Document Type : Research Paper

Authors

• Arezoo naghavi ¹
• Gholamreza Niknam ²
• Nasir Vazife ²

¹ Department of Plant Protection, Faculty of Agriculture, Lorestan University, Khorramabad, Iran

² Department of Plant protection-Faculty of Agriculture-University of Tabriz-Tabriz-Iran

Abstract

Keywords

• Aquatides
• fauna
• forest area
• Lorestan
• morphology
• 28S rDNA

Extended Abstract

Introduction

Free-living nematodes are one of the most diverse and abundant groups of invertebrates that are present in a variety of habitats. Among the phylum nematodes, the order of Dorylaimida, with more than 2500 valid species and 250 genera, is known as one of the most important taxa of nematodes (Jairajpuri & Ahmad, 1992).

The suborder Nygolaimina Ahmad & Jairajpuri, 1979, includes a group of free-living nematodes in the order Dorylaimida Pearse, 1942, which has a single superfamily, Nygolaimoidea Thorne, 1935, and three families, Nygolaimidae Thorne, 1935, Aetholaimidae Jairajpuri, 1965, and Nygellidae Andrássy, 1958. In the present study, one species from each of the three genera Aquatides Heyns, 1968, Clavicaudoides Heyns, 1968, and Paravulvus Heyns, 1968, belonging to the family Nygolaimidae and two genera from the subfamily Pungentinae of the family Nordiidae (Jairajpuri & Siddiqi, 1964) were identified and introduced.  

Materials and Methods

    During 2021 and 2022, about 120 soil samples were collected from the rhizosphere of oak trees and other forest trees in southeastern Khorramabad, Lorestan province, Iran. The nematodes were extracted using the modified Brown & Boag (1988) method, killed, fixed, and transferred to anhydrous glycerol by De Grisse’s (1969) technique and mounted on permanent glass slides. The species were identified under a light microscope equipped with Dino-Eye Eyepiece Camera based on the morphological and morphometric characteristics and valid identification keys. Photographs were taken with an Olympus BX41 light microscope equipped with a DP50 digital camera. Photoplates were made using CorelDRAW® software version 12. For molecular study, nematodes’ DNA was extracted from three to four individuals, and PCR assays were conducted as described by Archidona-Yuste et al. (2016). The D2-D3 segments were amplified using the forward D2A and reverse D3B primers (Nunn, 1992). PCR cycle conditions were as follows: denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 30 s, annealing of primers at 55°C for 45 s, and extension at 72°C for 3 min, followed by a final elongation step at 72°C for 10 min. PCR products were purified for sequencing in both directions by D2-D3 primers and sequenced by the Applied Biosystems® 3730/3730xl DNA Analyzer in South Korea. The obtained sequences were deposited in the GenBank database under accession numbers PP971542 https://www.ncbi.nlm.nih.gov/nuccore/PP971540, PP971539, PP971538, and PQ576734 for A. christicki, C. clavicaudatus, P. hartingii and P. sufiyanensis, respectively, and PQ576735, PQ576745 for P. engadinensis. The Basic Local Alignment Search Tool (BLAST) homology search program was used to compare the newly generated sequences with other available sequences in the GenBank database. The sequences were aligned using MEGA6 software, and phylogenetic analysis of the sequences was performed using MrBayes 3.1.2. BI analysis under the general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) model for the 28S rDNA gene was done. The tree was visualized using Fig Tree v.1.4.2

Results and Discussion

    In this study, three species belonging to Nygolaimidae, namely Aquatides christicki, Clavicaudoides clavicaudatus, and Pravulvus hartingii, were identified, of which A. christicki is the first record for Iran's nematode fauna. This species is characterized by having a cylindrical body, 1.45-1.99 mm long, tapering gradually toward anterior end, lip region asymmetrical, with a slight ventral incline, amphidial fovea cup-shaped, aperture slit-like, 8.5 to 9.5 µm wide, or 42 to 44% of lip region wide, guiding ring simple and single, at a distance of 9.5 to 10.5 µm from anterior end, mural tooth linear, 19.0-21.5 µm long, 0.9 to one times of lip region wide, odontophore, 31-38 µm long or 1.6 to 1.7 times of tooth length, basal expanded part of pharynx 158-230 µm long and occupying about 49 to 52% of the pharynx length, nerve ring surrounding the anterior slender part of the pharynx at 130 to 175 µm from anterior end of body, three cardia glands ovoid, vulva a transverse slit, female reproductive system didelphic-amphidelphic, tail short and rounded, dorsally convex conoid. No significant difference was found between the identified species and the original description. This species was first reported from India by Ahmad & Jairajpuri, 1982. The current study is the second report after the original description. The sequence of A. christicki was deposited for the first time in NCBI. Furthermore, in this study, Pungentus engadinensis and P. sufiyanensis, from the Nordiidae family, were also identified. The characteristics of these two species did not show any differences compared to the original description. Three nucleotide sequences obtained in this study for the species of A. christicki, C. clavicaudatus, and P. hartingii, along with other known sequences of the family Nygolaimidae, formed a single clade separated from other families of the order Dorylaimida with maximum posterior probability (BPP=1). Also, the sequences obtained for P. engadinensis, along with the sequences of the species in the GenBank, were placed in the same clade.