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Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?
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Short Communication
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Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?
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Charlotte E. Davies ⇑, Andrew F. Rowley
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Department of Biosciences, College of Science, Swansea University, Swansea SA2 8PP, Wales, UK
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Article history: Received 7 November 2014 Revised 6 February 2015 Accepted 16 February 2015 Available online xxxx Keywords: Pink crab disease Disease ecology Edible crab Endoparasite Dinoflagellate Inoculation
a b s t r a c t Hematodinium spp. infect over 40 species of crustaceans worldwide, but have not been reported to infect the European lobster, Homarus gammarus. In this study, Hematodinium parasites (a mixture of uni- and multinucleate trophont-like stages) were taken from donor crabs (Cancer pagurus) and injected into juvenile H. gammarus. Juvenile C. pagurus were also injected with the same inoculum. Haemolymph was taken at regular intervals and examined for the presence of Hematodinium using light microscopy and PCR, in two separate experiments of duration 4 and 8 months. All lobsters were negative for Hematodinium whilst the C. pagurus challenged became infected. It is concluded that European lobsters are not susceptible to infection with a clade of Hematodinium that infects C. pagurus. Ó 2015 Published by Elsevier Inc.
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1. Introduction Hematodinium spp. are endoparasitic dinoflagellates and the causative agents of the condition called ‘bitter crab disease’ in some majid crabs (Meyers et al., 1987; Taylor and Khan, 1995) and ‘pink crab disease’ in the European edible crab, Cancer pagurus (Stentiford et al., 2002; Stentiford and Shields, 2005). Hematodinium spp. have been detected in over 40 species of crustaceans worldwide (Small, 2012) since their first discovery in the 1930s by Chatton and Poisson (1931). In some instances, outbreaks of disease by these parasites have damaged commercial stocks of crustaceans including snow crabs, Chionoecetes opilio (Pestal et al., 2003), Tanner crabs, Chionoecetes bairdi (Meyers et al., 1987), American blue crabs, Callinectes sapidus (Messick, 1994) and velvet swimming crabs, Necora puber (Wilhelm and Mialhe, 1996). In the United Kingdom, it is has also been detected in commercially important species such as the European edible crab, C. pagurus (Latrouite et al., 1988; Stentiford et al., 2002). Although detected in the Norway lobster, Nephrops norvegicus (Field et al., 1992) there have been no reports of the presence of Hematodinium spp. in clawed lobsters such as the European lobster, Homarus gammarus or the American lobster, Homarus americanus. H. gammarus is an important commercial species, with worldwide landings of 4805 tonnes in 2012 (FAO, 2014). There are very ⇑ Corresponding author at: Department of Biosciences, Swansea University, Singleton Park, Swansea SA2 8PP, Wales, UK. Tel.: +44 7814161356. E-mail address: [email protected] (C.E. Davies).
few reports of diseases in this species although shell disease, gaffkaemia and parasitization of the gills by the copepod, Nicothoë astaci have been recently noted (Wootton et al., 2011, 2012; Davies et al., 2015a,b). However, this apparent lack of a wide range of pathogens in H. gammarus could simply be as a result of limited studies on disease monitoring. The European lobster shares the same general distribution and habitat as the edible crab, and considering the damage caused to commercial stocks of some crustaceans by Hematodinium spp. worldwide, it is pertinent to establish whether it is susceptible to parasitisation. This study was designed to determine if European lobsters are susceptible to infection by the same strain/genotype of Hematodinium that infects other crustaceans in the same environment, such as the edible crab, C. pagurus. This was achieved by taking these parasites from the haemolymph of Hematodiniuminfected edible crabs, challenging juvenile lobsters by intrahaemocoelic injection, and monitoring these for subsequent infection for up to 8 months post-challenge.
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2. Materials and methods
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2.1. Experimental lobsters
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Stage 5 H. gammarus (early benthic juveniles) were obtained from the National Lobster Hatchery (Padstow, U.K.) and grownup in an aquarium at Swansea University for ca. 11 months before experimentation. During this time, all lobsters were held in individual containers in one large tank within a 1132 L recirculating
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Please cite this article in press as: Davies, C.E., Rowley, A.F. Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/j.jip.2015.02.004
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seawater system. Water quality parameters were maintained at pH 7.8–8, with temperatures varying from 10.5 to 14.5 °C (according to seasonality). All lobsters were fed three times each week initially on Tetra marine mix gel (Spectrum Brands, Melle, Germany) for the first 6 months and live mussel, Mytilus edulis tissues thereafter. A preliminary experiment (Experiment 1) took place over an 8 month period, followed by a 4 month experiment (Experiment 2) in which lobsters were monitored every 28 days (see Table S1, Supplementary Materials for further detail of experimental design and sampling regime).
