Expression of amino acid transporter genes in ... - Springer Link

Polar Biol (2013) 36:1257–1267 DOI 10.1007/s00300-013-1345-1

ORIGINAL PAPER

Expression of amino acid transporter genes in developmental stages and adult tissues of Antarctic echinoderms Scott L. Applebaum • David W. Ginsburg Charles S. Capron • Donal T. Manahan

•

Received: 31 October 2012 / Revised: 13 May 2013 / Accepted: 14 May 2013 / Published online: 1 June 2013 Ó Springer-Verlag Berlin Heidelberg 2013

Abstract Epithelial cells in the body wall of adult and developmental stages of marine invertebrates absorb dissolved organic material directly from seawater. Despite over a century of study, little is known about the molecular biological mechanisms responsible for this transport process. Previous studies on embryonic and larval Antarctic echinoderms show that amino acid uptake could provide an important supplement of metabolic substrates. In the present study, partial cDNA sequences of 11 putative amino acid transporter genes were isolated from six species of Antarctic echinoderms including the Antarctic sea stars Acodontaster hodgsoni, Diplasterias brucei, Odontaster meridionalis, Odontaster validus, and Perknaster fuscus, and the Antarctic sea urchin Sterechinus neumayeri. Conserved domains of cDNA-deduced amino acid sequences characterized these genes as being members of a family of amino acid transporters (solute carrier family 6). Expression of these genes was detected throughout embryonic and larval development of two species that have contrasting developmental modes (A. hodgsoni: lecithotrophic; O. meridionalis: planktotrophic). In all six species studied, the expression of amino acid transporter genes was detected in tube feet and digestive organs of adult animals, demonstrating that members of a single amino acid transporter gene family are expressed during the entire life history of a marine invertebrate. The identification of these genes is an important step toward developing a mechanistic S. L. Applebaum (&) C. S. Capron D. T. Manahan Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-0371, USA e-mail: [email protected] D. W. Ginsburg Environmental Studies Program, University of Southern California, Los Angeles, CA 90089-0036, USA

understanding of amino acid transport capacities in Antarctic marine invertebrates. Keywords Antarctica Echinoderms Amino acid transporters Gene families

Introduction The highly seasonal nature of light availability in Antarctica limits primary production in the Southern Ocean to a narrow temporal window (Rivkin 1991; Clarke and Leakey 1996). Antarctic zooplankton that depend on microalgae as a primary food source must therefore be capable of enduring delays or fluctuations in food availability. The likely mismatch between feeding competence and food availability is a central focus in the study of the survival of many species of marine planktonic organisms (Edwards and Richardson 2004; Hays et al. 2005; Ji et al. 2010). For instance, larvae of some Antarctic echinoderms reach feeding stages before the annual phytoplankton bloom (Rivkin et al. 1986; Bowden et al. 2009). Experiments on Antarctic echinoderms have shown that their larval stages can maintain organic biomass for months in the absence of algal food, despite having metabolic rates that theoretically should deplete their endogenous biochemical stores (Shilling and Manahan 1994; Marsh et al. 1999). Studies of the ability of Antarctic larval echinoderms to transport dissolved amino acids from seawater have shown that *30 % of metabolic rate (Shilling and Manahan 1994) could be supported by the transport of these substrates from the low (50 nM) naturally occurring concentrations found in Antarctic waters (Welborn and Manahan 1991). When compared with temperate species, Antarctic echinoderm larvae have a greater capacity for amino acid transport relative to

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their metabolic rate, suggesting a unique transport physiology for supplemental energy intake (Shilling and Manahan 1994). The ability to absorb dissolved organic matter across the epithelial cells of their body wall is characteristic of most marine invertebrates (Stephens and Schinske 1961; Jorgensen 1976; Stephens 1988; Wright and Manahan 1989; Manahan 1990; Gomme 2001). Dissolved organic material in seawater is composed of a highly complex and variable suite of molecules (Benner 2002). Studies on the transport of dissolved organic material across body wall epithelia of marine invertebrates extend back over a century (Pu¨tter 1909), and a large percentage of these studies have focused on the transport of dissolved amino acids (op. cit.). Amino acids represent a significant component of the total pool of dissolved organic material, are readily identifiable and measureable in seawater, and are molecules with welldefined biological roles in metabolism and biosynthesis. Radiolabeled tracers and highly sensitive chemicaldetection methods (such as high-performance liquid chromatography) for identifying and quantifying individual substrates, combined with the culturing of bacteria-free larvae, allowed for convincing experimental demonstrations of the net uptake by marine invertebrates of dissolved amino acids from sub-micromolar concentrations typical of natural seawater (Manahan et al. 1982, 1983). Activation of amino acid transport occurs early in development—within minutes after fertilization (Epel 1972; Allemand et al. 1984). Characterization of transport kinetics during larval development has shown that different transport systems (defined by substrate affinities, Kt, and transport capacities, Jmax) contribute to the influx of amino acids from seawater (Epel 1972; Manahan et al. 1989). Progress toward understanding the molecular biological mechanism of transport has, however, been limited by a lack of knowledge about the identity of the genes underlying these transport processes. Recently, three amino acid transporter genes (SpAT1, SpAT2, and SpAT3), members of the solute carrier family 6 [SLC6, a.k.a. sodium-dependent neurotransmitter transporter family, SNF; or neurotransmitter sodium symporter family, NSS (Saier 1999; He et al. 2009; Boudko 2010)], were characterized in embryos and larvae of a temperate species of sea urchin, Strongylocentrotus purpuratus (Meyer and Manahan 2009). The protein encoded by the SpAT1 gene was localized to the ectoderm of larvae—body wall epithelial cells that are in direct contact with the seawater environment (Meyer and Manahan 2009). The question remains as to the developmental and physiological benefit these epithelial amino acid transporters might provide, as well as their relative contribution to metabolic stores and the maintenance of osmotic state (Jorgensen 1976; Gomme 2001; Meyer and Manahan 2009). Further

