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Journal of Shellfish Research, Vol. 24, No. 3, 719–731, 2005.

NUCLEAR SMALL-SUBUNIT RIBOSOMAL RNA GENE-BASED CHARACTERIZATION, MOLECULAR PHYLOGENY AND PCR DETECTION OF THE FROM WESTERN LONG ISLAND SOUND LOBSTER

THOMAS E. MULLEN,1† KATHLEEN R. NEVIS,1† CHARLES J. O’KELLY,2 REBECCA J. GAST3 AND SALVATORE FRASCA JR.1* 1Department of Pathobiology and Veterinary Science, 61 North Eagleville Road, University of Connecticut, Storrs, Connecticut 06269-3089; 2Bigelow Laboratory for Ocean Sciences, P.O. Box 475, 180 McKown Point Road, West Boothbay Harbor, Maine 04575; 3Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts 02543

ABSTRACT Western Long Island Sound (LIS) lobsters collected by trawl surveys, lobstermen and coastal residents during 2000 to 2002 were identified histologically as infected with a parasome-containing . Primers to conserved SSU rRNA sequences of parasome-containing amoebae and their nonparasome-containing relatives were used to amplify overlapping SSU rRNA fragments of the presumptive parasite from gill, antenna, antennal gland and ventral nerve cord of infected lobsters. The consensus sequence constructed from these fragments had 98% or greater nucleotide sequence identity with SSU rRNA gene sequences of strains of Neoparamoeba pemaquidensis and associated with high confidence in distance- and parsimony-based phylogenetic analyses with strains of Neoparamoeba pemaquidensis and not members of the family , e.g., eilhardi. Primers designed to SSU rRNA sequences of the lobster amoeba and other paramoebid/vexilliferid amoebae were used in a nested polymerase chain reaction (PCR) protocol to test DNA extracted from formalin-fixed paraffin-embedded tissues of lobsters collected during the 1999 die-off, when this amoeba initially was identified by light and electron microscopy and reported to be a paramoeba of the genera Paramoeba or Neoparamoeba (Mullen et al. 2004). All sequences amplified from 1999 lobsters, with the exception of one, had 98% to 99% identity to each other, and the 1999 PCR product consensus had 98% identity to Neoparamoeba pemaquidensis strains CCAP 1560/4 (AF371969.1) and 1560/5 (AF371970.1). Molecular characterization of the amoeba from western LIS lobsters by direct amplification circumvents a collective inability to culture the organism in vitro, provides insight into the molecular epidemiology of neoparamoebiasis in American lobster, and allows for PCR-based detection of infected lobsters for future research and diagnostics.

KEY WORDS: Homarus americanus, lobster, molecular phylogeny, Neoparamoeba pemaquidensis, paramoebiasis, PCR, small- subunit rRNA

INTRODUCTION and subsequent collapse of the natural lobster fishery in western Long Island Sound (LIS). The lobster die-off has been attributed, in part, to infection by a parasome-containing amoeba (Mullen et A few species of naked lobose amoebae (gymnamoebae) are al. 2004). The identity of this amoeba is unknown, yet knowledge considered to be parasitic to shellfish. Gymnamoebae belonging to of its identity is essential in determining its origin in LIS and in the paramoebid/vexilliferid (PV) lineage (Peglar et al. 2003); fami- lobsters. lies Paramoebidae and Vexilliferidae as defined by Page (1987) The identity of the amoebae infecting western LIS lobster is and Page & Siemensma (1991) have been associated with “grey unknown because criteria previously used to identify these amoe- crab disease” of blue crabs, Callinectes sapidus (Johnson 1977) bae have now been shown to be inadequate. The parasome, some- and with a wasting disease of green sea urchins, Strongylocentro- times referred to as a “secondary nucleus” because it contains tus droebachiensis (Jones & Scheibling 1985). Both diseases have DNA and superficially resembles the authentic cell nucleus (Grell caused significant, often catastrophic, economic losses. In addi- 1961, Grell & Benwitz 1970, Perkins & Castagna 1971), has been tion, one PV lineage species, Neoparamoeba pemaquidensis, is the considered diagnostic for the genera Paramoeba and Neopar- etiologic agent of amoebic gill disease, a commercially important amoeba (Page 1987). Both genera belong to the PV lineage (Peglar disease of aquaculture-reared finfish (Munday 1986, Munday et al. et al. 2003), and both genera contain species known or strongly 2001) including Atlantic salmon, Salmo salar (Roubal et al. 1989, suspected to be pathogens of fish and shellfish. However, the Zilberg et al. 2001, Adams & Nowak 2003, Adams & Nowak parasome has been shown to be a parasitic protozoon, genus Per- 2004, Butler & Nowak 2004), coho salmon, Oncorhynchus kisutch kinsella (Hollande 1980, Dyková et al. 2003), and species of this (Kent et al. 1988), turbot, Scophthalmus maximus (Dyková et al. parasite are present in amoebae that are unlikely to belong to the 1995, 1998, Fiala & Dyková 2003), European sea bass, Dicentrar- PV lineage (Hollande 1980). Moreover, species of Paramoeba and chus labrax (Dyková et al. 2000) and sea bream, Sparus auratus Neoparamoeba have been differentiated on the basis of submicro- (Athanassopoulou et al. 2002, as S. aurata). scopic structures on their cell surfaces (Page 1987, Page & Sie- Though infective gymnamoebae had previously been reported mensma 1991): scales on Paramoeba species (Grell & Benwitz in tissues of the American lobster, Homarus americanus (H. Milne 1970), glycostyles on Neoparamoeba species (Cann & Page 1982, Edwards, 1837) (Sawyer 1976, Sawyer & MacLean 1978), they Page 1987). The amoeba from lobster, however, has neither scales were not associated with either demonstrable pathology or epi- nor glycostyles on its cell surface (Mullen et al. 2004), a feature zootic disease. This changed in 1999, with the mass lobster die-off that it shares with tissue-borne forms of the pathogenic amoebae from blue crab (Perkins & Castagna 1971) and urchin (Jones †Current address: Department of Pathology, School of Medicine, Univer- 1985). Therefore, available characters from neither light nor elec- sity of North Carolina, Chapel Hill, North Carolina 27599 tron microscopy are sufficient to identify the lobster-borne amoeba *Corresponding author. E-mail: [email protected] or, for that matter, the crab- and urchin-borne amoebae.

719 720 MULLEN ET AL.