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2.2. Hematodinium donation
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For both Experiments 1 and 2, haemolymph (2.5 ml) was drawn into 2.5 ml sterile, ice-cold 3% NaCl solution from donor edible crabs (C. pagurus) with natural Hematodinium infections of grade 3–4 severity (see Smith et al., 2015 for criteria of severity rating). The mixture was transferred into a 9 cm plastic Petri dish and the haemocytes left to attach for 10 min at room temperature. After gentle mixing to dislodge any non-attached Hematodinium, the solution containing parasites was decanted into a new dish and a further 5 ml of sterile 3% NaCl solution added and the attachment process repeated on two more occasions. The solution was then centrifuged (850 g, 10 min at 4 °C) and the pellet re-suspended in 10 ml 3% sterile NaCl solution. Hematodinium were counted using an improved Neubauer haemocytometer.
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2.3. Challenge
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In the initial experiment (Experiment 1) 50 ll of inoculum containing 2 105 Hematodinium were injected into juvenile lobsters (n = 4, carapace length 51.7 ± 5.9 mm). Control animals were injected with the same volume of sterile 3% NaCl solution (n = 3,
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carapace length 47.0 ± 4.6 mm). At the same time, 3 juvenile edible crabs were also injected with the same dose of parasites. The lobsters were bled (ca. 100 ll dispensed into 100% ethanol) ca. every 100 days for 8 months and haemolymph preparations examined with phase contrast microscopy for the presence of Hematodinium. Following the final (day 251) bleed, all lobsters were fixed using Davidson’s sea water fixative (24 h), trisected transversely and incubated in decalcifying solution (10% formalin, 55 g l1 EDTA) for 7 days. Samples were processed for histology as described in Smith et al. (2015), wax sections cut at 7 lm and stained using Cole’s haematoxylin and eosin. Slides were viewed and photographed using an Olympus BX41 microscope with an Olympus SC30 digital camera. In Experiment 2, 50 ll of inoculum containing 1.2 105 Hematodinium parasites was injected into experimental lobsters known to be free of Hematodinium sp. (n = 6, carapace length 24.3 ± 1.3 mm) and sterile 3% NaCl solution was injected into the control group of lobsters (n = 4, carapace length 24.6 ± 2.3 mm). After 1 week, lobsters were bled (ca. 100 ll dispensed into 100% ethanol) every 28 days for up to 4 months post challenge. After the final bleed, all remaining lobsters were sacrificed, and haemolymph preparations examined with phase contrast microscopy for the presence of Hematodinium.
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2.4. DNA extraction and PCR conditions
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In Experiment 1, DNA was extracted from haemolymph using an adapted version of the Qiagen DNeasyÒBlood and Tissue Kit (Qiagen Ltd, Manchester, UK). Briefly, 400 ll ethanol-haemolymph mixture was centrifuged for 5 min at 7000g before 20 ll proteinase K, 220 ll phosphate buffered saline and 200 ll were added to the pellet, vortexed and incubated for 10 min at 56 °C. After this, the manufacturer’s instructions were followed according to the
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Fig. 1. Agarose gel showing PCR products from Experiment 2 haemolymph DNA, extracted every 28 days until 4 months post challenge, using universal decapod primers (A) and Hematodinium sp. specific primers (B). D = Donor crab infected with Hematodinium and N = No template control (with molecular grade water). Ladders are Hyperladder I (Bioline Reagents Ltd, London). The 700 bp band from the donor crab (D) following sequencing was confirmed as Hematodinium sp (see Supplementary Material for sequences).
Please cite this article in press as: Davies, C.E., Rowley, A.F. Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/j.jip.2015.02.004
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protocol for purification of total DNA from animal blood or cells (for nucleated blood – step 3 onwards). In Experiment 2, DNA was extracted with a simpler batch-version DNA extraction modified from Ivanova et al. (2006) using 96- well plates (see Supplementary Materials for details). All PCR reactions were carried out using primers synthesized by Eurofins MWG Operon (Ebersberg, Germany) and performed on a Bio-Rad PTC-100 Peltier thermal cycler before being visualized on a 1.5% agarose gel. All DNA was first tested for product using decapod-specific primers (143F 50 -TGCCTTATCAGCTNTCGATTGTAG-30 and 145R 50 -TTCAGNTTTGCAACCATACTTCCC-30 (Lo, 2014) and MangoMix (Bioline Reagents Ltd, London) in a 10 ll total reaction volume. Cycling conditions were as follows: 4 min at 94 °C followed by 40 cycles of 1 min at 93 °C, 1 min at 55 °C and 2 min at 72 °C, followed by 5 min at 72 °C.