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analysis of the genes responsible for amino acid transport will be a critical step toward understanding the biological functions of this process in marine invertebrates. Molecular biological analysis and genomic science is dramatically increasing our understanding of biological processes in Antarctic organisms (Peck et al. 2005; Cheng and Detrich 2007; Giordano et al. 2012). In this study, we present the discovery and characterization of 11 putative amino acid transporter genes in six species of Antarctic echinoderms. We show that this family of genes is expressed in early embryos and larvae of species with different developmental modes (lecithotrophic and planktotrophic), as well as in adult tissues and organs. The identification of this gene family is a critical step toward a mechanistic understanding of transport capacities of dissolved organic material by developmental stages of Antarctic marine invertebrates.

Materials and methods Adult individuals of the Antarctic sea urchin (Echinoidea: Sterechinus neumayeri) and five Antarctic sea star species (Asteroidea: Acodontaster hodgsoni, Diplasterias brucei, Odontaster meridionalis, Odontaster validus, and Perknaster fuscus) were collected by SCUBA divers from two shallow-water sites in McMurdo Sound, Antarctica (near McMurdo Station and Cape Evans). Animals were maintained in aquaria at McMurdo Station, supplied with fresh seawater, and held at a constant temperature of -1.0 °C. Adult tissues (tube feet and digestive tract) were excised and immediately frozen in liquid nitrogen or preserved in RNA laterÒ (Ambion, Inc., Austin, Texas), followed by storage at -80 °C. Developmental stages of A. hodgsoni and O. meridionalis were collected from cultures reared in the aquarium at McMurdo Station. A full description of broodstock collection, spawning, and rearing of these embryos and larvae is provided in Ginsburg and Manahan (2009). At several times during development, aliquots of between 50 and 100 individuals (depending on species and stage of development) were collected from cultures and frozen at -80 °C. Photomicrographs were taken to document embryonic and larval development of A. hodgsoni and O. meridionalis through 60 and 135 days post-fertilization, respectively. Images were taken using a Zeiss Axioscope microscope supplied with a Kontron KS 300 image analysis system (Kontron Elektronik, Munich, Germany). For all samples, RNA extraction was performed using TRIzolÒ Reagent (Life Technologies Corp., Carlsbad, California) according to the manufacturer’s instructions. Sample disruption was achieved by shaking for 3 min at 30 Hz in a Tissue Lyser II shaker (Quiagen, Inc., Valencia,

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California) in the presence of ceramic beads (Lysing Matrix D, MP Biomedicals, Solon, Ohio). Extracted RNA was treated with RNase-free DNase (Promega Corp., Madison, Wisconsin) for 1 h and re-extracted with TRIzolÒ Reagent. Total RNA (1 lg) was primed with random hexamers and reverse transcribed to create cDNA using MuMLV reverse transcriptase (RT; Promega Corp.) according to the manufacturer’s protocol. Prior to reverse transcription, RT-negative control reactions were prepared for each RNA sample. Failure of polymerase chain reaction (PCR) to amplify actin (primers in Table 1) from RTnegative templates was used to verify the absence of contaminating genomic DNA. Transcripts of putative amino acid transporters were amplified from reverse-transcribed cDNA templates by PCR using degenerate primers (forward primer = GTC TGG [A/C]G[A/C/G] TT[C/T] CC[C/T] TAC CT; reverse primer = GC[A/C/G/T] GTG AA[A/G] TA[G/C]

AC[A/C/T] ACC TT[G/T] CC) designed from previously characterized amino acid transporters cloned from a temperate species of sea urchin (Meyer and Manahan 2009). Primer design was also based on targeting regions of SLC6 genes that are highly conserved within the gene family, as well as across diverse phyla (Beuming et al. 2006). PCR amplifications intended for cloning and sequencing were conducted using Platinum PCR Supermix High Fidelity reagent (Life Technologies Corp.), according to the manufacturer’s instructions. Thermocycling conditions were as follows: 40 cycles of denaturing at 94 °C for 30 s; annealing over a gradient from 40 to 55 °C for 30 s; and extension at 68 °C for 60 s. Reaction products were visualized by agarose gel electrophoresis with ethidium bromide staining, and products of an appropriate size were gel purified, cloned into the pCR2.1-TOPO (Life Technologies Corp.) or pGEM (Promega Corp.) vector, and transformed into either JM109 (Promega Corp.) or TOP10E (Life

Table 1 Species-specific primers and size of diagnostic amplicons generated by reverse transcriptase polymerase chain reactions used to detect expression of putative SLC6 amino acid transporters and actin genes in adult tissues, embryos, and larvae of Antarctic echinoderms Species

Gene

Primer sequence (50 -30 )