Identifying the amoeba from lobster, comparing it with other hepatopancreas, gonad, stomach, intestine, gill, carapace and ven- amoebae responsible for diseases of fish and shellfish and under- tral nerve cord were fixed in 10% neutral buffered formalin, de- standing its pathogenicity are inhibited by the episodic nature of calcified using Bouin’s fixative and processed routinely for paraf- disease outbreaks, a lack of cultures and a lack of knowledge about fin embedding. the fundamental biology of marine gymnamoebae in general and of members of the PV clade in particular. To date, efforts to isolate Histopathology the lobster amoeba into in vitro culture have not been successful Soft tissues for histopathologic examination were fixed by im- (Mullen et al. 2004). Efforts to culture the blue crab pathogen have mersion in 10% neutral buffered formalin for 24 to 48 h. Hard likewise been unsuccessful (Johnson 1977). Cultures of the urchin tissues (e.g., antennae and eye) were placed in 5% trichloroacetic pathogen were achieved (Jones 1985, Jellett & Scheibling 1988a, acid solution overnight then returned to formalin. Tissues were Jellett & Scheibling 1988b) but not archived. The only cultures of routinely processed for paraffin embedding, sectioned at 4 ␮m, Paramoeba and Neoparamoeba species available when this study mounted on glass slides and stained using hematoxylin and eosin began were of free-living amoebae isolated from water and sedi- (H&E). Slides were examined using bright field microscopy, and ment samples (e.g., Grell 1961, Cann & Page 1982) or from the lobsters were identified as being infected with paramoebae based gills of finfish (e.g., Kent et al. 1988). on published morphologic criteria describing paramoebiasis in Efforts to characterize gymnamoebae at the molecular level are western LIS lobsters (Mullen et al. 2004). in their infancy, especially for marine species. Phylogenetic stud- ies, mostly based on sequences of the nuclear-encoded small- DNA Isolation subunit ribosomal RNA (SSU rRNA) gene, have discovered new ␮ lineages and significantly rearranged existing ones (Sims et al. Tissues for nucleic acid studies were placed into 800 L 1999, 2002, Amaral-Zettler et al. 2000, 2001, Bolivar et al. 2001). DEPC-treated water (ResGen, Carlsbad, California) at necropsy Peglar et al. (2003) were the first to demonstrate the existence of and snap frozen in liquid nitrogen for storage. Tissue samples from the PV lineage, phylogenetically distinct from all other groups of specimens diagnosed as infected with paramoebae were thawed on amoebae. Molecular tools are beginning to be used to probe ice, minced, and approximately 25–50 mg of tissue placed into 400 ␮ Neoparamoeba strains associated with amoebic gill disease (Fiala L ATL buffer (Qiagen Inc., Chatsworth, California) containing ␮ & Dyková 2003, Wong et al. 2004), but otherwise, the data and 40 L of 20 mg/mL (>600 mU/mL) proteinase K (Qiagen Inc., procedures that would permit DNA-based identifications of gym- Chatsworth, California). Gills received more rigorous mincing and namoebae in the environment, in mixed cultures or in infected host often sterile, plastic mortar and pestles (Fisher Scientific, Hamp- tissues, are still lacking or insufficient. ton, New Hampshire) were used to mechanically break down this This article reports efforts to characterize the amoeba present in tissue and facilitate digestion. Lysates were incubated at 55°Cina the tissues of western LIS lobsters based on direct amplification dry bath overnight and, if necessary, additional proteinase K was and sequence analysis of the amoebal nuclear SSU rRNA gene. In added and longer times were used to complete digestion. To im- addition to presenting the first epidemiologic data of this emerging prove the quality and quantity of DNA extracted from gill and disease of LIS lobster, this report communicates the identification antennae, these tissues were commonly allowed longer digestion of a consensus SSU rRNA gene sequence representative of the times, typically 48 h. Genomic DNA was isolated using silica-gel amoeba infecting western LIS lobsters, its molecular phylogenetic spin-column technology (DNeasy DNA Extraction System, characterization and the development and application of a nested Qiagen, Inc, Chatsworth, California), and total DNA was eluted ␮ PCR protocol to detect this and similar SSU rRNA gene sequences from spin columns in 100–200 L volumes. Total genomic DNA in lobster tissue. was quantified by either spectrophotometry using a Hoechst 33258 spectrophotometer (Bio-Rad Laboratories, Hercules, California) or MATERIALS AND METHODS by fluorometry using a Bio-Rad VersaFlour Fluorometer (Bio- Rad, Hercules, California). Sample Collection Genomic DNA Quality Control PCR Amplification Lobsters were collected during trawl surveys conducted by Connecticut Department of Environmental Protection officers as To determine the efficacy of genomic DNA extraction in iso- part of the State’s Long Island Sound Zoning Project (including lating amplifiable DNA for downstream experimentation, all lob- only zones 1 and 2), or by independent submission of dead and ster tissues were tested for positive PCR amplification using uni- dying, “limp,” lobsters by fishermen and biologists from autumn versal primers to eukaryotic small-subunit ribosomal RNA gene 2000 through autumn 2002. Animals were euthanized using 100 sequences. Primers 18e and 18h were used to amplify an approxi- mg of KCl/100 g of body weight as previously described (Battison mately 440-bp product (Hillis & Dixon 1991) in 50-␮L reactions et al. 2000). Tissues including antenna, antennal gland, append- containing 100–200 ng sample DNA, 1× Qiagen PCR Buffer

