Egypt. Acad. J. biolog. Sci., 5(1): 123– 129 (2013) C. Physiology & Molecular Biology Email: [email protected] ISSN: 2090-0767 Received: 29/5/2013 www.eajbs.eg.net

Ribosomal DNA sequences reveal a parasitic septate gregarine (: Gregarinia) in the scorpion Leiurus quinquestriatus in Egypt

Moustafa M. H. Sarhan Department of Zoology, Faculty of Science, Al-Azhar University, Assiut

ABSTRACT Ribosomal RNA genes are ubiquitously present and nucleotide sequences of many regions are relatively conserved among many organisms. Many specific PCR primers have been developed for amplification of the rDNA fragments from particular taxa. However, many authors use universal primers which can bind to conserved regions to detect protozoa pathogens that are obligate parasites of digestive tracts and body cavities in the analysed DNA sample of invertebrate animals. In this study, uncultured gregarine clones were detected by PCR and rDNA sequencing in the scorpion, Leiurus quinquestriatus (Scorpiones: Buthidae). Comparison of the amplified rDNA fragments with sequences deposited in GenBank revealed that analysed sequences corresponded to 18S rDNA from the genus Gregarina (88 % identity with Xiphocephalus ellisi). To our knowledge, this is the first record of scorpion infected with gregarines.

Keywords: rDNA, Leiurus quinquestriatus, Scorpion, Apicomplexa, gregarines

INTRODUCTION The Apicomplexa consists of unicellular protozoan parasites, infesting a wide range of Metazoa (Ellis et al. 1989). Included are numerous genera that attack humans or domesticated animals (e.g., , Toxoplasma, , , , , and ) (O'Donoghue 1995, Kim & Weiss 2004). These parasites cause great suffering and economic damage, and contribute to millions of human deaths each year (Hammarton et al. 2003). Gregarines are a diverse and successful group of protozoan parasites belonging to the phylum Apicomplexa (previously called Sporozoa), order Léger, 1900. They are all parasitic and are restricted to invertebrates (Clopton 2002). They are obligatory protozoan parasites of invertebrate animals, mainly of terrestrial arthropods. Most species of gregarines are found in the host gut, although a transition from intestinal to infect the body cavity and internal organs is observed in some families (Simdyanov & Kuvardina 2006). Although gregarines do not attack vertebrates, they harm arthropod vectors of human and animal diseases. This is because gregarines are extremely common in both field and laboratory arthropods (Johny et al. 1999; Pushkala & Muralirangan 1997). Gregarines are often considered to be sub-lethal or even harmless to their hosts, but in fact, they divert host nutrients to their own use, occupy space, alter host immune systems, and damage host cell walls when emerging, and thus foster microbial attack. As such, they can reduce longevity, vitality, or fecundity, or cause rapid mortality (Fukuda et al. 1997; Ball1 995). Recent studies demonstrate that gregarine infection significantly increases the effectiveness of both chemical and microbial control measures against insect pest (Lopes & Alves 2005).

124 Moustafa M. H. Sarhan

The gregarine parasites have been study revealed that, uncultured gregarine reported from a variety of arthropod clones have been identified in the hosts; adults and larval stages of , scorpion Leiurus quinquestriatus by this cockroaches, immature and adult simple approach. This typically scorpion common silverfish, adult differential species inhabits the Middle East is one of grasshoppers, field cricket, naiads of the the world's most dangerous scorpion common green darner, flat-backed species. In Africa, it occurs from greenhouse millipede, nymphs and adults Mauritania in the west to Sudan and of the brown-winged earwig and adults Egypt and goes far beyond the Middle of the common civil bluet damselfly East from Saudi Arabia to Iraq, and (Clopton 2002). They were isolated from perhaps even further east (Stockmann & penaeid shrimps of Bombay (Bower et Ythier 2010), Leiurus species secrete one al. 1994) and Kerala (Prasadan & of the most noxious venoms among Janardanan 2001). There is very little buthid scorpions in general, and are information available concerning the responsible for very serious human Gregarine pathogens of Arachnida. These incidents and have been the subject of pathogens were reported from mites, numerous biochemical studies (Simard & mainly from oribatids (Poinar & Poinar Watt 1990; Loret & Hammock 2001). It 1998; Loo et al. 2004) and water mites was proposed that each invertebrate (Issi & Lipa 1968). Although few species harbors at least 1 species of gregarine; were recorded from spiders such as therefore, the exact number of these Opiliones (Cokendolpher 1993), nothing protozoans is hard to precisely estimate. is known in scorpions. Here, simple and In fact, gregarines are reported from only rapid PCR and DNA sequencing of 18S about 3124 invertebrate species, which is rDNA methods were used for the < 0.3% of the named invertebrate fauna detection and identification of eukaryotic (Levine 1988).This study aims to explore pathogens in scorpion extracted DNA. gregarine parasites in unstudied The gene coding for the small subunit of arthropod group using simple molecular ribosomal RNA (SSU RNA) is the most techniques. This is, to our knowledge, the intensively sequenced marker for first record for parasitic gregarines in phylogenetic studies in all groups of scorpions. organisms. Newly obtained sequence data can be quickly and easily compared MATERIALS AND METHODS with all published sequences of this Scorpion collection marker deposited in GenBank (NCBI) Leiurus quinquestriatus were database. Moreover, the use of universal collected and transported alive to the primers that can amplify fragments of laboratory. They kept in separate plastic rDNA sequences of any boxes until DNA extraction. species in polymerase chain reaction DNA extraction, amplification, and (PCR) makes it possible not only to sequencing amplify rDNA from the studied Whole genomic DNA was specimens, but also from pathogens extracted using DNA easy Tissue Kit infecting them. The amplified 18S rRNA (Qiagen) following the standard gene fragments then individually manufacturer’s protocol. Total genomic sequenced (Valles & Pereira 2003). The DNA was extracted from a fragment of resultant sequence of the part of 18S the scorpion’s pedipalps, legs or rRNA gene was compared with metasoma. For PCR amplification the sequences deposited in GenBank for following primers were used in this study species identification. Results of this 18s rRNA F 5’- Ribosomal DNA sequences reveal a parasitic septate gregarine in the scorpion L. quinquestriatus 125

