Morphological and molecular characterization of a new isolate of entomopathogenic nematode, Steinernema feltiae (Filipjev) (Rhabditida: Steinernematidae) from the Arasbaran forests, Iran

Published Date
Journal of Asia-Pacific Biodiversity
30 June 2015, Vol.8(2):144–151, doi:10.1016/j.japb.2015.04.008
Open Access, Creative Commons license, Funding information
Original article

• Author 

• Mostafa Nikdel ^a,,
• Gholamreza Niknam ^b

• aForests and Rangelands Research Department, East Azarbaijan Agricultural and Natural Resources Research Center, AREEO, Tabriz, Iran
• bDepartment of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

Received 11 March 2015. Revised 29 April 2015. Accepted 30 April 2015. Available online 12 May 2015. 

Abstract

Steinernema feltiae isolate IRAZ13 was recovered from one soil sample collected from the Arasbaran forests, Iran. The new isolate can be distinguished from other isolates by morphologic and morphometric data, and DNA sequences. The first generation male is characterized by spicule length 79 ± 5 (73–87) μm, SW ratio [(spicule length/anal body diameter)*100] 160 ± 13 (137–177), GS ratio GS [(gubernaculum/spicule length)*100] 79 ± 8 (62–87), genital papillae arrangement and the second generation male by a mucron 10 ± 1 (9–11) μm long. For the infective juvenile, the body is 883 ± 82 (754–975) μm long, lateral fields with eight equally developed ridges, head smooth, slightly offset, large gonad primordium cells, a long hyaline tail portion (equal to half of tail length), and pore-like phasmid at 41% of the tail length to the semicircle anus are the differentiation factors. Additionally, molecular studies obtained from the 28S-D2/D3 region and ITS1 rDNA sequence analyses and phylogenetic reconstruction further support this nematode as a different isolate.

Keywords

Arasbaran forests

entomopathogenic nematode

morphology

morphometrics

Steinernema feltiae

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Introduction

Entomopathogenic nematodes (EPNs) infect many different types of insects living in the soil such as the larval forms of moths, butterflies, flies, and beetles as well as adult forms of grasshoppers and crickets. They are soil-inhabiting organisms and can be used effectively to control soil-borne insect pests, but are generally not effective when applied to control insects in the leaf canopy. The keys to success with EPNs are understanding their life cycles and functions, matching the correct nematode species with the pest species, applying them during appropriate environmental conditions, and applying them only with compatible pesticides. Because these agents are living organisms, they require careful handling to survive shipment and storage as well as appropriate environmental conditions to survive in the soil after application (Lawrence and Georgis 2012).

Finding indigenous EPN populations and species is an important step to attain effective biological control against pests due to their better adaptation to local environment. Currently, about 63 species of the genus Steinernema have been described worldwide and these have been divided into five groups according to their morphology and molecular characteristics (Nguyen and Hunt 2007). The most updated biogeographic account revealed that these nematodes have been isolated from all continents (except for Antarctica) and almost all regions of the world (Hominick 2002). So far, several species of Steinernema including S. bicornutum, S. carpocapsae, S. feltiae, S. kraussei, and S. glaseri (Nikdel et al., 2010 and Tanha maafi et al., 2006), plus a new species named as S. arasbaranense (Nikdel et al 2011) have been reported from north and north-west Iran. Herein, S. feltiae isolate IRAZ13 is described as a new isolate. The new isolate is separated from other S. feltiae isolates by differences in some morphological and morphometric characters, the 28S-rDNA D2/D3 region, and ITS-rDNA sequences.

Materials and methods

Nematode population

The nematode population was recovered by the Galleria-trap method from one soil sample collected during a survey conducted in the Arasbaran forests, a horn-beam (Carpinus betulus) and yew (Taxus baccata) dominated forest habitat, near the Kalaleh Research Forest Station (latitude N 38° 41.34′, longitude E 46° 34.07′, altitude 1208 m above sea level, annual average of temperature 13.7°C, precipitation 385 mm/year, soil type dark brown sandy clay) in the north of East Azarbaijan Province, Iran in May 2007. The nematodes were subsequently reared on G. mellonella larvae and established as a laboratory culture at the Nematology Lab, University of Tabriz, Iran and named as the IRAZ13 isolate.