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2.5. Hematodinium sp. detection
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DNA was amplified in 10 ll total reaction volume by adding 2 ll of 5 Coloured Reaction Buffer, 375 lM of each dNTP, 3 mM MgCl2, 2 lM of each primer, 0.05 ll of MangoTaq polymerase (1 U/ll, Bioline Reagents Ltd, London), 2 ll of diluted DNA (1:10), 10% BSA and sterile water to the final reaction volume. Hematodinium spp. specific primers shown to detect all Hematodinium spp. optimized by Hamilton et al. (2009) were used: forward primer DinoF 50 GTGGTGCATGGCCGTTCTTAGTT-3 (Kim et al., 2004) and reverse primer ITS1R1 50 -GAAGGGAAGGGGAGAAGAAGC-3 (Small et al., 2006). These primers are expected to produce a target sequence amplicon of 730 bp. Cycling conditions were as follows: 3 min at 95 °C followed by 34 cycles of 45 s at 94 °C, 45 s at 57 °C and 1 min at 72 °C, followed by 10 min at 72 °C. Positive controls from donor crabs were repeated, the PCR product cleaned up using the Wizard SV Gel and PCR Clean-Up System (Promega; Madison, USA) and sequenced by Eurofins MWG Operon (Ebersberg, Germany). Contigs from sequences were created using the CAP3 sequence assembly programme (Huang and Madan, 1999) and identity confirmed using matched positive controls via NCBI BLAST.
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3. Results and discussion
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In both the preliminary experiment (Experiment 1) and Experiment 2, all lobsters challenged with Hematodinium were found to be negative for the presence of these parasites using histology, PCR (Fig. 1) and by examination of live haemolymph preparations (Fig. 2). During Experiment 1, one control and two experimental lobsters died (one at 2 months, one at 7 months) but without any of the symptoms of disease caused by Hematodinium. In comparison, the edible crabs injected with the same batch of Hematodinium became infected after 3 months post-challenge. There was no mortality of lobsters in the second experiment. The PCR method used in the present study has been shown to be extremely sensitive and is capable of detecting the presence of Hematodinium of 10 parasite nuclei from pure parasite culture, and ca. 30 Hematodinium nuclei from N. norvegicus haemolymph (Hamilton et al., 2009). DNA-based diagnostic methods utilising PCR are now considered to be more sensitive to histology in terms of detection (e.g. Small et al., 2006) and have facilitated diagnosis of many marine pathogens of shellfish (for review see Cunningham, 2002). However, the use of both histological and molecular techniques gives the additional advantage of information on the pathology and severity of the infection, if any (Chualáin and Robinson, 2011). None of the histological or live haemolymph preparations examined in the current study showed any evidence for the presence of Hematodinium in the tissues of H. gammarus.
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Fig. 2. Haemolymph preparations examined for the presence of Hematodinium sp. using phase contrast microscopy. (A) Donor Cancer pagurus with Hematodinium (arrow) infection (clump colony) and (B) a time-final (112 days post- challenge), Hematodinium sp. – injected Homarus gammarus from Experiment 1. Note the lack of Hematodinium sp. parasites in this haemolymph preparation. Scale bars = 10 lm (A) and 50 lm (B).
Screening of wild H. gammarus populations in the Irish Sea region failed to find any lobsters that were positive to infection by Hematodinium spp. despite being found in the same area as infected edible crabs (C. pagurus) and velvet swimming crabs (N. puber); the former of which had disease prevalence values of up to 51% (Chualáin, 2010). The juvenile stages of C. pagurus that inhabit the intertidal and sub-tidal regions generally show the higher prevalence of Hematodinium infections (Stentiford, 2008; Chualáin et al., 2009; Smith et al., 2015), however it has also been recorded in adult crabs in deeper waters (Chualáin, 2010). The species/clade(s) of Hematodinium infecting C. pagrus used in this study, are yet to be fully determined. The parasites from the donor C. pagurus haemolymph used in both experiments, when sequenced, were found to be closely related to a Hematodinium sp. in N. norvegicus (NCBI accession number FJ844429). To date, two species of Hematodinium have been identified; Hematodinium perezi (Chatton and Poisson, 1931) in the Atlantic and Mediterranean and Hematodinium australis (Hudson and Shields, 1994) described from Australian waters. Small et al. (2012) further classified H. perezi into three genotypes: Genotype I in the sandy swimming crab, Liocarcinus depurator from the English Channel, Genotype II in the Japanese blue crab, Portunus trituberculatus and the mud crab, Scylla serrata from China, and Genotype III in Cal. sapidus from North America. Sequencing of 18S rDNA and the adjacent internal transcribed spacer 1 (ITS1) region of Hematodinium spp. isolated from 7 host species has given evidence for 2 potential clades of Hematodinium in the Northern Hemisphere (Jensen et al., 2010).