A. hodgsoni

AhATa

F: GCC TGC CCT TGT TCT TTC TCG

Diagnostic amplicon size (base pairs) 500

R: AGC CAG TTG CCA CTG TAC CTC AhATb

F: CTG GAC GGC TTT CTT CCT CTT C

256

R: TGG TCC CAG ATC TTC GAT ACC C D. brucei

DbAT

F: TAC AGC AGC AGT GGA CCC ATA C

462

R: CAG TGC CAA CTC CCA GAT GAT G O. meridionalis

OmATa

F: ACT TCA TCG CCT TGG CCT TTG

362

R: ACT ATC CAT CTC AAC CCC CGT C OmATb

F: AGG ACC AGT CAC CAT TTG GAG G

420

R: ATA CAG AGG AAG ACA ACG GCC C O. validus

OvATa

F: TGT TCA GAG GTG TTG GCT ACG G R: TGA TGA TCC AGG CAA GGA GGA G

447

OvATc

F: ATG CCG CTG TTC TTT CTG

384

R: GCT CTT GTC CAA GAC AAC C P. fuscus

PfATa

F: TCT TGA TGC TGA TCC TGG CTG C

392

R: AGT TCC ATT GCC CGT TCT GTT C PfATb

F: AGC CTT TCG CAA GAT TTG TCC C

324

R: AAC AGT TCC CAT CGC ATC TCC C S. neumayeri

SnATa

F: TCG TTC ACC AGT GAT CTC C

399

R: CTT TCC AGA TGA CTT GAT TCC C SnATb

F: TGT TGT GCC TCG CCG GTG TT

528

R: AAG CGA AGA GAA GGC ACA G A. hodgsoni

Actin

F: CAG AGC AAG AGA GGT ATC CTC A

*820

R: GGA GCA ATG ATC TTG ATC TTC

O. meridionalis O. validus S. neumayeri D. brucei P. fuscus

Actin

F: CAG AGC AAG AGA GGT ATC C

*860

R: ATG GAG CCT CCG ATC C

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Technologies Corp.) chemically competent Escherichia coli cells. Colonies from which inserts of the appropriate size were amplified by PCR were grown overnight in standard LB/antibiotic media. Plasmids were isolated with the PureYield Plasmid Miniprep System (Promega Corp.). Plasmid inserts were sequenced by the University of Southern California DNA Core facility using an ABI 3730 DNA Analyzer automated sequencer. At least three clones of each transcript were sequenced with 2-times, or greater, coverage to determine a consensus sequence. Additional nucleotide sequences of O. validus were obtained from searches of a transcriptomic database (provided by Dr. Ken Halanych of Auburn University). Partial transcripts obtained from O. validus by reverse transcription polymerase chain reaction (RT–PCR), as well as S. purpuratus sequences (SpAT1, EF538763.1; SpAT2, EF538764.1; SpAT3, EF538765.1) were used as queries in Basic Local Alignment Search Tool (BLAST, Altschul et al. 1990) searches of the transcriptomic database. A contiguous nucleotide sequence retrieved from this database overlapped with the O. validus Amino acid Transporter c (OvATc) sequence obtained by amplification with degenerate primers and contained additional 50 sequences. A second nucleotide sequence fragment was identified in the transcriptomic database that was highly similar to a region near the 30 end of the SpAT1 open reading frame. A reverse primer (GAC TGT CCA GTC CGA GGG TAA GCA ACA TG) specific to this fragment and a forward primer (TGT TCA GAG GTG TTG GCT ACG G) annealing within the O. validus Amino acid Transporter a (OvATa) sequence isolated by RT–PCR were used to amplify additional sequences from the 30 region of the transcript. Species-specific PCR primers were designed to amplify a portion of each transporter sequence from each species studied (Table 1). These diagnostic PCR products were used to assess the presence or absence of transcripts for amino acid transporters in tube feet and digestive tract. Expression of transporter genes was also assessed in embryos and larvae of A. hodgsoni and O. meridionalis, during different stages of development. RNA isolation and preparation of cDNA templates were conducted as described for adult tissues. PCRs were undertaken with GoTaq Hot Start Polymerase (Promega Corp.) reagents according to the manufacturer’s protocol. Thermocycling conditions were as follows: 94 °C for 120 s; 40 cycles at 94 °C for 30 s; 30 s at 60 °C; 60 s at 72 °C; 5 min at 72 °C for all primers and species combinations, with the exception of OvATa, which was amplified at an annealing temperature of 55 °C. The quality of reverse-transcribed cDNA templates was validated for each sample by PCR amplification of Actin (Table 1). Thermocycling conditions for Actin PCR were the same as those described for the amino acid transporter genes, with the exception of annealing

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temperature (52 °C for P. fuscus and 55 °C for all other species). PCR-amplified products were resolved by agarose gel electrophoresis and stained with ethidium bromide. Consensus nucleotide sequences were translated to deduced amino acid sequences and used to query the National Center for Biotechnology Information (NCBI) Conserved Domain Database Search (Marchler-Bauer et al. 2011). Similarity to other known sequences was determined by BLAST (BLASTX program) search of the NCBI non-redundant protein database. Amino acid multiple sequence alignments were created using ClustalW, followed by manual adjustment of the alignment in MacVector software (MacVector Inc., Cambridge, UK). Alignment of sequences with human and prokaryotic SLC6 transporter homologues was according to the structurebased alignment of Beuming et al. (2006).

