Morphomolecular identification and considerations of the infestation site adaptations of Pricea multae (Thoracocotylidae: Priceinae) from Scomberomorus commerson , off Arabian Gulf, Saudi Arabia

Morphomolecular


Introduction
Since the earliest times, fish have played an important role in the aquatic ecosystem and represent the main source of income for many countries, especially with great economic value (Mehanna, 2022). Presently, great consideration is paid to fish production as the replicable source of protein (Waite et al., 2014;Ganguly et al., 2022). Epipelagic and migratory marine fish, particularly those belonging to Scombridae fish, include species of high commercial interest. Scomberomorus commerson (Scombridae) is a predatory marine and one of the most expensive and high-quality fish due to its nutritious proteins (Claereboudt et al., 2005;Rajesh et al., 2017, Thai et al., 2021. Few and sporadic investigations spotlight fish parasites in the Arabian Gulf, which are more scarce than recorded on it, specially monogenean parasites (Bayoumy et al., 2012). The monogenean parasites are mainly external parasites infesting aquatic vertebrates, especially fish, where a few genera are endoparasites (Bayoumy et al., 2015;Klapper et al., 2017). Anemia and potential host mortality are primarily caused by the infestation of monogeneans (Ramasamy & Hanna, 1985;Woo & Bruno, 2011;Rigos et al., 2021). The Neothoracocotylidae classification includes Neothoracocotylinae Lebedev, 1969;Priceinae Chauhan, 1953;Scomberomorocotylinae;andThoracocotylinae Price, 1936 (Rohde &Hayward, 1999a, b). Furthermore, unlike other Neothoracocotylidae subfamilies, Priceinae has one or two rows of clamps, two pairs of large hamuli, and few male copulatory spines (Rohde & Hayward, 1999a, b).
Pricea multae (Neothoracocotylidae: Priceinae) was first recorded in the Indian Ocean as isolated from the gills of Cybium lanceolatus, and later; it was reported as present in the gills of several species of mackerel at different localities in the Indian Ocean, off the Australian coast and the South China Sea (Ramasamy et al., 1986, Rohde & Hayward, 1999a. For a short time, it has an optional intermediate host (Rohde & Hayward, 1999a, b).
In this investigation, we are concerned not only with the morpho-molecular description through light and scan electron microscopes of P. multae from the gills of S. commerson in the Arabian Gulf, off Jubail, but also with spotlighting on adaptations of this monogenean species.

Fish samples
Freshly caught S. commerson ('Spanish mackerel) samples (N=120) were collected weekly of local name: 'Kanad' from local fishermen along the Arabian Gulf, off Jubail, Saudi Arabia ('27 ° 57.9" N and '49° 40' 43.4" E) during the period from May 1 st , to August 31 st, 2018 and were brought to the laboratory of the Faculty of Science wrapped in ice bags. Fish used for parasite collection were dead, been commercially caught, and available for purchase at fish markets. Dissecting fish has complied with all relevant regulations.

Ethics statement
The fish used to collect the parasites were dead, commercially caught, and available on the fish market. All relevant regulations were observed when dissecting fish.

