Introduction
Pentastomida is a subclass of wormlike crustacean endoparasites with five anterior appendages one mouth, and two pairs of hooks for their attachment to the host. They have approximately 131 species distributed in two orders; Cephalobaenida and Porocephalida and seven families: Cephalobaenidae, Linguatulidae, Porocephalidae, Ralli- etiellidae, Reighardiidae, Sebekidae and Subtriquetridae, in addition to three Middle Cambrian fossil groups. The two orders can be distinguished mainly by differences in the disposition of the hooks relative to the mouth and by the form of the reproductive tract. All of the species of these groups attain sexual maturity in the respiratory tract of vertebrates.
Parasites of the order Cephalobaenida are characterized by the presence of a subterminal or ventral mouth situated anterior to the hooks disposed in a trapezium. Three enera of the order Cephalobaenida were recorded: (a) Cephalobaena, infecting the lung of American tree snakes; (b) Reighardia Ward, 1899 infecting the lung of marine birds as definitive hosts; (c) Raillietiella, the largest pen- tastome, worm-like, blood sucking parasites inhabiting the upper respiratory tract of insectivorous small lizards, which have piercing mouthparts surrounded by two pairs of compound hooks that embed into the lung to facilitate feeding on the host blood.
The life cycle of pentastomids includes two hosts; the eggs are either coughed out by the host or leave the host body through the digestive system. The eggs are then ingested by an intermediate host, which is commonly either a fish or a small herbivorous mammal. The definitive host becomes infected by pentastomids after eating the inter- mediate host, the parasite crawls within the host body into the respiratory tract from the esophagus2 . Humans can serve as an accidental definitive host after ingesting raw or poorly cooked viscera (i.e., liver, lungs, and trachea) of the intermediate hosts
The pathologic effect of pentastomid infection may be very serious and/or lethal, causing lesions in lung tissues and obstruction of the trachea that can result in interstitial subacute pneumonia with congestion In Egypt, rallietiellids were first recorded by Ali et al., who described two pentastomids, R. affinis and R. aegypti as a new species from Egyptian desert lizards, with much focus on R. affinis. Scant information with incomplete morphology and phylogeny is available worldwide for R. aegypti. The nuclear18S ribosomal DNA (18S rDNA) has proved to be a potential marker during the characteriza- tion of eukaryotes. Its highly conserved nature and slow rate of evolutionary mutation make 18S rDNA suitable for interspecies distinction at higher taxonomic levels.
In the present study, a morphological description of a pentastomid infecting the lungs of Berber skinks Eumeces schneideri (Scincidae) captured in Jizan, Saudi Arabia was carried out by light and scanning electron microscopy. Fur- thermore, the 18S rDNA molecular analysis of the parasite was conducted to determine the exact phylogenetic position of these parasites.
Materials and methods
Sample collection and parasitological study
Seventeen specimens of the berber skink Eumeces schnei deri (Reptilia: Scincidae), 10 males and 7 females, were collected by hand or noose from well -vegetated wadis at high altitudes, in Jizan, Saudi Arabia (17.6548° N, 42.8871°E) during the year 2017. Specimens were collected seasonally, during summer (5 males, 4 females) and winter (5 males, 3 females). Animals were kept alive in glass cages with sand and alluvium in the animal room at 25-30 °C and fed insect larvae. Identification of the examined species was carried out according to Arnold11and Al -Sadoon. When examined, a cotton ball soaked with isoflurane was placed inside a plastic bag containing the animals and left inside to allow sufficient time for the anaesthetic gas to cause euthanasia. Alternatively, the animals were left in the sealed bag for suf- ficient time to achieve anaesthesia, at which time they were removed from the bag and injected intraperitoneally with an overdose of sodium pentobarbital. These were in agreement with the regulatory laws regarding experimental ethics of animal use and collecting permits, Institute of Animal Care and Use Committee (protocol number BSU/FS/2015/10).
