Preliminary Studies on Habitat and Diversity of Some Sea Urchin Species (Echinodermata: Echinoidea) on the Southern Levantine Basin of Egypt

Home , Abu Qir Bay, Arbacia lixula, Arbacioida, Paracentrotus lividus, Parechinidae, Psammechinus

Egyptian Journal of Aquatic Research (2013) 39, 303–311

National Institute of Oceanography and Fisheries Egyptian Journal of Aquatic Research

http://ees.elsevier.com/ejar www.sciencedirect.com

Preliminary studies on habitat and diversity of some sea urchin species (Echinodermata: Echinoidea) on the southern Levantine basin of Egypt

Elzahrae Elmasry a, H.A. Omar a, F.A. Abdel Razek a,*, M.A. El-Magd b a Aquaculture, Invertebrate Aquaculture laboratory, National Institute of Oceanography and Fisheries, Alexandria, Egypt b Department of Biotechnology, Biotechnology Laboratory, Faculty of Veterinary Medicine, Kafr Elsheikh University, Kafr Elsheikh, Egypt

Received 20 November 2013; revised 24 December 2013; accepted 24 December 2013 Available online 22 January 2014

KEYWORDS Abstract For many years the sea urchin (Echinodermata: Echinoidea) diversity and habitat in the Mediterranean; Mediterranean Levantine basin lacked complementary data despite the critical role they have as Levantine basin; keystone species to any marine ecosystem. As the first step of the present study two stations were Sea urchin; selected along the Alexandria coast for investigation. These two stations are the Miami area and the Diversity; Abou Qir Bay. Around 200 individuals were collected monthly from each station from April 2012 Associated macrobenthic till April 2013 using Scuba diving. A comparison between the two stations was done that included a fauna and flora; comparison between the inhabiting sea urchin community and the present associated macrobenthic Genetic analysis fauna and flora. The results showed a great similarity between the two stations in relation to the sea urchin species present in these two areas as well as the surrounding habitat. This study aims to com- bine the morphology tool and the molecular tool along with data gathered from the habitat of the sea urchin present in these two stations to clear uncertainties in species identification of the sea urchin community found off the coast of Alexandria. The results showed four apparent morphologically different sea urchin specimens. To ensure our findings DNA extraction and Polymerase Chain Reaction (PCR) methods were used where the amplified 16S mitochondrial DNA products from all four sea urchin groups showed four sets of bands with different sizes which suggest that these four groups of sea urchins might be of four different species. Furthermore, the morphological characters along with the resulting DNA bands cleared uncertainties about two species the Paracentrotus lividus (Lamarck, 1816) and the Arbacia lixula (Linnaeus, 1758). The other two sea urchins that were thought to be Psammechinus microtuberculatus (Blainville, 1825) and Sphaerechinus granularis

* Corresponding author. Mobile.: +201006620222. E-mail address: [email protected] (F.A. Abdel Razek). Peer review under responsibility of National Institute of Oceanography and Fisheries.

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(Lamarck, 1816), remained unclear in identiﬁcation and this requires further biological and genetic studies. ª 2014 Production and hosting by Elsevier B.V. on behalf of National Institute of Oceanography and Fisheries.

