Accepted Manuscript
Elucidation of the first definitively identified life cycle for a marine turtle blood fluke (Trematoda: Spirorchiidae) enables informed control
Thomas H. Cribb, Jose L. Crespo-Picazo, Scott C. Cutmore, Brian A. Stacy, Phoebe A. Chapman, Daniel García-Párraga
PII: S0020-7519(16)30272-7 DOI: http://dx.doi.org/10.1016/j.ijpara.2016.11.002 Reference: PARA 3918
To appear in: International Journal for Parasitology
Received Date: 29 August 2016 Revised Date: 30 October 2016 Accepted Date: 3 November 2016
Please cite this article as: Cribb, T.H., Crespo-Picazo, J.L., Cutmore, S.C., Stacy, B.A., Chapman, P.A., García- Párraga, D., Elucidation of the first definitively identified life cycle for a marine turtle blood fluke (Trematoda: Spirorchiidae) enables informed control, International Journal for Parasitology (2016), doi: http://dx.doi.org/ 10.1016/j.ijpara.2016.11.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1 Elucidation of the first definitively identified life cycle for a marine
2 turtle blood fluke (Trematoda: Spirorchiidae) enables informed
3 control
4
5 Thomas H. Cribba,*, Jose L. Crespo-Picazob, Scott C. Cutmorea, Brian A. Stacyc , Phoebe A.
6 Chapmand, Daniel García-Párragab
7
8 aThe University of Queensland, School of Biological Sciences, St Lucia, Queensland, 4072,
9 Australia
10 bFundación Oceanogràfic, Veterinary Services & Research Department, Avanqua
11 Oceanogràfic-Ágora, C/ Eduardo Primo Yúfera 1B, 46013 Valencia, Spain
12 cNational Marine Fisheries Service, Office of Protected Resources and University of Florida,
13 College of Veterinary Medicine, Gainesville, FL, USA
14 dThe University of Queensland, Veterinary Marine Animal Research, Teaching and
15 Investigation Unit, School of Veterinary Science, Gatton, Queensland, 4343, Australia
16 17 * Corresponding author. Tel.: +61 7 3365 2581; fax: +61 7 3365 4699.
18 E-mail address: [email protected] (T.H. Cribb)
19 20 Nucleotide sequence data reported in this paper are available in GenBank under the
21 accession numbers: KX987107-KX987112.
22
1
23 Abstract
24 Blood flukes of the family Spirorchiidae are significant pathogens of both free-
25 ranging and captive marine turtles. Despite a significant proportion of marine turtle mortality
26 being attributable to spirorchiid infections, details of their life cycles remain almost entirely
27 unknown. Here we report on the molecular elucidation of the complete life cycle of a marine
28 spirorchiid, identified as Amphiorchis sp., infecting vermetid gastropods and captive bred
29 neonate Caretta caretta in the Oceanogràfic Aquarium, in Valencia, Spain. Specimens of a
30 vermetid gastropod, Thylaeodus cf. rugulosus (Monterosato, 1878), collected from the
31 aquarium filtration system housing diseased C. caretta, were infected with sporocysts and
32 cercariae consistent with the family Spirorchiidae. We generated rDNA sequence data
33 (internal transcribed spacer 2 (ITS2) and partial 28S rDNA) from infections from the
34 vermetid which were identical to sequences generated from eggs from the serosa of the
35 intestine of neonate C. caretta, and an adult spirorchiid from the liver of a C. caretta from
36 Florida, USA. Given the reliability of these markers in the delineation of trematode species,
37 we consider all three stages to represent the same species and tentatively identify it as a
38 species of Amphiorchis Price, 1934. The source of infection at the Oceanogràfic Foundation
39 Rehabilitation Centre, Valencia, Spain, is inferred to be an adult C. caretta from the western
40 Mediterranean being rehabilitated in the same facility. Phylogenetic analysis suggests that
41 this Amphiorchis sp. is closely related to other spirorchiids of marine turtles (species of
42 Carettacola Manter & Larson, 1950, Hapalotrema Looss, 1899 and Learedius Price, 1934).
43 We discuss implications of the present findings for the control of spirorchiidiasis in captivity,
44 for the better understanding of epidemiology in wild individuals, and the elucidation of
45 further life cycles.
46 Keywords: Trematoda; Spirorchiidae; Life cycle; Vermetidae; Conservation; Sea turtles,
47 Transmission
2
48 1. Introduction
49 The Spirorchiidae Stunkard, 1921 is an assemblage of 20 genera of blood flukes
50 which parasitise the circulatory system of freshwater and marine turtles globally (Platt, 2002;
51 Roberts et al., 2016). Spirorchiids have recently come to prominence as they have been
52 widely shown to cause extensive tissue injury and mortality in marine turtles (Glazebrook et
53 al., 1981; Gordon et al., 1998; Flint et al., 2010; Stacy et al., 2010a). As with blood fluke
54 infections in other taxa, disease in turtles results from both inflammation and vascular injury
55 caused by the trematodes and embolized ova, which affect a variety of tissues (Gordon et al.,
56 1998; Stacy et al., 2010a).
57 Despite their significance as a frequent cause of disease in some marine turtles, lack
58 of knowledge of their life cycles hampers progress in our understanding of the epidemiology
59 of marine spirorchiids. Life cycles are known for five freshwater spirorchiid species, all of
60 which infect pulmonate (heterobranch) gastropods as first intermediate hosts (Wall, 1941a, b,
61 1951; Pieper, 1953; Holliman and Fisher, 1968; Turner and Corkum, 1977). Numerous other
62 putative but unidentified freshwater spirorchiid cercariae are known from both pulmonate and
63 caenogastropod gastropods. In stark contrast, there exists just a single report of a marine
64 turtle spirorchiid life cycle, that of Stacy et al. (2010b) who reported a spirorchiid in a
65 fissurellid (vetigastropod) limpet from Florida, USA. However, this infection was detected by
66 molecular analysis alone; the parasite stages were not observed by microscopy.
67 Caretta caretta (the loggerhead turtle) is considered conservation-dependent globally,
68 including the Mediterranean population (Ullmann and Stachowitsch, 2015). In support of the
69 population, the Oceanogràfic Foundation in Valencia, Spain runs a head-starting program in
70 which cohorts of juvenile C. caretta are incubated and reared from eggs translocated from
71 nearby Mediterranean beaches. The facility also rehabilitates sick, bycaught and injured
3
72 juvenile and adult specimens of C. caretta from the nearby Mediterranean coast for eventual
73 reintroduction. Adult and juvenile turtles are always held in separate tanks, but the water
74 passing through the tanks is shared and recirculated. From January 2015 many captive-
75 hatched juvenile turtles began exhibiting debilitation and wasting disease that was
76 characterised by weight loss, malabsorption, anorexia, gastrointestinal stasis and occasionally
77 neurological signs of incoordination or swirling behaviour. Upon necropsy and subsequent
78 histopathology, spirorchiid eggs were found in several tissues and organs, and were
79 especially evident, even macroscopically, within the serosa of the intestine. Individual C.