ages, carapace, compound eye, gill, gonad, heart, hepatopancreas, (Tris-HCl, KCl, (NH4)2SO4, 1.5 mM MgCl2, pH 8.7; Qiagen Inc., intestine, mandibular apparatus and tail muscle were collected for Chatsworth, California), 200 ␮M dNTPs, 10 pmol each forward histopathologic examination, while antenna, antennal gland, gill and reverse oligonucleotide primer and 0.2 U HotStar Taq DNA and ventral nerve cord were subsampled for molecular biologic polymerase (Qiagen Inc., Chatsworth, California). Reactions were analysis. initiated by a 15-min incubation step at 94°C, followed by 35 Tissue samples of lobsters from the 1999 die-off were obtained cycles each consisting of denaturation at 94°C for 30 sec, anneal- from paraffin blocks prepared as described by Mullen et al. (2004). ing at 60°C for 30 sec, and extension at 72°C for 30 sec. A final Briefly, dead and moribund lobsters from western LIS (zone 1) extension at 72°C for 7 min completed the cycling protocol. Ge- were collected from late October through early December 1999, nomic DNA from Saccharomyces cerevisiae (American Type Cul- and tissues including antenna, antennal gland, compound eye, ture Collection, Manassas, Virginia) was used as an eukaryotic CHARACTERIZATION AND DETECTION OF LOBSTER PARAMOEBA 721 positive control. Products were separated on either 1.5% or 2% nus, (GenBank AF235971; Ragan et al. 1996), using the multiple agarose gels and visualized by ethidium bromide staining and UV sequence alignment functions of DNAMAN (Lynnon Biosoft, transillumination. Samples that resulted in no amplification were Quebec, Canada) and Vector NTI (InfoMax Inc., Frederick, Mary- eliminated from the study. land). Primer sequences were designed to hybridize with variable and conserved regions of SSU rRNA gene sequences from the Direct Amplification of Paramoeba SSU rRNA Gene Sequences amoebae that did not share significant nucleotide identity with the lobster SSU rRNA gene (Table 2). Three sets of nested primer Small-subunit rRNA gene sequences of the paramoeba infect- pairs were designed in this manner to amplify overlapping regions ing western LIS lobsters were amplified directly from tissues of of amoebal SSU rRNA genes that included a 5Ј, an internal 1.3-kb lobsters identified by histopathologic examination as infected with anda3Ј region (Table 2). paramoebae. SSU rRNA gene sequences from 8 strains of par- To avoid cross-reactivity with genomic DNA of actual and amoebid/vexilliferid amoebae, hemistylolepis, potential hosts, and to confirm amplification from representative O’Kelly et al. 2001 (ATCC 50804); Korotnevella stella (Schaeffer amoebae, specificity was assessed by testing first and second- 1926) Goodkov 1988 (CCAP 1547/6); Neoparamoeba aestuarina round primer pairs separately and then in tandem in PCR protocols (Page 1970) Page 1987 (CCAP 1560/7); N. pemaquidensis (Page using genomic DNA from lobster (Homarus americanus), blue 1970) Page 1987 (CCAP 1560/4, ATCC 30735, ATCC 50172); crab (Callinectes sapidus) and sea urchin (Strongylocentrotus Paramoeba eilhardi Schaudinn 1896 (CCAP 1560/2) and droebachiensis), along with genomic DNA from Neoparamoeba Pseudoparamoeba pagei (Sawyer 1975) Page 1979 (CCAP 1566/ pemaquidensis (ATCC 50172), Korotnevella stella (CCAP 1547/ 2), (Table 1), were aligned with each other and with the SSU 6) and Paramoeba eilhardi (CCAP 1560/2). Sensitivity was as- rRNA gene sequence of the American lobster, Homarus america- sessed by using serial 10-fold (10−2–10−7) dilutions of cloned Neoparamoeba pemaquidensis (CCAP 1560/4) SSU rRNA gene TABLE 1. added to infection-negative genomic lobster DNA. Amoebae representative of paramoebid/vexilliferid and vannellid Direct amplification of SSU rRNA gene sequences of parasitic clades used in small-subunit rRNA gene sequence comparisons for paramoebae used total genomic DNA isolated from lobsters de- primer design (*) and molecular phylogenetic analyses. SSU rRNA termined to be infected in one or more tissues by histopathologic gene sequences of the amoebae in boldface were determined in analysis and tested for DNA of sufficient quality to support host preliminary investigations in support of this study. Authorities are SSU rRNA gene amplification. Tissue samples were tested in trip- provided beneath the first-appearing entry for each species. licate using the three sets of nested primer pairs. Because the internal 1.3-kb region accounted for approximately 60% of the GenBank SSU rRNA gene sequence of the amoebae, and because it also Genus and Species Source Designation Number TABLE 2. Neoparamoeba aestuarina (Page, 1970) Page, 1987 ATCC 50744 AY121848 Forward (-F) and reverse (-R) primer sequences and combinations Neoparamoeba aestuarina ATCC 50805 AY121851.1 used in nested PCR to amplify SSU rRNA gene sequences from Neoparamoeba aestuarina ATCC 50806 AY121852.1 paramoebae infecting western LIS lobster. *Neoparamoeba aestuarina CCAP 1560/7 AY686574 *Neoparamoeba pemaquidensis Primer (Page, 1970) Page, 1987 CCAP 1560/4 AY183894.1 Target Region Name Primer Sequence (5؅ → 3؅) Neoparamoeba pemaquidensis ATCC 50172 AF371971.1 *Neoparamoeba pemaquidensis ATCC 50172 AY183889.1 5Ј-terminus 1st Neoparamoeba pemaquidensis ATCC 30735 AF371972.1 round 733-F CTGGTTGATCCTGCCAGTAGTC *Neoparamoeba pemaquidensis ATCC 30735 AY183887.1 5Ј-terminus 1st Neoparamoeba pemaquidensis CCAP 1560/5 AF371970.1 round 733-R CATGAAAGTCTAGTAAGGAGAWG Neoparamoeba pemaquidensis CCAP 1560/4 AF371969.1 5Ј-terminus 2nd Neoparamoeba pemaquidensis AVG8194 AF371968.1 round 673-F GCTTGTCTTAAAGACTAAGCC Neoparamoeba pemaquidensis PA027 AF371967.1 5Ј-terminus 2nd *Paramoeba eilhardi round 673-R CAAACATACCTTGTACAAGGCAGGTT Schaudinn, 1896 CCAP 1560/2 AY686575 Internal 1st *Pseudoparamoeba pagei round 1609-F ATRAACACTTTGTACTTGTG (Sawyer, 1975) Page, 1979 CCAP 1566/2 AY686576 Internal 1st *Korotnevella hemistylolepis round 1609-R GCCTCAAACTTCCCTTGGTTAAACA O’Kelly et al., 2001 ATCC 50804 AY121850.1 Internal 2nd Korotnevella monacantholepis round 1277-F GAGAGGGAGCCTGAGAAA O’Kelly et al., 2001 ATCC 50819 AY121854.1 Internal 2nd Korotnevella stella round 1277-R CCCTCTAAGAAGTCATTATCG (Schaeffer, 1926) 3Ј-terminus 1st Goodkov, 1988 CCAP 1547/6 AY686573 round 570-F AAAGCTTTGAGTTTGACTTGGAGA Korotnevella stella CCAP 1547/6 AY183893.1 3Ј-terminus 1st aberdonica Page, 1980 ATCC 50815 AY121853.1 round 570-R CGGATACCTTGTTACGACTT Vannella miroides Bovee, 1965 ATCC 30945 AY183888.1 3Ј-terminus 2nd armata Page, 1979 ATCC 50883 AY183891.2 round 405-F GTTAGAATTTTGATCTTTTCGG sp. ATCC 50884 AY183892.1 3Ј-terminus 2nd Clydonella sp. ATCC 50816 AY183890.1 round 405-R GCTCTACCCGAGAGGAAAACAAGTG 722 MULLEN ET AL.

included the majority of variable sites within the gene, this target were assembled from all host lobster yielding products to construct was amplified first. DNA samples were amplified in 50-␮L reac- an overall consensus SSU rRNA gene sequence of the paramoeba tions containing 100–300 ng sample DNA, 1× Qiagen PCR Buffer from western LIS lobster. This overall consensus SSU rRNA gene