Results Eggs of A. hodgsoni averaged 505 lm in diameter. Under the culturing conditions used in this study, embryos of A. hodgsoni reached the eight-cell stage by 2 day post-fertilization (dpf), wrinkled blastulae by 5 dpf, and gastrulae by 15 dpf, and non-feeding bilobed larvae appeared in the cultures between 31 and 50 dpf (Fig. 1). As evident in Fig. 1, eggs of O. meridionalis were smaller than those of A. hodgsoni, averaging 203 lm in diameter. By 4–5 dpf, blastulae had formed and gastrulae were observed after 10 dpf. Stomodaeal breakthrough occurred around 26 dpf, at which time early-stage bipinnaria larvae were competent to feed. By 135 dpf, feeding larvae reached the brachiolaria stage (Fig. 1). Using degenerate primers in RT–PCR, partial transcripts encoding putative amino acid transporters were successfully amplified and cloned from the reverse-transcribed RNA of all six species of Antarctic echinoderm. Additional 30 and 50 sequences of OvATa an OvATc, respectively, were isolated as described in ‘‘Materials and Methods.’’ Two transcripts were isolated from A. hodgsoni (AhATa, 625 base pairs; AhATb, 553 bp), O. meridionalis (OmATa, 625 bp; OmATb, 553 bp), O. validus (OvATa, 1,324 bp; OvATc, 982 bp), S. neumayeri (SnATa, 634 bp; SnATb, 619 bp), and P. fuscus (PfATa, 619 bp; PfATb, 511 bp), in addition to one transcript from D. brucei (DbAT, 622 bp), for a total of 11 putative amino acid transporter genes. All sequences are from protein-coding regions, with exception of OvATc, which includes 256 bp of 50 untranslated sequence. Names and notations assigned to genes (i.e., ATa, ATb, and ATc) denote the order in which sequences were isolated. The sequences isolated in this study have been deposited in GenBank under the following accession numbers: AhATa (KC155345), AhATb (KC155337), DbAT (KC155336),

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Fig. 1 Developmental stages of Acodontaster hodgsoni and Odontaster meridionalis and the detection of putative SLC6 amino acid transporter gene expression during development. A. hodgsoni: (from left to right) fertilized egg with raised fertilization membrane, \1 h post-fertilization (hpf); four-cell stage, 1 d post-fertilization (dpf); morula, 3 dpf; wrinkled blastula, 5 dpf; late blastula, 10 dpf; gastrula, 15 dpf; early-stage larva, 31 dpf; bilobed larva, 50 dpf. O. meridionalis: (from left to right) fertilized egg with raised fertilization

membrane, \1 hpf; morula, 3 dpf; blastula, 5 dpf; gastrula, 12 dpf; late gastrula showing opening of stomodaeum, 26 dpf; bipinnaria larva, 44 and 82 dpf; early brachiolaria larva, 135 dpf. Scale bar applies to all images and represents 100 lm. Detection of expression of genes by reverse transcriptase polymerase chain reaction indicated by ?; ND indicates transcript not detectable (see Fig. 3 for analysis of polymerase chain reaction products by agarose gel electrophoresis)

OmATa (KC155335), OmATb (KC155344), OvATa (KC155343), OvATc (KC155342), PfATa (KC155341), PfATb (KC155340), SnATa (KC155339), and SnATb (KC155338). Searches of conserved protein domain databases indicate that all 11 transcripts contain conserved domains of the SLC6 superfamily (all E-values \1910-45). BLASTX searches of the NCBI non-redundant database using deduced amino acid sequences for the Antarctic species show the highest similarity to SLC6 proteins from the purple sea urchin (S. purpuratus), the acorn worm (Saccoglossus kowalevskii), and the Pacific oyster (Crassostrea gigas) (Table 2). When BLASTX searches were constrained to only database entries for the temperate sea urchin, S. purpuratus, all 11 Antarctic putative transporter genes were found to be most similar to one of four proteins, each of which is categorized as an SLC6 member—namely SpAT1 (ABU41322.1 or its equivalent prediction from the S. purpuratus genome, XP_799073.3), SpAT2

(ABU41323.1), SpAT3 (ABU41324.1), or predicted protein XP_780120.1. The deduced amino acid sequences AhATb, OmATb, and OvATc, all of which have high similarity to SpAT2, were nearly identical ([98.9 %) to one another over the degenerate primer-amplified region analyzed (Table 3). Identity of deduced amino acid sequences AhATa, OvATa, and PfATb relative to SpAT1 and XP_780120.1 differed by less than 1 % within the region of mutual overlap (Table 3). SpAT1 and XP_780120.1 themselves are 50 and 50.8 % identical over the degenerate amplified region and the entire coding region, respectively. For all species in this study, except O. validus, the isolated sequences were amplified with degenerate primers specific to two conserved regions of SLC6 transcripts. As described within ‘‘Materials and Methods,’’ OvATa and OvATc sequences extending beyond the region amplified with degenerate primers were successfully isolated from O. validus with the aid of information from a transcriptomic

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Table 2 Results of BLASTX search for putative SLC6 amino acid transporter transcripts isolated from Antarctic echinoderms Species

Gene

Closest BLAST match

*E-value

A. hodgsoni

AhATa

Strongylocentrotus purpuratus PREDICTED: sodium- and chloride-dependent

2 9 10-69

AhATb

Saccoglossus kowalevskii PREDICTED: sodium- and chloride-dependent glycine transporter 1-like (XP_002741096.1)

glycine transporter 2-like isoform 1 (XP_780120.1) 2 9 10-74 4 9 10-61

Strongylocentrotus purpuratus SpAT2, sodium-dependent alanine transporter 2 (ABU41323.1) D. brucei

DbAT


2 9 10-86

glycine transporter 2-like isoform 1 (XP_780120.1) O. meridionalis

OmATa

Strongylocentrotus purpuratus PREDICTED: sodium- and chloride-dependent glycine transporter 2-like isoform 1 (XP_780120.1)

6 9 10-72

OmATb

Saccoglossus kowalevskii PREDICTED: sodium- and chloride-dependent glycine transporter 1-like (XP_002741096.1)

2 9 10-75 8 9 10-61

Strongylocentrotus purpuratus SpAT2, sodium-dependent alanine transporter 2 (ABU41323.1) O. validus