Light microscopy

First generation males and females were collected from 4–5 days postinoculated Galleria cadavers (dissected out in distilled water). Infective juveniles and second generation adults were collected during the week after their first emergence from Galleria cadavers and were killed using hot (50–60°C) Ringer's solution (Nguyen and Smart 1994). Dead nematodes were fixed in triethanolamine formalin, processed to anhydrous glycerin by a slow evaporation method (Woodring and Kaya 1988), and mounted on microscopic slides. In female specimens, cover slips were supported using pieces of hair to avoid flattening of nematodes. Morphological studies and morphometric measurements were made using an Olympus BX41 microscope (Olympus Corporation, Rochester, NY, USA) equipped with interference contrast, through a digital DP50 camera (Olympus, Shibuya-ku, Tokyo, Japan) and using UTHSCSA Image tool software, version 3.0 (Department of Dental Diagnostic Science at the University of Texas, San Antonio, TX, USA) (Vilcox et al 2002).

Scanning electron microscopy

Morphological features of adults and infective juveniles were examined using scanning electron microscopy (SEM). For this purpose, specimens were processed following protocols described by Nguyen and Smart (1995). In SEM examination, the first generation adults and infective juveniles were rinsed for 5 minutes each in Ringer's solution three times. The specimens were fixed in 3% glutaraldehyde buffered with 0.1M sodium cacodylate at pH 7.2 for 24 hours at 8°C. Then they were post-fixed with 2% osmium tetroxide (OsO₄) solution for 12 hours at 25°C, dehydrated in a graded ethanol series (5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, and 100%), mounted on aluminum SEM stubs, coated with gold powder (200 nm thickness) and studied using an LEO 440i scanning electron microscope (Oxford Microscopy, Oxford, UK).

Molecular characterization

Total genomic extraction of 28S-rDNA-D2/D3 and ITS-rDNA amplification were performed as described by Phan et al (2001). Amplified products were purified using a Qiagen Purification kit (Qiagen, Leusden, The Netherlands). Purified DNA was sequenced in IBMP-CNRS (Institute of Plant Molecular Biology, Le Centre National de la Recherche Scientifique, France). The DNA sequences were edited with Chromas 2.01 (developed by Technelysium Pty Ltd, South Brisbane, Queensland, Australia) and aligned using Clustal X version 1.64 software (European Bioinformatics Institute, Cambridgeshire, UK) provided by Thompson et al (1997) including the 28S-rDNA-D2/D3 and ITS-rDNA sequences of other S. feltie isolates obtained from GenBank.

28S-rDNA-D2/D3 and ITS-rDNA sequences for isolate IRAZ13 were deposited in GenBank under the accession numbers FJ860022 and FJ860035, respectively. DNA sequences were aligned by Clustal W (http://workbench.sdsc.edu, Bioinformatics and Computational Biology group, Dept. Bioengineering, UC San Diego, CA, USA). The model of base substitution was evaluated using MODELTEST (Posada and Crandall, 1998 and Huelsenbeck and Ronquist, 2001). The Akaike-supported model, the base frequencies, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 (Huelsenbeck and Ronquist 2001) running the chain for 1 × 10⁶generations and setting the “burn in” at 2500. The Markov Chain Monte Carlo method was used within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon 1999) using 50% majority-rule.