(Tris-HCl, KCl, (NH4)2SO4, 1.5 mM MgCl2, pH 8.7; Qiagen Inc., sequence, as well as one near-full-length SSU rRNA gene se- Chatsworth, California), 200 ␮M dNTPs, 10 pmol each forward quence obtained by contig alignment of products from one tissue and reverse primer and 0.2 U HotStar Taq DNA polymerase source of one host lobster, were used in nucleotide-nucleotide (Qiagen Inc., Chatsworth, California) using an initial 15-min heat BLAST searches of the National Center for Biotechnology Infor- activation step followed by cycling protocols individually opti- mation (NCBI) GenBank database and in molecular phylogenetic mized for each nested set of primer pairs. The following conditions analyses. for both first and second round reactions were used: (1) for primer pairs 733-F/733-R and 673-F/673-R, 40 cycles of denaturation at Nucleotide Sequence Accession Numbers 94°C for 45 sec, annealing at 50°C for 1 min, extension at 72°C for SSU rRNA gene sequences representing the amoeba infecting 1 min; (2) for primer pairs 1609-F/1609-R and 1277-F/1277-R, 40 western LIS lobster derived from single-source, lobster 01-9828-7 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 1 gill, or multisource, overall consensus, origins are available in the min, extension at 72°C for 2 min; (3) for primer pairs 570-F/570-R GenBank database under accession numbers AY686577 and and 405-F/405-R, 40 cycles of denaturation at 94°C for 30 sec, AY686578, respectively. SSU rRNA gene sequences of Par- annealing at 50°C for 45 sec, extension at 72°C for 45 sec. A final amoeba eilhardi CCAP 1560/2, Pseudoparamoeba pagei CCAP extension step at 72°C for 7 min concluded each PCR protocol. 1566/2, Neoparamoeba aestuarina CCAP 1560/7, Korotnevella Two microliters of the first round amplification was carried over to stella CCAP 1547/6 were determined previously in support of this ␮ the second round. From the second round amplification, 18 Lof study and are available in the GenBank database under accession amplified product was electrophoresed through a 10% polyacryl- numbers AY686575, AY686576, AY686574 and AY686573, re- amide gel, detected by means of ethidium bromide staining and spectively (Table 1). UV transillumination, then photodocumented. Genomic DNA from lobster, urchin and/or blue crab served as negative controls, Molecular Phylogenetic Analysis whereas purified plasmid DNA containing the complete SSU rRNA gene from Neoparamoeba pemaquidensis (CCAP 1560/4) Both the single-source and overall consensus SSU rRNA gene served as positive control. sequences representing the paramoeba from western LIS lobster were included in multiple sequence alignments constructed using ClustalX v1.81 (Thompson et al. 1997) and comprising the SSU Cloning, Sequencing and Sequence Assembly rRNA gene sequences of representative gymnamoebae of the par- amoebid/vexilliferid and vannellid clades (Peglar et al. 2003), Amplicons were purified into 10 ␮L volumes using commer- (Table 1). Phylogenetic trees were inferred by distance and parsi- cially available DNA purification reagents (QIAquick Mini-Elute mony algorithms using PAUP* 4.0b10 (Swofford 2002). The PCR purification/gel extraction kit, Qiagen Inc., Chatsworth, Cali- data set consisted of 25 taxa with 2,537 total characters, of which fornina). Purified products were ligated independently of one an- 893 were phylogenetically informative in parsimony analysis, and other into the pCR II-TOPO vector, transformed, and cloned using 244 were variable but parsimony-uninformative. A bootstrapped the TOPO TA cloning kit with dual promoter (Invitrogen Corp., maximum parsimony tree was generated using heuristic search Carlsbad, California). Multiple clones from each independent parameters. The confidence of branching was assessed using 1,000 cloning reaction were grown for at least 24 h in LB broth contain- bootstrap resamplings of the data set with a starting tree obtained ing 75 mg/mL ampicillin using a rotating (200 rpm) 37°C incu- via random stepwise addition and tree-bisection-reconnection bator. Plasmids were isolated using the QIAprep Spin Miniprep kit branch-swapping. A distance-based phylogenetic tree was recon- (Qiagen Inc., Chatsworth, California), screened by PCR for con- structed by 1,000 bootstrap resamplings of the dataset using the firmatory amplification and length polymorphism analysis of a minimum evolution optimality criterion and the HKY85 substitu- single product insert, then one clone representing each indepen- tion model (Hasegawa et al. 1985) with among site rate variation dently cloned PCR product was submitted for bidirectional se- assumed to follow a gamma distribution of 0.5. Missing data quencing by primer walking (HHMI Biopolymer/Keck Foundation (gaps) were ignored for sites affected in pairwise comparison, and Biotechnology Resource Laboratory, Yale University School of negative branch lengths were allowed, but collapsed to zero. Heu- Medicine, New Haven, Connecticut). Nucleotide sequences were ristic search parameters were used and starting tree(s) were ob- determined by oligonucleotide-directed dideoxynucleotide chain- tained by neighbor-joining followed by tree-bisection-reconnec- termination sequencing reactions utilizing 300 ng of template tion branch-swapping. DNA, forward (M13F) and reverse (M-13R) primers, fluores- cently-labeled dideoxynucleotides and AmpliTaq FS DNA poly- PCR Detection and Characterization of Paramoeba from merase in a cycling sequencing method. The resultant fragments 1999 Lobsters were subjected to electrophoresis and analyzed using an automated Applied Biosystems 377 DNA sequencer (Perkin Elmer, Applied Primers for PCR were designed to incorporate or span variable Biosystems Division, Foster City, California). regions of SSU rRNA sequences to detect known PV clade amoe- Sense and antisense sequence ABI files from each clone were bae and distinguish them by sequence analysis of amplicons. Com- assembled using Sequencher 4.1.1 for Macintosh (Gene Codes parative sequence alignments were performed using the SSU Corp., Ann Arbor, Michigan). A sequence for each clone from rRNA gene sequences representing the paramoeba from western each tissue of each host lobster was constructed, and contigs were LIS lobster along with the following PV clade amoebae, Neopar- assembled to yield consensus SSU rRNA gene sequences from amoeba pemaquidensis (ATCC 50172), Neoparamoeba pe- each host lobster. Additionally, contigs from each target region maquidensis (CCAP 1560/4), Neoparamoeba pemaquidensis CHARACTERIZATION AND DETECTION OF LOBSTER PARAMOEBA 723

