Bull 209 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Kamal et al. Bull. Iraq nat. Hist. Mus. (2024) 18 (1): 209-223. https://doi.org/10.26842/binhm.7.2024.18.1.0209 ORIGINAL ARTICLE MORPHOLOGICAL AND MOLECULAR STUDY OF THREE SPECIES OF ACROBELOIDES (COBB, 1924) THORNE, 1937 (RHABDITIDA, CEPHALOBIDAE) AS NEW RECORDS IN IRAQ Ruia Safwan Kamal*, Hayder Badri Ali* and Jawad B. Al-Zaidawi** *Department of Biology, College of Science, University of Baghdad, Baghdad, Iraq. **Ministry of Science and Technology, Directorate of Agricultural Research, Baghdad, Iraq. Corresponding author: hayder.badri@sc.uobaghdad.edu.iq Received: 3 January 2024, Revised: 1 March 2024, Accepted: 19 March 2024, Published:20 June 2024 This work is licensed under a Creative Commons Attribution 4.0 International License ABSTRACT Three species of nematodes within Acrobeloides (Cobb, 1924); Thorne, 1937 (Rhabditida, Cephalobidae) were collected and recorded for the first time in Iraq, based on morphometric and molecular data, these species, A. saeedi Siddiqi, Ley & Khan, 1992, A. apiculatus (Thorne, 1925), and A. bodenheimeri (Steiner, 1936), were molecularly characterized using the partial 28S rRNA gene sequences. The phylogenetic tree has been constructed to separate Acrobeloides species from closely related species. Keywords: Acrobeloides, Molecular, Morphometrics, Nematodes, New record. INTRODUCTION Despite having a vast variety of habitats and about 354 genera, the family Cephalobidae Filipjev (1934) is challenging to recognize and taxonomically describe based solely on morphological characteristics (De Ley and Blaxter, 2004). The morphology of the lip and stoma can be used to distinguish between different genera and species of Cephalobidae (Carta et al., 2022). Cephalobidae includes Acrobeloides (Cobb, 1924), which was raised to the rank of genus by Thorne (1937). These nematodes feed on bacteria and are found in almost all sand dunes across all land-dwelling environments, excluding steamy rainforests, agricultural land, deserts, and the barren soils of Antarctica (Timm, 1971; Waceke et al., 2005; Nadler et al., 2006; Rana et al., 2020; Bhat et al., 2021; Loulou et al., 2022). Sequence analysis suggests that the 26 valid species of Acrobeloides and the 5 species with uncertain taxonomic status represent at least two phylogenetically distinct groups that interact with species of Cephalobus and Chiloplacus, among other organisms (Smythe and Nadler, 2006; Nadler et al., 2006; Holovachov et al., 2009). Acrobeloides and other taxa can be difficult to distinguish morphologically from one another at times because of highly confusing patterns of intra- and interspecific variability (Janssen et al., 2017). Cobb (1924) made the first mention of the Acrobeloides taxonomy. BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Iraq Natural History Research Center & Museum, University of Baghdad https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home Copyright © Bulletin of the Iraq Natural History Museum Online ISSN: 2311-9799, Print ISSN: 1017-8678 https://doi.org/10.26842/binhm.7.2024.18.1.0209 https://orcid.org/0000-0003-4842-151X mailto:hayder.badri@sc.uobaghdad.edu.iq https://creativecommons.org/licenses/by/4.0/ https://jnhm.uobaghdad.edu.iq/index.php/BINHM/Home 210 Bull. Iraq nat. Hist. Mus. 18 (1): 209-223. Morphological and molecular study Some Acrobeloides species have been shown to exhibit insect association behavior when soil nematodes are isolated using the Galleria soil baiting method described by Bedding and Akhurst (1975), and Azizoglu et al. (2016). Grewal et al. (2003) have noted that in addition to their association with insects, there has been an infestation of some annelids, arthropods, and molluscs. While previously identified by Saeed et al. (1988) as Cephalobus litoralis (Akhtar, 1962). Bhat et al. (2021) and Rana et al. (2021) confirmed that A. saeedi was discovered by Siddiqi et al. (1992) in Pakistan. Nematodes that feed on soil-dwelling bacteria and fungus, parasitize plants and insects, and even eat other nematodes are among the species in this category that have been researched in great detail. The Cephalobidae are the most prevalent among them in fields of agriculture. Understanding and communicating each organism's ecological importance requires accurate species-level identification (Andrássy, 1967). According to previous observations (Bedding and Akhurst, 1975; Azizoʟglu et al., 2016; Rana et al., 2021), numerous