80 caretta that survived longest in the facility harboured the greatest numbers of eggs. Given
81 that the water supply of the aquarium facility is semi-closed and pretreated, that pre-hatching
82 transmission of spirorchiids is unknown, and the evidence of increasing egg loads in tissues
83 with age despite consecutive deworming treatment (Jacobson et al., 2003), it was concluded
84 that transmission was occurring within the facility. These circumstances created an
85 opportunity to identify the intermediate host within the filtration system. Here we report the
86 elucidation of the intermediate host of the spirorchiid parasite using novel ribosomal data and
87 detail the morphology of the stages associated with it.
88
89 2. Materials and methods
90 2.1. Morphological specimens
91 Examination of the aquarium system for potential intermediate hosts in September
92 2015 revealed the presence of just one gastropod species, a sessile form that was attached to
93 piping delivering and draining water from the turtle holding tanks. We dissected specimens of
94 this gastropod species under a stereomicroscope in a solution of 0.85% saline. When
95 observed, asexual stages and cercariae of trematodes were fixed either in near boiling saline
4
96 and then transferred immediately to 80% alcohol, or directly in cold alcohol for samples for
97 molecular analysis.
98 Specimens for morphological analysis were subsequently washed in fresh water,
99 stained with Mayer’s haematoxylin, destained in a solution of 1.0% HCl and neutralised in
100 0.5% ammonium hydroxide solution. Specimens were then dehydrated through a graded
101 ethanol series, cleared in methyl salicylate and mounted in Canada balsam. Measurements
102 were made using an Olympus SC50 digital camera mounted on an Olympus BX-53
103 compound microscope using cellSens Standard imaging software. Measurements are given in
104 µm and, where length is followed by breadth, the two measurements are separated by ‘×’.
105
106 2.2. Molecular sequencing
107 Total genomic DNA was extracted using phenol/chloroform extraction techniques
108 (Sambrook and Russell, 2001). We amplified the partial D1-D3 fragment of the 28S nuclear
109 rDNA region using the primers LSU5 (5'-TAG GTC GAC CCG CTG AAY TTA AGC A-3';
110 Littlewood, 1994) and 1500R (5'-GCT ATC CTG AGG GAA ACT TCG-3'; Snyder and
111 Tkach, 2001) and the internal transcribed spacer 2 (ITS2) region using the primers 3S (3S: 5'-
112 GGT ACC GGT GGA TCA CGT GGC TAG TG-3'; Morgan and Blair, 1995) and ITS2.2 (5'-
113 CCT GGT TAG TTT CTT TTC CTC CGC-3'; Cribb et al., 1998). The 28S rDNA region has
114 proven informative in phylogenetic analysis of blood flukes and the ITS2 rDNA region has
115 proven effective in species discrimination (Blasco-Costa et al., 2016).
116 PCR for both the 28S and ITS2 regions was performed with a total volume of 20 µl
117 consisting of 5 µl of 5x MyTaq Reaction Buffer (Bioline, United Kingdom), 0.75 µl of each
118 primer (10 pmols), 0.25 µl of Taq polymerase (Bioline MyTaq™ DNA Polymerase) and 2 µl
119 of DNA template (approximately 10 ng), made up to 20 µl with Invitrogen™ (United States)
5
120 ultraPURE™ distilled water. Amplification was carried out on an MJ Research (United
121 States) PTC-150 thermocycler. The following profile was used to amplify the 28S region: an
122 initial 95°C denaturation for 4 min, followed by 30 cycles of 95°C denaturation for 1 min,
123 56°C annealing for 1 min, 72°C extension for 2 min, followed by a single cycle of 95°C
124 denaturation for 1 min, 55°C annealing for 45 s and a final 72°C extension for 4 min. The
125 following profile was used to amplify the ITS2 region: an initial single cycle of 95°C
126 denaturation for 3 min, 45°C annealing for 2 min, 72°C extension for 90 s, followed by four
127 cycles of 95°C denaturation for 45 s, 50°C annealing for 45 s, 72°C extension for 90 s,
128 followed by 30 cycles of 95°C denaturation for 20 s, 52°C annealing for 20 s, 72°C extension
129 for 90 s, followed by a final 72°C extension for 5 min. Amplified DNA was purified using a
130 Bioline ISOLATE II PCR and Gel Kit according to the manufacturer’s protocol. Cycle
131 sequencing of purified DNA was carried out using ABI Big Dye™ v.3.1 chemistry following
132 the manufacturer’s recommendations, using the same primers used for PCR amplification as
133 well as the additional 28S primers 300F (5'-CAA GTA CCG TGA GGG AAA GTT G-3';
134 Littlewood et al., 2000), ECD2 (5'-CCT TGG TCC GTG TTT CAA GAC GGG-3';
135 Littlewood et al., 1997) and 1200R (5'-GCA TAG TTC ACC ATC TTT CGG-3'; Lockyer et
136 al., 2003a). Cycle sequencing was carried out at the Australian Genome Research Facility.
137 We extracted DNA from eggs found in the serosa of the small intestine of infected C.
138 caretta using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). The extraction
139 protocol was in accordance with the manufacturer’s instructions, with the exception that 1 g
140 of Silica/Zirconia 0.5 mm beads (Daintree Scientific, Tasmania, Australia) was utilised to
141 disrupt the eggs in a Biospec (United States) Mini-Beadbeater 16 for 3 min prior to
142 extraction, and the final elution was made in 100 µl of elution buffer as opposed to the
143 recommended 200 µl. PCRs for egg extractions were carried out using primers L3F and L2R
144 for 28S, and IF1 and IR1 for ITS2 (Chapman et al., 2015). Reactions and cycling conditions
6
145 were as described by Chapman et al. (2015). PCR products were visualised on a 1% agarose
146 gel and submitted to the Animal Genetics Laboratory (School of Veterinary Science,
147 University of Queensland, Gatton, Australia) for purification and sequencing using the same
148 primers as for PCR.
149 Sequencher™ version 4.5 (GeneCodes Corp., United States) was used to assemble
150 and edit contiguous sequences, and the start and the end of the ITS2 rDNA region were
151 determined by annotation through the ITS2 Database (Koetschan et al., 2012) using the
152 ‘Metazoa’ model.
153
154 2.3. Phylogenetic analysis
155 Partial 28S rDNA sequences generated during this study were aligned with those of
156 species of Schistosomatoidea available on GenBank using MUSCLE version 3.7 (Edgar,
157 2004) with ClustalW sequence weighting and UPGMA clustering for iterations 1 and 2
158 (Table 1). The resultant alignments were refined by eye using MESQUITE (Mesquite: a
159 modular system for evolutionary analysis. Version 2.72 http://mesquiteproject.org) and the
160 ends of each fragment were trimmed to match the shortest sequence in each alignment.
161 Bayesian inference and Maximum Likelihood analyses of the 28S rDNA dataset were
162 conducted to explore distinctions and relationships among these taxa. Bayesian inference
163 analysis was performed using MrBayes version 3.2.6 (Ronquist et al., 2012) and Maximum
164 Likelihood analysis was performed using RAxML version 8.2.8 (Stamatakis, 2014) , run on
165 the CIPRES portal (Miller et al., 2010). The software jModelTest version 2.1.10 (Darriba et
166 al., 2012) was used to estimate the best nucleotide substitution model for the dataset.