Please cite this article in press as: Davies, C.E., Rowley, A.F. Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/j.jip.2015.02.004
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These are classed as Clade A from Cal. sapidus, L. depurator and S. serrata, and Clade B from Pagurus bernhardus, Pagurus prideaux, Munida rugosa, N. norvegicus, Ch. opilio, C. pagurus and Carcinus maenas. This suggests that the Hematodinium sp. used in the present experiments is a species/clade infecting both C. pagurus and N. norvegicus. Some studies have employed molecular techniques in order to determine up-regulated genes in response to the presence of certain pathogens and immunostimulants in H. gammarus, suggesting that a certain specificity must be met in order for there to be a resulting immune response (e.g. Hauton et al., 2005, 2006). More recently, Clark et al. (2013a,b,c) have used microarrays to measure the transcriptomic changes occurring in genes of the American lobster, H. americanus following introduction of a range of pathogens. Large numbers of genes were found to be up-regulated following challenge, with some genes specific to each pathogen the lobsters were challenged with. Due to the closely related nature of H. americanus and H. gammarus, this may be worthwhile exploring further using artificial challenge with Hematodinium. In summary, based on field observations of European lobsters in the wild (Chualáin, 2010) and the current experimental attempts to infect juvenile lobsters, it is concluded that this species is highly refractory to infection by one species/clade of Hematodinium. Whilst these observations may provide us with an example of a crustacean in which the immune system can recognise and eliminate Hematodinium parasites, it would now be pertinent to determine why this particular Hematodinium species apparently fails to develop in the tissues of European lobsters, and whether it is eliminated and destroyed by the immune defenses of these animals.
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Acknowledgments
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These studies were partially supported by the Ireland-Wales Interreg IVA project, SUSFISH, to AFR. CED was part-funded by a tuition fee bursary from Swansea University’s Colleges of Science and Medicine.
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Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jip.2015.02.004.
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Meyers, T.R., Koeneman, T.M., Bothelho, C., Short, S., 1987. Bitter crab disease: a fatal dinoflagellate infection and marketing problem for Alaskan Tanner crabs Chionoecetes bairdii. Dis. Aquat. Organ. 3, 195–216. Pestal, G.P., Taylor, D.M., Hoenig, J.M., Shields, J.D., Pickavance, R., 2003. Monitoring the presence of the lethal parasite Hematodinium sp. in snow crabs from Newfoundland. Dis. Aquat. Organ. 53, 67–75. Small, H.J., 2012. Advances in our understanding of the global diversity and distribution of Hematodinium spp. – significant pathogens of commercially exploited crustaceans. J. Invertebr. Pathol. 110, 234–246. Small, H.J., Neil, D.M., Taylor, A.C., Atkinson, R.J.A., Coombs, G.H., 2006. Molecular detection of Hematodinium spp. in Norway lobster Nephrops norvegicus and other crustaceans. Dis. Aquat. Organ. 69, 185–195. Small, H.J., Shields, J.D., Reece, K.S., Bateman, K., Stentiford, G.D., 2012. Morphological and molecular characterization of Hematodinium perezi (Dinophyceae: Syndininiales), a dinoflagellate parasite of the harbour crab, Liocarcinus depurator. J. Euk. Microbiol. 59, 54–66. Smith, A.L., Hirschle, L., Vogan, C.L., Rowley, A.F., 2015. Parasitization of juvenile edible crabs (Cancer pagurus) by the dinoflagellate, Hematodinium sp.: pathobiology, seasonality and its potential effects on commercial fisheries. Parasitology 14, 1–11. Stentiford, G.D., 2008. Diseases of the European edible crab (Cancer pagurus): a review. ICES J. Mar. Sci. 65, 1578–1592. Stentiford, G.D., Shields, J.D., 2005. A review of the parasitic dinoflagellates Hematodinium species and Hematodinium-like infections in marine crustaceans. Dis. Aquat. Organ. 