Light microscopy
After separating the gills, they were placed in separate Petri dishes with seawater and examined for the presence of monogeneans using an Olympus SZ61 stereomicroscope. The monogeneans were examined alive or after being fixed in 70% alcohol under a coverslip. Others were preserved in 70% ethanol after being fixed in 4% formalin. After that, they were stained with Mayer's carmine and dehydrated with ethanol. Clove oil was used to clear dehydrated specimens before mounting them in gelatin-glycerol media. A light microscope was used to examine and photograph mounted specimens (Olympus CH40). Illustrations of stained specimens were created using a previous microscope with a drawing tube, then scanned and redrawn using Photoshop software on a computer. A few specimens (N=20) were mounted in ammonium picrate glycerin medium to note details of the hard parts of the haptor and the organization of the terminal genitalia. Measurements were taken with an ocular micrometer on flattened stained specimens and are presented as the mean (± St. error) followed by the range mean in parentheses. The opisthohaptor is included in the body length.
Scanning electron microscopy (SEM) Some isolated monogeneans specimens were fixed in a 4% aqueous glutaraldehyde solution (4°C for 48 Hrs.). They were then washed thoroughly with cacodylate buffer and post-fixed for 4 Hrs. with aqueous osmium tetroxide (Os O4) and dehydrated through alcohol. Later, they were dried in a Tousimis Autosamdri -815 Coater, E300 critical point drying apparatus using liquid CO 2 . The specimens were whole-mounted on an aluminum stub and fixed by a double-phase sticker. The specimens were then coated with gold-palladium in a Sputter Coating Evaporator unit (S.P.I. Model-Sputter Carbon/ Gold Coater) (Bayoumy et al., 2006). The specimens were examined using a JEOL JEM-2100 scanning electron microscope operating at 20 Kev. All the preparations for SEM were done at Electron Microscopy Unit at Mansoura University.
Molecular procedures for molecular identification, one specimen preserved in 70% ethanol was rinsed in ultrapure water. According to the manufacturer's procedure, the Genomic DNA. Purification Kit (Invitrogen, USA) was used to extract total genomic DNA. The primers C1 (5′-ACCCGCTGAATTTAAGCAT-3′) and D2 (5′-TGGTCCGTTTCAAGAC-3′) were used to amplify DNA. specific to the D1-D2 domain of the large subunit region of the 28S ribosomal gene (28S rRNA) (Hassouna et al., 1984). DreamTaq PCR master mix (Thermo Scientific) was used for PCR amplification, with an initial denaturation at 94°C for 3 minutes, followed by 35 cycles of 30 seconds at 93°C, 30 seconds at 58°C, and 90 seconds at 72°C, with a final 7-minute extension step at 72°C.
The PCR product was purified using the Purelink TM Quick Gel Purification Kit (Invitrogen, USA.) and submitted straight for sequencing on a 3730xl DNA sequencer (Applied Biosystems, USA) using the identical primers stated above. The nucleotide sequence has been submitted to GenBank. The nucleotide sequence acquired in this research was aligned with prior sequences submitted in GenBank using BLASTn (http://www.ncbi.nlm.nih.gov/blast).
MEGAX software was used to infer phylogenetic relationships based on incomplete 28S rRNA sequences using the maximum likelihood and the neighbor-joining methods. The trees' credibility was evaluated using the bootstrap technique with 1000 replicates, and the divergence time was calculated using the Tamura and Nei model.

Site of infestation: gills.
Locality: Arabian Gulf, Saudi Arabia. Description: (Based on 20 whole mounted specimens.) A total of 132 parasite specimens were collected from the sampled mackerel fish Body long, smooth, curving to the right, flattened dorso-ventrally, and pointed to the anterior end and the opisthaptor bifurcate ( Figures 1A and 2A). The fore and hind bodies with tegumental corrugations on dorsal and ventrolateral surfaces. The tegumental ridges run transversally, generating folds in dorsal and ventral surfaces of the body at regular intervals ( Figure 2B). Pit-like depressions, as well as microvilluslike tegumental projections, between the folds, densely packed with papillary-like sensory endings that are uniciliated ( Figures 2C and 2D). The total body length of compressed specimens was 2900 (±210), (2750-3940) in the uncompressed 2700 (±120), (2530-3240).
Anterior body length with 2450 (±120), (2250-2630). The average width at the germarium level 520 (±110), (420-650). The prohaptor with two septate, sub-terminal, sub-oval, and ventral buccal cavities. The buccal cavity was oval than circular and surrounded by a pre-oral pit (20 ± 0.8× 17±0.6) 18-22×16-19 length by width and 20-20 uniciliated sensory endings ( Figure 1B). Mouth subterminal, ventral, approximately small and surrounded by sensory receptors ( Figure 3A). Each one has knob-like cilium. Pharynx small, bulb-shaped, muscular, and sub-spherical 58 (±1.2), (54-62). The esophagus was short without diverticula. Intestine bifurcation behind the level of the vaginal opening; caeca with medial and lateral diverticula unbranched, extending into posterior body region but not into opisthohaptor and not confluent posteriorly. On each side of the body, note the two-row of 45(±2) (38-52) clamps with a maximum width of 49(±0.11), (46-53), and the brown or black pigment (haematin) derived from ingested The testes, 20 in number in the median field, arranged in two rows from behind the ovary to between the end of the caeca and the opisthaptor. Cirrus a circle of 14 (13-15) copulatory spines with a total length of 42.8 (±0.13), (41-44) ( Figures 1C; 1D and 4A). Gonopore a short distance behind the pharynx, approximately at the level of excretory openings, genital atrium unarmed. The germinal part of ovary is heavily branched, with a long coiled ovarian loop pointing anteriorly and germinal and terminal prats pointing posteriorly . Small follicular vitellaria in the lateral fields from the caecal bifurcation to the end of the caeca. The vaginal opening is armed with the sclerotized, hallow conical spine with an apical collar, in front of the caecal bifurcation. Egg of 580 (620-575; n = 2) in length; ends with long bipolar filaments ( Figures 4C and 5A).