Following dissection, pentastomids were recovered from lungs using a ZEISS Compact Greenough stereomicroscope (Model Stemi 305), heat fixed in 10% neutral buffered for- malin for 15 min and then preserved in 70% ethanol in 5% glycerol solution to avoid sudden drying. Finally, sam- ples were transferred to lactophenol for clearance. The prepared samples were examined using differential inter- ference contrast (DIC) light microscopy with digital image analysis system (analysis auto 5.0) and photographed by an Olympus research photomicroscope supplied with a built -in camera (Model BX53F, Tokyo, Japan). Drawings were made with the aid of a drawing tube.
For scanning electron microscopy, samples were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer, washed in the same buffer, and dehydrated in a graded alcohol series. Samples were then processed in a critical point drier ''Bomer-900’’ with freon 13, sputter-coated with gold-palladium in a Technics Hummer V, and finally examined with a Jeol scanning electron microscope (Model JSM7610F, Jeol Ltd, Japan). Identification of the recovered pentastomids was based on the key published by Rego and Ali et al.
Molecular analyses
Genomic DNA was extracted from the ethanol preserved samples of the isolated parasites using QIAmp DNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions, and further stored at -20°C until used. PCR reactions were carried out using the following primers (28Sa F: 5'-TGCTTGTCTCAAAGATTAAGCC-3', 28Sb R: 5'- TGCTTGTCTCAAAGATTAAGCC-3'). To target the 18S rRNA gene in a final reaction, a total volume of 50 was prepared containing 1 ^l of DNA templates (100 ng), 0.5 ^l (50pmol) of each primer, 2 of dNTPs (Advanced Bioenzymes, UK), 1 ^l DNA polymerase (2 U) (Advanced Bioenzymes, UK), 5 ^l of 10 x buffer (500mM KCl, 100mMTris HCl pH 9.0, 1.5mM MgCl2), and 40^l of dH2O. The temperature profile was as follows: Initial denaturation at 95 °C for 5min, followed by a denaturation step of 35 cycles at 94 °C for 45sec, anneal- ing at 49 °C for 45sec, extension at 72 °C for 1 min, and a final extension at 72 °C for 1 min. The amplification reac- tions were carried out in a PCR Thermocycler (PTC 100, MJR Research, USA). Two microliters of bromophenol blue were added to the aliquots of PCR products and the corre- sponding amplicons were electrophoresed on 2.5% agarose gel, stained with ethidium bromide, visualized using a UV transilluminator.
Sequencing and phylogeny
The PCR product was purified using a kit (Roche Diagnostics, Germany), and sequencing templates were prepared using a plasmid preparation kit (Machery-Nagel) and aBIO Dye Ter- minator v 3.1 Ready Sequencing Kit (Applied Biosystems) and 310 Automated DNA Sequencer (Applied Biosystems, USA) using the same primers. To identify related sequences, a BLAST search was carried out on the NCBI database. A BLAST rectangular tree was created from genetic distances calculated using the Jukes-Cantor model for nucleotide comparisons between the query sequence and arthropod sequences. The tree was built from the distance metrics using Fast Minimum Evolution (FastME) according to Des per and Gascuel. Moreover, data of DNA sequences were aligned using CLUSTAL-X multiple sequence alignment and compared with previously deposited sequences of pentas- tomids recovered from GenBank to analyze intra-specific differences. The alignment was corrected manually using the alignment editor on BIOEDIT software 4.8.9. A phyloge- netic tree was constructed using the MEGA program version 7.
Results
Among the 17 specimens of E. schneideri examined, twelve animals (prevalence 70.59%), eight males and four females, had their lungs infected by adult pentastomids. The infec- tion increased during summer (4 males, 4 females) with a percentage of 89% and decreased during winter (4 males, 0 males) with a percentage of 57%. Adults were identi- fied by the presence of tapered hooks devoid of chitinous accessories. The examined male hosts were generally more heavily infected (mean intensity of 4.3) than females (2.2).
Morphology
Raillietiella aegypti Ali et al.3Fig. 1 (A-G), Fig. 2 (A-H), Fig. 3 (A-H)
Measurements and description based on the holotype female and male and paratype females.