(ATCC 30735), Paramoeba eilhardi (CCAP 1560/2), Pseudopar- using ethidium bromide staining and UV transillumination, then amoeba pagei (CCAP 1566/2), Korotnevella hemistylolepis photodocumented. (ATCC 50804), Korotnevella stella (CCAP 1547/6), Neopar- PCR products were purified, cloned using the previously de- amoeba aestuarina (CCAP 1560/7), (Table 1), and the SSU rRNA scribed protocol, then submitted for oligonucleotide-directed gene sequence of lobster. SSU rRNA gene sequences were aligned dideoxynucleotide chain-termination sequencing reactions initi- using the multiple sequence alignment functions of DNAMAN ated by M13 forward and reverse primers (HHMI Biopolymer/ (Lynnon Biosoft, Quebec, Canada) and Vector NTI (InfoMax Inc., Keck Foundation Biotechnology Resource Laboratory, Yale Uni- Frederick, Maryland). Primers were designed to sequences that versity School of Medicine, New Haven, Connecticut). Nucleotide included or spanned selected variable regions. A nested PCR ap- sequences were determined by sense and antisense ABI sequence proach was adopted to accommodate density and distribution of files using Sequencher 4.1.1 for Macintosh (Gene Codes Corpo- paramoebae in tissue section. First and second round primer pairs ration, Ann Arbor, Michigan) and aligned using Clustal X v1.81 were selected based on the following criteria: (1) ability to amplify (Thompson et al. 1997). SSU rRNA genetic sequences from total genomic DNA of PV clade amoebae; (2) inability to amplify genomic DNA of lobster, RESULTS blue crab and sea urchin and (3) amplicon size less than 500-bp to accommodate amplification from formalin-fixed paraffin-em- Sample Analysis bedded (FFPE) tissues. A total of 240 lobsters were examined from autumn 2000 The study examined 170 paraffin-embedded tissue blocks from through autumn 2002. The disease during this period had a low 77 lobsters collected during the die-off of 1999; of these 170 prevalence, and in histopathologic terms was pauci-organismal and paraffin blocks, 144 blocks were identified by histopathologic ex- multifocal (i.e., a small number of amoebal cells in few and scat- amination as having one or more tissues infected with paramoebae. tered foci). Fifty-micron tissue sections were obtained from each paraffin Histologic examination resulted in the identification of 13 ani- block under study. DNA was extracted using the DNeasy DNA mals positive for the presence of parasome-containing amoebae in Extraction System (Qiagen Inc., Chatsworth, California) according tissues, providing a prevalence estimate of 5% for the 3-y period. to DNA isolation methods previously described for fresh tissues, Three additional lobsters had probable infections, in which amoe- with the following amendments to accommodate paraffin- bal cells had distinctive nuclei, but clear demonstration of para- embedded samples. FFPE tissues were deparaffinized using three somes could not be made. Two more lobsters had suspect infec- 800-␮L washes of xylene, followed by three 800-␮L washes of tions, in which amoeba-like bodies were found, but clear demon- 100% ethanol, then allowed to air dry in a biologic safety cabinet stration that these bodies contained either nucleus-like or until chalky white. After drying, tissues were digested using lysis parasome-like organelles could not be made. Paramoeba-positive buffer and proteinase K, and DNA was extracted using silica gel lobsters were identified in collections from both offshore and in- spin columns according to the same protocol used for DNA iso- shore waters. Seven of the 13 infection-positive lobsters were col- lation from fresh tissues. Extracted DNA from each paraffin block lected in autumn months (September to November), whereas the was individually quality-control tested by positive PCR amplifi- remainder were collected in late winter and spring (February to cation of a 440-bp eukaryotic SSU rRNA genetic sequence using May; Table 3). Paramoebae were identified most often in antennae primers 18e and 18h (Hillis & Dixon 1991) in 50-␮L reactions as (11 of 13 lobsters), followed by eye (5 of 13), gill (3 of 13), ventral previously described. DNA samples that failed quality control am- nerve cord (2 of 13) and antennal gland (1 of 13; Table 3). plification were eliminated from the study. Quantification of total genomic DNA and amplification of host DNA samples from 1999 FFPE tissues that passed quality con- SSU rRNA gene sequences for quality control assessment were trol assessment were tested for presence of paramoeba DNA using performed on all extracted lobster tissues. Most tissue extractions a nested PCR protocol that used Para A (5Ј-AACCTGCCTTGTA- yielded 5 to 20 ␮g (100–400 ng/␮lin50␮L) of genomic DNA, CAAGGTATGTTTG-3Ј) and Para C (5Ј-CGCCACTCCAAA- and most DNA extracts (approximately 90%) amplified using the GAGTGACT-3Ј) as first round primers and Para B (5Ј- eukaryotic SSU rRNA gene amplification protocol. However, it CATCTCCTTACTAGACTTTCATG-3Ј) and Mullen B2 (5Ј- was observed early in the study that DNA extractions from gill and GAACTAGATTTCTARTGAATA-3Ј) as second round primers. antenna yielded low quantities of total genomic DNA, and some DNA samples were tested in triplicate using 50-␮L reactions con- gill extractions resulted in no SSU rRNA gene amplification. The taining an estimated 10–50 ng sample DNA, 1× Qiagen PCR quality and quantity of DNA extracts from gill and antennae were Buffer (Tris-HCl, KCl, (NH ) SO , 1.5 mM MgCl , pH 8.7; 4 2 4 2 improved by longer digestion times with added proteinase K. Qiagen Inc., Chatsworth, California), 200 ␮M dNTPs, 10 pmol However, these samples and any others that resulted in no ampli- each forward and reverse primer and 0.2 U HotStar Taq DNA fication were eliminated from the study. polymerase (Qiagen Inc., Chatsworth, California). Reactions were initiated by a 15-min heat activation step followed by 40 cycles Direct Amplification of Paramoeba SSU rRNA Gene Sequences optimized for each round. First round reactions were cycled at 30 sec denaturation at 94°C, 45 sec annealing at 54°C, and 45 sec Three nested PCR primer sets were targeted to three overlap- extension at 72°C; second round reactions were cycled at 30 sec ping regions of the SSU rRNA gene of PV clade amoebae, a denaturation at 94°C, 45 sec annealing at 50°C, and 45 sec exten- 673-bp fragment from the 5Ј-terminus, an internal 1277-bp frag- sion at 72°C. Each round was completed by a 7-min extension at ment, and a 405-bp fragment from the 3Ј-terminus (Fig. 1). The 72°C. Two microliters of the completed first round reaction mix- nested PCR procedure targeted to the internal 1.3-kb region suc- ture was transferred to the second round PCR using the stated cessfully amplified SSU rRNA sequences from the several PV reaction conditions. A 12% polyacrylamide gel was used to sepa- clade amoebae tested (e.g., Korotnevella stella, Paramoeba eil- rate the second round PCR products, and bands were visualized hardi and Neoparamoeba pemaquidensis). Initial sequence analy- 724 MULLEN ET AL.

TABLE 3. Temporal and tissue-specific distribution of paramoeba-infected lobsters.

Month Collected Year Collected Antenna Antennal Gland Eye Gill Ventral Nerve Cord

01-943-2 February 2001 + + −− − 01-943-7 February 2001 + −−−− 01-943-9 February 2001 + −−−− 01-8041-2 September 2001 + −−−− 01-8041-3 September 2001 + −−−− 01-4758-10 November 2001 + −−−− 01-4758-12 November 2001 + −−−− 01-4758-14 November 2001 + − + −− 01-9828-7 November 2001 + − ++ + 01-9828-8 November 2001 + − ++ + 02-3383-8 April 2002 + − + −− 02-4202-6 May 2002 −−−+ − 02-4202-7 May 2002 −−+ −− ses of PCR products from this region suggested the paramoeba from one or two of these four tissues. Fifteen sequences were from western LIS lobster was a Neoparamoeba species. The nested obtained, 3 from the 5Ј fragment, 5 from the internal fragment, and PCR procedures targeted to the 5Ј and 3Ј termini of the SSU rRNA 7 from the 3Ј fragment. One lobster of the six, identified as 01- gene specifically amplified SSU rRNA sequences from Neopar- 9828-7, yielded amplicons for each of the three target regions from amoeba pemaquidensis; however, some PV clade amoebae were a single gill sample (Fig. 2), and a near-full-length SSU rRNA not available for testing (e.g., P. eilhardi). None of the nested PCR gene sequence was assembled from these amplicons. An overall procedures amplified regions from the total genomic DNA of lob- consensus SSU rRNA gene sequence (GenBank AY686578) was ster, sea urchin or blue crab. constructed from an alignment of all 15 sequence fragments, in- cluding the three overlapping fragments from lobster 01-9828-7 SSU rRNA Gene Sequence Assembly and Characterization gill tissue, which in turn were also assembled and treated as a Ribosomal RNA gene sequences referable to the three target single sequence from a single-source (GenBank AY686577). regions of amoebal DNA were amplified from 6 of the 13 lobsters Molecular Phylogenetic Analyses determined by histopathology to be infected with paramoebae. Sequences were obtained from antenna, antennal gland, gill and The single-source and overall consensus sequences represent- nerve cord tissues, with individual animals yielding sequences ing the SSU rRNA gene sequence of the amoebae from western