167 Bayesian inference and Maximum Likelihood analyses were conducted using the GTR+I
168 model predicted as the best estimators by the Akaike Information Criterion (AIC) in
7
169 jModelTest. Nodal support in the Maximum Likelihood analysis was estimated by
170 performing 100 bootstrap pseudoreplicates. Bayesian inference analysis was run over
171 10,000,000 generations (ngen = 10000000) with two runs each containing four simultaneous
172 Markov Chain Monte Carlo (MCMC) chains (nchains = 4) and every 1000th tree saved
173 (samplefreq = 1000). Bayesian inference analysis used the following parameters: nst = 6,
174 rates = invgamma, ngammacat = 4, and the priors parameters of the combined dataset were
175 set to ratepr = variable. Samples of substitution model parameters, and tree and branch
176 lengths were summarised using the parameters ‘sump burnin = 3000’ and ‘sumt burnin =
177 3000’. These ‘burnin’ parameters were chosen because the log likelihood scores ‘stabilised’
178 well before 3,000,000 replicates in the Bayesian inference analysis.
179
180 3. Results
181 3.1. Morphological analysis
182 The only gastropod found in the holding tanks in the Oceanogràfic Rehabilitation Centre was
183 identified by Dr Rüdiger Bieler of the Chicago Field Museum, Illinois, USA as Thylaeodus
184 cf. rugulosus (Monterosato, 1878) (Caenogastropoda, Vermetidae) (Fig. 1A). This vermetid
185 species occurs in the western Mediterranean and the central Atlantic (Bieler, 1995). We
186 lodged five voucher specimens in the Chicago Field Museum (FMNH 344699-700).
187 We found infections of spirorchiid sporocysts in 38 of the 45 T. cf. rugulosus
188 individuals dissected (infection prevalence 84%). Large and apparently fully developed
189 ocellate furcocercous cercariae were detected in just three infections; most of the sporocysts
190 had only immature cercariae which could be recognised as such by the presence of eye-spots
191 and developing tails.
192
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193 3.2. Description of intramolluscan stages
194 Sporocysts in digestive gland of T. cf. rugulosus. Body tubular, containing numerous
195 developing cercariae but no more than two close to fully developed. Maximum length
196 observed 1432 µm.
197 Cercaria (Figs. 1B, C) (based on single largest recovered specimen). Body 348 × 101
198 µm. Tegumental spines on body and tail, minute. Dorsal fin-fold not detected. Eye-spots
199 prominent, 159 µm from anterior extremity. Anterior organ 128 × 76 µm; head gland present,
200 prominent. Penetration glands in two groups of three with bodies filling posterior extremity.
201 Ventral sucker 260 µm from anterior extremity, 49 µm long. Tail 974 µm long in total,
202 maximum width 76 µm. Furcae 260 × 37 µm, surrounded by distinct fin-fold terminally.
203 Permanent preparations of cercariae and sporocysts have been lodged in the Queensland
204 Museum, Brisbane, Australia (Nos G 235497- G 235509).
205
206 3.3. Molecular analysis
207 ITS2 and partial 28S rDNA sequences were generated from asexual trematode stages
208 dissected from three individual T. cf. rugulosus and from spirorchiid eggs taken from the
209 serosa of the intestine of a necropsied neonate C. caretta from the Oceanogràfic. Sequences
210 from the intestinal eggs and the stages infecting vermetid gastropods were identical for both
211 markers. The new sequence data were compared with that available from GenBank and with
212 a library of unpublished sequences from marine turtles from off Florida, USA held by one of
213 the authors. No matches were identified from GenBank, but a perfect match (for both
214 markers) was found between samples from Valencia and an adult spirorchiid collected from
215 C. caretta from Florida. The voucher specimen relating to the Florida infection has been
216 deposited in the United States National Museum as no. 1422020. In the combination of
9
217 unfused intestinal caeca, two testes with the cirrus-sac and genital pore between, and slender
218 body with the vitelline follicles extending to the caecal extremities, the specimen is consistent
219 with the genus Amphiorchis Price, 1934. On this basis, we identify the form from the
220 Valencia rehabilitation centre as Amphiorchis sp. We refrain from attempting to identify the
221 specimen relative to the five recognised species of Amphiorchis because its poor condition
222 renders this unreliable.
223 Preliminary Bayesian inference and Maximum Likelihood analysis of the partial 28S
224 rDNA alignment of a wide range of Schistosomatoidea produced a topology (essentially
225 identical in both analyses) in which species of marine genera of Aporocotylidae formed a
226 well-supported clade sister to all spirorchiids and schistosomatids. The Spirorchiidae was
227 paraphyletic, forming one clade of freshwater spirorchiids and a second strongly supported
228 clade of four marine spirorchiids (including the current form, Amphiorchis sp.). The clade of
229 marine spirorchiids was sister to schistosomatids. These results are broadly consistent with
230 previous analyses (Snyder, 2004; Orelis-Ribeiro et al., 2014; Pinto et al., 2015) and are
231 therefore not reproduced here. In light of these preliminary results we realigned and analysed
232 a reduced data set comprising just sequences of marine spirorchiids (including Amphiorchis
233 sp.), using a range of schistosomatids as the outgroup. Maximum Likelihood and Bayesian
234 inference analyses of this reduced dataset resulted in phylograms with identical topologies
235 (Fig. 2), in which the marine spirorchiids formed a well-supported clade to the exclusion of
236 the schistosomatids. Within the marine spirorchiid clade, Carettacola hawaiiensis Dailey,
237 Fast & Balazs, 1991 was basal to the remaining taxa and Amphiorchis sp. was sister to
238 Hapalotrema pambanensis Mehrotra, 1973 (senior syn. of Hapalotrema mehrai Rao, 1976
239 according to Chapman et al. (2015)) and Learedius learedi Price, 1934.
240
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241 4. Discussion
242 The genetic match between sequences derived from infections from the vermetid
243 gastropod T. cf. rugulosus, from eggs from the intestine of neonate C. caretta at the
244 Oceanogràfic, and from adult worms from C. caretta from Florida allows us to conclude that
245 the life cycle of this species has been demonstrated.
246 The voucher specimen from C. caretta from Florida is consistent with the genus
247 Amphiorchis, but is too poorly preserved to allow reliable identification to species. Notably,
248 no species of Amphiorchis is known from C. caretta; all five described Amphiorchis spp. are
249 reported from either Chelonia mydas or Eretmochelys imbricata (Price, 1934; Oguro, 1938;
250 Simha, 1970; Fischthal and Acholonu, 1976; Gupta and Mehrotra, 1981; Werneck and Silva,
251 2013). The present form may thus prove to be a new species. Our phylogenetic analyses
252 suggest that the present Amphiorchis sp. is closely related to species of Hapalotrema Looss,
253 1899, Learedius Price, 1934 and Carettacola Manter & Larson, 1950. These four genera are
254 morphologically similar, and recent molecular analysis by Chapman et al. (2015) found that
255 the single species of Learedius nested within Hapalotrema. Further molecular
256 characterization of species of these genera is needed.