Acrobeloides species have been found to exhibit the insect association nature when they are isolated from soil samples employing the larvae of Galleria. For example, earthworm cocoons are infested by Acrobeloides nanus Anderson, 1968, (Kraglund and Ekelund, 2002). It has been earlier reported that they are complicated in the soil nitrogen cycle and soil mineralization. Anderson et al. (1981) and Hao et al. (2010) report that during these activities, they engage with a range of arthropods and invertebrate species, some of which can be phoretic and necronemic. A. saeedi and other morphologically similar species, like A. bodenheimeri, were to be included in the new genus Rafiqius, which Khan and Hussain (1991) proposed (Steiner, 1936; Thorne, 1937). Based on the morphology of the lip area and the presence of seta-like structures at the labial primary axils, this recently proposed genus was distinguished from Acrobeloides. Nevertheless, De Ley et al. (1999) felt that the genus's creation was not warranted. There is a description of two Indian cultures of A. saeedi. This specimen is consistent both morphologically and morphometrically with A. bodenheimeri, A. longiuterus, and A. maximus, as well as other species in the Maximus-group, particularly A. longiuterus. But according to Bharti et al. (2020), the Indian material can be more clearly distinguished from each of these species by molecular analyses using ITS, 28 S, and 18 S rDNA. To the best of our knowledge, no research has documented the morphological and molecular characteristics of the genus Acrobeloides in Iraq, thus, the aim of the current investigation was to use molecular, morphometric, and morphological data to culture and recognize nematode species that were taken from bitter gourd fields. The 28S ribosomal gene- based phylogenetic analysis was used to estimate their evolutionary relationship. The molecular and morphological database of Acrobeloides species. MATERIALS AND METHODS Isolation, cultivation, and processing of nematodes: Between January 2022 and April 2023, soil samples were gathered from several locations in the middle of Iraq (Map 1, Pl. 1); sampling was done according to Orozco (2014), and their nematode content was examined (Tab. 1). It is advisable to allocate a minimum of 2 - 4 m2 for every sampling site. At least five arbitrary soil samples should be taken in this area, with samples being taken at a 211 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Kamal et al. minimum depth of 15 cm. Using Galleria mellonella larvae in their last instar as bait, nematode specimens were extracted from five soil samples. The cadavers and afflicted larvae were subsequently moved to a white trap (White, 1927), following a thorough cleaning with distilled water and sterilization, with 1% NaOH. After being extracted from cadavers in a white trap, the nematodes were kept in 250 ml tissue culture flasks in an incubator at 8-10°C as described by Bhat et al. (2019). Map (1): Sampling areas. Plate (1): Soils sampling. 212 Bull. Iraq nat. Hist. Mus. 18 (1): 209-223. Morphological and molecular study Galleria mellonella (Linnaeus, 1758) larvae were inoculated with third-stage juveniles (≥100) for morphometric measurements and observations. After the deceased larvae were moved to a white trap, the adult stage (4-5 days) and third stage juveniles (7-8 days) were removed from the trap. After being killed with hot water, the specimens were placed in TAF (a solution of 2 % triethanolamine with 7 % formaldehyde) to be fixed. The fixed nematodes were mounted in pure glycerine on permanent glass slides after being processed to dehydrate glycerine according to Seinhorst's 1959 instructions (Siddiqi, 1964).The following details were recorded for each soil sample obtained: the location of the GPS coordinates, sample collection, the number of samples collected, the name of the strain, the insect host of the several nematode strains isolated, and the nematode species identified. Table (1): Geographical location of nematode sampling sites. Characteristics of Morphology: Nematodes were studied either alive or cooked to 60°C in Ringer's solution for morphological examinations. Every nematode employed in this investigation was raised as G. mellonella larvae in the final stage. A temperature-controlled (25±3◦C) petri-dish containing two moist filter sheets was used to expose ten G. mellonella larvae to roughly 1000 IJ. To separate the mature females of the first and second