OvATa

Strongylocentrotus purpuratus PREDICTED: sodium- and chloride-dependent glycine transporter 2 isoform 2 (XP_799073.3), genome prediction equivalent, or variant, of SpAT1 Saccoglossus kowalevskii PREDICTED: sodium- and chloride-dependent glycine transporter 1-like (XP_002741096.1)

OvATc

0.0 2 9 10-95 2 9 10-80

Strongylocentrotus purpuratus SpAT2, sodium-dependent alanine transporter 2 (ABU41323.1) P. fuscus

Crassostrea gigas Sodium- and chloride-dependent glycine transporter 2 (EKC19499.1)

2 9 10-54

Strongylocentrotus purpuratus SpAT1, sodium-dependent alanine transporter 1 (ABU41322.1)

1 9 10-45

Saccoglossus kowalevskii neurotransmitter transporter-like protein (ACY92591.1)

3 9 10-63


1 9 10-62

SnATa


2 9 10-121

SnATb


3 9 10-125

PfATa PfATb

S. neumayeri

glycine transporter 2-like isoform 1 (XP_780120.1) * The E-value for each result represents the number of matches with that degree of similarity expected by chance when querying a database containing the current number of sequences. For each transcript, the result with the highest similarity overall (i.e., lowest E-value) is given. The best match for any gene from the temperate sea urchin, Strongylocentrotus purpuratus, is also listed. Accession numbers for BLAST results are provided in parentheses

SpAT3

123

XP_780120.1

SpAT2

SpAT3

SpAT1

SpAT2

SnATb

SpAT1

SnATa

SnATb

PfATb

SnATa

PfATa

PfATb

OvATc

PfATa

OvATa

OvATc

OmATb

OvATa

OmATa

OmATb

DbAT

OmATa

AhATb

DbAT

AhATa

sequences identified in the temperate sea urchin, Strongylocentrotus purpuratus (columns shaded gray)

AhATb

Table 3 Percentage deduced amino acid sequence identity of region of mutual overlap between putative SLC6 amino acid transporter transcripts isolated from Antarctic echinoderms and the most similar

42.8

59.6

54.8

43.8

71.2

43.3

40.7

36.8

60.1

58.2

59.2

34.9

34.0

60.1

40.6

34.6

98.9

41.8

99.5

35.7

39.9

44.5

40.3

44.8

51.3

36.8

40.3

56.7

41.5

61.1

41.1

39.7

33.7

56.3

64.3

52.1

36.0

32.2

64.3

35.1

58.2

34.6

36.4

30.6

47.4

51.0

46.9

33.0

28.7

52.4

42.8

99.5

35.3

40.4

44.5

40.8

44.3

51.3

37.4

41.3

------------------------------

42.3

39.7

36.4

62.9

58.7

61.0

35.4

32.5

60.1

35.7

39.9

45.0

40.8

44.8

51.3

36.8

40.8

31.7

38.1

38.8

37.9

35.4

32.9

38.8

34.4

33.3

32.9

37.4

67.4

33.3

51.9

80.2

33.5

32.5

52.4

49.5

38.1

34.3

88.3

32.9

31.0

50.0

37.6

36.7 34.3

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database (access provided by Dr. Ken Halanych of Auburn University). Alignment of the amino acid sequences deduced from OvATa and OvATc with those deduced from SpAT1, SpAT2, SpAT3, a human SLC6 transporter (HsGlyt1), and a prokaryotic SLC6 orthologue (AaLeuT) reveals conserved structural elements (Fig. 2). The S1 central substrate-binding site, in which the transported substrate and co-transported Na? ions are positioned in AaLeuT, comprises transmembrane helical domains 1, 3, 6, and 8 (Kristensen et al. 2011). These segments are on average 54 % similar in OvATa and OvATc to AaLeuT, and 83 % similar to HsGlyt1. Lower sequence similarities are evident for the full nucleotide sequences analyzed, with mean values of 32 and 62 % to AaLeuT and HsGlyt1, respectively. Sequences isolated in this study were used to design species-specific primers for detection of transcript expression in tissues of adult, embryonic, and larval stages of Antarctic echinoderms (Table 1). Each of the 11 transcripts isolated was detected in the majority of tube foot and digestive tract RNA samples tested (Table 4), with the exception of OvATa transcripts, which were detected in only one of three RNA samples extracted from tube feet. During embryonic and larval development, both AhATa and AhATb were expressed in all developmental stages examined for A. hodgsoni, with the exception of 3- and 14-day-old embryos where AhATa was not detected (Figs. 1, 3). Both OmATa and OmATb were expressed in all developmental stages of O. meridionalis examined, with the exception of 78-day-old larvae in which OmATb was not detected (Fig. 1, 3).

Discussion Most phyla of marine invertebrates have the ability to absorb dissolved organic material (DOM) from seawater (Stephens 1988; Wright and Manahan 1989; Manahan 1990; Gomme 2001). Uptake of DOM can contribute a supplementary source of organic substrates for metabolism and also help maintain the intracellular concentrations of organic osmolytes (Wright and Manahan 1989; Manahan 1990; Gomme 2001). In Antarctic and temperate species, resistance to starvation and maintenance of organic mass has been observed in non-feeding (lecithotrophic) larvae as well as planktotrophic larvae deprived of particulate food, providing evidence for a role of exogenously dissolved nutrients (Shilling and Manahan 1994, Marsh et al. 1999; Moran and Manahan 2004; Meyer et al. 2007). For embryos and larvae of marine invertebrates that have high mass-specific metabolic rates and limited endogenous stores, the uptake of DOM may be an important exogenous