TTAAGCCATGCATGTCTAAG-3’ and was constructed with bootstrap analysis 18S rRNA R 5’- employed 1000 replicates. The TN93 GACTACGACGGTATCTAATC-3’. (Tamura-Nie) + G (gamma) model of Amplification was performed on a Mini nucleotide substitution was determined as Cycler (Techgene): initial step with 3 the most appropriate model according to min at 94° C followed by 35 steps with Bayesian Information Criterion (BIC) 30 sec at 95 °C, 30 sec at 52 °C and 3 and Akaike Information Criterion (AIC) min at 72 °C, and with final elongation (Nei M. and Kumar S. 2000). The 10 min at 72 °C. Reaction mix contained analysis involved 4 nucleotide sequences. 5 μl of DNA template, 12.5 µl of Codon positions included were GoTaq Green Master mix (Promega), 0.5 1st+2nd+3rd+Noncoding. All positions µl of primer F, 0.5 µl of primer R(10 containing gaps and missing data were pmoles) and the rest is nuclease free eliminated. There were a total of 919 water until it reaches a total volume of positions in the final dataset. Genetic 25 μl. Amplification products were distances were created in MEGA5 separated by electrophoresis on 1.5 % software (http://www.megasoftware.net). agarose gel, purified using QIAquick Gel Extractinion Kit (Qiagen), and sequenced RESULTS on genetic analyser ABI Prism 3500 Amplification of the rDNA (Applied Biosystems) using the BigDye fragment with the universal primers from Terminator v3.1 chemistry of the same the genomic DNA isolated from L. manufacturer. Chromatograms and quinquestriatus revealed specific strong sequences were analyzed and edited PCR product (Fig. 1). Comparison of the using BioEdit program®. A BLASTn nucleotide sequence of amplicons with search was used to infer the taxonomic sequences deposited in GenBank showed affiliation of these sequences. The that the 900 kb fragment was most gregarine-like sequences were compared similar to the Xiphocephalus ellisi 18S with corresponding sequences from the ribosomal RNA gene (Acc. FJ459762) GenBank database. For phylogenetic revealing 794/903(88%) identity; analysis 33 sequences in total were used, Stylocephalus giganteus 18S ribosomal including: The gregarine-like sequences RNA gene [Acc. FJ459761, 792/902 from this study, 28 sequences designated (88%) identity]; Xiphocephalus as Gregarinia and 4 other apicomplexan triplogemmatus 18S ribosomal RNA species (a close outgroup). For the 18S gene [Acc. FJ459763, 751/861 (87%) rRNA gene, Maximum Likelihood tree identity].

Fig. 1: PCR products (~900bp fragment) of 18S rRNA gene amplified by universal primers pair from Egyptian L. quinquestriatus (1), and 100 bp DNA Ladder Marker (2). 126 Moustafa M. H. Sarhan

To reveal the phylogenetic position that the isolate was deeply nested in of the isolated gregarine-like sequences, frame of the clades in Gregarinia tree corresponding 18S rRNA sequences that (Fig. 2). Garegrine that is found in have been annotated to Gregarinia in the scorpion is closely related and belong GenBank database were used as shown in most probably to Stylocephlidae group Clopton (2002). The ML analysis proved escpecially Xiphocephalus ellisi.