Figure 1. Location of primers used to amplify SSU rRNA gene sequences of paramoeba from tissues of infected lobsters and consensus SSU rRNA gene assembly. Primers were designed to amplify 3 overlapping subgenetic fragments including a 5؅-terminus (diagonal lines), an internal 1.3-kb region (vertical lines), and a 3؅-terminus (horizontal lines). Amplicon lengths are provided over each representative bar. The overall consensus SSU rRNA gene sequence of the paramoeba from western LIS lobster was assembled from overlapping subgenetic fragments; cross-hatched regions represent overlap between target regions. CHARACTERIZATION AND DETECTION OF LOBSTER PARAMOEBA 725

LIS lobsters shared 99% nucleotide sequence identity to each other and demonstrated 98% or greater nucleotide sequence identity in nucleotide BLAST searches with SSU rRNA gene sequences of Neoparamoeba pemaquidensis strains. Phylogenetic analyses (Fig. 3) also supported this result, robustly placing the two sequences as sister taxa to each other and in a clade consisting exclusively of Neoparamoeba pemaquidensis sequences. Within this clade, se- quences were very closely related but nonidentical. In fact, two nonidentical sequences were isolated, by different investigators, for each of three N. pemaquidensis strains, CCAP 1560/4, ATCC 30735 and ATCC 50172. The sequences from the ATCC strains are sister taxa, but the CCAP 1560/4 isolates are not (Fig. 3). Relationships among the strains of Neoparamoeba pemaquidensis were generally unresolved except for a clade that included the WLIS sequences plus those from strains CCAP 1560/4, CCAP 1560/5, and ATCC 50172. A clade composed of strains identified as N. aestuarina was also consistently recovered in the analysis. Distance and parsimony analyses, moreover, recovered a monophyletic PV clade, within which the Neoparamoeba se- quences were nested. Paramoeba eilhardi branched as a sister taxon to the neoparamoebae. Two other subclades were also re- constructed, one consisting of Korotnevella species, the other of the species Vexillifera armata and Pseudoparamoeba pagei.

PCR Detection and Characterization of Paramoeba from 1999 Lobsters

Primer pairs were selected based on comparative sequence alignments of PV amoebae and tested separately against genomic DNA of PV amoebae and hosts (lobster, blue crab and sea urchin) to determine possible combinations for nested PCR (Fig. 4A). The first round primer pair, Para A & Para C, amplified all the PV tested with the exception of Pseudoparamoeba pagei CCAP 1566/2 and Neoparamoeba pemaquidensis ATCC 50172, whereas second round primer pair, Para B & Mullen B2, amplified all PV amoebae tested with the exception of Korotnevella hemi- stylolepis ATCC 50804. Neither of these first or second round primers amplified genomic DNA from lobster, blue crab and sea urchin, which had been quality control tested for its ability to support PCR by successful amplification of SSU rRNA gene se- quences in preliminary PCRs. The resultant nested PCR protocol utilizing Para A/Para C and Para B/Mullen B2 generated a 165-bp product from the PV amoebae tested without cross-reactivity with genomic DNA of lobster, blue crab or sea urchin. Of the 170 paraffin blocks that underwent DNA extraction, -Figure 2. Amplification of all 3 SSU rRNA target regions (e.g., 5؅ representing 77 lobsters collected during the 1999 die-off, 39 sup- terminus, internal 1.3-kb fragment and 3؅-terminus) from gill of one western LIS lobster, ID#01-9828-7. gl = gill; ag = antennal gland; a = ported PCR amplification of a 440-bp eukaryotic SSU rRNA gene antenna; nc = nerve cord; NPC = Neoparamoeba pemaquidensis CCAP sequence during quality control testing. Of these 39, representing 1560/4 plasmid cloned SSU rRNA gene. A, Lane 1, PGEM marker; 30 lobsters, 11 yielded 165-bp amplicons using the nested PCR Lane 2, reagent blank (1st round); Lane 3, reagent blank (2nd round); protocol and 28 did not (Fig. 4B). All 39 paraffin blocks contained Lane 4, negative control (urchin); Lane 5, negative control (lobster); tissues that were histologically positive for parasome-containing Lane 6, positive control (NPC); Lanes 7–9, 01-9828-7 (gl); Lanes 10– amoebae. 12, 01-9828-7 (ag); Lanes 13–15, 01-9828-7 (nc). B, Lane 1, 50bp Nucleotide sequences of the 11 amplicons were determined marker; Lane 2, reagent blank (1st round); Lane 3, reagent blank (2nd individually and independently of each other. Single nucleotide round); Lane 4, negative control (lobster); Lane 5, positive control differences at several sites allowed separation of the 11 nested (NPC); Lane 6, 01-9828-7 (gl); Lane 7, 01-9828-7 (ag); Lane 8, 01- PCR product sequences into 6 groups. The nucleotide sequence 9828-7 (nc); Lane 9, 01-9828-7 (a); Lane 10, 01-9828-8 (ag); Lane 11, 01-9828-8 (nc); Lane 12, 01-9828-8 (a); Lane 13, PGEM marker. C, identity between the groups was 98% to 99%, with the exclusion Lane 1, PGEM marker; Lane 2, reagent blank (1st round); Lane 3, of one sequence that was 94% to 96% identical with the other 1999 reagent blank (2nd round); Lane 4, negative control (urchin); Lane 5, nested products (Fig. 5). The consensus nucleotide sequence, gen- negative control (lobster); Lane 6, positive control (NPC); Lanes 7–9, erated by alignment of all 11 samples, and the sequences of all six 01-9828-7 (gl); Lanes 10–12, 01-9828-7 (ag); Lanes 13–15, 01-9828-7 (nc). “groups,” from which the consensus was constructed, were most 726 MULLEN ET AL.