Results

Morphological characteristics

Infective juvenile

Body slender, habitus moderately curved ventrally upon heat-killed, sometimes enclosed in cuticle of second-stage juvenile, tapering regularly from base of pharynx to anterior end and from anus to tail terminus. Mouth and anus closed. Head broad, offset from body, labial papillae not observed, transverse slit-like amphidial apertures posterior to labial disc but at the level of four distinct cephalic papillae. Pharynx long, narrow, isthmus distinct, surrounded by nerve ring (Figure 1E), basal bulb elongate and valvate. Cardia prominent. Secretory–excretory pore at mid-pharynx level. Hemizonid distinct, anterior to the base of terminal bulb (Figure 1E). Bacterial vesicle elongate (42 μm long). Genital primordium clear and large. Lateral fields begin anteriorly with one line followed by two additional lines to form two ridges (Figure 2B). Near the level of secretory–excretory pore, two ridges separated into four, which increases the number of ridges to eight, the maximum number in the lateral fields (Figure 2C). The equally developed eight ridges (i.e,. 9 lines or incisures) pattern extends posteriorly close to three or four annules before the anus. Then, the number of ridges reduced to six then to four. Near the phasmid, the four ridges change to two ridges. With the above description, the formula for the arrangement of ridges from head to tail is: 2, 8, 6, 4, 2 (Figure 2B–2E). The portion with eight ridges is the longest part (compare with portions with 2, 6, 4 ridges) of the lateral fields. In the portion with six ridges the middle two are indistinct. Tail conical and often bent towards ventral side, tail terminus finely rounded, hyaline tail portion distinct, about 50% of tail length. Phasmids distinct, pore-like approximately at 41% of the tail length to the semicircle anus (Figure 2D).

Figure 1. Steinernema feltiae isolate IRAZ13. A–C, first generation female: A, anterior region, 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 second generation female; E, anterior region of third-stage infective juvenile; F, posterior region of third-stage infective juvenile. G–L, first generation male; G, anterior region; H and I, posterior region, spicules, gubernaculum, and mucron; J and K, gubernaculum; L, posterior region of male showing number and arrangement papillae and mucron. Scale-bars: A = 90 μm, B = 20 μm, C = 60 μm, D = 30 μm, E = 40 μm, F = 33 μm, G = 57 μm, H, I = 50 μm, J, K = 40 μm, L = 75 μm.

Figure 2. Scanning electron microscopy (SEM) photographs of infective juveniles of Steinernema feltiae isolate IRAZ13: A, head showing closed mouth, two of four prominent cephalic papillae (c.p.), and amphid (am.); B, lateral fields with one incisure and changes to two ridges; C, lateral field showing two and eight ridges; D, posterior portion showing six ridges (disappearance of two ridges) and position of anus and phasmid on tail, inset of higher magnification of anus (down, right corner); E, tail showing reduced number of ridges to four then two at the end of lateral fields. Scale bars: A = 2 μm, B and C = 3 μm, D and E = 6 μm.

Female, first generation

Body robust, habitus C-shaped. Cuticle appearing smooth under light microscopy, lateral fields not observed. Head broadly rounded, six pointed labial papillae and four cephalic papillae visible only under SEM. Amphids inconspicuous. Buccal cavity funnel- or cup-shaped, stoma shallow. Pharynx with cylindrical procorpus, metacorpus slightly swollen and nonvalvate, isthmus distinct, basal bulb pyriform and valvate. Nerve ring just anterior to basal bulb (Figure 1A). Secretory-excretory pore usually at mid-pharynx level and excretory duct cuticularized. Cardia prominent protruding into intestinal lumen. Genital system didelphic, reflexed, filled with eggs, vulva a median transverse slit, protruding from body, double flapped epiptygma present (Figure 1 and Figure 3A), vagina short, oblique with muscular walls. Tail dome-shaped, shorter than anal body diameter, with one terminal peg (Figure 1 and Figure 3D) and anus with a wide slit (Figure 3B).

Figure 3. Scanning electron microscopy (SEM) (A, B) and light microscope (C, D) photographs of first generation females of Steinernema feltiae isolate IRAZ13: A and C, vulva and double flapped epiptygma; B, beginning of tail showing anus; D, fornicated end of body with terminal peg and anus. Scale bars: A = 3 μm, B = 2 μm, C and D = 50 μm.

Female, second generation

Similar to first generation but smaller, body length approximately half that of the first generation and one-third of its body diameter. Eggs are arranged in one row. Tail conoid without mucron. Postanal region slightly swollen (Figure 1D).