Molecular identification
The nucleotide sequence has been submitted to GenBank under the accession number ON032998. This isolate sequence was identical to previously deposited P. multae isolates from Israel (accession numbers MT995137, MT995138, MT995139, MT995140) (Direct submission in 2020). Furthermore, this isolate has the highest resemblance (98.81%) to the specimen available for P. multae (accession number AF026111) collected from S. commerson from Heron Island, Australia.
Our phylogenies inferred using the maximum likelihood and the neighbor-joining method produced similar topologies. The maximum likelihood topology is shown in Figure 6. A phylogenetic analysis of 28S sequences revealed that the Pricea multae reported in this work belongs to the same clade as other Pricea multae isolates, Mexicotyle sp. and Paradewesia sp. (Neothoracocotylidae family), as well as Gotocotyla secunda and Gotocotyla bivaginalis (Gotocotylidae family). Furthermore, this isolate and P. multae isolates were the dataset's most closely related Neothoracocotylidae.

Discussion
Monogeneans are pathogenic parasites that live in a single host and are found in wild and captive fish. Priceinae members have been found in the gills of scombrid fishes of the genus Scomberomorus (and possibly Acanthocybium, Rastrelliger, and Katsuwonus) from warm to warm temperate seas around the world (Rohde & Hayward, 1999a). The parasites under investigation were previously recorded on the Shina sea, the Indian Ocean, and the Australian east coasts (Rohde, 1976;Rohde & Hayward, 1999a, b). The widespread of this monogenean species (P. multae) may be attributed to the wide range of the geographical distribution of their host (S. commerson) (Rohde, 1976). It is the first occurrence of the monogenean P. multae parasitizing S. commerson off Arabian Gulf, Saudi Arabia.
According to Rohde & Hayward (1999a, b), Priceinae members are characterized by one or two rows of clamps, two pairs of large hamuli, and a small number of male copulatory spines. Small hamuli of the present material   coexist with large hamuli on the terminal haptor lappet rather than in the typical prehaptoral position (Gupta & Chanana, 1976;Hayward & Rohde, 1999). So, the material under discussion has all Priceinae characteristic features. Comparing the morphometric data for the present material with the redescription of this species) Rohde, 1976;Lebedev, 1986;Rohde & Hayward, 1999a, b) reveals a few differences (Table 1). Some structures of present specimens were not measured in detail, such as the copulatory spine length, clamp parts, and hamuli measurements.
This study showed that this isolate and P. multae isolates (accession numbers MT995137, MT995138, MT995139, MT995140, and AF026111) were the dataset's most closely related Neothoracocotylidae. The phylogenetic analysis of 28S sequences showed that the species forms a clade with members of the family Neothoracocotylidae and the family Gotocotylidae. Similar to these results (Olson & Littlewood, 2002) found that the oligonchoinean families, Gotocotylidae and Neothoracocotylidae, were paraphyletic.
Despite the importance of confirming the taxonomic status of parasite, one of the goals of this study was to learn more about its functional appropriateness and the significance of its microstructures. The observations indicate that this monogenean species has a cylindrical body with multiple clamps and a pair of hamulus through which  attachment to the gills of the host is maintained. As a result, numerous tegument folds and perforations enhance surface area and facilitate absorption and excretion. The sensory ganglia present on the surface of parasite serve as sensory organs for detecting the host type, site of infestation, and water quality. The papilla-like uni-ciliated sensory endings found all over the body surface could be rheo-or tango-receptors. In contrast, the sensory endings around the oral apertures are thought to be involved with feeding (Hadied et al., 2015). Pits, folds, and a few scattered microvilli-like structures on the body surface may help with nutrient absorption, osmoregulation, and respiratory gaseous exchange (Ramasamy et al., 1986;Thompson & Geary, 2003). Moreover, the haptor's nonciliated sensory structures may help the fluke attach to the host's gills (Ramasamy & Brennan, 2000;Hadied et al., 2015). The posterior oriented curved hamulus spines are considered the primary attachment organ as they are highly modified, whereas a relatively unmodified internal spine supports the larger one.