Fish Sci (2011) 77:1–21 DOI 10.1007/s12562-010-0301-z REVIEW ARTICLE Mechanisms and control of vitellogenesis in crustaceans T Subramoniam Received: 13 May 2010 / Accepted: October 2010 / Published online: 16 November 2010 Ó The Japanese Society of Fisheries Science 2010 Abstract Crustaceans produce complex yolk proteins to meet the substrate and energy requirements of embryonic development Early electron microscopic investigations point to a biphasic yolk synthesis, first within the ovary, followed by heterosynthesis at extra-ovarian sites Recent advances in molecular techniques have enhanced our understanding of the genetic control of yolk synthesis in crustaceans Amino acid sequencing of crustacean vitellogenin (Vg) has enabled the elucidation of the cDNA sequence associated with it, the identification of genes, and the examination of their expression patterns in different tissues Yolk processing in crustaeans involves cleavage of the pro-Vg at consensus sites by subtilisin-like endoproteases within the hepatopancreas, hemolymph and oocytes The structural elucidation of crustacean yolk proteins, as well as the comparison of amino acid sequences of vitellogenins from various crustacean species, has revealed molecular phylogenetic relationships not only among them but also with other large lipid transfer lipoproteins of disparate function The combinatorial effects of eyestalk neuropeptides and a variety of trophic hormones achieve the hormonal coordination of molting and reproduction Biogenic amines secreted by the central nervous system may also play an integrative role by stimulating neuropeptide secretion Keywords Vitellogenesis Á Vitellogenin receptor Á Yolk processing Á Neuropeptides Á Methyl farnesoate Á Ecdysteroids Á 17b-Estradiol T Subramoniam (&) Marine Biotechnology Division, National Institute of Ocean Technology, Velachery, Tambaram Road, Pallikaranai, Chennai 600 100, India e-mail: thanusub@yahoo.com Introduction Many malacostracan crustaceans produce large numbers of yolk-laden eggs and brood them externally for extended periods Hence, vitellogenesis, the process of yolk formation, is central to oogenesis In Crustacea, vitellogenesis is a biphasic event consisting of autosynthesis and heterosynthesis [1] This contention is supported by recent molecular studies demonstrating yolk protein gene expression both in ovary and hepatopancreas Receptormediated endocytosis of the yolk precursor molecule, vitellogenin (Vg), into growing oocytes has been established in crustaceans [2] The molecular transformation of Vg into final yolk products for deposition in the mature oocyte is another crucial event in vitellogenesis A defining feature in the endocrine regulation of vitellogenesis in Crustacea is the occurrence of inhibitory hormones in the neurosecretory cells of the X-organ/sinus gland complex within the eystalk Conversely, many hormonal factors as diverse in nature as methyl farnesoate (MF) and vertebrate steroidal hormones have been implicated in the stimulation of vitellogenesis However, we are far from having achieved a clear understanding of the exact regulatory mechanisms relating to the vitellogenic processes in Crustacea, mainly because of the species-specific nature of the effector molecules Yet, recent molecular studies on the primary structure of the major vitellin molecules, as well as the deciphering of their gene sequences and the elucidation of their synthetic sites, are paving the way to an understanding of the transcriptional control of the vitellogenin gene in light of what is already known about insect and vertebrate vitellogenesis Homology searches and molecular phylogenetic analysis of various crustacean Vgs have revealed unexpected results on their closer relationship with several members of the large lipid transfer lipoprotein 123 superfamily as compared to their own orthologous Vg molecules This review undertakes a critical analysis of the various mechanisms involved in the vitellogenic process and their hormonal control Vitellogenesis Molecular composition of crustacean yolk proteins Crustacean yolk proteins, referred to as lipovitellin, are complex molecules comprising a high-density lipoprotein (HDL) conjugated to carbohydrates and carotenoid pigments [3] Crustacean lipovitellin differs from that of vertebrates in that it lacks protein phosphates and has high lipid content In the mole crab Emerita asiatica, purified lipovitellin contains neutral lipids, glycolipids and phospholipids, among which phospholipids are the dominant lipid class, with phosphatidyl choline and phosphatidyl serine being the major species [4, 5] However, the proportion of lipid to protein seems to be higher in the precursor protein, Vg Crustacean lipovitellin characteristically contains a variety of carotenoid pigments They include betacarotene, astaxanthin, canthaxanthin and cis-canthaxanthin, among other minor intermediary metabolites [1] Crustacean lipovitellin also possesses a higher carbohydrate content than vertebrate vitellins In E asiatica, most protein-bound carbohydrates found in lipovitellin are hexosamines and hexoses [5] Emerita lipovitellin also contains galactosamine as well as O-linked oligosaccharides with N-acetyl hexosamine as the terminal residue, whereas sialic acid is specifically absent Khalaila et al [6] have identified the glycosylation sites in the vitellogenin of the crayfish Cherax quadricarinatus and characterized the glycan moieties Besides providing an important source of carbohydrates for the developing embryos, the glycosylation of Vg has an important role in the folding and subunit assembly of these molecules The glycan moieties may also play an equally important role in the recognition of the Vg membrane receptor during yolk accumulation After uptake into the oocytes, they may also be involved in packaging and compressing the yolk precursor proteins into the yolk bodies [5] Biogenesis of yolk In Crustacea, vitellogenesis occurs in two stages: a primary vitellogenesis or previtellogenic phase characterized by the differentiation of endoplasmic reticulum and the formation of endogenous yolk stored in vesicles; and a secondary vitellogenesis corresponding to an intensive phase of uptake and storage of exogenous yolk precursor molecules, which accumulate into large yolk globules [7] Early electron microscopic investigations point to this biphasic 123 Fish Sci (2011) 77:1–21 yolk synthesis, first within the ovary, followed by a heterosynthetic yolk formation in somatic tissues such as the hepatopancreas or subepidermal fat body [1] Support for autosynthetic yolk formation came from in vitro incubation studies using ovaries of crayfish Procambarus sp and of the crab Pachygrapsus crassipes [8] In the shrimp, the yolk content of the egg is meager, and hence oocytes may be in a position to synthesize most of them, with only a very limited contribution deriving from extra-ovarian sites In the kuruma prawn Marsupenaeus japonicus, under in vitro conditions, only the ovary incorporated radioactive amino acids into a protein immunologically identical to lipovitellin [9] Similar in vitro studies on the vitellogenic ovaries of another shrimp Penaeus semisulcatus also revealed that the ovary is the primary organ of vitellin synthesis [10] Egg maturation in penaeid shrimp is characterized by vitellogenesis and cortical rod protein (CRP) formation In M japonicus, Kim et al [11] showed that CRP mRNA is highly expressed before the onset of vitellogenesis and that Vg mRNA exhibited high expression during intense vitellogenesis, suggesting that different genes are involved in the ovarian synthesis of CRP and Vg proteins Okumura et al [12] provided further evidence that eyestalk ablation induced both Vg and CRP synthesis within the ovary Khayat et al [13] demonstrated high levels of Vg mRNA in the vitellogenic ovary of P semisulcatus, as evidenced by its ability to direct the cell-free synthesis of large amounts of Vg However, unlike the other decapods, where autosynthesis of yolk has been shown to occur within the oocytes [8], in penaeid shrimp, the ovarian synthesis of yolk probably takes place in the follicle cells Thus, in M japonicus, an immunofluorescence study with antivitellin IgG was suggestive of yolk protein synthesis by the follicular epithelium rather than by the oocytes Northern blot analysis and in situ hybridization have revealed that mRNA encoding vitellogenin was expressed in the follicle cells of the vitellogenic females [14] Tsang et al [15] also showed the expression of the vitellogenin gene, MeVg1, in the ovary and hepatopancreas of Metapenaeus ensis, suggesting equal contributions from both tissues In recent years, several gene expression studies, using quantitative real-time PCR techniques, have demonstrated that the ovary remains the principal organ that synthesizes yolk proteins in several penaeid shrimp species Interestingly, in species such as M japonicus and P semisulcatus, one and the same Vg is expressed in the ovary and hepatopancreas [16, 17] However, in other species, such as Litopenaeus merguiensis, M ensis and P monodon, more than one Vg may be involved in the tissue-specific expression of the gene in both the ovary and hepatopancreas [18–20] Especially in L merguiensis, the patterns of Vg mRNA expression between the hepatopancreas and ovary Fish Sci (2011) 77:1–21 differ in that the expression level in the hepatopancreas is much lower than that in the ovary at all stages of ovarian development [18] Evidently, the relative contributions of the ovary and hepatopancreas to overall yolk production may differ among various shrimp species Vitellogenin In addition to being the precursor of ovarian lipovitellin, crustacean vitellogenin is considered to be an important transporter of lipids to the ovary from the hemolymph during vitellogenesis In general, lipid transport through the hemolymph is accomplished by two HDLs and a very high-density lipoprotein (VHDL) [21, 22] Female-specific vitellogenin is one of the HDLs, with its production being correlated with ovarian development in female crustaceans, whereas the other HDL as well as VHDL are found in both males and females In the penaeid shrimp, P semisulcatus, the non-sex-specific hemolymph lipoprotein, LP I, consists of one 110-kDa peptide unit, whereas the sex-specific LP II consists of subunits of 200, 120, and 80 kDa [23] Interestingly, the same subunits were also present in the lipovitellin of this shrimp Furthermore, the lipid compositions of these two HDLs in P semisulcatus also differ: LP II (Vg) has a lower lipid content than does LP I, in addition to differences found in lipid classes linked to the apolipoprotein Apparently, vitellogenin and lipovitellin have similar protein structures, but show differences in their lipid contents, with the lipovitellin having more percentage lipid acquired through adsorption within the oocytes LP I is also different from LP II in its protein composition, as