257 Despite the absence of previous reports of species of Amphiorchis from C. caretta,
258 this species hosts a rich fauna of spirorchiids from five genera (Luhman, 1935; Manter and
259 Larson, 1950; Jacobson et al., 2006; Stacy et al., 2010a; Chen et al., 2012). Strikingly, as far
260 as we can detect, spirorchiids have not previously been reported from C. caretta nor other
261 turtle species from the Mediterranean despite several surveys (e.g. Manfredi et al., 1998;
262 Gracan et al., 2012). However, it is likely this population does harbour spirorchiids, given
263 that the ultimate source of the infection of Amphiorchis sp. in the neonate turtles housed at
264 the Oceanogràfic rescue centre is inferred to have been mature turtles taken from the western
11
265 Mediterranean. Caretta caretta from the western Mediterranean are part of a trans-Atlantic
266 population, genetically distinguishable from the eastern Mediterranean population (e.g.
267 Carreras et al., 2011). Possibly there is a distinction in the parasites of these populations,
268 which might explain the absence of spirorchiids in previous reports of parasites of some
269 Mediterranean C. caretta.
270 Our studies provide direct evidence that marine spirorchiids can be transmitted in
271 closed-system aquaria. Notably, the Amphiorchis specimens collected from Florida, which
272 were genetically identical to the specimens from Valencia, was also from a captive juvenile
273 C. caretta. This individual had been raised in an aquarium from hatching and was believed to
274 have acquired fatal spirorchiidiasis in captivity (unpublished observations). Glazebrook and
275 Campbell (1990) reported spirorchiids in hatchling-raised individuals of both C. mydas and
276 E. imbricatus in Queensland, Australia. Furthermore, the study of Stacy et al. (2010b) was
277 based on material from a Cayman Island turtle farm where transmission of L. learedi occurs
278 (Greiner et al., 1980). Given the threatened status of many marine turtles and the frequent
279 rearing of young turtles and rehabilitation of older turtles in aquarium facilities, our work
280 demonstrates the need for vigilance to ensure that pathogenic spirorchiid infections are not
281 transmitted within these systems. Elimination of vermetids is now one clear approach to the
282 control of spirorchiidiasis in turtle hatchery and rehabilitation centres. Notably, many
283 vermetids produce non-planktonic “crawl-away juveniles” (e.g. Calvo et al., 1998), and this
284 reproductive habit may make them especially liable to multiply in marine aquaria.
285 We do not know whether T. cf. rugulosus is the only host for the present Amphiorchis
286 sp., however, given the general pattern of specificity of trematodes for molluscan
287 intermediate hosts (Cribb et al., 2001), it seems unlikely that the host range is wider than a
288 range of vermetids. As far as we can determine, no spirorchiid has been reported from more
289 than a single gastropod family. A potentially parallel system may be exemplified by an
12
290 aporocotylid (fish blood fluke), Cardicola forsteri Cribb, Daintith & Munday, 2000. The tuna
291 hosts of this species are highly mobile marine animals that, similar to marine turtles, travel
292 large distances. This trematode infects different polychaete intermediate hosts in Australia
293 and Japan (Cribb et al., 2011; Shirakashi et al., 2016). Thus, further examination of vermetids
294 in other geographical locations may well expand the intermediate host range of this
295 Amphiorchis sp..
296 There is extensive literature relating to the life cycles of spirorchiids, but almost all of
297 it relates to freshwater species. A series of classical and often highly detailed experiment-
298 based studies characterised life cycles for five freshwater species from North America –
299 Spirorchis artericola (Ward, 1921), Spirorchis elephantis (Cort, 1918), Spirorchis parvus
300 (Stunkard, 1923), Spirorchis scripta Stunkard, 1923 and Vasotrema robustum (Stunkard,
301 1928) by Wall (1941a, b, 1951), Pieper (1953), Holliman and Fisher (1968), and Turner and
302 Corkum (1977). All these species infect Planorbidae (“Pulmonata”: Planorboidea). The
303 cercariae of these species are morphologically similar and were recognised by Wall (1941b)
304 as a distinct group among furcocercous cercariae, united by a range of features including
305 being large, distomate, and having a distinctive arrangement of eye-spots. No complete new
306 life cycle has been described for a spirorchiid for many years. However, five further
307 spirorchiid cercariae have recently been characterised as such by molecular analyses (Brant et
308 al., 2006; Kraus et al., 2014; Pinto et al., 2015). Table 2 summarises the reports of these and
309 the five classically elucidated cycles. There are also descriptions of several other cercariae
310 likely to be spirorchiids (e.g. Nasir and Diaz, 1973; Bayssade-Dufour et al., 1989). The status
311 of these species as spirorchiids cannot be considered certain (in the absence of experimental
312 or molecular data) and they do not modify our understanding of the apparent intermediate
313 host range of the Spirorchiidae. Before the present study, the only report of marine
314 spirorchiid asexual stages was that by Stacy et al. (2010b), who generated sequences
13
315 consistent with the marine spirorchiid L. learedi from samples from the tissues of a fissurellid
316 limpet, Fissurella nodosa, collected from a marine turtle mariculture facility. Notably, the
317 approach did not lead to observation of the parasitic stages and vermetids were not among the
318 potential host taxa examined. The details of this report are included in Table 2.
319 The limited material available for examination in the present study did not allow the
320 detailed observations on the anatomy of the intramolluscan stages made by earlier studies
321 (e.g. Wall, 1941a, b, 1951). However, the present specimens are broadly consistent with
322 previously described spirorchiid cercariae. All typical features of previously described
323 spirorchiid cercariae were present – substantial size, well-developed eye-spots and ventral
324 sucker, penetration glands occupying space posterior to eye-spots, and a well-developed head
325 organ. We did not detect a dorsal finfold or “cuticular crest” in the present form. In this, it
326 resembles the cercariae of S. artericola, S. elegans, S. elephantis and S. scripta which lack
327 finfolds, rather than those of V. robustum and S. parvus which have them. Overall, it appears
328 that the morphology of spirorchiid cercariae is conservative.
329 The new data reported here makes the distribution of intermediate hosts of
330 spirorchiids especially striking. Freshwater species overwhelmingly infect planorbids
331 (Heterobranchia: “Pulmonata”), with just a single species known from an ampullariid
332 (Caenogastropoda) from South America. The two reported marine infections are from
333 gastropods from separate subclasses, a caenogastropod (present study) and a vetigastropod
334 (Stacy et al., 2010b). Although understanding of the deep branches of gastropod evolution
335 remains unstable (e.g. Aktipis and Giribet, 2010), it is clear that the Heterobranchia,
336 Caenogastropoda and Vetigastropoda represent deeply distinct and ancient lineages within
337 the Gastropoda. It is surprising that L. learedi and the present form (Amphiorchis sp.), which
338 are evidently closely related, have intermediate hosts that are deeply phylogenetically distant.
339 Such a host distribution implies dramatic host-switching. However, there is a precedent for
14
340 such a contrast in that African and Asian species of Schistosoma Weinland, 1858, which
341 represent distinct clades in the same genus (Lockyer et al., 2003b), infect gastropods as
342 phylogenetically distinct as caenogastropods and vetigastropods.