Introduction Materials and methods

Sea urchins (Echinodermata: Echinoidea) are a very important Sample collection group because they play a major role as cleaners of the ocean bottom. The regular sea urchin has a round, flattened and As a part of a research plan, a survey for the presence of sea sometimes globular calcareous test. It has long, sharply urchin community was done along the Alexandria coast for se- pointed spines which have a wide variety of colors. The gonads lected sites. These sites were selected according to testimonies are considered a delicacy in the Mediterranean. They inhabit of fishermen who collect commercial size sea urchins for sale. rocky areas and vary in their distribution starting from 1 m till Starting from April 2012 to April 2013, monthly samples of 17 m deep. Despite their major importance, little attention has around 200 individuals of sea urchin were collected by SCUBA been paid to them in Egypt with regard to their biological, eco- diving from two near shore rocky reef stations along the Alex- logical and reproductive studies. andria coast. The First station is the Miami area and the sec- Egypt’s coasts are part of the Levantine basin which is sit- ond station is the Abu Qir bay around Nelson Island as shown uated in the eastern part of the Mediterranean Sea. Its north- in Fig. 1. The depths from which the samples were collected ern end is defined by Cyprus and the Larnaca Thrust Zone, ranged between 3 and 17 m. In the Miami area, the substrate and its northwestern margin by the Eratosthenes Seamount. is rocky and has assemblages of sea urchins in depths that ran- The Nile Delta Cone and the East Mediterranean coast define ged between 3 and 17 m, while in Abu Qir the substrate was its southwestern and eastern margins. sandy rocky and has assemblages of sea urchin in depths that Taxonomy and phylogeny of echinoids have been always ranged between 6 and 9 m. based on data from comparative morphology and paleontol- ogy. Recently due to the lack of a reliable key of identifications Sampling analysis that delineates by clear-cut morphological differences some problems persist in classification and phylogeny of many gen- Sea urchin individuals were examined with the naked eye for era of echinoids (Manchenko and Yakovlev, 2000). Some liter- external morphological features that might help in the process atures described difficulties to identify different species of identification of the species. The morphological array of belonging to the same echinoid genus such as: Strongylocentro- characters used to delineate differences comprised: color of tus sp. (Manchenko and Yakovlev, 2000), Diadema sp. (Yokes spines and the spines tips, length of spines, test diameter and and Galil, 2006) and Echinometra sp. (Bronstein and Loya, height, color and wideness of the skin around the peristome 2013). and the color of the milled ring (a flange at the base of the Recent surveys along the Mediterranean coast show new spine that indicates the outer muscle attachment to the spine data about sea urchin species diversity and new species being base and it is sometimes of different color than the spine) introduced due to the processes of the Lessepsian migration (Bronstein and Loya, 2013). from the Suez Canal (Netos et al., 2005; Yokes and Galil, Measurements of the test and spines were done to the near- 2006). Turkey and Israel made several records of newly intro- est 0.5 mm using a Vernier caliper to prevent interference by duced sea urchin species from the Red Sea to the Levantine ba- the spines. Different colors of the sea urchin were recorded ¨ sin during the past years as mentioned by Ozgu¨r et al. (2008), and they were divided into four groups that ranged from black, Yokes and Galil (2006) and Por (2009). purple, brown and green as shown in Fig. 2. The morphological similarity between the sea urchin species Following external examination sea urchins were dissected most common in the Mediterranean Levantine basin along in order to find more distinctive features in the color of gonads with the lack of studies in Egypt that tackles the biology and and test. Many calcareous tests were left to dry for a couple of ecology of these organisms have resulted in a confused nomen- days in the sun where they lost their spines and revealed their clature and uncertainties in classification. Furthermore, the different colors which were also used in identification. uncertain geographical extent due to continuous introduction The sampling for macrobenthic fauna and flora analysis in- of new species of sea urchin from the Red Sea through the Suez volved all organisms present in the same area from where the Canal as well as the introduction from the Atlantic Ocean sea urchin was collected in the two stations. The samples was through Gibraltar has resulted in inconclusive reliability on made to cover the whole area of the sea urchin habitat in each biogeography as a base for correct species identification station so whenever a different organism was found it was (Yokes and Galil, 2006). sampled by taking some adult individuals and if the juvenile The purpose of this study is: firstly, to increase our under- stage of the same organism was present it was sampled also standing of sea urchins diversity patterns by observing their to show the degree of presence of both stages the juvenile habitat and describing their associated macrobenthic fauna and the adult. All samples were kept alive in the lab in fiber and flora in front of the Alexandria coast, Egypt. Secondly, tanks with continuous aeration and sea water exchange on a revealing genetic variation between four species using 16S daily basis. The data were collected and tabulated to show mitochondrial rRNA gene. Preliminary studies on habitat and diversity of some sea urchin species 305