Male, first generation

Body ventrally curved, habitus C- or J-shaped, much smaller and more slender than female. Cuticle smooth under light microscope, lateral fields not observed. Head rounded, slightly depressed from body. Six pointed labial papillae and four cephalic papillae visible under SEM. Amphids inconspicuous. Buccal cavity funnel- or cup-shaped, stoma shallow. Pharynx muscular, procorpus cylindrical, metacorpus slightly swollen, nonvalvate, isthmus distinct, basal bulb pyriform and valvate. Nerve ring just anterior to the basal bulb. Cardia prominent protruding into intestinal lumen, deirids not seen. Secretory-excretory pore at middle of pharynx, excretory duct cuticularized. Testis monorchic, reflexed, with short reflection. Spicules paired, slightly brownish in color, strongly curved, length/width 6.2, head (manubrium) width is approximately equal to length (Figure 1 and Figure 4), blade arcuate with straight tip, dorsal lobe well curved, terminating at spicule tip, lateral lobe prominent, terminating at spicule tip, ventral lobe enlarged anteriorly at dorsal and ventral side to form prominent apex and rostrum, terminating posterior to rounded spicule tip, velum large, not covering spicule tip. Gubernaculum approximately 75% of spicule length, boat-shaped in lateral view, swollen at middle, with slightly ventrally curved knob at proximal end, in ventral view, cuneus short, pointed posteriorly, wing of corpus expanding laterally (Figure 1 and Figure 4). There are 23 genital papillae comprising 11 pairs of papillae and a single ventral papilla located anterior to cloacal opening. Of these, seven pairs are located precloacal (3 pairs lateral and 4 pairs subventral), one pair adcloacal and three pairs postcloacal (1 pair subdorsal and 2 pairs subterminal nearly subventral) (Figures 1L and 5E). Tail conoid with 8 μm long mucron that always present (Figure 5E). Phasmids inconspicuous.

Figure 4. Light microscope photographs of first generation males of Steinernema feltiae isolate IRAZ13: A–F, variation in spicule and gubernaculum shape. Scale bars: A = 17.2 μm, B = 15 μm, C = 29 μm, D = 25.5 μm, E and F = 14 μm.

Figure 5. Scanning electron microscopy (SEM) photographs of first and second generation males of Steinernema feltiae isolate IRAZ13: A, anterior region with secretory-excretory pore position (EP) in the second generation male; B and C, posterior region of the second generation male, arrangement of genital papillae and long mucron; D, head of the first generation male with cephalic papillae (C.P.), labial papillae (L.P.), and amphid; E, posterior region of the first generation male showing number and distribution of genital papillae. Scale bars: A = 13 μm, B = 5.5 μm, C = 16 μm, D = 33 μm, E = 40 μm.

Male, second generation

Similar to first generation male but more slender, body diameter approximately half that of the first generation male (Figure 5A). Testis flexure extending more posteriorly. Mucron more distinct, approximately 10 ± 1 μm long (Figure 5B and 5C).

Molecular characterization

Sequences of 28S-rDNA-D2/D3 and ITS1 regions were used as molecular data to differentiate the new isolate from other S. feltiae isolates and examine phylogenetic relationships. In the 28S-rDNA-D2/D3 region, the sequenced fragment of the new isolate is 724 bp and its nucleotide usage composition is as follows: 24.19% A, 19.27% C, 30.10% G, and 26.44% T. All the S. feltiae isolates from GenBank with the same region after a Blast search were selected for phylogenetic analysis. Using some of the other species, it belongs to the ‘feltiae-kraussei-oregonensis’ group as outgroups (Nadler et al., 2006, Nguyen and Hunt, 2007 and Spiridonov et al., 2004), the multiple alignment generated many ambiguous sites. Thus, they are removed from further tree construction. Figure 6 presents a phylogenetic tree based on the 28S-rDNA-D2/D3 region from a multiple alignment of 35 taxa. Among them, many taxa with IRAZ numbers in Figure 6 were Steinernema isolates collected from Iran (Nikdel et al 2010). This alignment revealed S. ashiuense was the most distant species and therefore used as the root in the tree (Figure 6). This tree inferred many highly supported monophyletic clades which had similar topology with other Steinernema phylogenetic studies (Spiridonov et al., 2004 and Nadler et al., 2006).

