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Research/Investigación Addition of a New Insect Parasitic Nematode, Oscheius Tipulae, to Iranian Fauna

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Research/Investigación Addition of a New Insect Parasitic Nematode, Oscheius Tipulae, to Iranian Fauna RESEARCH/INVESTIGACIÓN ADDITION OF A NEW INSECT PARASITIC NEMATODE, OSCHEIUS TIPULAE, TO IRANIAN FAUNA J. Karimi1*, N. Rezaei1, and E. Shokoohi2 1Biocontrol and Insect Pathology Lab., Department of Plant Protection, Ferdowsi University of Mashhad, PO Box 91775-1163, Mashhad, Iran; 2Unit for Environmental Sciences and Management, Potchefstroom, North West University, South Africa; *Corresponding author: [email protected] ABSTRACT Karimi, J., N. Rezaei, and E. Shokoohi. 2018. Addition of a new insect parasitic nematode, Oscheius tipulae, to Iranian fauna. Nematropica 48:45-54. On behalf of an ongoing project on diversity of insect pathogenic and insect parasitic nematodes of Iran, a new species was collected and characterized. This species was collected in soil from the Mashhad, Arak, and Mahalat regions of Iran through 2011-2012 using Galleria larvae baits. Based on morphologic and morphometric traits as well as SEM images, the species tentatively has been identified as Oscheius tipulae. Phylogenetic analysis based on ITS and 18S rDNA genes confirmed the species delimitation. This is the first record of this species from Iran. Key words: 18S rDNA, insect parasitic nematode, Iran, ITS, Oscheius tipulae, SEM RESUMEN Karimi, J., N. Rezaei, y E. Shokoohi. 2018. Adición de un nuevo nematodo parásito de insectos, Oscheius tipulae, a la fauna Irán. Nematropica 48:45-54. En nombre de un proyecto en curso sobre diversidad de patógenos de insectos y nematodos parásitos de insectos de Irán, se recolectó y caracterizó una nueva especie. Esta especie fue recolectada en suelo de las regiones de Mashhad, Arak y Mahalat de Irán durante el período 2011-2012 utilizando cebos Galleria. En base a los rasgos morfológicos y morfométricos, así como a las imágenes SEM, la especie tentativamente ha sido identificada como Oscheius tipulae. El análisis filogenético basado en los genes ITS y 18S rDNA confirmó la delimitación de especies. Este es el primer registro de esta especie de Irán. Palabras claves: 18S rDNA, Irán, ITS, nemátodo parásito de insectos, Oscheius tipulae, SEM INTRODUCTION and are potent biocontrol agents against a variety of insect pests that are widely distributed in soils Nematodes comprise a tremendously diverse throughout the world (Kaya and Gaugler, 1993; group that have the most abundance in marine and Gaugler, 2002; Grewal et al., 2005; Ye et al., 2010; terrestrial habitats. In the latter case, they represent Torres-Barragan et al., 2011). Moreover, other a significant component of the soil community members of the Rhabditida have shown potential to (Kaya and Gaugler, 1993). The interest in the use infect insects and are promising as new candidates of nematodes as biocontrol agents for pest control for biocontrol of insect pests. The Rhabditidae are has increased exponentially over last decade (Kaya important in nematological studies because of their and Gaugler, 1993; Grewal et al., 2005). The most great ecological diversity and association with important groups of nematodes for use as insects. These bacterial feeding nematodes are biocontrol agents are the families Steinernematidae suitable models for understanding the evolution of and Heterorhabditidae (Rhabditia). parasitism in diverse lineage groups of Secernentea Entomopathogenic nematodes (EPN) in both (Sudhaus, 2011; Liu et al., 2012). families are symbiotically associated with bacteria The broad range of association of members of 82 Oscheius tipulae from Iran: Karimi et al. 83 the Rhabditidae with insects can be divided in Iran. We applied a routine sampling of soil using different ways. Some species of Rhabditids have the Galleria mellonella baiting technique (Bedding mutualistic association with insects. The and Akhurst, 1975) to soil from apple and apricot Rhabditidae have various feeding behaviours and orchards. This work led to the isolation of several are usually specialized to feed on bacteria EPN species and a Rabditid nematode. The (decaying organic matter) and are often associated aforementioned rhabditid was collected from with animal hosts or vectors. Caenorhabditis, Mashhad (Razavi Khorasan Province, 36°15'N, Mesorhabditis, Pellioditis, Rhabditis, 59°38'E), Arak (Markazi Province, 34°4'N, Phasmarhabditis, and Oscheius are the main 49°46'E), and Mahalat (Markazi Province, genera of this family. Andrássy (2005) described 50°41'N, 33°91'E). The killed larvae of G. the genus Oscheius and subsequently O. mellonella with the nematode had visible insectivorus was assigned as type species. Sudhaus colour.The nematodes emerging in the White trap (1976) placed Oscheius in the Rhabditidae and were collected (Kaya and Stock, 