generations, the infected larvae were dissected in Ringer's solution 4 and 7 days after infection, respectively. Following heat-killing, the nematodes were preserved in triethanolamine formalin (TAF) fixative before being processed using a gradual evaporation method with anhydrous glycerin for mounting (Poinar, 1976; Kaya and Stock, 1997). A drawing tube and differential interference components were added to an Olympus BX41 microscope for use in Sampling site GPS coordinates Number of soil sample Nematodes species isolated Accession number of Design strain name Insect host Wasit, Al- Suwaira 32°56′25″N 044°38′04″E 10 Acrobeloides saeedi OR272345 Galleria mellonella Baghdad, Al- Zafaraniya 33° 15' 2" N 44° 28' 58" E 10 Acrobeloides apiculatus OR363452 G. mellonella Salahuddin / samarra 34°11'32.4" N 43°52'16.1" E 10 Acrobeloides bodenheimeri OR363692 G. mellonella Wasit, Al- Hafriya 32°58'59" N 44°49'60" E 10 none - - Wasit, Badra 33°06'60.0" N 45°56'59.99" E 10 none - - Baghdad, Al- Yusufiyah 33° 3' 29" N, 44° 22' 11" E 10 none - - Baghdad, Al-Mansour 33.3114° N, 44.3497° E 10 none - - 213 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Kamal et al. morphometric and morphology research. The nematode specimens were photographed using a Canon camera (Japan) with magnifications of 10x and 40x. Male and female measurements were made for the following characters: Overall body length, maximum body diameter, anal body diameter, excretory pore position, and distance between the anterior end and position of the nerve ring, pharynx position, tail length, and vulva positions. Ratios a, b, c, D, and E (Tab. 2), a represent (total body length divided by maximum body diam.); b represents (all body length divided by space from the anterior end to the base of the pharynx); c represents (the entire body length divided by tail length); D represents (the excretory pore position divided by distance from the anterior end to the base of the throat), and E represents (excretory pore position divided by tail length). Using the taxonomic standards recommended by Stock and Kaya (1996), morphological identification was completed. Additionally, scanning electron microscopy was used to analyze the morphological characteristics of female representative isolates of each species group. Specimens were handled in accordance with Nguyen and Smart's (1995) methods for this purpose. Characterization via morphology and morphometry: An optical microscope fitted with a micrometric eyepiece was used for morphological and morphometric identification. The IJs were taken from the white trap (Pl. 2), and the nematodes were handled by Nguyen and Smart (1996). Wax worm larvae were dissected. Plate (2): White trap. Nematode molecular characterization: Using a genomic DNA isolation kit from Geneiad BioTEK and according to the manufacturer's instructions, DNA was extracted from the four isolated worms. PCR was utilized to amplify distinct sections of the rRNA genes and mtCO1 using isolated genomic DNA. ITS regions. Polymerase Chain Reaction is used for DNA amplification: PCR Using primer forward (F2), the isolated DNA was utilized as a template for PCR amplification of a fragment 214 Bull. Iraq nat. Hist. Mus. 18 (1): 209-223. Morphological and molecular study containing the D2 and D3 regions of the 28S rR A gene. Reverse rDNA 5'- CGATAGCGAACAAGTACCGAGAG-3 (R2) 5-(900 bp amplification size) CCTGCTCAGGCATAGTTCACCATC-3 28S rDNA (Joyce et al., 1994). PCR cycling conditions used were: A heated lid Techne PHC-3 thermocycler was used to do amplifications. In the thermocycler, the samples were put in and heated to 95 °C. They were then incubated for 2 minutes at 94 °C, then for 40 cycles at 94 °C for 30 seconds, 50 °C for 1 minute, and 72 °C for 1.5 minutes. To make sure all of the final amplification products were full length, a last step of 5 min at 72 °C was added (Yadav et al., 2022). The total amount of the 40 μl PCR reaction consisted of 20 μl of the 2xTaq PCR Master mix that was ready to use, 1 μl of each forward and reverse primer (10 pml), 10 μl of genomic DNA (10–20 μg/μl), and 8 μl of sterilized water. Electrophoresis was used to separate the PCR products in a 1% TAE (Tris-acetic acid- EDTA) buffered agarose gel, stained with GelRed nucleic acid gel dye (Biotium), for 45 minutes at 100 V. The Macrogene firm received the PCR products for Sanger sequencing. Sequences were cut and curated by hand. Every sequence was uploaded to the NCBI databank (National