Figure 1 Map showing locations of collection sites retrieved from website maps.google.com. the name of the group or taxa of the associated macrobenthic of phenol:chloroform:isoamyl alcohol, 25:24:1, until the inter- fauna and flora that were found and their abundance in each face became clear. After two final phenol/chloroform extrac- of the two studied areas. tions, the resultant aqueous phase was adjusted to 0.5 M NaCl and to 1% cetyltrimethylammonium bromide (CTAB) DNA extraction and PCR protocol and was incubated for a further 20 min at 65 C. This step is necessary to get rid of any mucopolysaccharide impurities All ongoing tasks in this section were done in the fully which interfere with successful extraction of genomic DNA. equipped Biotechnology Laboratory, Faculty of Veterinary Following cold centrifugation, the aqueous phase (containing Medicine, Kafr Elsheikh University. Two genetic methods DNA) was transferred to the new microtubes and the genomic were used which were the DNA extraction and the Polymerase DNA was precipitated by adding absolute ethanol or isopropyl Chain Reaction (PCR) method. alcohol followed by sedimentation at 13,000 rpm for 20 min in a microcentrifuge. The DNA pellet was then rinsed with 055 llof DNA extraction 75% ethanol, air-dried, and resuspended in 255 ll of TE buffer (10 mM Tris–HCl, pH 7.5, 1 mM EDTA). Whenever extrac- tions showed impurities they were immediately discarded and Whole sea urchins are dissected and kept in liquid nitrogen in the extraction was repeated. 2 ml eppendorf tube till used. Upon arrival in the lab 50 or 70 mg of tissue was collected from the 4 groups of sea urchins. Each tissue specimen, from gonads and gut, was then homoge- PCR amplification nized using a Teflon homogenizer in TES buffer (10 mM Tris– HCl, 140 mM NaCl, 25 mM EDTA, pH 7.8). The suspensions Primer design were mixed vigorously by vortex to release the nuclei from the To amplify partial 16S rRNA region (approximately 480–500 cells. Equal volumes from TES buffer containing 0.4% SDS base pairs (bp)), two universal primers (F:50GACGAGA and 0.5 mg/ml proteinase K were added to the solution, and AGACCCTGTGGAGC30 and R:50ACTTAGATAGAAACT the solution was mixed gently and incubated in a 50 C water GACCTG30) were used. These primers have been designed bath overnight until the tissue was completely digested. The di- using Primer 3.0 software based on conserved regions in gested samples were repeatedly extracted with an equal volume published sea urchin and sea star sequence data (Amos and 306 E. Elmasry et al.

Figure 2 Four apparent morphologically different sea urchin individuals presented in the study area of the Alexandria coast during 2012–2013. 1. Paracentrotus lividus;2.Sphaerechinus granularis;3.Arbacia lixula;4.Psammechinus microtuberculatus.