66, 47–70. Stentiford, G.D., Green, M., Bateman, K., Small, H.J., Neil, D.M., Feist, S.W., 2002. Infection by a Hematodinium-like parasitic dinoflagellate causes Pink Crab Disease (PCD) in the edible crab Cancer pagurus. J. Invertebr. Pathol. 79, 179– 191. Taylor, D.M., Khan, R.A., 1995. Observations on the occurrence of Hematodinium sp. (Dinoflagellata: Syndinidae): the causative agent of bitter crab disease in the Newfoundland snow crab (Chionoecetes opilio). J. Invertebr. Pathol. 65, 283–288. Wilhelm, G., Mialhe, E., 1996. Dinoflagellate infection associated with the decline of Necora puber crab populations in France. Dis. Aquat. Organ. 26, 213–219. Wootton, E.C., Pope, E.C., Vogan, C.L., Roberts, E.C., Davies, C.E., Rowley, A.F., 2011. Morphology and pathology of the ectoparasitic copepod, Nicothoë astaci (’lobster louse’) in the European lobster, Homarus gammarus. Parasitology 138, 1285–1295. Wootton, E.C., Woolmer, A.P., Vogan, C.L., Pope, E.C., Hamilton, K.M., Rowley, A.F., 2012. Increased disease calls for a cost-benefits review of marine reserves. PLoS ONE 7, e51615.
Please cite this article in press as: Davies, C.E., Rowley, A.F. Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/j.jip.2015.02.004
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Short Communication
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Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?
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Charlotte E. Davies ⇑, Andrew F. Rowley
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Department of Biosciences, College of Science, Swansea University, Swansea SA2 8PP, Wales, UK
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Article history: Received 7 November 2014 Revised 6 February 2015 Accepted 16 February 2015 Available online xxxx Keywords: Pink crab disease Disease ecology Edible crab Endoparasite Dinoflagellate Inoculation
a b s t r a c t Hematodinium spp. infect over 40 species of crustaceans worldwide, but have not been reported to infect the European lobster, Homarus gammarus. In this study, Hematodinium parasites (a mixture of uni- and multinucleate trophont-like stages) were taken from donor crabs (Cancer pagurus) and injected into juvenile H. gammarus. Juvenile C. pagurus were also injected with the same inoculum. Haemolymph was taken at regular intervals and examined for the presence of Hematodinium using light microscopy and PCR, in two separate experiments of duration 4 and 8 months. All lobsters were negative for Hematodinium whilst the C. pagurus challenged became infected. It is concluded that European lobsters are not susceptible to infection with a clade of Hematodinium that infects C. pagurus. Ó 2015 Published by Elsevier Inc.
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1. Introduction Hematodinium spp. are endoparasitic dinoflagellates and the causative agents of the condition called ‘bitter crab disease’ in some majid crabs (Meyers et al., 1987; Taylor and Khan, 1995) and ‘pink crab disease’ in the European edible crab, Cancer pagurus (Stentiford et al., 2002; Stentiford and Shields, 2005). Hematodinium spp. have been detected in over 40 species of crustaceans worldwide (Small, 2012) since their first discovery in the 1930s by Chatton and Poisson (1931). In some instances, outbreaks of disease by these parasites have damaged commercial stocks of crustaceans including snow crabs, Chionoecetes opilio (Pestal et al., 2003), Tanner crabs, Chionoecetes bairdi (Meyers et al., 1987), American blue crabs, Callinectes sapidus (Messick, 1994) and velvet swimming crabs, Necora puber (Wilhelm and Mialhe, 1996). In the United Kingdom, it is has also been detected in commercially important species such as the European edible crab, C. pagurus (Latrouite et al., 1988; Stentiford et al., 2002). Although detected in the Norway lobster, Nephrops norvegicus (Field et al., 1992) there have been no reports of the presence of Hematodinium spp. in clawed lobsters such as the European lobster, Homarus gammarus or the American lobster, Homarus americanus. H. gammarus is an important commercial species, with worldwide landings of 4805 tonnes in 2012 (FAO, 2014). There are very ⇑ Corresponding author at: Department of Biosciences, Swansea University, Singleton Park, Swansea SA2 8PP, Wales, UK. Tel.: +44 7814161356. E-mail address: [email protected] (C.E. Davies).