the former does not cross-react with anti-vitellin antiserum In the crayfish Cherax quadricarinatus, Yehezkel et al [24] observed that the hemolymph lipoprotein II, equivalent to Vg, appears only at the onset of secondary vitellogenesis In the mole crab E asiatica, Subramoniam and Gunamalai [25] have described three hemolymph lipoproteins: LP1, LP2, and LP3 LP1 is non-sex-specific, but is accumulated into the oocytes along with LP2, which is the female-specific Vg LP3, which appears only during the premolt of male and female crabs, plays a role in the transport of lipids to the epidermis for the purposes of cuticle formation In addition to transporting a variety of lipophilic compounds such as triglycerides and phospholipids, crustacean Vgs are known to transport steroidal hormones like ecdysteroids and vertebrate steroids, including estradiol 17b and progesterone [26, 27] These hormones are stored within the oocytes as conjugates of yolk proteins and serve regulatory functions during embryogenesis Site of vitellogenin synthesis Initial electrophoretic and isotope tracer studies have implicated several organs such as the hemocytes in crabs [28, 29], the fat body in isopods and the amphipods [30, 31], and the subepidermal adipose tissue in Palaemon serratus [32], and Scylla serrata [33] as the synthetic sites of Vg However, the hepatopancreas has proven to be the most important organ synthesizing Vg outside of the ovary in the majority of crustacean species analyzed The crustacean hepatopancreas is the functional homolog to the fat body in insects and the liver in vertebrates Subsequent investigations employing molecular techniques have revealed that the hepatopancreas is the sole site of Vg synthesis in the giant freshwater prawn, Macrobrachium rosenbergii Chen et al [34] cloned a cDNA fragment encoding Vg in this species and found its expression in the hepatopancreas of the vitellogenic female In addition, Yang et al [35] obtained cDNA fragments for four vitellins; using these cDNA fragments as probes, they found the exclusive expression of Vg mRNAs for the four vitellins in the hepatopancreas of vitellogenic female M rosenbergii Using quantitative real-time PCR techniques, Jayasankar et al [36] measured the expression levels of mRNA in the hepatopancreas of this species and also determined Vg levels using enzyme immunoassay Vg mRNA expression in the hepatopancreas and hemolymph Vg levels showed a gradual increase concomitant with increasing gonadosomatic index Vg mRNA expression was, however, negligible in the ovary, confirming that the hepatopancreas is the principal site of Vg synthesis in M rosenbergii In general, Vg expression may occur at multiple sites, but expression patterns nevertheless vary according to species That one and the same gene for vitellin and Vg can be simultaneously expressed both in the ovary and hepatopancreas was shown in P semisulcatus [37] Multiple genes may also show tissue-specific expression of Vg in the ovary and hepatopancreas, as demonstrated in another penaeid shrimp, Metapenaeus ensis, where two Vgs (MeVg1 and MeVg2) have been identified [15] The MeVg1 gene is expressed equally in the ovary and hepatopancreas, whereas MeVg2 is expressed only in the hepatopancreas Furthermore, the MeVg2 gene gives rise to smaller transcripts, resulting in the production of many smaller MeVg2 subunits destained for ovarian uptake [19] Evidently, the ovary is the primary site of yolk synthesis in penaeid shrimp, as indicated by gene expression studies enumerated above; on the contrary, large-bodied decapods such as crabs and lobsters seem to rely largely on extraovarian organs such as the hepatopancreas for the synthesis of Vg Using molecular techniques, Li et al [38] have demonstrated that in the Chinese crab Eriocheir sinensis, the hepatopancreas is the main site of Vg synthesis, although immunocytochemical studies have suggested a parallel role for ovary However, in the red crab Charybdis feriatus, northern blot analysis revealed that the crab expresses the Vg precursor only in the hepatopancreas 123 In addition to the major 8.0-kb transcript, a large proportion of smaller C feriatus Vg-specific transcripts are also detected in the hepatopancreas These transcripts most likely result from the alternative splicing and alternative use of promoter and/or termination signals [39] The occurrence of many Vg subunits in the crab hemolymph may also result from autoproteolysis due to intrinsic protease activity in Vg itself [40] In a recent study using quantitative real-time PCR techniques, Zmora et al [41] found evidence that Vg is primarily expressed in the hepatopancreas of the vitellogenic females, with only minor expression in the ovary of the blue crab C sapidus Furthermore, Vg expression in the hepatopancreas of this brachyuran anecdysic crab is correlated with ovarian maturation, with a remarkable 8000-fold increase in expression from stage to of ovarian development Recent cloning and expression studies on the Vg in the lobster Homarus americanus also adduced further evidence that the hepatopancreas is the primary organ for yolk precursor synthesis in lobsters [42] The lobster HaVg1, expressed mainly in the hepatopancreas, comprises 14 introns and 15 exons This study also revealed that the sizes and locations of the exons and introns of Vg are conserved among crustaceans The HaVg1 precursor contained the lipoprotein domain at the N-terminus, followed by a domain of unknown function in the middle The von Willebrand factor type-D domain is located at the C-terminus of the precursor A unique feature of crustacean Vg is that it contains several cleavage sites, resulting in increased subunit composition More numbers of Vg subunits may also arise from smaller transcripts, as reported for the crab C feriatus [43] All these studies lead to the compelling conclusion that the hepatopancreas is the principal site of Vg synthesis in brachyuran crabs, lobsters, and probably other representative species under the suborder Pleocyamata Conversely, in Dendrobranchiata, including mainly the penaeid shrimp, both the hepatopancreas and ovary provide equal contributions towards Vg synthesis The Vg synthesized at extraovarian sites such as the hepatopancreas undergoes several modifications, such as glycosylation and lipid addition, bringing about changes in molecular weight when compared with the final yolk products accumulated within the ovary [41] To sum up, besides being the precursor protein molecule that supplies the amino acid pool for the developing embryo, vitellogenin can also serve other subfunctions, such as the transport of a variety of organic and inorganic molecules required for embryonic development Phylogenetic analysis of crustacean vitellogenin Crustacean Vg is a multidomain apolipoprotein that is cleaved into distinct yolk proteins Multiple alignments of 123 Fish Sci (2011) 77:1–21 all known crustacean Vg sequences have revealed almost similar cleavage sites ClustalW alignment of M rosenbergii Vg with that of 17 other crustacean species has shown that the first common cleavage site RXRR occurs at amino acid residues 707–710, and the homology for the first segment is high when compared with the rest of the module The results from BLAST searches indicate that the N-terminal region of crustacean Vgs is conserved, as in the apolipoproteins that are involved in lipid transport This property is in accord with the fact that Vg, insect apolipophorin II/I, apoB, and MTP are members of the same multigene superfamily of large lipid transfer proteins (LLTP) [44] Next to the N-terminal segment, the middle segment is comparable to a lipovitellin domain of unknown function called DUF1943 The C-terminal domain of M rosenbergii Vg harbored a von Willebrand-factor type D domain (YGP4) found in mammals Similarity in amino acid sequence of the von Willebrand factor at the C-terminal region has also been reported for another LLTP protein, the insect apolipophorin [45] A phylogenetic tree constructed based on the alignment of amino acid sequences of 18 crustacean Vgs using the ClustalW programme shows six distinct lineage groups: Penaeidea (A), Brachyura (B), Astacidea (C), Caridea (D), Copepoda, and Brachiopoda (E), and Thalassinidea (F) (Fig 1; Table 1) The Vgs of the penaeidian species seem to be highly homogeneous, with [92% identity in amino acid sequence, except in the case of M ensis In M ensis, the two Vgs (MeVg1 and MeVg2), identified by Tsang et al [15] and Kung et al [19], are expressed independently in the ovary and hepatopancreas, with only a sequence identity of 56% between them These MeVgs also showed less homology with Vgs of other penaeid shrimp, revealing a greater evolutionary distance from other penaeid species [46] As seen from Fig 1, Upogebia major, representing Thalassinidea, has taken a separate lineage near the Brachyura In addition to all the above decapods, the two copepods and a branchiopod, Daphnia, formed a separate clad in-between Caridia and Thalassinidea in the radial tree The structural elucidation of Vg from different crustacean species has also been helpful in solving phylogenetic relationships with other arthropod groups In a primitive brachiopod, Daphnia magna, two Vgs, DmagVg1 and DmagVg2, have been isolated Interestingly, the lipid transport module in the N-terminal region of DmagVg1 is more closely related to those of insect Vgs than to those of decapod crustacean Vgs [47] Yet again, the intergenic region of the two genes contains sequences resembling juvenile hormone-responsive and ecdysone-responsive elements, typical of insect Vgs [48] The close homology found between Daphnia and insect LLT Vg modules may be due to either divergence or convergence Fish Sci (2011) 77:1–21 D E A F C B Fig Phylogenetics of eighteen crustacean Vgs with two dominant domains, (A) penaeoidean and (B) brachyuran, at both ends of the radial tree Other major domains such as Astacidea (C) and Caridea (D) are found on either side of the tree Copepods and brachiopods form a common grouping (E) along the brachyuran side Thalassinidea (F) also has a separate lineage along the brachyuran crabs The protein sequence accession numbers for all the Vgs are given in Table In relation to other invertebrates, crustacean Vg has shown homology in amino acid sequence with molluscan and coral Vgs [49, 50], although homology among the coral Vg, GfVg, and the shrimp Vg (L vannamei) is much closer These homology studies purportedly point to the emergence of Vg as an egg protein precursor before the cnidarian–bilaterian divergence The origin and evolutionary progression of Vgs from a common ancestral molecule at the cnidarian–bilatrian divergence denotes a landmark interception in crustacean arthropods, giving rise to other lipid-carrying apolipoproteins that perform disparate physiological functions Even among crustaceans, we find a number of lipoproteins, such as crustacean clotting proteins and hemocyanin, that show limited amino-acid sequence homology with vitellogenin [2] The inclusion of crustacean Vg among other LLTP proteins is also justified by the immunological relatedness found between Vg of the crab S serrata and apoB, the major protein component of LDL and VLDL [40] Warrier and Subramoniam [40] demonstrated the recognition of Vg by antibodies to apoB-containing mammalian lipoproteins LDL and VLDL, and not to HDL (Fig 2) Furthermore, the apoB antibodies reacted with greater efficacy to S serrata Vg, thereby providing corroborative evidence for the structural identity of apoB with Vg Avarre et al [51] conducted a homology study between crustacean Vgs and other members of the LDL superfamily of lipoproteins, and arrived at the conclusion that crustacean Vgs are closer to mammalian LDL and insectan apolipophorins However, the vertebrate apo-lipo B line of proteins is thought to have diverged from the vertebrate Vg line, which, in turn, arose from the ancient egg yolk storage proteins of invertebrates [52] The closer relationship between apoB and crustacean Vg discussed above not only indicates the high conservancy in the lipid-binding domains of both these proteins, but may also point to the evolutionary derivation of vertebrate apo-lipo B proteins at the crustacean Vg level Vitellogenin receptors and yolk protein uptake In crustaceans, only a few studies have been carried out with reference to Vg receptors In the giant freshwater 123 Fish Sci (2011) 77:1–21 Table Vg accession numbers of 18 crustaceans, along with their taxonomic classifications Species Accession no Systematics Penaeus monodon ABB89953.