Figure 3. Distance-based phylogram reconstructed using SSU rRNA gene sequences representing the amoeba from western LIS lobster and gymnamoebae from paramoebid/vexilliferid and vannellid clades. Numbers at nodes indicate confidence values after 1000 bootstrap replicates for parsimony and distance analyses, respectively. Nodes lacking numbers had values below 50%, or were not represented in the bootstrap consensus trees. Both the amoeba from 01-9828-7-gill (single source) and the amoeba from WLIS lobster (overall consensus) branch with strains of Neoparamoeba pemaquidensis. closely related on the basis of BLAST search scores and percent most closely related to the consensus sequence from 2001 to 2002 nucleotide identity with strains of N. pemaquidensis (93% to 98% lobsters (Fig. 3). The consensus nucleotide sequence from the identity), the next nearest matches being with strains of N. aes- 1999 samples had 95% identity with the single- and multi-source tuarina (<90% identity). The closest matches (98% identity) were SSU rRNA gene sequences from the 2001 to 2002 lobsters; how- to sequences from CCAP strains 1560/4 and 1560/5, the sequences ever, the sequence of the exceptional group, group 4 (Fig. 5), from CHARACTERIZATION AND DETECTION OF LOBSTER PARAMOEBA 727

Figure 4. Paramoebid-Vexilliferid (PV) nested PCR primer alignments and PCR products from archival 1999 formalin-fixed paraffin-embedded (FFPE) lobster tissues. A, Comparative sequence alignments used to design PV primers. Dots (.) represent sites wherein there is exact nucleotide identity with the lobster parasite and spaces (-) represent gaps due to the absence of nucleotides at those sites. B, PCR products typical of FFPE tissues of histologically infection-positive lobsters. Lanes 1&2, 99-8438-2B; Lanes 3–5, 99-8438-3A; Lane 6, 50 bp marker; Lane 7, reagent blank; Lane 8, negative control (lobster); Lane 9, positive control (N. pemaquidensis CCAP 1560/4 plasmid cloned SSU rRNA gene); Lanes 10–12, 99-8438-5B; Lanes 13–15, 99-7374-21H; Lanes 16–18,99-8438-3B; Lanes 19&20, 99-8438-4B. 728 MULLEN ET AL.

Figure 5. Sequences of nested PCR products from 1999 lobsters infected with parasome-containing amoebae can be separated into 6 distinct groups based on nucleotide differences at a small number of sites (bold letters). Sequences have 98% to 99% nucleotide identity, with the exception of the group 4 sequence, which has 94% to 96% identity with the other nested PCR products and 98% identity with the amoebal SSU rRNA sequences amplified from 2001 to 2002 western LIS lobster. Nucleotide differences were resolved by majority rule (bold italic letters) in the consensus sequence, which had 98% nucleotide identity with the SSU rRNA gene sequences of Neoparamoeba pemaquidensis stains CCAP 1560/4 [AF371969.1] and CCAP 1560/5 [AF371970.1], and 95% nucleotide identity with the amoebal SSU rRNA sequences amplified from 2001 to 2002 western LIS lobster. the 1999 PCR products had 98% identity with the 2001 to 2002 belong to the species Neoparamoeba pemaquidensis, a species amoebal SSU rRNA sequences. already known to be pathogenic to other shellfish species and to certain marine finfish. No sequences belonging to other species of DISCUSSION amoebae were obtained, even though the protocols used were ca- pable of amplifying at least other members of the amoebal PV Identity of the Lobster Amoeba clade. Instead, partial SSU rRNA sequences from 1999 and 2001 All DNA sequences obtained, whether from paraffin-embedded to 2002 lobsters had the highest degree of nucleotide identity with tissues collected from 1999 lobsters or from fresh tissues obtained the same subset of N. pemaquidensis strains, CCAP strains 1560/4 from lobsters collected in 2001 to 2002, indicate that the amoebae and 1560/5. The occurrence of Neoparamoeba SSU rRNA se- present in western LIS lobsters during and after the 1999 die-off quences in 1999 and 2001 to 2002 lobster, and their relatively high CHARACTERIZATION AND DETECTION OF LOBSTER PARAMOEBA 729 nucleotide identity (95% to 98%), is evidence of continued, en- Infectivity of the Amoeba demic, infection of a subpopulation of western LIS lobster in the Only in the case of amoebic gill disease of salmon has disease years after the die-off with one or more similar strains of N. pe- been recreated through experimental exposure to cultured Neopar- maquidensis. amoeba pemaquidensis (Zilberg et al. 2001); however, Mullen et The Neoparamoeba sequences isolated from lobster tissues are al. (2004) induced paramoebiasis in healthy Maine lobsters closely related but nonidentical. The most straightforward inter- through cohabitation with moribund, “limp,” lobsters obtained pretation of this result is that several closely related N. pe- from western LIS, thus providing circumstantial evidence for the maquidensis strains have participated in the lobster infection, as pathogenicity of one or more LIS strains of N. pemaquidensis to many as six in the 1999 samples. This interpretation, however, lobsters and the ability of the disease to be transmitted from one assumes that all populations of nuclear-encoded small-subunit ri- lobster to another. From the results of this study, and examination bosomal RNA genes in a clonal isolate of N. pemaquidensis are of N. pemaquidensis infections in other animals, some additional identical in primary sequence. If sequence microheterogenity ex- inferences are possible. ists, possibly because SSU rDNA loci have not evolved in concert, It is unlikely that strains of N. pemaquidensis can be separated as is the case in certain other protists (e.g., Reddy et al. 1991, into “pathogenic” and “nonpathogenic” cohorts. In the phyloge- Scholin et al. 1993), then the observed sequence variation will netic trees reported here, strains identified as having been isolated overstate the number of strains present. The isolation of two non- from animals with paramoebiasis do not cluster together, but rather identical SSU rRNA gene sequences from each of three cultured N. are scattered within the clade. Moreover, in both the sea urchin pemaquidensis strains suggests that rRNA gene sequence micro- (Jellett & Scheibling 1988b, as Paramoeba invadens) and the fin- heterogeneity is commonplace in this species, and ongoing work fish (e.g., Adams & Nowak 2004) systems, virulence of cultured (O’Kelly & collaborators, unpublished) is consistent with this in- amoebae is lost over time. Adams and Nowak (2004) speculated terpretation. The number of amoebal strains infecting western LIS that virulence factors are downregulated in the amoeba when they lobster during the 1999 die-off, and thereafter, may have been as are no longer exposed to target host cells/tissues. Jellett and many as 6 or as few as one. Scheibling (1988b) reported that the loss of virulence was in part Species of Neoparamoeba, including N. pemaquidensis, have dependent on the bacterial food source used to maintain the cul- been characterized at the ultrastructural level by the presence of tures and that virulence could be restored by passaging the amoe- glycostyles on the cell surface (Cann & Page 1982, Page 1987). bae through sea urchins. In this view, pathogenesis of N. pe- Glycostyles are absent from the surface of the lobster-borne maquidensis is an inducible phenomenon, and any strain of N. amoeba (Mullen et al. 2004), as they are from the blue crab-borne pemaquidensis is potentially pathogenic to any susceptible host. (Perkins & Castagna 1971) and urchin-borne (Jones 1985) amoe- bae. This finding would appear to be inconsistent with the mo- Progression of Paramoebiasis in Western Long Island Sound lecular results. However, O’Kelly (unpublished) isolated two strains of amoebae from moribund sea urchins in the Gulf of During the mass mortality event of 1999, paramoebae were Maine in the autumn of 2002. Partial SSU rRNA gene sequences identified in neural hemocytic infiltrates in 29 of 31 animals (94%) from these amoebae belong to N. pemaquidensis, with high (98% examined from October 27 to November 10, 1999, and 11 of 38 to 99%) BLAST similarity scores to a sequence obtained from (29%) animals examined from November 18 to December 3, 1999 strain PA027, a Tasmanian strain isolated from salmon gill lesions. (Mullen et al. 2004). However, only approximately 5% of lobsters These amoebae produce glycostyles in monoprotist culture. This examined during the 2000 to 2002 sampling period for this study result suggests that Neoparamoeba amoebae can suppress the pro- were identified as infected with paramoebae by histologic exami- duction of glycostyles when they are pathogenic within tissues, nation. Furthermore, although qualitative and subjective in its as- and therefore the glycostyle character is not reliable for taxonomic sessment, densities of paramoeba infections in 2000 to 2002 lob- or clinical diagnosis of tissue-invasive stages. It also suggests that ster appear to have been much less than those described in 1999 the species name Paramoeba invadens Jones 1985, created for the (Mullen et al. 2004). Nevertheless, the observation that 5% of pathogen of sea urchins, is a junior synonym of N. pemaquidensis. lobsters sampled after the die-off were infected supports the pre- liminary proposition that this parasite represents an emerging dis- Origin of the Amoeba in Western Long Island Sound ease in American lobster. Low prevalence and reduced density of Neoparamoeba pemaquidensis is considered an amphizoic pro- paramoeba infections in 2000 to 2002 lobster were factors that tozoon, one that is normally free living but becomes pathogenic limited intersample diversity in generation of single-source PV under certain circumstances (Scholz 1999). The species was ini- SSU rRNA gene sequences and were the principal reasons for tially isolated as a free-living bacterivorous amoeba from surface adopting a nested PCR approach to direct amplification from in- sediments (Page 1970), and it is considered to be among the most fected lobster tissues. An accurate prevalence of paramoebiasis in ubiquitous of marine gymnamoebae (Page 1983). In an examina- western LIS after the 1999 mortality is not known and may be tion of the exoskeletal microbiota of lobsters in the Gulf of Maine precluded by an inability to properly assess subpopulations of during July and August 2003, N. pemaquidensis strains were iso- infected lobster not reliably sampled by conventional trawl survey lated frequently (O’Kelly, unpublished). It is possible that N. pe- (e.g., dead or dying lobster in burrows). maquidensis strains were not only common in western LIS during Preliminary Model for the Paramoebiasis Outbreak in Western Long 1999 but were intimately associated with lobsters. Island Sound Sequences of western LIS lobsters were most closely related to sequences from two strains of N. pemaquidensis isolated from Douglas-Helders et al. (2003) examined environmental factors sediment samples in Wales. It is likely, however, that this circum- associated with amoebic gill disease in salmonids. They found that stance reflects undersampling of this species in sequence datasets, Neoparamoeba densities increased with temperature and with and unlikely that it represents strain migration from one locality to numbers of bacteria in the water column, and that the numbers of another. bacteria in the water column also increased with temperature. 730 MULLEN ET AL.

