 S. abbasi CS17

KU187261 S. abbasi CS18 99

AF331890 S. abbasi 99 99

AF331904 S. bicornutum 91 92 92

AF331888 S. ceratophorum 90 91 91 96

AF331893 S. riobrave 89 89 90 92 91

AY748451 S. yirgalemense 94 94 95 92 91 91

JQ838179 S. bifurcatum 99 100 100 92 91 89 95

KJ913708 S. papillatum 89 89 89 91 89 95 90 90

KR781039 S. goweni 90 90 91 93 92 95 91 91 94


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Table 4. Sequence lengths and nucleotide composition of ITS-rDNA, D2-D3 and coxI regions of CS17 and CS18 isolate of S. abbasi and

earlier described species of Steinernema of bicornutum group. Data for Steinernema abbasi CS17 and CS18 in bold.

Species Molecular composition ITS length

(bp) Seq. length (bp)

ITS1

(bp)

5.8S

(bp)

ITS2

(bp)

G+C

(%)

A+T

(%)

A

(%)

C

(%)

G

(%)

T

(%)

ITS region

S. abbasi CS17 268 157 314 0.369 0.631 0.237 0.15 0.219 0.394 739

S. abbasi 268 157 314 0.369 0.631 0.237 0.15 0.219 0.394 739

S. bicornutum 281 157 330 0.375 0.625 0.262 0.16 0.215 0.363 768

S. ceratophorum 243 157 341 0.362 0.638 0.259 0.158 0.204 0.379 741

S. yirgalemense 270 157 284 0.357 0.643 0.269 0.131 0.226 0.374 711

S. pakistanense 291 157 300 0.369 0.631 0.289 0.158 0.211 0.342 748

S. riobrave 281 157 316 0.348 0.653 0.271 0.13 0.218 0.382 754

S. papillatum 314 157 320 0.355 0.645 0.279 0.14 0.215 0.365 791

S. goweni 283 157 324 0.387 0.613 0.257 0.155 0.233 0.356 764

D2-D3 region

S. abbassi CS17

0.452 0.548 0.271 0.163 0.29 0.277

922

S. abbasi CS18

0.458 0.542 0.265 0.169 0.289 0.277

920

S. abbasi

0.467 0.533 0.253 0.164 0.302 0.281

870

S. bicornutum

0.469 0.531 0.254 0.167 0.302 0.277

870

S. ceratophorum

0.457 0.543 0.264 0.168 0.289 0.279

871

S. yirgalemense

0.486 0.514 0.246 0.18 0.307 0.268

646

S. riobrave

0.475 0.52 0.25 0.176 0.304 0.27

869

S. bifurcatum

0.468 0.532 0.248 0.169 0.299 0.284

895

S. papillatum

0.473 0.528 0.251 0.171 0.301 0.276

836

S. goweni

0.483 0.519 0.249 0.178 0.302 0.271

837

coxI

S. abbasi CS17 0.341 0.659 0.464 0.208 0.133 0.196

634

S. abbasi CS18

0.346 0.654 0.459 0.212 0.134 0.195

636


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S. abbasi

0.342 0.659 0.197 0.137 0.204 0.461

568

S. carpocapsae

0.303 0.695 0.236 0.134 0.169 0.46

568

S. bicornutum

0.352 0.648 0.206 0.157 0.195 0.442

568

S. ceratophorum

0.347 0.653 0.203 0.157 0.19 0.451

568

KP219885 S. abbasi L PAK.S.H.16

JN571086 S. abbasi B PAK.S.S.15

KR029843 S. abbasi CS18

AY230158 S. abbasi

EF469773 S. abbasi

KF573496 S. abbasi

GQ377417 S. abbasi

KP219885 S. abbasi CS17

EF431958 S. thermophilum

KF312236 S. thermophilum CICR-NewB1

KC633189 S. abbasi

KC633188 S. abbasi

AY748450 S. yirgalemense

JX989267 S. bifurcatum

AY230181 S. sp.