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Dendrobranchiata; Penaeoidea; Penaeidae; Penaeus Fenneropenaeus chinensis ABC86571.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Dendrobranchiata; Penaeoidea; Penaeidae; Fenneropenaeus Fenneropenaeus merguiensis AAR88442.2 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Dendrobranchiata; Penaeoidea; Penaeidae; Fenneropenaeus Litopenaeus vannamei AAP76571.2 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Dendrobranchiata; Penaeoidea; Penaeidae; Litopenaeus Marsupenaeus japonicus BAD98732.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Dendrobranchiata; Penaeoidea; Penaeidae; Marsupenaeus Metapenaeus ensis AAT01139.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Dendrobranchiata; Penaeoidea; Penaeidae; Metapenaeus Homarus americanus ABO09863.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Astacidea; Nephropoidea; Nephropidae; Homarus Cherax quadricarinatus AAG17936.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Astacidea; Parastacoidea; Parastacidae; Cherax Portunus trituberculatus AAX94762.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Brachyura; Eubrachyura; Portunoidea; Portunidae; Portunus Callinectes sapidus ABC41925.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Brachyura; Eubrachyura; Portunoidea; Portunidae; Callinectes Charybdis feriatus AAU93694.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Brachyura; Eubrachyura; Portunoidea; Portunidae; Charybdis Scylla serrata ACO36035.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Brachyura; Eubrachyura; Portunoidea; Portunidae; Scylla serrata Macrobrachium rosenbergii BAB69831.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Caridea; Palaemonoidea; Palaemonidae; Macrobrachium Pandalus hypsinotus BAD11098.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Caridea; Pandaloidea; Pandalidae; Pandalus Upogebia major BAF91417.1 Malacostraca; Eumalacostraca; Eucarida; Decapoda; Pleocyemata; Thalassinidea; Callianassoidea; Upogebiidae; Upogebia Daphnia magna BAE94324.1 Branchiopoda; Diplostraca; Cladocera; Anomopoda; Daphniidae; Daphnia Lepeophtheirus salmonis ABU41135.1 Maxillopoda; Copepoda;Siphonostomatoida; Caligidae; Lepeophtheirus Tigriopus japonicus ABZ91537.1 Maxillopoda; Copepoda; Neocopepoda; Podoplea; Harpacticoida; Harpacticidae; Tigriopus; Tigriopus japonicus Fig Dot blot analysis of crab Vg (1), rat LDL (2), VLDL (3), and HDL (4) using anti-crab Vg antibodies (dilution 1:2000) Anti-Lv antibodies are seen to react well with Vg, LDL, and VLDL, but there is no reaction with HDL (from Warrier and Subramoniam [40]) prawn M rosenbergii, Jugan and Soyez [53] demonstrated Vg uptake by the oocytes by employing colloidal goldconjugated vitellin The labeling was visualized in the microvilli, coated pits, and intraooplasmic vesicles These authors further observed that a sinus gland neuropeptide 123 inhibited vitellogenin endocytosis, possibly by blocking the membrane receptors Laverdure and Soyez [54] solubilized the vitellogenin receptor from the oocyte membrane of H americanus, and characterized it using an enzymelinked immunosorbent assay Binding of Vg with the solubilized receptors increased at the onset of vitellogenesis, but decreased in older oocytes of the freshwater crayfish Orconectus limosus [55] The solubilized oocyte membrane receptor with a molecular weight of 28–30 kDa binds specifically to Vg of O limosus Warrier and Subramoniam [56] purified the vitellogenin receptor in the mud crab Scylla serrata using HPLC and found a still higher molecular weight of 230 kDa In direct binding studies using 125I-labeled Vg, crab VgR was observed to have increased affinity to its ligand in the presence of Ca2? and was inhibited by suramin, a polysulfated polycyclic Fish Sci (2011) 77:1–21 hydrocarbon These authors also showed an immunological relatedness between VgR of S serrata and LDLR by virtue of the ability of VgR to bind rat LDL and VLDL In a recent study, the cloning and characterization of a cDNA encoding a putative Vg receptor from the tiger prawn P monodon (PmVgR) has been reported [57] PmVgR has a molecular weight of 211 kDa, and is ovary specific It consists of conserved cysteine-rich domains, EGF-like domains and YWTD motifs, similar to the mammalian LDL receptor as well as to the Vg receptors of insects and vertebrates PmVgR expression in the ovary coincides with the rapid pace of Vg production by the hepatopancreas Immunological detection of PmVgR in the oocyte membrane during intense vitellogenesis has also been done in this prawn Further, PmVgR expression was knocked down in animals after they were injected with PmVgR dsRNA, leading to a decrease in vitellin content in the ovary, and at the same time elevating the levels of hemolymph Vg A similar molecular characterization of VgR has also been reported for the kuruma prawn M japonicus [58] The expression dynamics of MjVgR during vitellogenesis have been found to be similar to those of P monodon Furthermore, structural analysis of the VgR of this shrimp also reconfirmed its inclusion in the LDLR superfamily The results of these studies are comparable with those of S serrata with respect to molecular weight and functional characteristics [56] It would be of interest to know whether crustacean VgR also facilitates the endocytosis of other hemolymph lipoproteins into the ovary, similar to avian VgRs [59] and insectan lipophorin receptor [60] Receptor-mediated internalization of Vg into the oocytes has been demonstrated by an immunogold electron microscopic study using anti-Vg as the primary antibody in S serrata [56] Immunogold labeling against Vg antibody was first visualized in the coated pits found on the plasma membrane of the vitellogenic oocytes This is followed by their appearance in the pinched-off coated vesicles as well as in early endosomes, which fuse together to form the mature electron-dense late endosomes (Figs 3, 4) Such an endocytotic entry of Vg into the oocytes to form the yolk body is similar to that described for insects [61] In P monodon, after the binding of Vg with VgR, the complex moves into the oocyte cytoplasm, aided by internalization signals present in VgR [57] Interestingly, the VgR of P monodon has two putative internalization signals (i.e., FANPGFG and FENPFF) found in vertebrate VgRs as well as several IL and LI sites characterizing the insect VgR and Drosophila yolk peptide receptors [57] This redundancy with the internalization signals present in the shrimp oocytes could increase the efficiency of receptor-ligand binding during crustacean vitellogenesis Fig Immunogold labeling of Vg in ultrathin sections of the ovary of Scylla serrata, examined with a Philips CM10 transmission electron microscope to demonstrate the endocytosis of Vg Vg labeling is seen along the luminal surface of the coated vesicle (cv), which fuses into a mature endosome Electron-dense particles representing Vg molecules are densely packed within the endosomes Scale bar 0.5 lm (from Warrier and Subramoniam [56]) Fig Immunogold labeling of Vg in ultrathin sections of the ovary of Scylla serrata In this micrograph, fusion of an early endosome (ee) with a mature endosome is observed (indicated by an arrow) Electron-dense particles of Vg are extensively labeled in the mature endosome compared to the early endosome The mature endosomes finally form the yolk bodies Scale bar 0.5 lm (from Warrier and Subramoniam [56]) Yolk processing In general, Vg undergoes post-translational proteolytic cleavage at the site of synthesis (e.g., insects [61]) or after sequestration into the ovary (e.g., amphibians [62]) In crustaceans, SDS-PAGE analysis of hemolymph and ovary yolk proteins has indicated the occurrence of varying numbers of Vg and vitellin (Vn) fractions, suggesting that Vgs are already fragmented at the time of endocytotic uptake into the ovary In the isopod Armadillidium vulgare, four female-specific glycoprotein bands in hemolymph, detected on SDS-PAGE, 123 were found to be the same in the ovarian extract [63] In this isopod, an anion-exchange HPLC separation has yielded vitellins from the ovary, ranging in molecular weight from 112 to 205 kDa [64] The N-terminal sequencing of these proteins showed identical amino acids except for the 112 and 59 kDa proteins PCR-assisted cloning of the 50 region of a cDNA encoding Vg revealed the presence of an amino-terminal sequence identical to those of the 112 and 122 kDa yolk proteins, suggesting that the Vg gives rise to the Vn fractions by cleavage either in the hemolymph or ovary In M rosenbergii, Vg, after being synthesized as a single precursor protein, undergoes initial cleavage at amino acids 707–710 by a subtilisin-like endoprotease to give rise to two subunits, A and pro-B, within the hepatopancreas [65] After secretion into the hemolymph, subunit A is sequestered as is into the ovary, whereas pro-B is cleaved by another processing enzyme to give rise to subunits B and C/D (Fig 5) The ovary subsequently takes them up to give rise to the yolk proteins, VnA, VnB, and VnC/D Examination of subunit composition of Vg in hemolymph and Vn into the ovary by SDS-PAGE and Fig Schematic representation of synthesis and processing of vitellogenin in Macrobrachium rosenbergii Vg is synthesized as a single precursor molecule, A–B–C/D, in