Summer water temperatures in western LIS in 1999 were well death of a subpopulation of lobsters in western LIS during the 1999 above the 30-y-mean, providing ideal conditions for Neopar- die-off, and that N. pemaquidensis is present in western LIS lobster at amoeba growth (cf. Scheibling & Hennigar 1997) and borderline low levels after the die-off. Still to be determined are the identification conditions for survival and growth of lobsters. Moreover, lobster of virulence factors in pathogenic Neoparamoeba, the transcriptional populations in 1999 were at an historic maximum, suggesting that regulation of putative virulence-associated genes and the full spec- lobsters were not only heat-stressed but also crowded. trum of immunomodulatory environmental conditions leading to Based on this evidence, one possible model accounting for the the inability of lobsters to cope with Neoparamoeba infection. occurrence of paramoebiasis as a contributing factor to the 1999 ACKNOWLEDGMENTS lobster die-off is the following. Under conditions of maximum crowding, maximum temperature stress and maximum Neopar- The authors appreciate the assistance of Drs. Richard French amoeba pemaquidensis growth experienced by western LIS lob- and Sylvain De Guise in sharing facilities, samples and resources sters in the summer of 1999, one or more strains or groups of toward completion of this research, acknowledge Michael Peglar, strains of the amoeba, perhaps those already associated with the Thomas Nerad and Patrick Gillevet for their support of the se- lobster exoskeleton, were able to establish competent infections in quencing efforts of the PV amoeba and are grateful to Meghan stressed lobsters. Further environmental insults, such as those cir- Tucker and Jennifer Maratea for assistance in postmortem prepa- cumstantially associated with the major rain events of August and ration of lobster and to Andrew Draghi and Spencer Russell for September 1999, contributed to a larger percentage of stressed and technical support (University of Connecticut). They thank Wendy crowded lobsters susceptible to paramoebal infection and presum- Bellows for amoebal cultivation and DNA isolation and sequenc- ably, a subpopulation of lobsters that died during the mass mor- ing, Dr. Brian Wysor for DNA sequencing and analysis, and Glo- tality died with paramoebiasis (Mullen et al. 2004). The exact rya Laughton for field and culture work related to lobster exoskel- percentages of lobsters with paramoebiasis during the 1999 die-off eton sampling (O’Kelly Laboratory). They also thank Harry Ya- will never accurately be known, given the practical discrepancy malis of the Connecticut Department of Environmental Protection between the real numbers of lobsters sampled and the theoretical for his tireless support in the management of grant-related issues numbers necessary to achieve significant prevalence and incidence impacting this research. Funding for this work was provided by the values relative to the geographic area of LIS and the number of Connecticut Department of Environmental Protection under Long dead lobsters reported, which was in the millions. Lower levels of Island Sound Research Fund Grant No. CWF 333-R to S. Frasca; paramoebiasis in subsequent years, 2000 to 2002, reflect declining and by the Connecticut Sea Grant College Program, Grants No. transmission due to a combination of factors. First, reduced lobster LR/LR-4 to R. Gast and No. LR/LR-5 to P. Gillevet and C. population densities after the die-off have presumably resulted in O’Kelly, through the US Department of Commerce, National fewer encounters between lobsters and fewer opportunities for Oceanic and Atmospheric Administration (NOAA), Award transmission. Second, the relative reduction in host animals may NA16RG1364. Any opinions, findings, and conclusions or recom- have progressively altered survival pressures on strains of the mendations expressed in this publication are those of the author(s) amoebae, leading to a reduction in the expression of virulence- and do not necessarily reflect the views of the Connecticut De- associated genetic traits. This model, however, supports the asser- partment of Environmental Protection or the National Oceanic and tion that Neoparamoeba pemaquidensis is the proximate cause of Atmospheric Administration, US Department of Commerce.

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