few reports of diseases in this species although shell disease, gaffkaemia and parasitization of the gills by the copepod, Nicothoë astaci have been recently noted (Wootton et al., 2011, 2012; Davies et al., 2015a,b). However, this apparent lack of a wide range of pathogens in H. gammarus could simply be as a result of limited studies on disease monitoring. The European lobster shares the same general distribution and habitat as the edible crab, and considering the damage caused to commercial stocks of some crustaceans by Hematodinium spp. worldwide, it is pertinent to establish whether it is susceptible to parasitisation. This study was designed to determine if European lobsters are susceptible to infection by the same strain/genotype of Hematodinium that infects other crustaceans in the same environment, such as the edible crab, C. pagurus. This was achieved by taking these parasites from the haemolymph of Hematodiniuminfected edible crabs, challenging juvenile lobsters by intrahaemocoelic injection, and monitoring these for subsequent infection for up to 8 months post-challenge.
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2. Materials and methods
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2.1. Experimental lobsters
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Stage 5 H. gammarus (early benthic juveniles) were obtained from the National Lobster Hatchery (Padstow, U.K.) and grownup in an aquarium at Swansea University for ca. 11 months before experimentation. During this time, all lobsters were held in individual containers in one large tank within a 1132 L recirculating
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Please cite this article in press as: Davies, C.E., Rowley, A.F. Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/j.jip.2015.02.004
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seawater system. Water quality parameters were maintained at pH 7.8–8, with temperatures varying from 10.5 to 14.5 °C (according to seasonality). All lobsters were fed three times each week initially on Tetra marine mix gel (Spectrum Brands, Melle, Germany) for the first 6 months and live mussel, Mytilus edulis tissues thereafter. A preliminary experiment (Experiment 1) took place over an 8 month period, followed by a 4 month experiment (Experiment 2) in which lobsters were monitored every 28 days (see Table S1, Supplementary Materials for further detail of experimental design and sampling regime).
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2.2. Hematodinium donation
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For both Experiments 1 and 2, haemolymph (2.5 ml) was drawn into 2.5 ml sterile, ice-cold 3% NaCl solution from donor edible crabs (C. pagurus) with natural Hematodinium infections of grade 3–4 severity (see Smith et al., 2015 for criteria of severity rating). The mixture was transferred into a 9 cm plastic Petri dish and the haemocytes left to attach for 10 min at room temperature. After gentle mixing to dislodge any non-attached Hematodinium, the solution containing parasites was decanted into a new dish and a further 5 ml of sterile 3% NaCl solution added and the attachment process repeated on two more occasions. The solution was then centrifuged (850 g, 10 min at 4 °C) and the pellet re-suspended in 10 ml 3% sterile NaCl solution. Hematodinium were counted using an improved Neubauer haemocytometer.
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2.3. Challenge
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In the initial experiment (Experiment 1) 50 ll of inoculum containing 2 105 Hematodinium were injected into juvenile lobsters (n = 4, carapace length 51.7 ± 5.9 mm). Control animals were injected with the same volume of sterile 3% NaCl solution (n = 3,
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carapace length 47.0 ± 4.6 mm). At the same time, 3 juvenile edible crabs were also injected with the same dose of parasites. The lobsters were bled (ca. 100 ll dispensed into 100% ethanol) ca. every 100 days for 8 months and haemolymph preparations examined with phase contrast microscopy for the presence of Hematodinium. Following the final (day 251) bleed, all lobsters were fixed using Davidson’s sea water fixative (24 h), trisected transversely and incubated in decalcifying solution (10% formalin, 55 g l1 EDTA) for 7 days. Samples were processed for histology as described in Smith et al. (2015), wax sections cut at 7 lm and stained using Cole’s haematoxylin and eosin. Slides were viewed and photographed using an Olympus BX41 microscope with an Olympus SC30 digital camera. In Experiment 2, 50 ll of inoculum containing 1.2 105 Hematodinium parasites was injected into experimental lobsters known to be free of Hematodinium sp. (n = 6, carapace length 24.3 ± 1.3 mm) and sterile 3% NaCl solution was injected into the control group of lobsters (n = 4, carapace length 24.6 ± 2.3 mm). After 1 week, lobsters were bled (ca. 100 ll dispensed into 100% ethanol) every 28 days for up to 4 months post challenge. After the final bleed, all remaining lobsters were sacrificed, and haemolymph preparations examined with phase contrast microscopy for the presence of Hematodinium.