Hoelzel, 1992; Jacobs et al., 1988; Smith et al., 1993). The gel in 1· TAE buffer containing ethidium bromide (0.5 lg/ml) validity of these primers was checked and succeeded to isolate was prepared. The gel was cast and allowed to set before being 16S rRNA clones with size ranges from 480 bp to 500 bp from submerged in 1· TAE buffer in the gel tank. Samples (0.25– the 4 different groups of Sea urchins. 2 ll) were mixed with 9 ll gel loading buffer (6· stock solution) and DNase-free water to make the total volume up to PCR technique 9 ll. These samples were then loaded on the gel and run at PCR was carried out following the manufacturer’s protocol 100 V. The bands were visualized under UV trans-illuminator (Fermentas, #K1071) in TC-plus thermal cycler (Techne, and the location of the predicted products was confirmed by UK). The partial coding sequences of 16S rRNA gene were using 100 bp molecular ladder. For future work, the most in- amplified in 25 ll volume of the reaction mixture containing tense products will be selected for sequencing after their 1· PCR buffer, 0.2 mM each dNTP, 0.4 lM of each primer, purification. 2 mM MgCl2, 1 ng of genomic DNA and 0.5 unit of Taq DNA polymerase or 2· Master Mix Taq kit. Thermal cycler Results and discussion conditions consist of an initial denaturation step at 95 C for 5 min, 28 cycles of 30 s at 95 C, 90 s at Ta 58 C, and 30 s at Morphologically-based identification 72 C, with a final extension step of 30 min at 68 C, and cooling to 4 C until the PCR products were removed from the The four sea urchin specimens were examined according to thermocycler. FAO (1987). From the first observations of the samples collected of the sea urchin, the black sea urchin was identified Analysis of PCR products by DNA gel electrophoresis as being the Arbacia lixula (Linnaeus, 1758) see Fig. 2.It was very different and very distinctive from the rest of the The PCR products from all sea urchins genomic DNA were other three urchins. The most important feature in the black fractionated on 1% agarose gels. In brief, a 1% (w/v) agarose urchin was that in all individuals throughout the year of Preliminary studies on habitat and diversity of some sea urchin species 307 sample collection, upon any irritation or offensive manipula- pale or juvenile P. lividus when the latter has a lighter green tion (for example dissection) the black sea urchin starts to color (Doris, 2013). However, it’s occurrence is quite rare in release its gametes immediately (personal observation). Such the Mediterranean and not as frequent or as abundant as behavior was never observed in the other three sea urchins: P. lividus. the purple, the brown and the green. Another important fea- The fourth sea urchin Sphaerechinus granularis (Lamarck, ture that differentiated the black sea urchin was that it was 1816) as shown in Table 1 and Fig. 2 is of brown to reddish not edible and this was also recorded by (Vielmini et al., brown color. The spine tips are sometimes of white color. 2005; Wangensteen, 2013) as its gonads tasted bitter. The gonads of this sea urchin are smaller in size than that of The black sea urchin A. lixula lived in deeper areas reaching the commercial P. lividus (FAO, 1987). S. granularis (Lamarck, around 17 m depth in station one. It was larger in size exceed- 1816) is known to feed on encrusting algae and soft algae. It is ing 6.0 cm in diameter and had a flat bottomed shape. It has considered as a bioeroder of the coralline substrate as men- thicker longer spines and a wide area void of spines around tioned by Martıńez-Pita et al. (2008). Its test when void of the mouth. The test of the black sea urchin A. lixula (Linnaeus, spines is of pink to violet color. A juvenile S. granularis is often 1758) is pinkish in color as seen in Fig. 3 and as mentioned by confused with P. lividus (Doris, 2013) and both are collected Guidetti and Mori (2005). It is sometimes found within the together within the same catch. same ground of the other three species of sea urchin found The presence of the four species P. lividus (Lamarck, 1816), in this study. The juvenile individuals of