hepatopancreas, which is then cleaved into two subunits, A and proB Subunits A and proB are released into the hemolymph, and proB is cleaved to form two subunits B and C/D The three processed subunits A, B, and C/D are incorporated into the ovary (From Okuno et al [65]) 123 Fish Sci (2011) 77:1–21 western blotting has also supported the above sequence of Vg conversion to Vn fractions Furthermore, identity in the N-terminal amino acid sequences of these Vg and vitellin fractions that appear in hemolymph and ovary has also provided final support to the scheme of Vg processing in this freshwater prawn [65] Further studies on the processing of other decapod crustacean vitellogenins have revealed conservancy in the first cleavage site at amino acids 707–710, although the subsequent cleavage sites may differ among many species In Litopenaeus vannamei, Raviv et al [66] predicted an N-terminal sequence of 78 kDa, with the first cleavage site occurring at an RTRR consensus cleavage for subtilisin-like endoprotease These authors isolated five HDL polypeptides of masses 179,113, 78, 61, and 42 kDa from the ovary and found that all of these polypeptides are derived from the 179 kDa second fraction of the premature Vg of L vannamei These results are in accord with those described for M rosenbergii yolk protein processing In a recent study on the mud shrimp, Upogebia major, belonging to the infraorder Thalassinidea of Decapoda, Kang et al [67] found three polypeptides in the oocytes These subunits were found to be derived from a single long polypeptide translated from the Vg transcript in the hepatopancreas This precursor polypeptide of 289 kDa is cleaved to produce two Vg subunits at the consensus cleavage site, RLRR, which is recognized by subtilisinlike endoproteases These two subunits are also suggested to undergo further processing upon or immediately after incorporation into oocytes Evidence for the secondary cleavage of vitellogenin after its uptake into the ovary is given in other decapods such as the freshwater crayfish, Ibacus ciliates [68] A low-density lipoprotein isolated from the ovary of this crayfish degraded Vg into apolipoprotein fragments, which are similar to the lipovitellin subunits of the egg Furthermore, the Vg digested by LDL exhibited proteinase activity whereas the native Vg did not have it The instability of Vg and its susceptibility to undergo proteolytic cleavage may be a general feature, but in a brachyuran crab Scylla serrata, Vg itself possesses proteinase activity [40] Warrier and Subramoniam [40] demonstrated that conformational changes in the native Vg could bring about such proteolytic cleavage, as indicated in a study using urea as a destabilizer Whereas Vg showed a spectral change with M-urea treatment due to exposure of the hydrophobic core containing aromatic residues (absorption at 274 nm), lipovitellin did not show such a spectral shift Clearly, Vg is a relatively unstable lipoprotein, but the ovarian lipovitellin is more stable Yolk utilization Yolk proteins primarily evolved to supply both energy as well as organic building blocks to support embryonic Fish Sci (2011) 77:1–21 growth in oviparous animals Understandably, yolk utilization is the central event of embryogenesis, and is accomplished by a host of hydrolytic enzymes acting on the complex yolk molecules Subramoniam [69] has reviewed the existing information on crustacean embryonic nutrition from the perspective of yolk utilization During yolk utilization, the complex lipovitellins are dismantled by esterases, proteases and glycosidases, resulting in the release of conjugated steroidal hormones [70] The regulated release of active ecdysteroids from their conjugates by nonspecific esterases at specific times in embryogenesis may not only trigger embryonic cuticle formation but may also accomplish larval molting and egg hatching [26, 71] Direct utilization of lipovitellins in the egg by way of proteolytic cleavage in the developing embryos has also been documented in the blue crab C sapidus [72] As much as the yolk proteins meet the metabolic demands of embryonic development, they are also used in early larval development In an extreme case of cirripede development, a new protein is expressed during the nonfeeding cypris stage of the barnacles This protein, called cypris major protein, is interestingly related to the heavy chain of barnacle yolk protein both structurally and immunologically [73] Another glycoprotein, called settlement-inducing protein complex (SIPC), which is found in juvenile and cyprid larvae of the barnacle Balanus amphitrite, also showed immunological and peptide sequence similarity with cirripede yolk proteins [74] Evidently, cirripede larval storage protein and the SIPC may share a common ancestor with yolk protein Alternatively, crustacean yolk protein genes would have undergone duplication to give rise to different proteins necessary for larval metamorphosis and gregarious larval settlement in these sessile barnacles Endocrine regulation of vitellogenesis In most malacostracan crustaceans, except the diecdysic crabs, vitellogenic activities are sandwiched between two molt cycle stages Such an inextricable linkage between molting and vitellogenesis is accomplished by a delicate multihormonal interaction unique to crustaceans Egg brooding within the pleopods of several malacostracans provides another intervention in the coordinated control of molting and reproductive cycles Essentially, the hormonal controlling mechanisms enabling the temporal separation of these two processes involve principally the inhibitory neuropeptides—vitellogenesis-inhibiting hormone (VIH) and molt-inhibiting hormone (MIH)—originating from the X-organ/sinus gland complex in the optic ganglia Thus, the hormonal coordination of both molting and vitellogenesis becomes vital to accomplishing continued body growth and increased fecundity [75] The endocrine factors that control vitellogenesis can be considered under two categories: gonad-inhibiting and gonad-stimulating hormones Gonad-inhibiting hormones Gonad-inhibiting hormones of Crustacea mainly reside in the eyestalk X-organ/sinus gland complex The crustacean hyperglycemic hormone (CHH) superfamily of neuropeptides that mainly originate from this neuronal complex include important regulatory molecules to control somatic growth and reproduction CHHs themselves play a pivotal role in the regulation of glucose metabolism However, they also exhibit considerable cross-functional activities with other peptides such as MIH, VIH, and mandibular organ inhibitory hormone (MOIH) The application of peptide sequencing as well as PCR-based cloning techniques has resulted in the isolation of many cDNA sequences of CHH family members involved in diverse regulatory functions In addition, these investigations have facilitated sequence homology studies to establish structural relationships among them Their neuronal distribution outside eyestalk ganglia implicates other parts of CNS such as supraesophageal ganglia, thoracic ganglia and ventral nerve cord in the regulatory roles of molting and reproduction Vitellogenesis-inhibiting hormone Vitellogenesis-inhibiting hormone belongs to the CHH family of neuropeptides, and shows inhibitory effects on ovarian growth and vitellogenesis Our present understanding of endocrine regulation of crustacean vitellogenesis per se is mainly based on experimental studies involving the removal of VIH by way of eyestalk extirpation VIH was first characterized in the American lobster H americanus as a 78-residue peptide that exists as two enantiometric isoforms, both of which have a molecular mass of 9135 Da, an amidated C-terminus and a free N-terminus [76, 77] However, the vitellogenesis-inhibiting effect was found in only one isoform when tested with an in vivo heterologous assay developed in the grass shrimp Palaemonetes varians VIH has been subsequently isolated and characterized from many malacostracans, and has been shown to play a prominent role in the regulation of reproduction, especially vitellogenesis [78] Amino acid sequence homology studies on the VIH of several crustacean species have uncovered considerable similarities with other CHH family peptides such as MIH and MOIH, claiming a separate subgroup (Type II) from the CHH molecules [79] Bioassay studies to test VIH activity have been carried out either using an ovarian growth index [80, 81] or by in vitro culturing of ovarian tissue and monitoring the inhibition of protein synthesis [82, 83] Inhibition of 123 144 protein-accelerated TMAO degradation [9, 12] In contrast, other studies have revealed no effect of hemoglobin on DMA generation [13] Thus, although dark muscle contains a large amount of iron [19], the types of iron that affect DMA formation in fish muscle have not yet been defined The purpose of the current research was to determine the reason that DMA is conspicuously generated in dark muscle of gadoid species during frozen storage, and the factors that accelerate its production in fish muscle Thus, we first monitored DMA formation during chilled and frozen storage, and then conducted a detailed analysis of the muscle components of the three gadoid species to directly investigate the factors that accelerate DMA formation in fish muscle Identifying the factors that impact DMA generation in dark muscle of gadoid species may facilitate the development of effective procedures to suppress DMA and FA formation and lead to better stability of fish meat during frozen storage Materials and methods Fish samples Walleye pollock Theragra chalcogramma were caught by trawling in the Bering Sea and immediately stored in refrigerated seawater (RSW) at ± 2°C until samples for TMAO and DMA determination were taken Southern blue whiting Micromesistius australis (SBW) and hoki Macruronus magellanicus were caught by trawling off the coast of Chile and immediately kept in RSW at ± 2°C until samples were taken When preparing frozen fish samples, the fish were first beheaded and gutted by hand, and then the samples were frozen in a contact freezer until it reached -25°C, after which they were stored at -23°C until analysis Initial analysis started within month TMAO and DMA determination Fresh or frozen fish muscle samples were kept in 5% trichloroacetic acid and analyzed within month TMAO content in muscle samples was determined as described by Hashimoto and Okaichi [20] using a modified version of the original method of Dyer et al [4, 21] TMAO was reduced to trimethylamine (TMA) using Devarda’s alloy The amount of TMA was determined colorimetrically TMAO content was calculated from the TMA results The copper dithiocarbamate procedure was used to measure DMA [22] TMAO for calibration standard was purchased from the Aldrich Chemical Company, Inc (Milwaukee, WI, USA) All other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan) 123 Fish Sci (2011) 77:143–149 Proximate component analysis Although certain substances