Center for Biotechnology Information). The phylogenetic trees provide accession numbers. Abbreviations: The following terms are shortened in the tables or text: L is the body length, ABD is the anal or cloacal body diameter, EP is the excretory pore position, ES is the pharynx length, GS is the GuL/SpL, GuL is the gubernaculum length, MBD is the maximum body diameter, NR is the nerve ring position, and the ratios are a = L/MBD, b = L/ES, c = L/T, D% is the EP/ES ×100, E% is the EP/TL × 100, SpL is the spicule length (measured along the curved median line), SW is SpL/ABD, T is the tail length, and ratio v indicates the distance from head end to vulva/ L x 100. RESULTS AND DISCUSSION Three species of the genus Acrobeloides were found in soil samples taken from several locations in the center of Iraq. Isolates exhibit the physical traits of the genus Acrobeloides, as reported by Thorne (1937) and Steiner (1936), according to microscopic investigations. An explanation of the three new records from Iraq (A. saeedi, A. apiculatus, and A. bodenheimeri) is given. Measurements: (Tab. 2). The ventral length of the female is slightly bent. The cuticle annulated; the inner labial papillae are six and four outer cephalic papillae and are present in the lip’s region, which is continuous with the body's contour. In A. saeedi, the body length varied from1050 to 1448 μm, the maximum body width was 75 to 112, and the tail length was 48 to 62 μm, a value of 13.7, b value of 6.7, and c value of 22.2. While in Acrobeloides bodenheimeri, the body length was from 635 to 775 μm, the maximum body width was 30 to 40 μm, and the tail length was 35 to 44 μm. a value of 19.8, b value of 5.1, and c value of 17.7, on the other hand, in Acrobeloides apiculatus The body length ranged from 445 to 655 215 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Kamal et al. μm, the maximum body width was 12 to 16.6μm, and the tail length was 41 to 51 μm, a value of 36.5, b value of 3.7, and c value of 11.1 (Pls. 3, 4, 5). Sequencing of 28S and phylogenetic tree: Selected specimens were subjected to DNA analysis to verify the morphological identification of the isolated nematodes. A large subunit ribosomal RNA gene (28S) partial sequence of Acrobeloides isolates sequenced revealed 902 bp of unique DNA fragments. The sequences were then matched to rDNA sequences found in Genbank using the BLAST search algorithm. The results of the phylogenetic analysis revealed that 98% of the sequences had similarity with a bootstrap phylogenetic tree based on 28S sequences from local Acrobeloides isolates constructed with morphological structure supported 28S gene identification and was constructed using the neighbor joining (NJ) method. Table (2): Morphometric data for female of Acrobeloides spp. included in this study and isolated from Galleria culture. [All measurements are in μm (except ratio, and percentage) and in the form: mean ± SD (range)]. Characters A. apiculatus A. bodenheimeri A. saeedi Body length 502.77 ±72.49 (445-655) 690.13±54.79 (635-775) 1.189.37±140.16 (1055-1448) Mid body diameter 13.76 ± 1.47 (12-16.6) 34.9± 4.14 (30-44) 86.46 ±12.48 (75-112) EP 91.28 ±10.56 (83-115) 134.29±13.43 (118-151) 144.27 ±17.04 (125.6-171) ES 133.27 ±10.56 (122-155) 133.53± 3.99 (128-140) 170.42 ±10.38 (155.5-185) NR 88.87 ±7.28 (82-104) 113.58±6.66 (107-126) 112.07 ±8.12(105- 128) T 44.89 ±3.48 (41-51) 39.75± 3.14 (35-44) 53.50 ±4.62 (48.2-62) ABD 11.24 ±1.17 (10.5-13.5) 19.49±1.29 (18-22) 27.97±4.24 (22.8-34.0) a =L/MBD 36.50 ±1.61 (34.92-39.46) 19.86± 1.06 (17.61-21.17) 13.78 ±0.38 (13.45-14.28) v= vulva distance/L * 100 37.58 ±2.10 (33.47-39.69) 76.44±5.54 (68.68-83.44) 76.01 ±2.37 (72.77-78.96) b = L/ES 3.76 ±0.26 (3.54-4.23) 5.16± 0.27 (4.91-5.54) 6.97±0.48 (6.39-7.83) c= L/T 11.17 ±0.72 (10.70-12.84) 17.37± 0.51 (16.20-18.14) 22.20 ±0.96 (20.61-23.85) D=EP/ES *100 68.45 ±3.19 (65.58-74.19) 100.39± 7.25 (92.19-109.16) 84.48 ±5.56 (76.60-92.43) E%= EP/T * 100 203.18 ±8.78 (195.83-225.49) 337.39± 9.57 (320.53-352.25) 269.11 ±11.08 (249.02-283.45) Vulva distance 187.98 ±25.66 (176-260) 528.8±67.96 (445-631) 902.17 ±81.73 (833-1045) The tree (Diag. 1) shows a strong relationship among Acrobeloides saeedi and A. apiculatus isolates collected from different regions in Iraq more than A. bodenheimeri and clustered together with high similarity with A. saeedi from India ( MN101167.1, 216 Bull. Iraq nat. Hist. Mus. 18 (1): 209-223. Morphological and molecular study