Gregarina polymorpha Gregarina niphandrodes Gregarina cloptoni Gregarina diabrotica Gregarinidae group 2 Gregarina coronata Gregarina basiconstrictonea Gregarina cuneata Protomagalhaensia granulosae Protomagalhaensia wolfi Gregarina cubensis Blabericolidae Leidyana haasi Leidyana migrator Gregarina tropica Gregarina blattarum Amoebogregarina nigra Gregarinidae group 1 Leidyana erratica Gregarina kingi Pyxinia crystalligera S tenophora robusta Hoplorhynchus acanthatholius Paraschneideria metamorphosa Actinocephalidae Prismatospora evansi Geneiorhynchus manifestus Xiphocephalus ellisi Gregarina sp. HOST SCORPION Stylocephalus giganteus St ylocephalidae Xiphocephalus triplogemmatus Colepismatophila watsonae Cryptosporidium muris Cryptosporidium serpentis Cryptosporidium baileyi Out group Cryptosporidium wrairi Cryptosporidium parvum

0.1

Fig. 2: Phylogenetic position of the isolated gregarine-like sequences. Maximum likelihood tree inferred from nucleotide sequences of 18S rRNA gene fragments of the isolate from scorpion (black circle), and corresponding sequences from the GenBank database annotated to Gregarinia. Representatives of other Apicomplexa were used as outgroups.

DISCUSSION Michiels 2006: Valigurová & Koudela Gregarines are protozoa belonging 2006). to the phylum Apicomplexa that live in The current genetic analysis digestive tracts, Malpighian tubules, fat showed that the scorpion gregarine is tissue, hemolymph, or reproductive only distantly closely related to organs of marine and terrestrial Gregarina. The phylogeny constructed by invertebrates (Chen et al. 1997; Field & rRNA gene sequences revealed that the Ribosomal DNA sequences reveal a parasitic septate gregarine in the scorpion L. quinquestriatus 127 found gregarine sequence is Gromphadorhina portentosa. J. phylogenetically distinct from all Invertebr. Pathol., 65:311-312. available Gregarina species. Actually, Bower, S.M., McGladdery, S.E. & Price, molecular phylogenetic approaches are I.M. 1994: Synopsis of infectious very useful for the systematics of diseases and parasites of gregarine parasites because their life commercially exploited shellfish. histories involve several developmental Ann Rev Fish Dis 4:1–199 stages (e.g., cysts, sprorozoites, and Chen, W.J., Wu, S.T., Chow, C.Y. & trophozoites) that complicate species Yang, C.H. 1997: Sporogonic identification based on morphology alone development of the gregarine (Leander 2008; Rueckert et al. 2011; Ascogregarina taiwanensis (Lien Wakeman & Leander 2012). On and Levine) (Apicomplexa: contrary, Clopton 2012 argued strongly ) in its natural host against the massive use of molecular Aedes albopictus (Skuse) phylogenetic data for describing (Diptera: Culicidae). J. Eukaryot. gregarine species in favor of a set of Microbiol., 44: 326-331. ideological rules based on detailed Clopton, R.E. 2002: Phylum analyses of morphometric data. Apicomplexa Levine, 1970: The finding of a gregarine infection Order Eugregarinorida Lé ger, in the scorpions may shed light on further 1900. In: Lee JJ, Leedale G, pest control regimes of this dangerous Patterson D, Bradbury PC (eds) species. Recent studies demonstrate that Illustrated guide to the protozoa, gregarine infection significantly 2nd ed. Society of increases the effectiveness of both Protozoologists, Lawrence, chemical and microbial control measures Kansas, pp 205–288. against insect pest (Lopes & Alves Clopton, R. E. 2012. Synoptic revision of 2005). Hence, controlling an arthropod Blabericola vector may hinge on the presence of (Apicomplexa:Eugregarinida: gregarines. Blabericolidae) parasitizing Finally, molecular methods including blaberid cockroaches DNA isolation, PCR and sequencing (Dictyoptera: Blaberidae), with seem to be a promising approach to study comments on delineating protozoan diversity by providing a rapid gregarine species boundaries. J. and easy screening of host organisms for Parasitol., 98:572–583. which traditional microscopic techniques Cokendolpher, J. C. 1993: Pathogens and could be ineffective. parasites of Opiliones (Arthropoda: Arachnida). J. ACKNOWLDGEMENT Arachn., 21: 120 – 146 The author is grateful to Prof. Dr. Ellis, J., Morrison, D.A., Jeffries, A.C. Mohamed Moussa Ibrahim, Professor of 1989: The phylum Apicomplexa: parasitology, Zoology Dept., Faculty of an update on the molecular Science, Suez Canal University, Ismailia, phylogeny. In Evolutionary Egypt, for reviewing the manuscript. Relationships Among Protozoa Edited by: Coombs G, Vickerman REFERENCES K, Sleigh M, Warren A. Kluwer Ball, S., Cunningham, A.A., Clarke, D. Academic Publishers, Boston; & Daszak, P. 1995: Septate 255-274. gregarines associated with a Field, S.G. & Michiels, N.K. 2006: disease of the hissing cockroach Acephaline gregarine parasites (Monocystis sp.) are not 128 Moustafa M. H. Sarhan

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