such as Fe2?, reducing agents, sulfur dioxide, and so on are known to accelerate DMA generation [8, 9, 12–15], there has been no report on the relationships between the concentrations of these accelerators in fish muscle and the rate of DMA generation during frozen storage Therefore, we carried out a detailed analysis of the muscle components of the three gadoid species Ordinary and dark muscle was carefully separated The moisture content of each muscle type was determined after drying the samples at 115°C for h The protein content was measured using the Kjeldahl method (nitrogen coefficient was 6.25) Crude fat content was determined gravimetrically using acid hydrolysis and diethyl ether extraction [23] Free amino acid composition of the samples was measured with an automatic amino acid analyzer (model L8900; Hitachi, Tokyo, Japan) after the samples had been deproteinized with picric acid solution Total lipids were extracted using the modified method of Bligh and Dyer [24] and then used to determine lipid classes Lipid classes were assigned based on analysis by an Iatroscan TLC–FID using a TH-10 Iatroscan thin-layer chromatograph with a flame ionization detector (Iatron Laboratories Inc., Tokyo, Japan) Iron analysis Total iron content was measured using an atomic absorption spectrometer (model Z2000; Hitachi) Nonheme iron analysis was performed as described in the preceding research [25] Effects of various types of iron on DMA generation using an in vitro system An in vitro system was used to assess the reactions between various iron substances and TMAO Two types of iron, namely nonheme iron and heme iron, were evaluated in order to elucidate the relationship between iron state and DMA generation Table lists the components included in various reactions used to measure TMAO degradation Ferrous sulfate (FeSO4; Wako Pure Chemical Industries) was selected as the nonheme iron, and hemin (Sigma– Aldrich, St Louis, MO, USA), oxymyoglobin, and metmyoglobin were selected as the heme irons Oxymyoglobin and metmyoglobin from big eye tuna were purified as described [26] Only the pure oxymyoglobin with a ratio of the a peak (577 nm) to the b peak (542 nm) of over 0.95 was used in the in vitro system The concentrations of oxymyoglobin and metmyoglobin were determined using the pyridine–hemochromogen method [27] The reaction mixtures were frozen in an air-blast freezer at -40°C for Fish Sci (2011) 77:143–149 145 Table Components in reactions used to measure TMAO degradation in vitro Tris–acetate (pH 7) (mM) TMAO (mM) EDTA (mM) Cysteine (mM) Fe substances FeSO4 10 50 – 0.12 mM FeSO4(II) Fe-EDTA 10 50 1 0.12 mM FeSO4(II) MbO2 10 50 1 0.12 mM MbO2(II) metMb 10 50 1 0.12 mM metMb(III) Hemin 10 50 1 0.12 mM hemin(III) TMAO trimethylamine-N-oxide, EDTA disodium ethylenediaminetetraacetate, Fe iron, FeSO4 ferrous sulfate, Fe-EDTA ferrous sulfate and ethylenediaminetetraacetic acid, MbO2 oxymyoglobin, metMb metmyoglobin 18 h and then held at -10°C for weeks Reactions were terminated using a 5% trichloroacetic acid solution The amount of DMA generated was determined as the difference between the DMA amount immediately after initial freezing and the DMA amount after the storage period Effects of various types of iron on DMA generation in pollock mince Only fresh fish was used for this analysis to eliminate potential changes in properties of fish meat caused by frozen storage Pollock could only be purchased as fresh from a local (Tokyo, Japan) fish wholesale market and stored on ice Skinless fillets were minced using a mincer (BK-220, Bonny Co Ltd., Osaka, Japan) with a mm diameter mesh size Five substances were added individually to the minced meat, including the nonheme iron FeSO4 (1 mM), the heme irons oxymyoglobin (0.1 mM), and hemin (0.1 mM), disodium ethylenediaminetetraacetate (disodium EDTA, 10 mM; Wako Pure Chemical Industries), and mM ascorbic acid (Wako Pure Chemical Industries) Each substance was dissolved in water weighing 5% of the meat weight The meat was then added and mixed for 90 s with a silent cutter (SCP-2A, Hanaki Manufacturing Co Ltd., Tokyo, Japan) The control sample contained the same amount of water without any test substance The samples were packed in plastic bags, frozen in an air-blast freezer at -40°C for 18 h, and then kept at -10°C for weeks were carefully separated Muscle was homogenized in a solution (pH 7.0) containing 20 mM Tris–HCl, 0.5 M sodium chloride, 1% sodium dodecyl sulfate (SDS), and 1% 2-mercaptoethanol, and passed through triple-layer gauze The filtrates were centrifuged at 80009g at 25°C for 20 The supernatants obtained were subjected to chromatography at 25°C with a Sepharose 6B gel column (1.5 cm 100 cm; GE Healthcare, Piscataway, NJ, USA) and a mobile phase (pH 7.0) of 20 mM Tris–HCl, 0.5% NaCl, and 1% SDS at 0.5 ml/min Measuring DMA generation activity in gel filtration fractions To assess the DMA generation activity in gel filtration fractions, reaction mixtures (pH 7.0) containing 10 mM Tris–acetate, 50 mM TMAO, mM disodium EDTA, 0.12 mM FeSO4, mM cysteine, and a gel filtration fraction sample (40% v/v) in a total volume of 2.5 ml were used Control reactions contained the eluent buffer instead of a fraction The mixtures were prepared on ice, frozen in an air-blast freezer at -40°C, and then kept at -10°C for week Reactions were terminated by adding ml of trichloroacetic acid to give a final percentage of 5% The amount of DMA generated was determined as the difference between the DMA amount immediately after initial freezing and the DMA amount after the storage period Results Gel filtration chromatography of muscle extracts To directly investigate the factors that accelerate DMA formation from TMAO in fish muscle, homogenized pollock ordinary and dark muscle were fractionated separately by gel filtration chromatography Only fresh fish was used for this analysis in order to eliminate potential changes in properties of fish meat caused by frozen storage Fresh pollock purchased from a local (Tokyo, Japan) fish wholesale market was used, and ordinary and dark muscle Changes in the concentrations of TMAO and DMA during RSW storage TMAO and DMA in each fish were monitored immediately after catching to establish a baseline of TMAO and DMA content before being frozen Immediately after the fish were caught, ordinary muscle TMAO concentrations in the meat samples were as follows: 78 ± mM in pollock, 57 ± mM in SBW, and 62 ± mM in hoki (Fig 1) 123 146 Fish Sci (2011) 77:143–149 100 TMAO (mM) 100 60 Pollock SBW Hoki 60 40 20 25 50 75 100 Pollock Time kept in RSW (h) Fig Changes in the trimethylamine-N-oxide (TMAO) content in ordinary muscle from fish that were held in refrigerated seawater (RSW) at ± 2°C Data represent the mean ± SD values for five independent determinations SBW southern blue whiting Dark Dark SBW Hoki Fig Trimethylamine-N-oxide (TMAO) content in ordinary and dark muscle in gadoid species stored at -23°C for 15 months Results are averages ± SD of triplicate measurements SBW southern blue whiting Open bars initial, filled bars after storage at -23°C for 15 months 1.2 14 Pollock 1.0 12 0.8 DMA (mM) SBW Hoki 0.6 0.4 10 25 50 75 100 Time kept in RSW (h) Pollock Dark Dark SBW Ordinary Ordinary 0.0 Dark 0.2 Ordinary DMA (mM) Ordinary 20 Ordinary Dark 40 80 Ordinary TMAO (mM) 80 Hoki Fig Dimethylamine (DMA) formation in ordinary muscle from fish that were stored in refrigerated seawater (RSW) at ± 2°C Data represent the mean ± SD values for five independent determinations SBW southern blue whiting Fig Dimethylamine (DMA) formation in samples of ordinary and dark muscle Results are averages ± SD of triplicate measurements SBW southern blue whiting Open bars initial, filled bars after storage at -23°C for 15 months TMAO content in each of the fish types decreased slightly during RSW storage The initial concentration of DMA in ordinary muscle was below 0.1 mM in all three species, but the concentration gradually increased, particularly in hoki, where 0.9 mM DMA was formed after 100 h (Fig 2) samples stored for 15 months at -23°C (Fig 4), in agreement with much of the literature [2, 5, 17, 18] DMA generation in hoki ordinary and dark muscle was significantly greater than that in the other two species Changes in the concentrations of TMAO and DMA during frozen storage The rate of TMAO degradation differs significantly depending on the fish species [5, 17, 18], fish part [17, 18], temperature [2, 14, 28], and processing method [28] Here we compared the TMAO contents and the formation of DMA during frozen storage in the ordinary and dark muscle of the three gadoid fish species In all three species, the TMAO concentration in dark muscle was 19–46% lower than in ordinary muscle at the initial stage (Fig 3) Substantial amounts of DMA were formed in dark muscle 123 Proximate component analysis of ordinary and dark muscle As shown in Table 2, ordinary muscles of the three species had similar moisture, protein, and total fat contents For dark muscle, however, hoki had the lowest moisture and protein contents but the highest fat content compared with the other two species Triglycerides accounted for 97% of the total lipid in hoki dark muscle, whereas phospholipid was the major component in the other muscle, including ordinary muscle The iron content of dark muscle was 3–4 times higher than that of ordinary muscle in all three species, and all three species had comparatively high nonheme iron contents in dark muscle Fish Sci (2011) 77:143–149 147 0.6 Table Comparison of ordinary and dark muscle components from gadoid species SBW Hoki 0.4 Ordinary Dark Ordinary Dark Ordinary Dark Moisture (%) 80.3 80.1 79.6 80.3 80.1 56.3 Protein (%) 17.8 16.9 18.6 16.2 18.2 11.5 Total fat (%) 0.7 Total iron (lM) 108 1.7 287 0.6 91 2.3 358 0.5 77 31.3 287 Nonheme iron (lM) 85 20 78 30 215 24 DMA (mM) Pollock FeSO4 Fe-EDTA MbO2 metMb Hemin 0.2 0 14 Time (days) Free amino acid (mg/100 g) 107 Aspartic acid 245 84 281 47 211 5 Threonine 5 3 Serine Glutamic acid 12 20 16 11 13 Glycine 23 12 11 Alanine 14 14 Valine Cysteine 4 Methionine 3 Isoleucine 3 Leucine 4 Tyrosine 2 2 Phenylalanine 3 2 Lysine 13 Histidine 0 0 Anserine 110 88 31 105 Arginine 5 0 Asparagine 11 10 10 Proline 0 Lipid composition (%) Wax Trace Trace Trace Trace Trace Trace Triglyceride Free fatty acid Trace 12.5 Trace 1.1 13.0 20.7 Trace 1.4 9.3 8.8 96.8 0.9 Cholesterol 3.6 3.6 3.1 2.8 3.5 Trace Phospholipid 83.9 83.4 75.1 87.9 86.3 2.3 compared with ordinary muscle, particularly in hoki dark muscle, where 215 lM was measured Each fish species had a characteristic free amino acid composition in each muscle type Dark muscle contained an abundance of nonprotein taurine Free cysteine was not detected in either muscle type; this is notable because it has been reported that DMA formation is stimulated when cysteine and Fe2? coexist [14] Effects of various types of iron on DMA generation DMA formation was only observed when the chelated iron, Fe-EDTA, was