343 The identification of a vermetid as an intermediate host for marine spirorchiids has
344 clear implications for the search for further intermediate hosts of marine spirorchiids. Stacy et
345 al. (2010b) observed that this search is challenging due to the immense range of potential
346 hosts and because infections might be rare or localised. We consider that it now makes sense
347 to focus future studies on fissurellid limpets and vermetids. In this context, we note that both
348 families of gastropods have been surveyed for trematode infections previously and shown to
349 be infected. Fissurellids are known to be infected by a hemiuroid and an opecoelid (Cable,
350 1956, 1963). Vermetids are known to be infected by an echinostomatid, a hemiurid, a
351 mesometrid, a microphallid and a possible opecoelid (Prévôt, 1969, 1971a, b; Jousson and
352 Bartoli, 1999). Despite these reports, we suspect that the nature of these gastropod families,
353 especially the particularly unconventional Vermetidae, has led to them being less studied than
354 many other groups of gastropods. In some parts of the world, vermetids occur in such large
355 numbers that they comprise reef-building organisms (Shier, 1969; Safriel, 1975). Should the
356 vermetid species involved prove to be intermediate hosts for spirorchiids, then such localities
357 may be especially important in spirorchiid transmission.
358 The sessile habit of the Vermetidae raises the intriguing possibility that they could
359 attach to the carapaces of turtles so that the intermediate hosts of spirorchiids are carried
360 around with the turtles; this idea was suggested by Frazier et al. (1985) before any marine
361 spirorchiid life cycle was known. There is extensive literature on the epibiotic fauna of turtle
362 carapaces (e.g. Frazier et al., 1985; Kitsos et al., 2005; Pfaller et al., 2008; Domenech et al.,
363 2015). However, apart from a few references to unidentified ‘tubeworms’ by Frazier et al.
364 (1985), which might refer to several kinds of animals, we have not detected any reports of
15
365 Vermetidae on turtles. Vermetids are easily confused with some groups of polychaetes
366 (especially Serpulidae) to the extent that some species of the two groups were described in
367 the wrong phylum. We think that vermetids may well occur on turtles, and such infestation is
368 worthy of a specific search.
369 The present study is just the second to report information on marine spirorchiid life
370 cycles, following that of Stacy et al. (2010b), and the first evidence of sporocysts and
371 cercariae formation in a marine intermediate host. The intermediate hosts identified in the
372 two studies represent completely unrelated gastropods. Additionally, this is the first known
373 report of spirorchiid infection in sea turtles in the Mediterranean Sea. Thus, despite recent
374 progress, we do not yet have a broad understanding of the transmission of marine
375 spirorchiids. Increased knowledge is bound to prove valuable in explaining the epidemiology
376 of spirorchiids over the complex life cycles of their hosts, which incorporate pelagic, inshore
377 and migratory phases. Also, as seen here, improved life cycle knowledge can enhance the
378 well-being of captive turtle populations.
379
380 Acknowledgements
381 We thank Dr Rüdiger Bieler of the Chicago Field Museum, USA for his help with the
382 identification of the vermetid gastropod. We also thank all professionals, students and
383 volunteers at the Oceanogràfic [Spain] and Fundación Oceanografic [Spain], especially at the
384 ARCA Rehabilitation Centre [Spain], for their many efforts and complete dedication to the
385 best animal care. In particular, we are grateful to the Conselleria de Agricultura, Medio
386 Ambiente, Cambio Climático y Desarrollo Rural of the Valencia Community Regional
387 Government, Spain, especially to Juan Jiménez, Juan Antonio Gómez y Juan Eymar who
388 made this project possible with their invaluable support. We thank the ‘Institut Cavanilles de
16
389 Biodiversitat i Biologia Evolutiva,’ University of Valencia, Spain for their collaboration and
390 Fundación Bios CV, [Spain] for their advice on the head starting program. This research did
391 not receive any specific grant from funding agencies in the public, commercial or not-for-
392 profit sectors.
393
394
17
395 References
396 Aktipis, S.W., Giribet, G., 2010. A phylogeny of Vetigastropoda and other
397 "archaeogastropods": re-organizing old gastropod clades. Invertebr. Biol. 129, 220–
398 240.
399 Bayssade-Dufour, C., Kudo, S.D., Albaret, J.L., 1989. Morphologie et chétotaxie d’une
400 cercaire de Spirorchiidae du Togo. Ann. Parasitol. Hum. Comp. 64, 323–331.
401 Bieler, R., 1995. Vermetid gastropods from São Miguel, Azores: comparative anatomy,
402 systematic position and biogeographic affiliation. Açoreana Supp. 1995, 173–192.
403 Blasco-Costa, I., Cutmore, S.C., Miller, T.L., Nolan, M.J., 2016. Molecular approaches to
404 trematode systematics: 'best practice' and implications for future study. Syst. Parasitol.
405 93, 295–306.
406 Brant, S.V., Morgan, J.A.T., Mkoji, G.M., Snyder, S.D., Rajapakse, R., Loker, E.S., 2006.
407 An approach to revealing blood fluke life cycles, taxonomy, and diversity: Provision
408 of key reference data including DNA sequence from single life cycle stages. J.
409 Parasitol. 92, 77–88.
410 Cable, R.M., 1956. Marine cercariae of Puerto Rico. Sci. Surv. Porto Rico & Virgin Islands
411 16, 491–577.
412 Cable, R.M., 1963. Marine cercariae from Curaçao and Jamaica. Z. Parasitenkd. 23, 429–469.
413 Calvo, M., Templado, J., Penchaszadeh, P.E., 1998. Reproductive biology of the gregarious
414 Mediterranean vermetid gastropod Dendropoma petraeum. J. Mar. Biol. Assoc. U. K.
415 78, 525–549.
416 Carreras, C., Pascual, M., Cardona, L., Marco, A., Bellido, J.J., Castillo, J.J., Tomas, J., Raga,
417 J.A., Sanfelix, M., Fernandez, G., Aguilar, A., 2011. Living together but remaining
418 apart: Atlantic and Mediterranean loggerhead sea turtles (Caretta caretta) in shared
419 feeding grounds. J. Hered. 102, 666–677.
18
420 Chapman, P.A., Cribb, T.H., Blair, D., Traub, R.J., Kyaw-Tanner, M.T., Flint, M., Mills,
421 P.C., 2015. Molecular analysis of the genera Hapalotrema Looss, 1899 and Learedius
422 Price, 1934 (Digenea: Spirorchiidae) reveals potential cryptic species, with comments
423 on the validity of the genus Learedius. Syst. Parasitol. 90, 67–79.
424 Chen, H.C., Kuo, R.J., Chang, T.C., Hus, C.K., Bray, R.A., Cheng, I.J., 2012. Fluke
425 (Spirorchiidae) infections in sea turtles stranded on Taiwan: prevalence and
426 pathology. J. Parasitol. 98, 437–439.
427 Cribb, T.H., Adlard, R.D., Hayward, C.J., Bott, N.J., Ellis, D., Evans, D., Nowak, B.F., 2011.
428 The life cycle of Cardicola forsteri (Trematoda Aporocotylidae), a pathogen of
429 ranched southern bluefin tuna. Int. J. Parasitol. 41, 861–870.
430 Cribb, T.H., Anderson, G.R., Adlard, R.D., Bray, R.A., 1998. A DNA-based demonstration
431 of a three-host life-cycle for the Bivesiculidae (Platyhelminthes: Digenea). Int. J.