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source of energy to supplement metabolism when particulate foods are limiting. The experimental approaches applied in this field of study have usually employed supplementation or depletion of DOM from seawater to assess the importance of this mode of nutrition (Jaeckle and Manahan 1992; Johnson and Wendt 2007). Another approach to the study of the role of DOM update could be achieved, not by manipulation of the exogenous organic chemical environment, but by manipulating biological functions of the organism (e.g., specific transporters). By identifying and characterizing the genes responsible for transport of dissolved amino acid from seawater across body wall epithelial cells, experiments that specifically inhibit the function of these genes become possible (e.g., antibody inhibition, antisense RNA knockdown). Identification and sequencing of these genes is a necessary first step toward a fuller understanding of the physiological role of DOM transport across body wall epithelial cells in marine invertebrates. Here we report the partial mRNA sequences of 11 putative amino acid transporters from six Antarctic echinoderm species. Conserved domains within transcripts identify these genes as members of the SLC6 family of transporters (Fig. 2). Most of our understanding of the physiology and molecular biology of SLC6 transporters is derived from the study on mammalian intestinal, renal, and neural systems (Ferraris and Diamond 1989; Palacin et al. 1998; Pacha 2000; Chen et al. 2004; Bro¨er 2008; Stevens 2010). SLC6 proteins couple energy stored in sodium ion concentration gradients to the movement of amino acids, osmolytes, and neurotransmitters against concentration gradients (Boudko et al. 2005; Hoglund et al. 2005). In this study, the transcripts were expressed throughout the development of both lecithotrophic and planktotrophic Antarctic echinoderm species (A. hodgsoni and O. meridionalis, respectively). Transcripts were also present in tissues from digestive and hydrovascular systems (tube feet) of adults. The expression of these transporters in all life history stages suggests that this family of genes may have different functions in developmental stages, adults, and diverse tissues. For instance, transporters in the external body wall of embryos would be exposed to low amino acid concentrations in ambient seawater; transporters in the adult digestive system would experience higher concentrations of amino acids liberated from digested foods. The photomicrographs of developing A. hodgsoni and O. meridionalis (Fig. 1) extend upon the available information regarding the mode, timing, and pattern of development in these species (Bosch and Pearse 1990; Pearse et al. 1991; Stanwell-Smith and Peck 1998; Ginsburg and Manahan 2009). As illustrated in Fig. 1, eggs of A. hodgsoni are substantially larger than those of O. meridionalis. Eggs of A. hodgsoni have a *66-fold greater lipid content (Moore and

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Fig. 2 Amino acid alignment of SLC6 amino acid transporter orthologues from O. validus (OvATa, OvATc), S. purpuratus (SpAT1, ABU41322.1; SpAT2, ABU41323.1; and SpAT3, ABU41324.1), human (HsGlyt1, NP_001020016), and the prokaryote, Aquifex aeolicus (AaLeuT, NP_214423). Black arrows denote the location of reverse transcriptase polymerase chain reaction primer sites used in this study. Transmembrane domains (TM1-TM10) follow the structural-based alignment of prokaryotic and eukaryotic sequences by Beuming et al. (2006)

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Table 4 Proportion of adult tissue samples from Antarctic echinoderm species in which expression of putative SLC6 transporter transcripts was detected (e.g., ‘‘2/3’’ indicates that a gene was detected in two of three animals analyzed) Species

Gene

Digestive tract

Tube feet

A. hodgsoni

ATa

2/3

2/2

ATb

3/3

2/2

D. brucei

AT

3/3

3/3

O. meridionalis

ATa

5/5

5/7

ATb

5/5

6/7

ATa

2/3

1/3

ATc

2/3

3/3

ATa

4/4

2/2

ATb

4/4

2/2

ATa

3/3

4/4

ATb

3/3

4/4

O. validus P. fuscus S. neumayeri

Fig. 3 Detection of expression of SLC6 transporters (AhATa, AhATb, OmATa, and OmATb) or reverse transcription control (Actin) by reverse transcriptase polymerase chain reaction in developmental stages of the Antarctic sea stars, a Acodontaster hodgsoni and b Odontaster meridionalis. Age of embryos or larvae (day postfertilization) is indicated within each lane

Manahan 2007) relative to O. meridionalis, presumably reflecting the importance of these energy reserves for lecithotrophic development. Despite these contrasting energetic strategies of early development, expression of putative SLC6 transporter genes was detected throughout development of A. hodgsoni and O. meridionalis. The expression of these

genes during development suggests a possible mechanism for supplementing endogenous energy reserves, regardless of whether developmental stages use particulate sources of food or rely on maternally supplied reserves. While lecithotrophic larvae cannot consume exogenous particulate food (e.g., algae), such developmental forms are, however, not energetically ‘‘closed systems’’—lecithotrophic larvae have been shown to absorb energetically significant quantities of dissolved organic matter directly from seawater (Jaeckle and Manahan 1989; Shilling and Manahan 1994). In lecithotrophs, dissolved nutrient sources could augment maternally deposited energetic reserves thereby providing resilience to fluctuations in food availability once exogenous feeding begins in juvenile stages. Although the expression of transporter genes was detected in embryonic and larval Antarctic echinoderms, localization of transcript expression, and the presence of proteins encoded by these genes remain to be demonstrated. However, the physiology of amino acid transport has previously been studied in developmental stages of A. hodgsoni and O. meridionalis (Shilling and Manahan 1994; Ginsburg and Manahan 2009), and those results support the gene expression patterns reported in the present study. In a limited number of developmental stages for both species (Figs. 1, 3: A. hodgsoni and O. meridionalis), expression of a specific transporter gene was not detected. Based upon expression patterns of SLC6 amino acid transporter genes during embryogenesis in other echinoderm species (Meyer and Manahan 2009), it would be surprising to find expression to be present at day 12 of development, then absent at day 14, and expressed again at day 18 (Figs. 1, 3). It is possible that the occasional lack of detection of AhATa was due to low amounts of that transcript in some samples. Solute carrier family 6 amino acid transporter genes have been cloned and characterized in developmental stages of the temperate sea urchin, S. purpuratus (Meyer and Manahan 2009). In that study, transporter proteins were shown to be present in ectoderm of larvae. In the present study, SLC6 members, likely orthologous to those previously identified in S. purpuratus, are expressed in embryos, larvae, and also in adult tissues. Sites of expression in adults include tissues that could facilitate direct absorption of amino acids from low ambient concentrations in seawater, as well as from the higher concentrations of amino acid present in the digestive tract (following digestion of food). This finding raises interesting new questions regarding the substrate affinity of this group of amino acid transporters in Antarctic echinoderms, which function in tissues exposed to dramatically different substrate environments. Further analysis of amino acid transporter genes in body wall epithelial cells will provide new opportunities to understand the physiological role and