added to the in vitro system, and DMA was Fig Relationship between dimethylamine (DMA) formation and the type of iron substance added to fish meat during frozen storage (at -10°C) Reactions were conducted using the trimethylamine-N-oxide in vitro degradation system Results are averages ± SD of triplicate measurements FeSO4 ferrous sulfate, Fe-EDTA ferrous sulfate and ethylenediaminetetraacetic acid, MbO2 oxymyoglobin, metMb metmyoglobin 15 Control FeSO4 EDTA DMA (mM) Taurine 10 MbO2 Hemin Ascorbic acid 0 14 28 Time (days) Fig Relationship between dimethylamine (DMA) formation and the type of iron substance added to fish meat during frozen storage (at -10°C) Reactions were conducted using pollock minced meat Results are averages ± SD of triplicate measurements FeSO4 ferrous sulfate, EDTA ethylenediaminetetraacetic acid, MbO2 oxymyoglobin not produced in the presence of FeSO4 or heme irons such as myoglobin, metmyoglobin and hemin (Fig 5) On the other hand, a large quantity of DMA was formed when either FeSO4 or ascorbic acid was added to pollock mince (Fig 6), although FeSO4 did not accelerate DMA formation in the in vitro system Gel filtration chromatography of muscle extracts, and DMA generation activity in the fractions Extracts of pollock ordinary and dark muscle were subjected to gel filtration chromatography to investigate factors that could accelerate DMA formation The first absorbance peak in the gel filtration chromatogram of pollock ordinary muscle was lower than the corresponding peak for dark muscle, but the second peak for each muscle type showed a similar elution pattern (Fig 7) Fractions from both ordinary and dark muscle chromatography were 123 148 Fish Sci (2011) 77:143–149 Absorbance at 280 nm 2.5 Ordinary muscle Dark muscle 2.0 1.5 1.0 0.5 0.0 20 40 60 80 100 120 Fraction Number Fig Sepharose 6B gel filtration chromatography of the supernatant obtained from pollock ordinary and dark muscle homogenates DMA (mM) 3.0 Ordinary muscle Dark muscle 2.0 1.0 0.0 control 58 73 87 92 96 Fraction Number Fig Dimethylamine (DMA) formation activity in selected fractions obtained from the Sepharose 6B gel filtration of pollock ordinary and dark muscle homogenates The amount of DMA generation is shown as the difference between the DMA amount immediately after freezing and the DMA amount after storage at -10°C for week Results are averages ± SD of triplicate measurements added to the DMA formation model system described in ‘‘Materials and methods.’’ Dark muscle fraction 92 significantly accelerated DMA formation (Fig 8) The amount of iron in this fraction was below detectable limits Discussion Previous studies have shown that a higher amount of DMA forms in dark muscle than ordinary muscle in gadoid species during frozen storage [5, 17] Although several factors have been reported to accelerate DMA formation [8, 9, 12–15], few studies have compared the relationship between the rate of DMA formation and the content of potential accelerating factors in fish muscle The present study aimed to clarify the mechanism that causes DMA to be produced so rapidly in dark muscle in three gadoid species Toward this end, we determined the extent of DMA formation in gadoid species and attempted to directly identify factors that accelerate DMA formation 123 Ordinary muscle in all of the species formed DMA during RSW storage, and hoki ordinary muscle in particular formed 0.9 mM DMA after 100 h Previous studies have shown that millimolar levels of FA decrease fish protein quality [2, 9, 29] Therefore, fish meat quality may decline during frozen storage if fish are not dealt with properly in the chilled state All dark muscle assessed in the present study formed more DMA than ordinary muscle during frozen storage Our results suggest that accelerated DMA formation in dark muscle results from higher amounts of nonheme iron compared to ordinary muscle Hoki dark muscle formed the largest amount of DMA compared with the dark muscle from other species One possible reason for this could be that hoki dark muscle had the highest amount of nonheme iron among the dark muscle of the various species, but there may be other factors Although FeSO4 did not accelerate DMA formation in vitro, DMA was formed when the iron was chelated by EDTA The reason for this is that when Fe2? is chelated by EDTA, the potential of the system changes from -0.77 to ?0.10 V and an electron is liberated [8] On the other hand, DMA formation was accelerated when FeSO4 by itself was added to pollock mince These results suggest that pollock mince may contain components that act similarly to EDTA with regard to its observed effect on the addition of FeSO4 in our in vitro system In our study, both nonheme iron and heme iron compounds were used Although these substances are commonly used in DMA formation research [8, 11–14], the effects of the counterion, such as SO42-, need to be carefully considered The taurine content in dark muscle was higher than that in ordinary muscle in each gadoid species As taurine has been reported to accelerate DMA formation [14], taurine could be one of the reasons why dark muscle forms more DMA than ordinary muscle It has also been reported that DMA formation is stimulated when both cysteine and Fe2? are present [14], but we did not detect free cysteine in either muscle type Extracts of ordinary and dark muscle from pollock were fractionated by gel filtration, and DMA formation was accelerated by a low-molecular-weight fraction from dark muscle Although the factor(s) in this fraction that accelerates DMA formation has/have not yet been identified, past studies suggest that reductants or EDTA-like substances could be candidates for DMA formation acceleration factor(s) [8, 9, 11, 12, 14], and further studies will be necessary to determine the factor(s) The gel filtration mobile phase contained SDS and 2-mercaptoethanol; therefore, the DMA formation-accelerating activity of the low-molecular-weight component present in dark muscle may not be affected by these chemical reagents Previous studies have shown that substantial amounts of DMA are formed when fish muscle, which has been dried and Fish Sci (2011) 77:143–149 subjected to frozen storage, is heated to 100°C [8] These results suggest that the factor(s) in the fraction that promote DMA formation may exhibit stable activity In conclusion, the amount and redox state of nonheme iron are critical to DMA formation in fish meat during frozen storage Although fish muscle nonheme iron, such as that in ferritin and transferrin, is in the Fe3? state, these iron-containing compounds may have been able to catalyze DMA formation if the Fe3? was released 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and formaldehyde in Alaska pollack muscle during frozen storage (in Japanese with English abstract) Bull Hokkaido Reg Fish Res Lab 29:108–122 123 Fish Sci (2011) 77:151–157 DOI 10.1007/s12562-010-0306-7 ORIGINAL ARTICLE Food Science and Technology PCR-DGGE analysis of bacterial communities in funazushi, fermented crucian carp with rice, during fermentation Tateo Fujii • Shoko Watanabe • Masako Horikoshi Hajime Takahashi • Bon Kimura • Received: 19 March 2010 / Accepted: 18 October 2010 / Published online: 27 November 2010 Ó The Japanese Society of Fisheries Science 2010 Abstract Funazushi (fermented Crucian Carp with rice) is a fermented fish product found only around Lake Biwa in Shiga Prefecture, Japan It is characterized by a unique cheese-like flavor and characteristic sour taste We analyzed the changes in the microbial community during funazushi fermentation by denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA fragments (PCRDGGE) and by plate counts The plate counts showed that lactic acid bacteria reached 8.0 log10 CFU/g within days of fermentation initiation before decreasing slowly to 4.0 log10 CFU/g during the remainder of 1-year study period PCR-DGGE revealed that the dominant bacteria in the initial (days 14 and 30) and latter (days 90, 180, and 360) periods of fermentation were Lactobacillus plantarum and L acetotolerans This is the first identification of L acetotolerans in funazushi as traditional cultivation techniques have not been sufficiently sensitive This is the first report of PCR-DGGE being used to assess the microbial community in funazushi This technique was also found to be effective in profiling microbial diversity T Fujii Á S Watanabe Á H Takahashi Á B Kimura Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan M Horikoshi Faculty of Education, Shiga University, Otsu, Shiga 520-0862, Japan Present Address: T Fujii (&) Department of Domestic Science, Tokyo Kasei University, 1-18-1 Kaga, Itabashi-ku, Tokyo 173-8602, Japan e-mail: fujii@tokyo-kasei.ac.jp Keywords Funazushi Á Bacterial community Á PCR-DGGE Á Lactobacillus plantarum Á L acetotolerans Introduction Funazushi (fermented Crucian Carp with rice), a fermented fish product, has been produced for an extended period only around Lake Biwa in Shiga Prefecture It is characterized by a unique cheese-like flavor and unusual sour taste and can be considered a typical fermented fish product in the truest sense, namely, the fermentation of carbohydrate by lactic acid bacteria results in the production of lactic acid [1] The annual production volume of funazushi in the last 20 years has been less than 100 t, with a decreasing trend due to a shortage of raw materials There are two stages to the manufacturing process of funazushi [2]: (1) immediately after being caught, crucian carps are salted for at least months; (2) after the initial salting stage, the fish are washed and subsequently fermented with boiled rice in barrels, under the pressure of stones, for more than year Pickling of the fish in boiled rice during the manufacturing process is thought to be primarily responsible for the ripening of funazushi During this process, the unique flavor and taste of funazushi are produced by the autolytic enzymes from the fish [3, 4] and by microorganisms, such as lactic acid bacteria and yeasts, among others [5, 6], which produce various kinds of compounds, such as organic acids and alcohols, that contribute to the product’s flavor and taste and, by lowering pH, inhibit the growth of pathogenic bacteria, such as Clostridium botulinum and other putrefactive bacteria [6–8] As it is important to stimulate the rapid growth of lactic acid bacteria to inhibit undesirable bacteria, the pickling of fish in boiled rice is carried out during the summer Furthermore, in order to 123 152 maintain anaerobic conditions during fermentation, layers of fish and rice in the pickling barrel are weighted with stones and, in some cases, the barrel is also topped up with water Lactic acid bacteria hitherto isolated from funazushi or its fermentation processes have been identified as Lactobacillus plantarum, L pentoaceticus, L kefir, L alimentarius, L sake, Streptococcus faecium, S thermophilus, Pediococcus parvulus, among others [6, 7] However, in