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2.4. DNA extraction and PCR conditions
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In Experiment 1, DNA was extracted from haemolymph using an adapted version of the Qiagen DNeasyÒBlood and Tissue Kit (Qiagen Ltd, Manchester, UK). Briefly, 400 ll ethanol-haemolymph mixture was centrifuged for 5 min at 7000g before 20 ll proteinase K, 220 ll phosphate buffered saline and 200 ll were added to the pellet, vortexed and incubated for 10 min at 56 °C. After this, the manufacturer’s instructions were followed according to the
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Fig. 1. Agarose gel showing PCR products from Experiment 2 haemolymph DNA, extracted every 28 days until 4 months post challenge, using universal decapod primers (A) and Hematodinium sp. specific primers (B). D = Donor crab infected with Hematodinium and N = No template control (with molecular grade water). Ladders are Hyperladder I (Bioline Reagents Ltd, London). The 700 bp band from the donor crab (D) following sequencing was confirmed as Hematodinium sp (see Supplementary Material for sequences).
Please cite this article in press as: Davies, C.E., Rowley, A.F. Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/j.jip.2015.02.004
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protocol for purification of total DNA from animal blood or cells (for nucleated blood – step 3 onwards). In Experiment 2, DNA was extracted with a simpler batch-version DNA extraction modified from Ivanova et al. (2006) using 96- well plates (see Supplementary Materials for details). All PCR reactions were carried out using primers synthesized by Eurofins MWG Operon (Ebersberg, Germany) and performed on a Bio-Rad PTC-100 Peltier thermal cycler before being visualized on a 1.5% agarose gel. All DNA was first tested for product using decapod-specific primers (143F 50 -TGCCTTATCAGCTNTCGATTGTAG-30 and 145R 50 -TTCAGNTTTGCAACCATACTTCCC-30 (Lo, 2014) and MangoMix (Bioline Reagents Ltd, London) in a 10 ll total reaction volume. Cycling conditions were as follows: 4 min at 94 °C followed by 40 cycles of 1 min at 93 °C, 1 min at 55 °C and 2 min at 72 °C, followed by 5 min at 72 °C.
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2.5. Hematodinium sp. detection
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DNA was amplified in 10 ll total reaction volume by adding 2 ll of 5 Coloured Reaction Buffer, 375 lM of each dNTP, 3 mM MgCl2, 2 lM of each primer, 0.05 ll of MangoTaq polymerase (1 U/ll, Bioline Reagents Ltd, London), 2 ll of diluted DNA (1:10), 10% BSA and sterile water to the final reaction volume. Hematodinium spp. specific primers shown to detect all Hematodinium spp. optimized by Hamilton et al. (2009) were used: forward primer DinoF 50 GTGGTGCATGGCCGTTCTTAGTT-3 (Kim et al., 2004) and reverse primer ITS1R1 50 -GAAGGGAAGGGGAGAAGAAGC-3 (Small et al., 2006). These primers are expected to produce a target sequence amplicon of 730 bp. Cycling conditions were as follows: 3 min at 95 °C followed by 34 cycles of 45 s at 94 °C, 45 s at 57 °C and 1 min at 72 °C, followed by 10 min at 72 °C. Positive controls from donor crabs were repeated, the PCR product cleaned up using the Wizard SV Gel and PCR Clean-Up System (Promega; Madison, USA) and sequenced by Eurofins MWG Operon (Ebersberg, Germany). Contigs from sequences were created using the CAP3 sequence assembly programme (Huang and Madan, 1999) and identity confirmed using matched positive controls via NCBI BLAST.
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3. Results and discussion
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In both the preliminary experiment (Experiment 1) and Experiment 2, all lobsters challenged with Hematodinium were found to be negative for the presence of these parasites using histology, PCR (Fig. 1) and by examination of live haemolymph preparations (Fig. 2). During Experiment 1, one control and two experimental lobsters died (one at 2 months, one at 7 months) but without any of the symptoms of disease caused by Hematodinium. In comparison, the edible crabs injected with the same batch of Hematodinium became infected after 3 months post-challenge. There was no mortality of lobsters in the second experiment. The PCR method used in the present study has been shown to be extremely sensitive and is capable of detecting the presence of Hematodinium of 10 parasite nuclei from pure parasite culture, and ca. 30 Hematodinium nuclei from N. norvegicus haemolymph (Hamilton et al., 2009). DNA-based diagnostic methods utilising PCR are now considered to be more sensitive to histology in terms of detection (e.g. Small et al., 2006) and have facilitated diagnosis of many marine pathogens of shellfish (for review see Cunningham, 2002). However, the use of both histological and molecular techniques gives the additional advantage of information on the pathology and severity of the infection, if any (Chualáin and Robinson, 2011). None of the histological or live haemolymph preparations examined in the current study showed any evidence for the presence of Hematodinium in the tissues of H. gammarus.