A. lixula are some- A. lixula (Linnaeus, 1758), S. granularis (Lamarck, 1816) and times confused with Paracentrotus lividus as mentioned by Psammechinus microtuberculatus (Blainville, 1825) were re- FAO (1987). corded from adjacent western Mediterranean countries such The second species of the present study is the European sea as: Algeria (Soualili et al., 1999), Spain (Martıńez-Pita et al., urchin or common sea urchin P. lividus (Lamarck, 1816), as 2008) Greece (Pantazis, 2006), Italy (Vielmini et al., 2005) shown in Table 1 see Fig. 2 and as reported by Guidetti and Tunisia (Sellem and Guillou, 2007; El Lakhrach et al., (2004). It is edible, having orange colored gonads with a deli- 2012). cious taste and is well recognized by the Egyptian consumers. While the Eastern Mediterranean countries such as: Turkey The external morphology of this species is characterized by (Yokes and Galil, 2006), Lebanon (Nader and Indary, 2011), having a green colored test when void of spines as shown in and Israel (Fishelson, 2000) show the presence of other differ- Fig. 3. The diameter of the test does not exceed 6.0 cm in all ent echinoids such as Diadema setosum (Leske, 1778) which is a measured individuals of the present study. This sea urchin recent invader species from the Red Sea. Furthermore, in the inhabited the lower shores within a depth range from 3 to Egyptian records, (Elhaweet et al., 2011) recorded the presence 5 m on the rocky substrate. It was often found on the under- of S. granularis (Lamarck, 1816) in Sallum Gulf which was de- sides and crevices of rocks. Egyptian fishermen have tradition- clared a protected area by the Egyptian’s prime minister in ally thought of these two species the purple urchin P. lividus 2010. (Lamarck, 1816) and the black urchin A. lixula (Linnaeus, Eventhough A. lixula is in a different order (Arbacioida) 1758) as the female and male respectively of a single species than the other three species. S. granularis is of a different fam- as mentioned by Wangensteen (2013). ily (Toxopneustidae) than P. lividus and P. microtuberculatus. The third urchin was identified as being the Psammechinus Although P. lividus and P. microtuberculatus are in the same microtuberculutus (Blainville, 1825). It is small in size less than family (Parechinidae) but of different genera, still much con- 4 cm in diameter and its spines are green in color as in Table 1 fusion occurs in their identification as mentioned previously. and Fig. 2. It has short thicker spines. Its test when void of spines is green with ten white bands. It overlaps within the Associated groups of macrobenthic fauna and flora habitat and the depth ranges of the purple sea urchin P. lividus but not commercially important as the P. lividus due the small size of its gonads. P. microtuberculatus is easily confused with a The examination of the two study areas gave insights about the identity of the present sea urchin species. The community of sea urchin in both stations showed great similarity in the species composition of the sea urchin and the habitat surrounding the urchins including all the associated macrobenthic fauna and flora. A list of most important associated macrobenthic fauna and flora found in association with the sea urchin habitat is enlisted in Table 2 and shown in Fig. 4. Such findings were supported by the work of (Sala et al., 2012; Bella et al., 2013; Netos et al., 2005). In station one, the Miami area, the substrate is basically a rocky reef ecosystem, providing structural complexity for juvenile sea urchins to hide from predators such as fish, gastropods and octopus. Most species inhabiting these areas are those characteristic of the intertidal zone in the Mediterranean Sea. Sala et al. (2012), reported similar findings in the Western and the Northeastern Mediterranean Sea. Among the major groups of macrobenthic fauna associated with the sea urchin Figure 3 Morphological differences between two sea urchin community in station one were Crustacea (i.e. crab, Gammarus species: a. A. lixula & b. P. lividus. sp., snapping shrimp, barnacles). They were found on and 308 E. Elmasry et al.