MK935147.1), while A. bodenheimeri clustered together with A. bodenheimeri isolates from the USA (AF147065.1 and DQ145625.1). Thakar et al. (2022) showed the same result with the participation of species A. apiculatus and A. bodenheimeri within the same clade. This result was achieved by the 18S small subunit rRNA gene partial sequence. According to phylogenetic analyses based on 18S rDNA sequences published by Rana et al. (2020), isolates of A. saeedi and other unidentified Acrobeloides species from Iran formed a group that was clearly monophyly. These isolates were likely conspecific isolates within a clade that had 100% support, and they joined forces to form a sister clade with other species of the "Maximus" group that were from different geographical regions, including A. maximus and A. bodenheimeri. A comparative phylogenetic framework of the morphological characteristics of Acrobeloides was presented by Smythe and Nadler (2006); however, during their investigation, they were unable to identify a common ancestor (Smythe et al., 2006). By using ribosomal small subunit sequence alignment, visually comparable specimens of A. saeedi and A. maximus were shown to be conspecific in another molecular characterization of A. saeedi (Rana et al., 2020). Phylogenetic analysis can be particularly useful in identifying conspecific or closely related worm species, based on these findings (Thakar et al., 2022). Diagram (1): Phylogenetic tree of Acrobeloides species collected from different localities of Iraq based on partial 28S rRNA gene sequences and other genera and species from GenBank. 217 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Kamal et al. Plate (3): Acrobeloides saeedi; (A) Anterior end of female, (B) Entire female, (C) Female posterior end, (D) Vulva of female. Plate (4): A. bodenheimeri; (A) Anterior end of female, (B) Entire female, (C) Female posterior end, (D) Vulva of female. 218 Bull. Iraq nat. Hist. Mus. 18 (1): 209-223. Morphological and molecular study Plate (5): A. apiculatus; (A) Anterior end of female, (B) Entire female, (C) Female posterior end, (D) Vulva of female. CONCLUSIONS In the present article, three nematode species from the genus Acrobeloides (Cobb, 1924); Thorne, (1937) are described for the first time in Iraqi fauna. This study characterizes the first molecular study of these species in Iraq. The phylogenetic relationship is determined by the sequence of the partial 28S rDNA gene, and a phylogenetic tree was also constructed. The analysis resulted in recording of the species A. saeedi, A. apiculatus, and A. bodenheimeri for the first time in Iraq. CONFLICT OF INTEREST STATEMENT We would like to point out that the practical research procedures were carried out by the first and second researchers, while the third researcher analyzed the genetic tree and registered it in GenBank. The results of this study are part of the requirements of the Ph. D. in Zoology, Department of Biology, College of Science-University of Baghdad for the first author. LITERATURE CITED Anderson, R. V. 1968. Variation in taxonomic characters of a species of Acrobeloides (Cobb, 1924) Steiner and Buhrer, 1933. Canadian Journal of Zoology, 46: 309-20. [CrossRef] Anderson, K. J., Read, P. A., Matthews, J. E. and Watson, P. G. 1981. An investigation of the effects of Edinburgh City Sewage Scheme on the ecology of the Firth of Forth. Report of Napier College, Edinburgh, Survey commissioned by Lothian Regional https://doi.org/10.1139/z68-048 219 BULLETIN OF THE IRAQ NATURAL HISTORY MUSEUM Kamal et al. Council 1973–1981. 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(2024) 18 (1): 209-223. دراسة مظهرية و جزيئية لثالث انواع من جنس Acrobeloides (COBB, 1924) Thorne, 1937 (Rhabditida, Cephalobidae) في العراق كتسجيالت الول مرة الزيداوي ** بلبلجواد حيدر بدري علي * و, *رؤيا صفوان كمال داد, العراق.كلية العلوم, جامعة بغداد, بغ-الحياة علوم * قسم .وزارة العلوم والتكنولوجيا, مديرية البحوث الزراعية** 20/6/2024، النشر: 19/3/2024القبول: ، 1/3/2024املراجعة: ، 3/1/2024االستالم: الخالصة Acrobeloides (Cobb, 1924)ثالثة أنواع من الديدان الخيطية الجنسُعِزلْت Thorne, 1937 (Rhabditida, Cephalobidae) ألول مرة في العراق. استناًدا إلى ُسجلْت و التالية: األنواع وِصَفْت البيانات املورفومترية والجزيئية, A. saeedi Siddiqi, Ley & Khan, 1992 A. apiculatus (Thorne, 1925) A. bodenheimeri (Steiner, 1936) نِشأْت والجزئي. S 28باستخدام تسلسل جينات الرنا الرايبوس ي جزيئًيا ُ شجرة النشوء أ .املسجلة عن األنواع ذات الصلة الوثيقة بها Acrobeloides جنس لفصل أنواع