432 Parasitol. 28, 1791–1795.
433 Cribb, T.H., Bray, R.A., Littlewood, D.T.J., 2001. The nature and evolution of the association
434 between digeneans, molluscs and fishes. Int. J. Parasitol. 31, 997–1011.
435 Darriba, D., Taboada, G.L., Doallo, R., Posada, D., 2012. jModelTest 2: more models, new
436 heuristics and parallel computing. Nat. Meth. 9, 772–772.
437 Devkota, R., Brant, S.V., Thapa, A., Loker, E.S., 2014. Sharing schistosomes: the elephant
438 schistosome Bivitellobilharzia nairi also infects the greater one-horned rhinoceros
439 (Rhinoceros unicornis) in Chitwan National Park, Nepal. J. Helminthol. 88, 32–40.
440 Domenech, F., Badillo, F.J., Tomas, J., Raga, J.A., Aznar, F.J., 2015. Epibiont communities
441 of loggerhead marine turtles (Caretta caretta) in the western Mediterranean: influence
442 of geographic and ecological factors. J. Mar. Biol. Assoc. U. K. 95, 851–861.
443 Edgar, R.C., 2004. MUSCLE: multiple sequence alignment with high accuracy and high
444 throughput. Nucleic Acids Res. 32, 1792–1797.
19
445 Fischthal, J.H., Acholonu, A.D., 1976. Some digenetic trematodes from the Atlantic
446 Hawksbill turtle, Eretmochelys imbricata imbricata (L.), from Puerto Rico. Proc.
447 Helm. Soc. Wash. 43, 174–185.
448 Flint, M., Patterson-Kane, J.C., Limpus, C.J., Mills, P.C., 2010. Health surveillance of
449 stranded green turtles in southern Queensland, Australia (2006-2009): an
450 epidemiological analysis of causes of disease and mortality. EcoHealth 7, 135–145.
451 Frazier, J., Margaritoulis, D., Muldoon, K., Potter, C.W., Rosewater, J., Ruckdeschel, C.,
452 Salas, S., 1985. Epizoan communities on marine turtles. 1. Bivalve and gastropod
453 mollusks. Mar. Ecol. 6, 127–140.
454 Glazebrook, J.S., Campbell, R.S.F., 1990. A survey of the diseases of marine turtles in
455 northern Australia. II. Oceanarium-reared and wild turtles. Dis. Aquat. Org. 9, 97–
456 104.
457 Glazebrook, J.S., Campbell, R.S.F., Blair, D., 1981. Pathological changes associated with
458 cardiovascular trematodes (Digenea: Spirorchidae) in a green sea turtle Chelonia
459 mydas (L). J. Comp. Pathol. 91, 361–368.
460 Goodchild, C.G., Kirk, D.E., 1960. The life history of Spirorchis elegans Stunkard, 1923
461 (Trematoda, Spirorchiidae) from the painted turtle. J. Parasitol. 46, 219–229.
462 Gordon, A.N., Kelly, W.R., Cribb, T.H., 1998. Lesions caused by cardiovascular flukes
463 (Digenea: Spirorchidae) in stranded green turtles (Chelonia mydas). Vet. Path. 35,
464 21–30.
465 Gracan, R., Bursic, M., Mladineo, I., Kucinic, M., Lazar, B., Lackovic, G., 2012.
466 Gastrointestinal helminth community of loggerhead sea turtle Caretta caretta in the
467 Adriatic Sea. Dis. Aquat. Org. 99, 227–236.
20
468 Greiner, E.C., Forrester, D.J., Jacobson, E.R., 1980. Helminths of mariculture-reared green
469 turtles (Chelonia mydas mydas) from Grand Cayman, British West-Indies. Proc.
470 Helm. Soc. Wash. 47, 142–144.
471 Gupta, N.K., Mehrotra, V., 1981. On two blood flukes (Trematoda) of the family
472 Spirorchidae Stunkard, 1921 from Indian marine turtles. Acta Parasit. 28, 11–20.
473 Holliman, R.B., Fisher, J.E., 1968. Life cycle and pathology of Spirorchis scripta Stunkard,
474 1923 (Digenea - Spirorchiidae) in Chrysemys picta picta. J. Parasitol. 54, 310–318.
475 Jacobson, E.R., Harman, G.R., Maxwell, L.K., Laille, E.J., 2003. Plasma concentrations of
476 praziquantel after oral administration of single and multiple doses in loggerhead sea
477 turtles (Caretta caretta). Am. J. Vet. Res. 64, 304–309.
478 Jacobson, E.R., Homer, B.L., Stacy, B.A., Greiner, E.C., Szabo, N.J., Chrisman, C.L., Origgi,
479 F., Coberley, S., Foley, A.M., Landsberg, J.H., Flewelling, L., Ewing, R.Y., Moretti,
480 R., Schaf, S., Rose, C., Mader, D.R., Harman, G.R., Manire, C.A., Mettee, N.S.,
481 Mizisin, A.P., Shelton, G.D., 2006. Neurological disease in wild loggerhead sea
482 turtles Caretta caretta. Dis. Aquat. Org. 70, 139–154.
483 Jousson, O., Bartoli, P., 1999. The life-cycle of three species of the Mesometridae (Digenea)
484 with comments on the taxonomic status of this family. Syst. Parasitol. 44, 217–228.
485 Kitsos, M.S., Christodoulou, M., Arvanitidis, C., Mavidis, M., Kirmitzoglou, I., Koukouras,
486 A., 2005. Composition of the organismic assemblage associated with Caretta caretta.
487 J. Mar. Biol. Assoc. U. K. 85, 257–261.
488 Koetschan, C., Hackl, T., Müller, T., Wolf, M., Förster, F., Schultz, J., 2012. ITS2 database
489 IV: interactive taxon sampling for internal transcribed spacer 2 based phylogenies.
490 Mol. Phylogenet. Evol. 63, 585–588.
491 Kraus, T.J., Brant, S.V., Adema, C.M., 2014. Characterization of trematode cercariae from
492 Physella acuta in the middle Rio Grande. Comp. Parasitol. 81, 105–109.
21
493 Littlewood, D.T.J., 1994. Molecular phylogenetics of cupped oysters based on partial-28S
494 ribosomal-RNA gene-sequences. Mol. Phylogenet. Evol. 3, 221–229.
495 Littlewood, D.T.J., Curini-Galletti, M., Herniou, E.A., 2000. The interrelationships of
496 Proseriata (Platyhelminthes: Seriata) tested with molecules and morphology. Mol.
497 Phylogenet. Evol. 16, 449–466.
498 Littlewood, D.T.J., Johnston, D.A., 1995. Molecular phylogenetics of the four Schistosoma
499 species groups determined with partial 28S ribosomal RNA gene sequences.
500 Parasitology 111, 167–175.
501 Littlewood, D.T.J., Rohde, K., Clough, K.A., 1997. Parasite speciation within or between
502 host species? - phylogenetic evidence from site-specific polystome monogeneans. Int.
503 J. Parasitol. 27, 1289–1297.
504 Lockyer, A.E., Olson, P.D., Littlewood, D.T.J., 2003a. Utility of complete large and small
505 subunit rRNA genes in resolving the phylogeny of the Neodermata (Platyhelminthes):
506 implications and a review of the cercomer theory. Biol. J. Linn. Soc. 78, 155–171.