Figure 6. Phylogenetic relationship of Steinernema feltiae isolate IRAZ13 with other Steinernemaspecies and S. feltiae isolates based on 28S-rDNA D2/D3 region. The 10001st Bayesian tree was performed under GTR + I + G model [lnL = 6292.5405; freqA = 0.2435; freqC = 0.1725; freqG = 0.2941; freqT = 0.2899; R(a) = 0.6376; R(b) = 3.8809; R(c) = 1.278; R(d) = 0.2515; R(e) = 6.0674; R(f) = 1; Pinva = 0.3049; Shape = 0.7368].

The sequenced ITS1 region of S. feltiae isolate IRAZ13 includes partial ITS1 + 5.8S + ITS2 + partial 28S sequences. The size including primers TW81 and AB28 is 759 bp and its nucleotide usage composition is as follows: 25.55% A, 17.45% C, 39.19% G, and 35.16% T. There were numerous ITS sequences of S. feltiae available in GenBank. In order to reduce the ambiguous sites in multiple alignment and limit the number of taxa in the tree construction, 42 taxa in the ‘feltiae-kraussei-oregonensis’ group from GenBank were selected for phylogenetic analysis. Figure 7 presented a phylogenetic tree based on rDNA-ITS1 sequences from a multiple alignment. Rooted by S. hebeiense, a basal species in Figure 7, this tree generated many highly supported monophyletic clades and generally agreed with the topology inferred by 28S-rDNA-D2/D3 sequences in Figure 6. S. feltiae isolate IRAZ13 is unique and separated from several other isolates of S. feltiae in a monophyletic clade.

Figure 7. Phylogenetic relationship of Steinernema feltiae isolate IRAZ13 with other Steinernemaspecies and S. feltiae isolates in the ‘feltiae-kraussei-oregonensis’ group based on ITS-rDNA. The 10001st Bayesian tree was performed under GTR + G model [lnL = 6315.3257; freqA = 0.2507; freqC = 0.1762; freqG = 0.2161; freqT = 0.3571; R(a) = 1.0881; R(b) = 2.4811; R(c) = 1.3951; R(d) = 0.7959; R(e) = 3.1808; R(f) = 1; Pinva = 0; Shape = 0.4765].

Discussion

S. feltiae isolate IRAZ13 is morphologically characterized by a combination of the features of various developmental stages of the nematode (Table 1). Infective juveniles are distinguished by their body length of 883 ± 82 (754–975) μm, smooth head and slightly offset, lateral fields with eight equally developed ridges (9 lines or incisures) at mid-body area, hyaline tail portion long (equal to half of tail length) and large gonad primordium cells. The first generation males are characterized by long, head rounded, slightly brownish spicules with 79 ± 5 (73–87) μm long, developed velum, spicule length/width 6.2 ± 0.8 (5.2–6.9), ratio SW (%) 160 ± 13 (137–177), GS ratio (%) 79 ± 11 (65–90), posterior region with 23 genital papillae (11 pairs and a single ventral preanal). The second-generation males have a longer mucron, 10 ± 1 (9–11) μm long. The infective juvenile of the new isolate is distinguished from all other isolates by a clear genital primordium, lateral field morphology at mid body, with eight equally developed ridges versus eight ridges with submarginal pair indistinct in other isolates, and a distinct hyaline tail portion about half of the tail length. Males of isolate IRAZ13 are separated from type species and other Iranian isolates by a longer spicule, 79 (73–87) versus 70 (65–77); SW, 160 (137–177) versus 113 (99–130); GS, 79 (65–90) versus 59 (52–61) (Table 2), developed velum, and round head spicule. In addition, the gubernaculum in the new isolate is longer than that of other isolates of S. feltiae (62 vs. 51 μm). The mentioned differences can be considered as intraspecific variations and discriminate this isolate from others. Furthermore, infective juveniles of isolate IRAZ13 are completely coiled under 4–7°C temperatures, whereas none of the other Iranian species (belonging to the ‘feltiae-kraussei-oregonensis’group species) show similar behavior.

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