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metabolic significance of the uptake of dissolved organic material from seawater by Antarctic organisms. Acknowledgments This research was undertaken, in part, during the Antarctic Biology Course (National Science Foundation Grant 05-1234124) and by National Science Foundation Grant 01-0980987 to DTM. The authors wish to thank the diving team (Addie Coyac and Jim Leichter) and students (Dafne Eerkes-Medrano, Rob Ellis, Nimrod Kiss, Josh Osterberg, and Sam Rastrick) who assisted with collecting samples from adult echinoderms in McMurdo Sound and Michael Sheng for laboratory assistance. Our thanks to Dr. Ken Hallanych, Alexis Janosik, and Johanna Cannon for sharing information from their RNA analysis of O. validus.

References Allemand D, Derenzis G, Ciapa B, Girard JP, Payan P (1984) Characterization of valine transport in sea urchin eggs. Biochim Biophys Acta 772:337–346 Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410 Benner R (2002) Chemical composition and reactivity. In: Hansell DA, Carlson CA (eds) Biogeochemistry of marine dissolved organic matter. Academic Press, San Diego, pp 59–90 Beuming T, Shi L, Javitch JA, Weinstein H (2006) A comprehensive structure-based alignment of prokaryotic and eukaryotic neurotransmitter/Na? symporters (NSS) aids in the use of the LeuT structure to probe NSS structure and function. Mol Pharmacol 70:1630–1642 Bosch I, Pearse JS (1990) Developmental types of shallow-water asteroids of McMurdo Sound, Antarctica. Mar Biol 104:41–46 Boudko DY (2010) Molecular ontology of amino acid transport. In: Gerencser GA (ed) Epithelial transport physiology. Humana Press Inc., New Jersey, pp 379–472 Boudko DY, Kohn AB, Meleshkevitch EA, Dasher MK, Seron TJ, Stevens BR, Harvey WR (2005) Ancestry and progeny of nutrient amino acid transporters. PNAS 102:1360–1365 Bowden DA, Clarke A, Peck LS (2009) Seasonal variation in the diversity and abundance of pelagic larvae of Antarctic marine invertebrates. Mar Biol 156:2033–2047 Bro¨er S (2008) Amino acid transport across mammalian intestinal and renal epithelia. Physiol Rev 88:249–286 Chen NH, Reith MEA, Quick MW (2004) Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6. Pflugers Arch Eur J Physiol 447: 519–531 Cheng C–C, Detrich HWI (2007) Molecular ecophysiology of Antarctic notothenioid fishes. Philos Trans R Soc B Biol Sci 362:2215–2232 Clarke A, Leakey RJG (1996) The seasonal cycle of phytoplankton, macronutrients, and the microbial community in a nearshore Antarctic marine ecosystem. Limnol Oceanogr 41:1281–1294 Edwards M, Richardson AJ (2004) Impact of climate change on marine pelagic phenology and trophic mismatch. Nature 430: 881–884 Epel D (1972) Activation of an Na? -dependent amino acid transport system upon fertilization of sea urchin eggs. Exp Cell Res 72:74–89 Ferraris RP, Diamond JM (1989) Specific regulation of intestinal nutrient transporters by their dietary substrates. Annu Rev Physiol 51:125–141 Ginsburg DW, Manahan DT (2009) Developmental physiology of Antarctic asteroids with different life-history modes. Mar Biol 156:2391–2402