507 Lockyer, A.E., Olson, P.D., Ostergaard, P., Rollinson, D., Johnston, D.A., Attwood, S.W.,
508 Southgate, V.R., Horak, P., Snyder, S.D., Le, T.H., Agatsuma, T., McManus, D.P.,
509 Carmichael, A.C., Naem, S., Littlewood, D.T.J., 2003b. The phylogeny of the
510 Schistosomatidae based on three genes with emphasis on the interrelationships of
511 Schistosoma Weinland, 1858. Parasitology 126, 203–224.
512 Luhman, M., 1935. Two new trematodes from the loggerhead turtle (Caretta caretta). J.
513 Parasitol. 21, 1–3.
514 Manfredi, M.T., Piccolo, G., Meotti, C., 1998. Parasites of Italian sea turtles. II. Loggerhead
515 turtles (Caretta caretta Linnaeus, 1758). Parassitologia 40, 305–308.
516 Manter, H.W., Larson, M.I., 1950. Two new blood flukes from a marine turtle, Caretta
517 caretta. J. Parasitol. 36, 595–599.
22
518 Miller, M.A., Pfeiler, E., Schwartz, T., 2010. Creating the CIPRES Science Gateway for
519 inference of large phylogenetic trees. In: Proceedings of the Gateway Computing
520 Environments Workshop (GCE), 14 Nov. 2010, New Orleans, LA, USA, pp. 1–8.
521 Morgan, J.A.T., Blair, D., 1995. Nuclear rDNA ITS sequence variation in the trematode
522 genus Echinostoma: an aid to establishing relationships within the 37-collar-spine
523 group. Parasitology 111, 609–615.
524 Nasir, P., Diaz, M.T., 1973. Freshwater larval trematodes. 31. Two new species of
525 Venezuelan cercariae. Proc. Helm. Soc. Wash. 40, 69–73.
526 Oguro, Y., 1938. A new blood fluke, Amphiorchis lateralis nov. sp. (Spirorchidae) found in a
527 marine turtle in Japan. J. Sci. Hiroshima Univ., Ser. B, Div. 1 6, 1–4.
528 Orelis-Ribeiro, R., Arias, C.R., Halanych, K.M., Cribb, T.H., Bullard, S.A., 2014. Diversity
529 and ancestry of flatworms infecting blood of nontetrapod craniates "fishes". Adv.
530 Parasitol. 85, 1–64.
531 Pfaller, J.B., Frick, M.G., Reich, K.J., Williams, K.L., Bjorndal, K.A., 2008. Carapace
532 epibionts of loggerhead turtles (Caretta caretta) nesting at Canaveral National
533 Seashore, Florida. J. Nat. Hist. 42, 1095–1102.
534 Pieper, M.B., 1953. The life history and germ cell cycle of Spirorchis artericola (Ward,
535 1921). J. Parasitol. 39, 1–13.
536 Pinto, H.A., de Melo, A.L., Brant, S.V., 2015. Where are the South American freshwater
537 turtle blood flukes (Trematoda: Spirorchiidae)? The first morphological and
538 molecular analysis of spirorchiid cercariae from freshwater snails in Brazil. Parasitol.
539 Int. 64, 553–558.
540 Platt, T.R., 2002. Family Spirorchiidae Stunkard, 1921, in: Gibson, D.I., Jones, A., Bray,
541 R.A. (Eds.), Keys to the Trematoda. CAB International and The Natural History
542 Museum, Wallingford, UK, pp. 453–467.
23
543 Prévôt, G., 1969. Les trématodes larvaires parasites de Vermetus triqueter Bivone
544 (gastéropode prosobranche marin) de Golfe de Marseille. Bull. Soc. Zool. Fr. 94,
545 463–470.
546 Prévôt, G., 1971a. Contribution á l'étude des Microphallidae Travassos, 1920 (Trematoda).
547 Cycle évolutif de Microphallus pachygrapsi Deblock et Prevot, 1969 parasite du
548 Goeland a pieds jaunes (Larus argentatus). Ann. Parasitol. Hum. Comp. 46, 453–461.
549 Prévôt, G., 1971b. Cycle évolutif d'Aporchis massiliensis Timon-David, 1955 (Digenea
550 Echinostomatidae), parasite du goéland Larus argentatus michaellis Naumann. Bull.
551 Soc. Zool. Fr. 96, 197–208.
552 Price, E.W., 1934. New genera and species of blood flukes from a marine turtle, with a key to
553 the genera of the family Spirorchidae. J. Wash. Acad. Sci. 24, 132–141.
554 Roberts, J.R., Platt, T.R., Orelis-Ribeiro, R., Bullard, S.A., 2016. New genus of blood fluke
555 (Digenea: Schistosomatoidea) from Malaysian freshwater turtles (Geoemydidae) and
556 its phylogenetic position within Schistosomatoidea. J. Parasitol. 102, 451–462.
557 Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Hohna, S., Larget, B.,
558 Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: Efficient Bayesian
559 phylogenetic inference and model choice across a large model space. Syst. Biol. 61,
560 539–542.
561 Safriel, U.N., 1975. The role of vermetid geastropods in the formation of Mediterranean and
562 Atlantic reefs. Oecologia 20, 85–101.
563 Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual. Cold Spring
564 Harbor Laboratory Press, Cold Spring Harbor, New York, USA.
565 Shier, D.E., 1969. Vermetid reefs and coastal development in the Ten Thousand Islands,
566 Southwest Florida. Bull. Geol. Soc. Am. 80, 485–508.
24
567 Shirakashi, S., Tani, K., Ishimaru, K., Shin, S.P., Honryo, T., Uchida, H., Ogawa, K., 2016.
568 Discovery of intermediate hosts for two species of blood flukes Cardicola orientalis
569 and Cardicola forsteri (Trematoda: Aporocotylidae) infecting Pacific Bluefin tuna in
570 Japan. Parasitol. Int. 65, 128–136.
571 Simha, S.S., 1970. A new genus and species of a blood fluke, Squaroacetabulum solus, from
572 the ventricle of the heart of a marine turtle, Chelone mydas. Zool. Anz. 184, 290–294.
573 Snyder, S.D., 2004. Phylogeny and paraphyly among tetrapod blood flukes (Digenea :
574 Schistosomatidae and Spirorchiidae). Int. J. Parasitol. 34, 1385–1392.
575 Snyder, S.D., Tkach, V.V., 2001. Phylogenetic and biogeographical relationships among
576 some holarctic frog lung flukes (Digenea: Haematoloechidae). J. Parasitol. 87, 1433–
577 1440.
578 Stacy, B.A., Foley, A.M., Greiner, E., Herbst, L.H., Bolten, A., Klein, P., Manire, C.A.,
579 Jacobson, E.R., 2010a. Spirorchiidiasis in stranded loggerhead Caretta caretta and
580 green turtles Chelonia mydas in Florida (USA): host pathology and significance. Dis.
581 Aquat. Org. 89, 237–259.
582 Stacy, B.A., Frankovich, T., Greiner, E., Alleman, A.R., Herbst, L.H., Klein, P., Bolten, A.,
583 McIntosh, A., Jacobson, E.R., 2010b. Detection of spirorchiid trematodes in
584 gastropod tissues by polymerase chain reaction: preliminary identification of an
585 intermediate host of Learedius learedi. J. Parasitol. 96, 752–757.