Female (based on 5 specimens): body pyriform, annu- lated 25 ±2 (24-28), widened anteriorly between 3-14 annuli and tapered posteriorly to a bilobed terminal seg- ment. Body was 16.22 ±4 (15.25-18.31) mm long and 4.16 ±0.21 (3.6-5.9) mmwide. Cephalothorax trapezoidal, slightly wider and indistinct from abdomen, rostrum not prominent, with a sclerotized U- shaped buccal cadre and a thick-framed terminal mouth. Two pairs of hooks, a pair of sharp anterior hooks with AB 135 ±5 (110-146) ^m and BC 158 ±5 (150-169) ^m and a pair of sharp poste rior hooks with AB 221 ±5 (200-236) ^m and BC 286 ±6 (280-289) ^m. Four dorsal and prominent apical papillae present, one adjacent to each hook. Abdomen with dis- tinct annuli, parapodial lobes present, anus ventral; caudal papillae indistinct; uterus straight, tube-like, and opened anteriorly.
Male (based on 5 specimens): body morphology was sim ilar to that of the female; with a pyriform body, annulated 19±2 (16-21), widest anteriorly, 12.7±3 (10.5-13.3) mm long and 3.45 ±0.13 (3.2-5.8) mm wide, anterior hook with AB 73 ±3 (72-75) ^m and BC 102 ±5 (100-103) ^m; pos terior hook larger with AB 190.6 ±5 (190-191) ^m and BC 221 ±5 (280-289) ^m. Dorsal papillae present; cephalotho rax trapezoidal, widest at annuli 3-12, indistinct from abdomen. Two copulatory spicules curved with absolute uni- form width; anus ventral; testis single, rounded, opened anteriorly.
Molecular study
Sequence alignment by the maximum likelihood analysis for the data obtained from 18S rDNA analysis of the parasite iso- lated from the host examined yielded 1405 bp, which exhibit identities ranging between 88-95% with pentastomid gen era recovered from GenBank. BLAST computed a pairwise alignment between a query and the database sequences searched; a tree was constructed and constituted differ- ent clades from arthropod species (Figs. 4 and 5). It was observed that a separate monophyletic clade of Pentas- tomida was constructed including the present sequences and pentastomid species. Moreover, the results obtained strongly support that pentastomes are included within crus- taceans and most closely related to branchiurans. The constructed phylogenetic tree by the maximum likelihood method based on the Tamura 3-parameter model among different species of the class Pentastomida arranged the aligned sequences and the present one into two main clades; the first clade (clade I) included species of the order Porocephalida while the second clade (clade II) included species of the order Cephalobaenida which was subdivided into two subclades: the first subclade (subclade A) arranged the species of family Reighardiida7: Hispania vulturis and Reighardiasternae exhibited 94.90% and 94.76 identities respectively with the query sequences. The second subclade (subclade B) arranged the species of family Raillietiellida7 to which the present sequences were included, the subclade B species and their identities being: R. orientalis, 95.33% and Raillietiella sp. , 95.89% showed maximum identi ties with the query sequences. Estimates of Evolutionary Divergence between Sequences were shown in Table 1. The recovered sequences from the present study were deposited in GenBank under Accession number MK970649.1.
DISCUSSION
Studying parasite diversity worldwide is important for at least two major reasons. First, parasites are now recog- nized as playing important roles in ecosystem fractions by influencing the populations and communities of their hosts. Second, many parasite species are agriculturally and medically important. Therefore, the identification of parasitic worms based on microscopic observations and PCR amplification/sequencing of 18S rDNA from isolated para sites is highly recommended for successful investigations and taxonomy. There are many parasitic crustaceans, such as pentastomes, whose adult morphology is devoid of crus- tacean features.