123

Polar Biol (2013) 36:1257–1267 Giordano D, Russo R, Di Prisco G, Verde C (2012) Molecular adaptations in Antarctic fish and marine microorganisms. Mar Genomics 6:1–6 Gomme J (2001) Transport of exogenous organic substances by invertebrate integuments: the field revisited. J Exp Zool 289: 254–265 Hays G, Richardson A, Robinson C (2005) Climate change and marine plankton. Trends Ecol Evol 20:337–344 He L, Vasiliou K, Nebert DW (2009) Analysis and update of the human solute carrier (SLC) gene superfamily. Hum Genomics 3:195–206 Hoglund PJ, Adzic D, Scicluna SJ, Lindblom J, Fredriksson R (2005) The repertoire of solute carriers of family 6: identification of new human and rodent genes. Biochem Biophys Res Commun 336: 175–189 Jaeckle W, Manahan D (1989) Feeding by a ‘‘nonfeeding’’ larva: uptake of dissolved amino acids from seawater by lecithotrophic larvae of the gastropod Haliotis rufescens. Mar Biol 103:87–94 Jaeckle W, Manahan D (1992) Experimental manipulations of the organic composition of seawater—Implications for studies of energy budgets in marine invertebrate larvae. J Exp Mar Biol Ecol 156:273–284 Ji R, Edwards M, Mackas DL, Runge JA, Thomas AC (2010) Marine plankton phenology and life history in a changing climate: current research and future directions. J Plankton Res 32:1355–1368 Johnson CH, Wendt DE (2007) Availability of dissolved organic matter offsets metabolic costs of a protracted larval period for Bugula neritina (Bryozoa). Mar Biol 151:301–311 Jorgensen CB (1976) August Pu¨tter, August Krogh, and modern ideas on the use of dissolved organic matter in aquatic environments. Biol Rev Camb Philos 51:291–328 Kristensen AS, Andersen J, Jorgensen TN, Sorensen L, Eriksen J, Loland CJ, Stromgaard K, Gether U (2011) SLC6 neurotransmitter transporters: structure, function, and regulation. Pharmacol Rev 63:585–640 Manahan DT (1990) Adaptations by invertebrate larvae for nutrient acquisition from seawater. Am Zool 30:147–160 Manahan DT, Wright SH, Stephens GC, Rice MA (1982) Transport of dissolved amino acids by the mussel, Mytilus edulis: demonstration of net uptake from natural seawater. Science 215: 1253–1255 Manahan DT, Davis JP, Stephens GC (1983) Bacteria-free sea urchin larvae: selective uptake of neutral amino acids from seawater. Science 220:204–206 Manahan DT, Jaeckle WB, Nourizadeh SD (1989) Ontogenic changes in the rates of amino acid transport from seawater by marine invertebrate larvae (Echinodermata, Echiura, Mollusca). Biol Bull 176:161–168 Marchler-Bauer A, Lu S, Anderson JB, Chitsaz F, Derbyshire MK, DeWeese-Scott C, Fong JH, Geer LY, Geer RC, Gonzales NR, Gwadz M, Hurwitz DI, Jackson JD, Ke Z, Lanczycki CJ, Lu F, Marchler GH, Mullokandov M, Omelchenko MV, Robertson CL, Song JS, Thanki N, Yamashita RA, Zhang D, Zhang N, Zheng C, Bryant SH (2011) CDD: a conserved domain database for the functional annotation of proteins. Nucleic Acids Res 39:D225–D229 Marsh AG, Leong PKK, Manahan DT (1999) Energy metabolism during embryonic development and larval growth of an Antarctic sea urchin. J Exp Biol 202:2041–2050 Meyer E, Manahan DT (2009) Nutrient uptake by marine invertebrates: cloning and functional analysis of amino acid transporter genes in developing sea urchins (Strongylocentrotus purpuratus). Biol Bull 217:6–24 Meyer E, Green AJ, Moore M, Manahan DT (2007) Food availability and physiological state of sea urchin larvae (Strongylocentrotus purpuratus). Mar Biol 152:179–191

Polar Biol (2013) 36:1257–1267 Moore M, Manahan DT (2007) Variation among females in egg lipid content and developmental success of echinoderms from McMurdo Sound, Antarctica. Polar Biol 30:1245–1252 Moran AL, Manahan DT (2004) Physiological recovery from prolonged ‘starvation’ in larvae of the Pacific oyster Crassostrea gigas. J Exp Mar Biol Ecol 306:17–36 Pacha J (2000) Development of intestinal transport function in mammals. Physiol Rev 80:1633–1667 Palacin M, Estevez R, Bertran J, Zorzano A (1998) Molecular biology of mammalian plasma membrane amino acid transporters. Physiol Rev 78:969–1054 Pearse JS, McClintock JB, Bosch I (1991) Reproduction of Antarctic benthic marine invertebrates: tempos, modes, and timing. Am Zool 31:65–80 Peck L, Clark M, Clarke A, Cockell C, Convey P, Detrich H, Fraser K, Johnston I, Methe B, Murray A, Romisch K, Rogers A (2005) Genomics: applications to Antarctic ecosystems. Polar Biol 28:351–365 Pu¨tter AFR (1909) Die Erna¨hrung der Wassertiere und der Stoffhaushalt der Gewasser. Fischer, Jena Rivkin RB (1991) Seasonal patterns of planktonic production in McMurdo Sound, Antarctica. Am Zool 31:5–16 Rivkin RB, Bosch I, Pearse JS, Lessard EJ (1986) Bacterivory: a novel feeding mode for asteroid larvae. Science 233:1311–1314

1267 Saier MH Jr (1999) A functional-phylogenetic system for the classification of transport proteins. J Cell Biochem Suppl 32–33: 84–94 Shilling FM, Manahan DT (1994) Energy metabolism and amino acid transport during early development of Antarctic and temperate echinoderms. Biol Bull 187:398–407 Stanwell-Smith D, Peck LS (1998) Temperature and embryonic development in relation to spawning and field occurrence of larvae of three Antarctic echinoderms. Biol Bull 194:44–52 Stephens GC (1988) Epidermal amino acid transport in marine invertebrates. Biochim Biophys Acta 947:113–138 Stephens GC, Schinske RA (1961) Uptake of amino acids by marine invertebrates. Limnol Oceanogr 6:175–181 Stevens BR (2010) Amino acid transport by epithelial membranes. In: Gerencser GA (ed) Epithelial transport physiology. Humana Press Inc., New Jersey, pp 353–378 Welborn JR, Manahan DT (1991) Seasonal changes in the concentration of amino acids and sugars in McMurdo Sound, Antarctica: uptake of amino acids by asteroid larvae. Antarct J US 26: 60–162 Wright SH, Manahan DT (1989) Integumental nutrient uptake by aquatic organisms. Annu Rev Physiol 51:585–600

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