586 Stamatakis, A., 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of
587 large phylogenies. Bioinformatics 30, 1312–1313.
588 Turner, H.M., Corkum, K.C., 1977. New snail host for Spirorchis scripta Stunkard, 1923
589 (Digenea - Spirorchiidae) with a note on seasonal incidence. Proc. Helm. Soc. Wash.
590 44, 225–226.
25
591 Ullmann, J., Stachowitsch, M., 2015. A critical review of the Mediterranean sea turtle rescue
592 network: a web looking for a weaver. Nat. Conserv.-Bulgaria 10, 45–69.
593 Wall, L.D., 1941a. Life history of Spirorchis elephantis (Cort, 1917), a new blood fluke from
594 Chrysemys picta. Am. Midl. Nat. 25, 402–412.
595 Wall, L.D., 1941b. Spirorchis parvus (Stunkard) its life history and the development of its
596 excretory system (Trematoda: Spirorchiidae). Trans. Am. Microsc. Soc. 60, 221–260.
597 Wall, L.D., 1951. The life history of Vasotrema robustum (Stunkard 1928), Trematoda:
598 Spirorchiidae. Trans. Am. Microsc. Soc. 70, 173–184.
599 Werneck, M.R., Silva, R.J., 2013. Occurrence of Amphiorchis indicus Mehrotra, 1973
600 (Digenea, Spirorchiidae) infecting green turtle Chelonia mydas Linnaeus, 1758
601 (Testudines, Cheloniidae) in Brazil. Braz. J. Biol. 73, 225–227.
602
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603 Captions to figures
604 Fig. 1. Life cycle of Amphiorchis sp. (A) Thylaeodus cf. rugulosus (Vermetidae), the
605 intermediate host of Amphiorchis sp ex Caretta caretta. Scale bar = 3 mm. (B) Whole
606 cercaria of Amphiorchis sp. ex T. cf. rugulosus. Scale bar = 100 µm. (C) Cercarial body of
607 Amphiorchis sp. Scale bar = 50 µm.
608
609 Fig. 2. Relationships of marine Spirorchiidae, with Schistosomatidae as outgroup, based on
610 Maximum Likelihood and Bayesian inference analysis of the partial 28S rDNA dataset.
611 GenBank accession numbers are indicated in parentheses. Nodal support values for each node
612 are indicated as posterior probabilities (Bayesian inference) above the node and bootstrap
613 support (Maximum Likelihood) below the node. Support values < 70 are not shown.
614 Sequences generated in this study are in bold.
615
616
617
618
619
27
620
621
622 Fig. 1
623
624
28
625
626
627
628
629 Fig. 2
630
29
631 Table 1 28S rDNA sequences from GenBank analysed in this study.
Species Host Locality GenBank Accession # Reference
Spirorchiidae
Carettacola hawaiiensis Chelonia mydas Hawaii, Pacific Ocean AY604709 Snyder (2004)
Hapalotrema mehrai Chelonia mydas Hawaii, Pacific Ocean AY604708 Snyder (2004)
Learedius learedi Chelonia mydas Hawaii, Pacific Ocean AY604707 Snyder (2004)
Schistosomatidae
Austrobilharzia variglandis Larus delawarensis Delaware, USA AY157250 Lockyer et al. (2003b)
Bivitellobilharzia nairi Rhinoceros unicornis Nepal JQ975005 Devkota et al. (2014)
Dendritobilharzia pulverulenta Gallus gallus New Mexico, USA AY157241 Lockyer et al. (2003b)
Heterobilharzia americana Mesocricetus auratus Louisiana, USA AY157246 Lockyer et al. (2003b)
Ornithobilharzia canaliculata Larus delawarensis Texas, USA AY157248 Lockyer et al. (2003b)
Schistosoma japonicum Mus musculus Philippines Z46504 Littlewood and Johnston (1995)
Schistosoma mansoni Mus musculus Puerto Rico Z46503 Littlewood and Johnston (1995)
Schistosomatium douthitti Mesocricetus auratus Indiana, USA AY157247 Lockyer et al. (2003b)
30
632 Table 2. Hosts of spirorchiid intermediate hosts demonstrated as such either experimentally or by molecular analysis.
Species Host Family Superfamily Higher taxon Basis Reference Freshwater Spirorchis elephantis Helisoma trivolvis Planorbidae Planorboidea SCl Heterobranchia Experimental Wall (1941a) Spirorchis parvus Helisoma trivolvis Planorbidae Planorboidea SCl Heterobranchia Experimental Wall (1941b) Vasotrema robustum Physa spp Physidae Planorboidea SCl Heterobranchia Experimental Wall (1951) Spirorchis artericola Helisoma trivolvis Planorbidae Planorboidea SCl Heterobranchia Experimental Pieper (1953) Spirorchis elegans Helisoma anceps Planorbidae Planorboidea SCl Heterobranchia Experimental Goodchild and Kirk
(1960) Menetus dilatatus Planorbidae Planorboidea SCl Heterobranchia Experimental Goodchild and Kirk (1960) Spirorchis scripta Helisoma anceps Planorbidae Planorboidea SCl Heterobranchia Experimental Holliman and Fisher (1968) Ferrissia fragilis Planorbidae Planorboidea SCl Heterobranchia Experimental Turner and Corkum (1977) W1120 Biomphalaria Planorbidae Planorboidea SCl Heterobranchia Mol. + Morphology Brant et al. (2006) sudanica Spirorchiidae sp. 1 Biomphalaria spp Planorbidae Planorboidea SCl Heterobranchia Mol. + Morphology Pinto et al. (2015) Spirorchiidae sp. 2 Biomphalaria Planorbidae Planorboidea SCl Heterobranchia Mol. + Morphology Pinto et al. (2015) glabrata Spirorchiidae sp. 3 Pomacea sp. Ampullariidae Ampullarioidea SCl Caenogastropoda Mol. + Morphology Pinto et al. (2015) Marine Learedius learedi Fissurella nodosa Fissurellidae Fissurelloidea SCl Vetigastropoda Mol. only Stacy et al. (2010b) Amphiorchis sp. Thylaeodus cf. Vermetidae Vermetoidea SCl Caenogastropoda Mol. + Morphology Present study rugulosus
31
633 Mol., molecular studies.
634
635
32
636 Highlights 637
638 • Spirorchiidae blood flukes are significant pathogens of marine turtles but their life
639 cycles remain almost entirely unknown.
640 • We show that Amphiorchis sp. of Caretta caretta infects a vermetid gastropod,
641 Thylaeodus cf. rugulosus.
642 • We matched DNA from infections from the vermetid, eggs from neonate C. caretta,
643 and an adult spirorchiid from C. caretta.
644 • The Amphiorchis sp. is closely related to marine turtle spirorchiid of the genera
645 Carettacola, Hapalotrema and Learedius.
646 • The findings enable control of spirorchiidiasis in captivity and better understanding of
647 epidemiology in wild individuals.
648 649
650
651
33
652
653
654
655 Graphical abstract
656
657
34