Table 1 Four apparent morphological differences between the four sea urchin adult specimens collected from the Alexandria coast at depths ranging between 3 and 17 m (size ranges between 3 and 6 cm). Species Color Size, cm Spines Spines tips Test Gonads Milled fringe Paracentrotus lividus Purple Purple Green Orange Purple/green 3–4 Sphaerechinus granularis Brown/reddish Brown/white Pink/ Brownish Purple 3–4 brown violet orange Arbacia lixula Black Black Pink Dark purple Black 4–6 Psammechinus Green Green/Light Green Orange Green/light 2–3 microtuberculatus purple purple

Table 2 A list of the main groups of macrobenthic fauna and flora associated with the sea urchin community found in two stations of the Alexandria coast at depths ranging between 3 and 17 m. Group/Taxon Abundance in the 2 stations Miami area Abu Qir Bay Stage Macroalgae Green Algae (Codium, Ulva)+ + – Red Algae +– Brown seaweed + + Coral Lace coral + + – Sponge Encrusting/erect sponge – + – Brachiopods ++ +++ A/J Bryozoa (Hard) +++ +++ – Bivalves Clams ++ + A/J Oysters +++ +++ A/J Mussels +++ +++ A/J Gastropods Whelks/winkle (Thais sp.) +++ ++ A Limpets ++ + A/J Crustacea Barnacles +++ +++ A/J Snaping Shrimp + – Crabs ++ + A/J Amphipods – ++ A/J Gammarus sp. ++ ++ A Tunicates Ascidia sp. + + A/J Polychaetes Nereids ++ +++ A/J Small Tube worms +++ +++ A/J Nematods + ++ A/J Other Echinoderms Irregular sea urchin + – A Sea cucumber + ++ A Brittle Star ++ +++ A/J Cephalopods Octopus sp. + + A +, low abundance; ++, moderate abundance; +++, high abundance; A, Adult and J, Juvenile. Preliminary studies on habitat and diversity of some sea urchin species 309 between rocks crevices, such crevices enhance the juvenile and abundant fauna by providing refuge from predation for recruit of sea urchins to survive. small individuals and increased food abundance or survivor- A heavy black matt of mussels of different species was ship for small macroinvertebrates. found covering the whole bottom of station one in the form On the other hand, the sea urchins in station one had major of adhered bundles of the small bivalve with different sizes groups of associated macroflora such as macroalgae that were ranging from juvenile to adult. These bundles were shelters basically seaweeds (red tuft seaweeds (Rhodophyta), Cordy- for many polychaetes, nematods and other smaller mussels lecladia sp., Amphiroa sp., Gracilaria sp., Corallina elongata) (i.e. Modiolus barbatus). The dead shells of these mussels are and green algae such as (Codium sp.) which was recorded as collected by the sea urchin using their spines as a sort of orna- an invader species by (Netos et al., 2005). Codium sp. is con- mentation for their test or for camouflage. sumed by the sea urchin if its preferable seaweed Ulva sp. is The sea urchin community was accompanied as well by missing as stated by Kitching and Thain (1983). three other groups of mollusks: gastropods (Thais sp., limpets, In station two, the Abu Qir Bay around Nelson Island, the conch sp. and whelks), cephalopods (octopus sp.) and oysters substrate was sandy rocky. It encompasses the same species of (Pinctata radiata) which is a migratory species. The rocks from sea urchins found in station one. Also station two shared great where sea urchins were collected hold many species of the similarities with station one in what concerns the macrobenthic phylum echinodermata: adult and juvenile stages of many fauna and flora associated with the sea urchin community as ophiuroids (brittle star), some irregular echinoids (Stapangus shown in Table 2. However, there were minor differences ob- sp.) and the sea cucumber (Holothuria sp.). Over the rocks served like the frequent presence of hermit crabs inhabiting many lace corals are found such as (Oculina patagonica) which the shells of diversified gastropods. Small red sea squirts (tuni- was also recorded by Bitar and Zibrowius (1997) as being an cates: Ascidiacea) were also observed that were not recorded in exotic lace coral of the eastern Mediterranean after their intro- station one. Higher abundance of Polychaetes in station two duction from the Atlantic Ocean. included large tube worms/beard worms, fire worms, common Two other groups of intertidal macrobenthic fauna were clam worms and flat worms. Finally, chiton sp. was recorded found which were the encrusting brachiopods and many spe- which was not present in station one. As for the flora present cies of tunicates such as the Ascidia sp. as well as other types in station two the peculiar brown seaweed the turkish towel al- of tunicates that are found encrusted on rocks forming an gae (Chondracanthus exasperates) was observed. ‘‘overgrowing mat tunicates’’. The chicken liver sponge From previous findings, these two stations are optimum (Chondrilla nucula) was also recorded in station one having a feeding grounds for all four sea urchin species. The presence slimy brown appearance. Many hard encrusting bryozoa were of the encrusting macroalgae and hard bryozoa in both sta- found on rocks and the shells of many large bivalves. The tions, Miami area and Abu Qir Bay, is optimum for the feed- nematods and polychaetes were a dominant group in station ing of A. lixula. As for P. microtuberculatus and S. granularis, one with the frequent presence of encrusting colonies of very they prefer encrusting and soft macroalgae which are also pres- small white tube worms, fireworms and the most peculiar sea ent in both stations. S. granularis is also considered in many mouse worm. literatures as a bioeroder as it feeds on any coralline substrate The presence of both the juvenile stage and adult stage of as reported by Ine´s Martıńez-Pita et al. (2008). Finally, the many different species such as ophiuroidae, bivalves, Crusta- presence in both stations of benthic soft algae was observed, cea and tunicates suggest strongly that both studied areas as well as different species of sponge and turf seaweeds serving are good nursery grounds providing shelter and protection as optimum feed for P. lividus (Pinna et al., 2012; FAO, 1987). from predation for the first most vulnerable stages of an animal life cycle. Other major observations were also the abun- Results of DNA extraction and PCR methods dance of clams, mussels and oysters. Mussels support a diverse Polymerase chain reaction (PCR) methods are suitable to compare between populations and individuals as reported by Selkoe and Toonen (2006). The results of amplified 16S mitochondrial DNA products for the four sea urchin species showed four major sets of bands with different sizes (ranged from 480 to 500 base pairs (bp)) see (Fig. 5). As the resulting PCR products are of different sizes, this suggests that these four sea urchins may be of different species. As previously stated, the external examination of the four sea urchins showed that the black sea urchin A. lixula was the most morphological distinctive species. The analysis of the four resulting bands supports this finding as the first band belonged to the purple urchin P. lividus and the third band belonged to the black urchin A. lixula as shown in Fig. 5. These two bands (1 and 3) showed the most genetic divergence, while the other two bands (2 and 4) of the urchins identified from external morphological examination as P. microtuberculatus and S. granularis, show similar level of genetic variation. Such close similarity between these two sets of bands (2 and 4) Figure 4 Associated macrobenthic fauna and flora with the sea might suggest that these urchins are somehow related. urchin community in the studied areas. However, their degree of relation cannot be determined by 310 E. Elmasry et al.

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