these previous studies dealing with fermented fishes, including funazushi, traditional microbiological methods, such as plate counts, microbial isolation, and biochemical identification, were used However, various researchers have reported different results in similar fermented fishes [6, 7, 9], likely due to biases directly associated with the medium used for isolation and the taxonomic criteria of the researcher To avoid these biases, molecular identification techniques, particularly sequencing analyses of the 16S rRNA gene for the identification of bacteria, have become standard procedures in various areas of microbiology [10] In addition, denaturing gradient gel electrophoresis (DGGE), a culture-independent procedure, has been developed and is being widely used in the study of the microbial flora of fermented foods [11–13] To date, however, there have been no reports on the molecular biological analysis of the microflora of funazushi In this paper, we describe the use of PCR–DGGE to assess changes in the microbial flora of funazushi during fermentation Materials and methods Samples Funazushi was prepared by traditional techniques from female Crucian Carp (Carassius carpio), salt, and boiled rice in Shiga Prefecture, Japan About 30–40 freshwater Crucian Carp (body length approx 30 cm, weight 250–300 g) were degutted, leaving the ovaries inside the body, salted with a saturated salt solution for approximately months (from April to July 2003), and washed with tap water to remove the salt Fish were then placed in a layer on top of a rice bed at the bottom of a barrel (30-l volume) and covered with rice; the layering process was repeated three times thereafter (4 layers of fish), with the top layer of fish covered with a thick layer of rice In total, 10 kg of fish, 14 kg of boiled rice, and 200 g of salt were used for the production of funazushi samples The layered rice and fish were subjected to pressure by being weighted with stones (two layers, 40 kg each) and fermented at ambient temperature ranging from approximately 10–35°C for year Samples of fish and rice were collected on days 123 Fish Sci (2011) 77:151–157 0, 3, 7, 14, 30, 90, 180, and 360 for the measurement of pH, salt, and organic acids and for microbiological analyses (plate count and DGGE) On day 0, the washed salted fish and boiled rice were sampled for analysis Analysis of pH, salt concentration, and organic acid content A 10-g sample of Funazushi was placed in a stomacher bag with 10 ml of sterilized water and mixed by a Stomacher Lab-blender 80 (Seward, London, UK) A portion of the mixture was used for the analysis of pH, salt concentration, and organic acid content The pH was measured using a M— pH meter (Horiba, Kyoto, Japan), and salt concentration was measured using a C121 compact salt analyzer (Horiba) The organic acids in the funazushi samples were analyzed by high-performance liquid chromatography (HPLC) as described previously [14] Briefly, ml of funazushi homogenate was filtered through a filter (pore size 0.2 lm; Toyo Roshi, Tokyo, Japan) and analyzed on a LC-9A chromatograph system (Shimadzu, Kyoto, Japan) using a Shim-pack SCR-102H column (Shimadzu) Plate count method Five grams of each sample was homogenized in 45 ml of sterilized saline containing 2.5% (w/v) NaCl, 0.25% MgSO4Á7H2O, 0.1% KCl, and 1% polypepton with a Stomacher On each sampling day, individual funazushi samples were obtained from both the rice portion and fish portion Diluted homogenate was plated onto trypticase soy agar (TSA) (BBL Becton Dickinson, Sparkes, MD) for the total viable count (TVC), onto MRS medium (Merck, Darmstadt, Germany) supplemented with 0.1% (w/v) sodium azide and 0.3% CaCO3 for lactic acid bacteria (LAB), and onto potato dextrose agar (PDA; Eiken Chemical, Osaka, Japan) containing 0.01% chloramphenicol for yeast and mold The cultures on TSA and MRS medium were incubated at 30°C for days, and those on PDA medium were incubated at 20°C for days Cultures on MRS medium were incubated under anaerobic conditions using the AnaeroPak system (Mitsubishi Gas Chemical, Tokyo, Japan) Direct extraction of bacterial DNA and PCR amplification Bacterial DNA from ml of homogenate from each sample described above was extracted using phenol–chloroform and ethanol precipitation [15] Briefly, bacterial cells in ml of the funazushi homogenate were centrifuged at 15,000 g for and the pellet suspended in 567 ll of TE (10 mM Tris-HCl, mM EDTA) buffer containing Fish Sci (2011) 77:151–157 DGGE analysis of PCR products The DGGE analysis of PCR amplification products was performed as described previously [18] using the DCode System apparatus (Bio-Rad Laboratories, Hercules, CA) Polyacrylamide gels [8% (w/v) acrylamide–bisacrylamide (37.5:1)] in 19 Tris-acetate-EDTA buffer (40 mM Trisacetate, mM EDTA) with a denaturing gradient ranging from 30 to 60% denaturant [100% denaturation corresponds to M urea and 40% (v/v) formamide] were prepared using the Bio-Rad model 475 Gradient Delivery System Polymerization was achieved by adding 0.9% (v/v) ammonium persulfate (10% solution) and 0.09% (v/v) N,N,N,N-tetra methyl ethylene diamine (TEMED) The gels were electrophoresed at a constant voltage of 200 V at 60°C for h The DNA fragments were stained with ethidium bromide and washed with distilled water prior to UV transillumination Recovery of bands from DGGE gels and sequencing analysis The main DGGE fragments were selected for nucleotide sequence determination Each band was excised with a sterile razor The DNA of each fragment was eluted in 50 ll TE buffer at 100°C for 10 The extracts were re-amplified by PCR using the same primers and purified with the Ultrafree-MC 30,000 NMWL filter unit (Millipore, Bedford, MA) according to the manufacturer’s instructions Purified DNA fragments were ligated in pT7blue-vectors (Novagen, Darmstadt, Germany) and transformed into Escherichia coli JM109 The transformants were grown on LB agar containing ampicillin and subsequently screened for b-galactosidase activity using a specific b-galactosidase assay [15] Plasmid DNA of selected transformants was isolated using a Plasmid Miniprep kit (Bio-Rad) The inserted DNA sequence, approximately 200 bp of 16S rDNA (E coli position 389–530) [19], was determined using a SQ5500E DNA Sequencer (Hitachi High-Technologies, Tokyo, Japan) with the Thermo Sequenase Primer Cycle Sequencing kit (Amersham Biosciences, Piscataway, NJ) To identify the inserted sequences, the BLAST 2.0 algorithm was used to compare the derived sequence to 16S rDNA sequences in the DNA Data Bank of Japan (DDBJ) database Species identification was made on the basis of percentage similarity to database sequences (98.0–100.0% similarity) Results Changes in chemical composition Immediately after the initiation of pickling, the salt concentrations in the rice and fish portions were 1.3% (w/v) and 14.5%, respectively (Fig 1) The salt concentration NaCl concentrations (%) lysozyme (2 mg/ml) After incubation for h at 37°C, 30 ll of 10% (w/v) sodium dodecyl sulfate (SDS) and ll of 20-mg/ll proteinase K were added and the cells lysed for h at 37°C A 100-ll aliquot of M NaCl was then added and the DNA subsequently purified by extraction with chloroform–isoamyl alcohol (24:1) followed by extraction with phenol–chloroform–isoamyl alcohol (25:24:1) The DNA was then precipitated with isopropanol, washed with 70% ethanol, and dried Purified DNA was dissolved in 100 ll of TE buffer and used as the DNA template in PCR The primer pair chosen for the amplification of the V3 region (approximately 220 bp) of the 16S rRNA gene was the forward primer with GC clamp [16] (GC-339f; 50 -CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GCT CCT ACG GGA GGC AGC AG-30 ) and the reverse primer V3-53r (50 -GTA TTA CCG CGG CTG CTG G-30 ) PCR amplification was performed with 50 ng of template DNA in a 100-ll reaction mixture containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 50 pmol each of primer, 0.2 mM each of dNTPs, and 2.5 U of TaKaRa Taq DNA polymerase (Takara Bio, Shiga, Japan) To minimize the amplification of nonspecific products and to obtain a large amount of PCR products, ‘touchdown’ PCR [17] was performed wherein the initial annealing temperature was set at 8°C above the expected annealing temperature and decreased by 0.8°C every second cycle until the expected annealing temperature, 62°C, was reached (total of 20 cycles), and then five additional cycles were carried out Amplification was carried out in a GeneAmp 9600 thermal cycler (Applied Biosystems, Foster City, CA) using the following cycling program: denaturation at 94°C for 30 s, annealing for 30 s, and primer extension at 72°C for 10 s Aliquots (5 ll) of the PCR products were initially analyzed by electrophoresis on 2% (w/v) agarose gels 153 16 14 12 10 0 20 90 200 400 Aging period (days) Fig Changes in NaCl concentration during the aging process of the rice (filled squares) and fish (open circles) portions of funazushi 123 154 Fish Sci (2011) 77:151–157 20 90 200 400 Aging period (days) 1000 10 800 Fig Changes in pH during the aging process of the rice (filled squares) and fish (open circles) portions of funazushi 1600 A (rice) 1400 1200 Organic acids (mg/100g) Viable counts (Log10CFU/g) pH 10 600 A (rice) 100 200 300 400 B (fish meat) 100 200 300 400 Aging period (days) 400 200 0 1600 50 100 150 200 250 300 350 400 B (fish meat) 1400 Fig Viable counts of samples taken during the aging process of funazushi Total viable (open circles), lactic acid bacteria (filled squares), and yeasts (filled triangles) were counted on trypticase soy agar (TSA), MRS agar, and potato dextrose agar (PDA), respectively a Rice portion, b fish portion 1200 1000 800 600 400 200 0 50 100 150 200 250 300 350 400 Aging period (days) Fig Changes in organic acids during the aging process of the rice (a) and fish (b) portions of funazushi Symbols indicate content of lactic acid (filled squares), phosphoric acid (filled diamonds), acetic acid (open triangles), propionic acid (open squares), and butyric acid (asterisks) equilibrated to approximately 4.0% in both the rice and fish portions within days The pH value was 6.5 in the rice portion and 5.8 in the fish portion on day (Fig 2) The value in both portions decreased to 3.7 after 30 days and was constant thereafter As summarized in Fig 3, the amount of lactic acid in the rice portion increased from day to day 30 On day 360, large amounts of lactic acid had accumulated in both the rice portion (1,500 mg/100 g) and fish portion (1,070 mg/100 g) Changes in plate counts Changes in the viable counts of funazushi samples during the fermentation are shown in Fig The TVC of the 123 0-time sample was very low, being 2.5 log10 colony-forming units (CFU)/g in fish muscle on TSA medium LAB and yeasts on MRS medium and PDA medium, respectively, could not be detected at this time Three days later, the TVC in the sample had increased to 108 CFU/g and continued to