Family Raillietiellidae represented by the single pen- tastomid genus Raillietiella is characterized by a quite wide diversity of hosts. The taxonomy and systemat- ics of the pentastomid genus Raillietiella were reviewed by Ali et al. in the description of R. cartagenensis and in the redescription of R. amphiboluri, R. kochR. shipleyi, and R. indica. Ali et al. reorganized the known and valid species of Raillietiella into five groups (Table 2): small lizards, varanid lizards, amphisbaenians, snakes, and amphibians, which are mainly based on host characteristics (host type, ecology, and zoogeography). Two of these groups (groups I and II; after Ali et al.) include species infecting small insectivorous lizards and are easily differentiated by the initial ideas of Self into species with sharp-tipped posterior hooks (group I) and blunt-tip posterior hooks (group II). Species differentia- tion in these two groups is mainly based on a combination of characteristics, including body size, annulus number, posterior-hook dimensions, and size and shape of the male copulatory spicule. The sharp-tipped posterior-hook of Raillietiella spp. includes six well-characterized species: R. amphiboluriinfecting the Australian bearded lizard Amphibolurus barbatus; R. chamaeleonis 28 in Chamaeleo oustaleti and Chamaeleo verrucosus from Madagascar; R. mottae from Tropidurus hispidus from Northeastern Brazil; R. morenoi 1 from Gallotia atlantica in the Canary Islands, R. aegyptiin different small lizards from Egypt; and R. cartagenensis in Hemidactylus sp. and Gonatodes sp. from Colombia. The reported presence of R. affinis in Lepidactylus lugutris from the British Salomon Island still awaits confirmation and also that in Lioheterodon modestus from Madagascar due to the inclusion of R. chamaeleonis as a host. The present Raillietiella sp. could be dis- tinguished from Raillietilla sharp-hooked species of small lizards through different combinations of characteristics (Table 3), such as measurements of body length, annu- lus number, anterior-hook and posterior-hook size, and dimensions of the male copulatory spicule. Furthermore, it can be differentiated from similar species in group I. The taxonomic and systematic morphological characteristics stated by Ali et al. and others referring to male hooks are reliable in distinguishing and separating the present Raillietiella sp. from the rest. Based on light and scan- ning electron microscopy, the present parasite should be classified morphologically as R. aegypti according to the following criteria: the same host with a new type locality; it is a group I type of Raillietiella spp. and the dimen- sions of anterior and posterior hook. This is the first report of a pentastomid in a reptile species from Saudi Arabia. The congruence between molecular and morphological data in the taxonomic justification of pentastomids was demon- strated by Mohanta and Itagaki.
Our analysis corroborates the monophyly of Pentastomida and the results thus provide strong support for Wingstrand’s proposal that pentastomes are highly modified crustaceans most closely related to branchiurans. Data derived from the DNA analysis coincide with those from morphological studies in accordance with Wagele and Nielsen; the maximum identity obtained for the present sequences was 95.89% with Raillietiella sp. recovered from different hosts and localities with some morphological differences that exclude the suggestion to classify the present species as being the same. Moreover, since the present parasite is morpholog- ically the same as R. aegypti described previously with no molecular confirmation, the present study represents the first one to confirm the taxonomy of R. aegypti. The results of molecular analyses vary according to the selected species, sample size, and particular methods of analysis used, where the sequences of 18S rRNA may contain consis- tent phylogenetic information for cladogenetic events as old as the Median Cambrian. This morphology-based taxon omy is consistent with present molecular phylogenies, which are supported by previous observations. Consequently, revi- sion on the morphology-based taxonomy or inclusion of molecular data from an extensive range of taxa should be necessary to elucidate the phylogenetic relationships of the Pentastomida.
Animal Rights Statement
Authors declare that the experiments on animals were con- ducted in accordance with local Ethical Committee laws and regulations as regards care and use of laboratory animals.
Financial Disclosure Statement
The Deanship of Scientific Research at King Khalid Univer- sity through Research group Project under grant number (R.G.P.1-112 -40).
Conflict of Interests Statement
No conflict of interests for the publication of this article was reported.
Acknowledgment
The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University for funding this work through Research group Project under grant number (R.G.P.1-112 -40).