1997). These divided the genus into two groups. “Insectivora- extracted nematodes were tested for ability to group” with leptoderan bursa, comprising 14 infect wax moth larvae. Mixed stages of nematodes species and “Dolichura-group” with peloderan were emerged from the larvae cadavers 10 days bursa, comprising 13 species (Sudhaus, 2011). The after inoculation and were collected for 10 more species of Oscheius are common soil nematodes, days. all reproducing hermaphroditically. Oscheius tipulae reproduces through XX hermaphrodites Light microscopy and XO males such as C. elegans (Félix et al., 2001; Felix, 2006; Pervez et al., 2013). Species of For morphological characterization, 50 adults Oscheius in the “insectivorus” group represent and infective juveniles (IJs) were randomly various associations with invertebrate hosts selected from G. mellonella cadavers, fixed using ranging from facultative to obligate parasitism (Liu hot (80°C) 4% formaldehyde solution, and et al., 2012). Torrini et al. (2015) described the first processed to anhydrous glycerin for mounting (De EPN from the Dolichura group and later Campos- Grisse, 1969). Fixed nematodes were mounted on Herrera et al. (2015) provided more data about the a glass slide using cover glass and glass rod Oscheius genus. O. insectivorus (Andrássy, 1976) supports to avoid flattening. Measurements and and O. maqbooli were described as insectivorous microphotographs assured quality and accuracy. species (Tabassum and Shahina., 2010), O. Drawings were made using a drawing tube attached carlianonsis as EPN (Ye et al., 2010), O. amsactae to an Olympus light microscope CH-2. as a necromenic associate (Liu et al., 2012) and O. gingeri as an EPN (Pervez et al., 2013). Oscheius Scanning electron microscopy (SEM) tipulae was described for the first time by Lam and Webster (1971) and redescribed by Sudhaus First-generation adults were fixed in 3% (1993). This species has been associated with glutaraldehyde buffered with 0.1 M phosphate leatherjackets, larvae of a tipulid dipteran, Tipula buffer then fixed with 2% osmium tetroxide paludosa. The nematode specific epithet refers to solution for 12 hr at 25°C. The samples were the etymology of its host name (Sudhaus, 2011). washed with sterile water three times and DNA sequence analysis of several loci including dehydrated in a graded ethanol series (10, 20, 50, 18S and ITS rRNA as well the D2-D3 expansions 70 and 100%), mounted on SEM stubs, and coated of 28S rRNA (De Ley et al., 2005; Stock, 2003; with gold using CamScan MV2300 microscope Stock et al., 2009) is widely accepted for nematode operating at 15 mA. Images of the species were identification and assessing phylogenetic obtained with LEO 1450VP scanning electron relationships (Dorris et al., 1999; Kumari and microscope (LEO Co. Ltd., Oberkochen, Lišková, 2009; Stock, 2009). The objective of this Germany). research was to identify a new Rhabditid species from Irainian fauna using classic approach, as well DNA extraction, PCR, and sequencing molecular analysis of two genes. The SEM images of the Iranian population of the new species and Genomic DNA was extracted from an their drawings are provided (Fig. 1-4). individual adult nematode. The adult nematode was picked and transferred into a microtube after MATERIALS AND METHODS grinding in 50 μl 5% Chelex-100 (Sigma Aldrich, Germany) and 2 μl proteinase K, incubated at 60°C Nematodes isolation for 3 hr, followed by 10 min at 95°C. After centrifugation at 13,000 rpm for 3 min, the During 2010 to 2013, a project was conducted extracted DNA was stored at –20°C. A molecular to identify native EPNs in different locations of approach, including sequences analysis of ITS and 84 NEMATROPICA Vol. 48, No. 1, 2018 18S genes were considered for molecular PCR based RFLP characterization of the isolate. The primers for amplification of ITS gene were 5ʹ-TTG ATT ACG We checked genetic diversity among three TCC CTG CCC TTT-3ʹ (forward), and 5ʹ-TTT populations of the species studied. The selected CAC TCG CCG TTA CTA AGG-3ʹ (reverse) restriction enzymes were Bgl II and Hinc II. After (Vrain et al.,1992) and the primers used for 18S PCR, the amplicons of ITS gene were subjected to rDNA amplification were 5ʹ-GGT GAA ACT GCG digestion (Felix et al., 2001). We also checked the AAC GGC TCA-3ʹ (forward) and 5ʹ-CCG GTT digestion results by in silico digestion using CAA GCC ATT GCG ATT-3´ (reverse) (Blaxter et Webcutter 2.0. al.,1998). The PCR mixture was carried out in a reaction volume of 25 μl, containing 2.5 μl of 10× RESULTS AND DISCUSSION PCR-buffer, 15.7μl of H2O, 1 μl of MgCl2 (25mM), 0.5 μl of dNTPs (10 mM), 0.3μl Taq polymerase (5 Three isolates of collected Rhabditid units/μl), 1μl of forward primer (10 pmol/μl), 1μl belonged to Oscheius. These Oscheius nematodes of reverse primer (10 pmol/μl), and 3 μl of template were collected from Arak in 2010, Mashhad in DNA. After DNA
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