AY748449 S. pakistanense

KF312236 S. ceratophorum

KR781038 S. goweni

DQ835613 S. riobrave

KJ913707 S. papillatum LPVO23

KM211574 S. papillatum LPV723

X03680 C. elegans

61

94

92

82

77

100

100

84

64

76

55

97

100

50

Fig. 9. Phylogenetic relationships of CS17 and CS18 isolates of S. abbasi in the „bicornutum‟ group of Steinernema based on analysis of

ITS rDNA regions. C. elegans was used as the out-group taxon. The percentages of replicate trees in which the associated taxa clustered

together in the bootstrap test (10,000 replicates) are shown next to the branches. Branch lengths indicate evolutionary distances and are

expressed in units of number of base differences per site.


1576




AF331890 S. abbasi

JQ838179 S. bifurcatum

AY748451 S. yirgalemense

KR781039 S. goweni

AF331893 S. riobrave

KJ913708 S. papillatum

KM229421 S. papillatum

AF331904 S. bicornutum

AF331888 S. ceratophorum

HQ190043 S. surkhetense

HQ190045 S. nepalense100

77

92

100

99

100

97

99

72

5

Fig. 10. Phylogenetic relationships of CS17 and CS18 isolates of S. abbasi in the

„bicornutum‟ group of Steinernema based on analysis of D2–D3 expansion segments of

the 28S rDNA. S. sukhetense and S. nepalense were used as the outgroup taxon. The

percentages of replicate trees in which the associated taxa clustered together in the

bootstrap test (10,000 replicates) are shown next to the branches. Branch lengths

indicate evolutionary distances and are expressed in units of number of base differences

per site.

AY943976 S. abbasi

JN572121 S. abbasi NBAII EN01

GU569060 S. abbasi S01

GU569064 S. bicornutum

AY943980 S. bicornutum

AY943982 S. ceratophorum

AY943998 S. riobrave

JN683831 Steinernema sp. 15G

JN683830 Steinernema sp. 59F

AY943999 S. scapterisci

AY508069 Bursaphelenchus xylophilus



20

Fig. 11. Phylogenetic relationships of CS17 and CS18 isolates of S. abbasi in the

„bicornutum‟ group of Steinernema based on analysis of cox1 gene of mtDNA.

Bursaphelenchus xylophilus was used as the outgroup taxon. The percentages of

replicate trees in which the associated taxa clustered together in the bootstrap test

(10,000 replicates) are shown next to the branches. Branch lengths indicate evolutionary

distances and are expressed in units of number of base differences per site.

The phylogenetic relationships between 13 nucleotide sequences with CS17 and CS18 isolates

of S. abbasi were carried out (Fig. 11) and the tree length was 490 with consistency index

0.749465; retention index 0.768317 and composite index 0.584862 (0.575826). All positions

containing gaps and missing data were eliminated and a total of 400 positions in the final

dataset. In this consensus tree, the newer two isolates form a separate clade with 100%


1577


bootstrap value but don‟t show clustering with other described species thus proving non-

utility of coxI gene for identification of EPN (Fig. 11). By the aforementioned critical

analysis it was concluded by the authors that the newly recovered worms CS17 and CS18

proposed to be the isolates of S. abbasi but not identical one due to various epimorphic as

well molecular heterogeneity as an impact of composite environmental (extrinsic) as well as

intrinsic factors responsible for their existence and survival to be as natural biocontrol agents.

ACKNOWLEDGEMENTS

Authors are grateful to Nematology Section, Indian Agricultural Research Institute, New

Delhi for Scanning Electron Microscope facility for ultra topology of worms. Aashaq

Hussain Bhat is thankful to the Department of Science and Technology (DST), New Delhi,

India for providing the financial assistance through DST INSPIRE Fellowship/2014/76.

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