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Fig. 2. Haemolymph preparations examined for the presence of Hematodinium sp. using phase contrast microscopy. (A) Donor Cancer pagurus with Hematodinium (arrow) infection (clump colony) and (B) a time-final (112 days post- challenge), Hematodinium sp. – injected Homarus gammarus from Experiment 1. Note the lack of Hematodinium sp. parasites in this haemolymph preparation. Scale bars = 10 lm (A) and 50 lm (B).
Screening of wild H. gammarus populations in the Irish Sea region failed to find any lobsters that were positive to infection by Hematodinium spp. despite being found in the same area as infected edible crabs (C. pagurus) and velvet swimming crabs (N. puber); the former of which had disease prevalence values of up to 51% (Chualáin, 2010). The juvenile stages of C. pagurus that inhabit the intertidal and sub-tidal regions generally show the higher prevalence of Hematodinium infections (Stentiford, 2008; Chualáin et al., 2009; Smith et al., 2015), however it has also been recorded in adult crabs in deeper waters (Chualáin, 2010). The species/clade(s) of Hematodinium infecting C. pagrus used in this study, are yet to be fully determined. The parasites from the donor C. pagurus haemolymph used in both experiments, when sequenced, were found to be closely related to a Hematodinium sp. in N. norvegicus (NCBI accession number FJ844429). To date, two species of Hematodinium have been identified; Hematodinium perezi (Chatton and Poisson, 1931) in the Atlantic and Mediterranean and Hematodinium australis (Hudson and Shields, 1994) described from Australian waters. Small et al. (2012) further classified H. perezi into three genotypes: Genotype I in the sandy swimming crab, Liocarcinus depurator from the English Channel, Genotype II in the Japanese blue crab, Portunus trituberculatus and the mud crab, Scylla serrata from China, and Genotype III in Cal. sapidus from North America. Sequencing of 18S rDNA and the adjacent internal transcribed spacer 1 (ITS1) region of Hematodinium spp. isolated from 7 host species has given evidence for 2 potential clades of Hematodinium in the Northern Hemisphere (Jensen et al., 2010).
Please cite this article in press as: Davies, C.E., Rowley, A.F. Are European lobsters (Homarus gammarus) susceptible to infection by a temperate Hematodinium sp.?. J. Invertebr. Pathol. (2015), http://dx.doi.org/10.1016/j.jip.2015.02.004
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These are classed as Clade A from Cal. sapidus, L. depurator and S. serrata, and Clade B from Pagurus bernhardus, Pagurus prideaux, Munida rugosa, N. norvegicus, Ch. opilio, C. pagurus and Carcinus maenas. This suggests that the Hematodinium sp. used in the present experiments is a species/clade infecting both C. pagurus and N. norvegicus. Some studies have employed molecular techniques in order to determine up-regulated genes in response to the presence of certain pathogens and immunostimulants in H. gammarus, suggesting that a certain specificity must be met in order for there to be a resulting immune response (e.g. Hauton et al., 2005, 2006). More recently, Clark et al. (2013a,b,c) have used microarrays to measure the transcriptomic changes occurring in genes of the American lobster, H. americanus following introduction of a range of pathogens. Large numbers of genes were found to be up-regulated following challenge, with some genes specific to each pathogen the lobsters were challenged with. Due to the closely related nature of H. americanus and H. gammarus, this may be worthwhile exploring further using artificial challenge with Hematodinium. In summary, based on field observations of European lobsters in the wild (Chualáin, 2010) and the current experimental attempts to infect juvenile lobsters, it is concluded that this species is highly refractory to infection by one species/clade of Hematodinium. Whilst these observations may provide us with an example of a crustacean in which the immune system can recognise and eliminate Hematodinium parasites, it would now be pertinent to determine why this particular Hematodinium species apparently fails to develop in the tissues of European lobsters, and whether it is eliminated and destroyed by the immune defenses of these animals.
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Acknowledgments
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These studies were partially supported by the Ireland-Wales Interreg IVA project, SUSFISH, to AFR. CED was part-funded by a tuition fee bursary from Swansea University’s Colleges of Science and Medicine.
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Appendix A. Supplementary material
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jip.2015.02.004.
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