increase until the 14th day of fermentation; thereafter, it decreased gradually, falling to approximately 4.0 log10 CFU/g after year The number of yeasts was lower than the TVC and number of LAB (approximately 3.0 log10 CFU/g) until the 180th day The number of yeast cells after day 90 was approximately 3.0 log10 CFU/g, and cell numbers remained consistent thereafter These results are in agreement with those reported earlier by our group [6, 7] Changes in bacterial flora analyzed by PCR–DGGE PCR products originating from funazushi preparations during the course of the fermentation were divided into three to eight fragments by DGGE analysis (Fig 5) The banding patterns differed greatly from day to the day of fermentation initiation, whereas the variety of patterns decreased after day 14 and the bacterial flora gradually stabilized Seven to eight bands were observed at the first stage of fermentation, decreasing to three bands with increasing fermentation time The banding patterns obtained from the rice and fish portions were not significantly different after day 180 Fish Sci (2011) 77:151–157 (Day) F R 155 14 30 90 F R F R F R F R F R 180 360 F R F R Table Identification of dominant fragments in denaturing gradient gel electrophoresis patterns during the aging process of funazushi Sample Fragmenta Closest relative Rice, Day Uncultured bacterium 100 Fish, Day Staphylococcus epidermidis 100 10 Rice, Day Fish, Day S warneri S epidermidis 100 99 Fish, Day S epidermidis 100 Rice, Day S epidermidis Rice, Day 7 Lactobacillus curvatus Fish, Day 14 L plantarum Fish, Day 90 L acetotolerans Fish, Day 90 10 Similarity (%) Fig Denaturing gradient gel electrophoresis (DGGE) profiles of DNA amplicons obtained directly from fermented funazushi Profiles obtained at time and after 3, 7, 14, 30, 90, 180, and 360 days of fermentation are shown F Fish portion, R rice portion Bands (1–10) were identified by BLAST searching in the DNA Data Bank of Japan (DDBJ; see Table 1) DGGE-gel band identification To identify the main bands, each band was recovered from the DGGE-gel and sequenced The results obtained from clone sequences are shown in Table At the initial stage of fermentation (day 3), two bands of strong intensity (band and 3), suggesting the dominant microbial flora, were observed The sequences of band and were concordant with those of Staphylococcus epidermidis and S warneri, respectively, in the DDBJ DNA database However, the intensity of band 3, corresponding to S warneri, weakened gradually during the middle stage of fermentation, and the band was barely detectable after day 90 Samples obtained from the 7th to 30th day showed a band at the same position (band 7), identified as Lactobacillus curvatus After day 14, band 8, identified as L plantarum, was detected and clearly observed until the day 30 A high-intensity band (band 9), identified as L acetotolerans, was detected on day 90, and its stable presence was observed until day 360 The only bacterium, identified as belonging to genus Haloanaerobium, was detected only in the 90-day fish portion sample (Fig 5, band 10) Discussion The pH value in both the rice and fish portion decreased to 3.7 after 30 days, with accompanying large amounts of lactic acid in both portions The taste and flavor of funazushi may be affect by the organic acid composition The majority of spoilage/pathogenic bacteria, including Clostridium botulinum, cannot grow under such a low pH condition [20, 21], even though it is believed that anaerobic a Haloanaerobium sp 98 100 98 100 97 Numbers correspond to bands in Fig conditions occur at the bottom of the funazushi pickling barrel For the DGGE analysis, we selected the V3 region of 16S rDNA as the target region This region has been widely used in analyses of bacterial communities or the identification of isolated bacteria [12, 13, 22] At the initial stage of fermentation, two Staphylococcus species were detected as main flora Staphylococcus spp are widely distributed in the natural environment and on human skin, and they are tolerant of high salt concentrations Therefore, they may have become dominant during the fermentation of funazushi, during which time salt concentration is one of the factors determining microbial growth Yamazaki et al [23] reported the predominance of both S warneri and S epidermidis and their dominant role in the starter culture in ika-shiokara, a salted-fermented squid Two lactic acid bacterial species were identified between days and 30 The presence of these species might contribute to the decrease in pH during fermentation Previous studies on funazushi reported the isolation of L plantarum, L alimentaris, L sake, L sanfrancisco, L kefir, L fermentam, and Pediococcus parvulus at high frequency [6, 7] However, these bacteria, with the exception of L plantarum, were not present in our samples, based on the DGGE profile This difference in results is thought to be due to the high selectivity of conventional cultivation conditions Lactobacillus plantarum is known to be an economically valuable microorganism in the commercial production of various lactic acid fermented foods [24], including funazushi [6, 7], and it grows well on MRS agar In contrast, L acetotolerans was first detected in fermented fish only on the basis of DGGE bands and could not be isolated in our experiment (data not shown) Although L acetotolerans is known to be difficult to isolate and is characterized by slow 123 156 growth, it is an important microorganism for the production of food products that require long periods of fermentation, such as nukadoko, a pickling bed of rice bran [25], and tsubosu, a traditional vinegar produced following a long fermentation in a pot [26] Therefore, further studies are required to elucidate the complete fermentation system of funazushi; specifically, the isolation and characterization of L acetotolerans and other slow-growing lactobacilli A bacterium belonging to the genus Haloanaerobium was detected only in the 90-day fish portion sample This is consistent with previous reports of Haloanaerobium isolates being present in fermented foods, such as H praevalens (a halophilic anaerobe) from surstromming, a canned Swedish fermented herring [27], and H fermentans (a novel, strictly anaerobic fermentative halophile) from puffer fish ovaries [28] Therefore, these microorganisms, which have the ability to ferment a variety of carbohydrates to produce significant amounts of acetate, lactate, butyrate, ethanol, etc., are expected to be widely distributed among fermented seafoods and to contribute to the characteristics of these foods In conclusion, we report here the first use of PCRDGGE to assess the microbial community in funazushi This technique was effective in profiling microbial diversity, revealing—for the first time—the presence of L acetotolerans in funazushi fermented for a long period, which traditional culture techniques had been unable to Further studies on the isolation and properties of L acetotolerans inhabiting funazushi are currently in progress Acknowledgments This study was supported in part by a Grant-in Aid for Scientific Research (B 16380143) from the Ministry of Education, Science, Sports and Culture of Japan References Kubo K, Nishi K, Horikoshi M (2007) Flavor of smell of fermented sushi (in Japanese) J Jpn Assoc Odor Environ 38:173–178 Fujii T (1992) Shiokara Kusaya and Katsuobushi (in Japanese) Koseisha Koseikaku, Tokyo Makinodan Y, Nakagawa T, Hujita M (1991) Participation of muscle cathepsin D in ripening of funazushi (fermented 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PCR-mediated denaturing gradient gel electrophoresis Int J Food Microbiol 109:79–87 157 27 Kobayashi T, Kimura B, Fujii T (2000) Strictly anaerobic halophiles isolated from canned Swedish fermented herrings (Surstromming) Int J Food Microbiol 54:81–89 28 Kobayashi T, Kimura B, Fujii T (2000) Haloanaerobium fermentans sp nov., a strictly anaerobic fermentative halophile isolated from fermented puffer fish ovaries Int J Syst Evol Microbiol 50:1621–1627 123 Fish Sci (2011) 77:159–160 DOI 10.1007/s12562-010-0315-6 ACKNOWLEDGMENT Ó The Japanese Society of Fisheries Science 2010 The Editorial Board is grateful to the reviewers listed below for their cooperation in reviewing manuscripts for Fisheries Science, Vol 76, 2010 Abe, Syuiti Adachi, Kohsuke Akamine, Tatsuro Amakasu, Kazuo Ando, Hironori Anraku, Kazuhiko Arakawa, Osamu Asakawa, Manabu Asakawa, Shuichi Awaji, Masahiko Ban, Shuhei Biswas, Amal Boudry, Pierre Castilla Espino, David Chiba, Susumu Chung, J Sook Dunham, Jason Ebihara, Akihiko Elizur, Abigail Endo, Masato Fausch, Kurt Fujii, Tetsuo Fujimori, Yasuzumi Fujita, Masaki Fukada, Haruhisa Fukuda, Yutaka Fukushima, Hideto Fukuwaka, Masa-aki Funabara, Daisuke Funatsu, Yasuhiro Furuita, Hirofumi Futami, Kunihiko Goshima, Seiji Gotoh, Naohiro Haga, Yutaka Hagiwara, Tomoaki Hamaguchi, Masami Harada, Kazuki Harada, Yasushi Hasegawa, Eiichi Hasegawa, Koh Hayakawa, Youichi Hayashizaki, Ken-ichi Higano, Junya Hinata, Hirofumi Hiramatsu, Kazuhiko Hiramatsu, Naoshi Hirono, Ikuo Hori, Masakazu Horiguchi, Toshihiro Horinouchi, Masahiro Hoshino, Kouichi Hosoi, Masatomi Hosoi-Tanabe, Shoko Iguchi, Keiichiro Ijiri, Shigeho Ikeda, Minoru Ikejima, Kou Imai, Chifumi Imai, Ichiro Imamura, Shintaro Inagake, Denzo Inoue, Akira Ishibashi, Yasunori Ishihara, Kenji Ishizaki, Munechika Itoi, Shiro Iwata, Nakahiro Jasmani, Safiah Kagawa, Hirohiko Kai, Yoshiaki Kakinuma, Makoto Kanaiwa, Minoru Kaneko, Gen Kaneniwa, Masaki Kanno, Manami Kasai, Hisae Katano, Osamu Katow, Hideki Kawabata, Atsushi Kawamura, Koichi Kawamura, Shoji Kawase, Hiroshi Kazeto, Yukinori Kikko, Takeshi Kikuchi, Kotaro Kimura, Ikuo Kitagawa, Takashi Kobayashi, Kazuhiko Kobayashi, Raita Kobayashi, Yasuhisa Kohno, Hiroshi Koizumi, Itsuro Komaru, Akira Kondo, Hidehiro Konno, Kunihiko Korsu, Kai Kotani, Tomonari Koyama, Jiro Kudo, Hideaki Kurashima, Akira Kurata, Osamu Kurita, Yutaka Kurokawa, Tadahide Lapesa, Sara Li, Qi Maeda, Hiroto Maeda, Toshimichi Mano, Nobuhiro Masuda, Reiji Masuma, Shukei Masumoto, Toshiro Matsubara, Hajime Matsubara, Kiminori Matsubara, Takahiro Matsuda, Hirokazu Matsumoto, Kazunori Matsushita, Teruo Matsuyama, Yukihiko Miura, Takeshi 123 160 Miyasaki, Taiko Miyashita, Kazushi Mochida, Kazuhiko Morioka, Shinsuke Morita, Kentaro Morita, Tamaki Muramoto, Koji Murashita, Koji Nagasawa, Toru Nagasawa, Hiromichi Nagler, James Nakagawa, Yoshizumi Nakai, Toshihiro Nakajima, Masamichi Nanami, Atsushi Neilson, John Nishimura, Akira Nomura, Kazuharu Nozawa, Hisanori Ogawa, Hiroshi Ohara, Kenichi Ohira, Tsuyoshi Ohizumi, Hiroshi Ohshima, Toshiaki Ohta, Hiromi Ohtomi, Jun Ojima, Takao Oka, Masakazu Okada, Shigeru Okamura, Akihiro Okamura, Hiroshi Okinaka, Yasushi Okino, Tatsufumi Oku, Hirosuke Okumura, Seiichi Onduka, Toshimitsu O’Neill, Barry Onikura, Norio Oohara, Ichiro Osada, Makoto Osako, Kazufumi Osatomi, Kiyoshi Oshima, Yuji Oshimo, Seiji Otake, Tsuguo Pankhurst, Ned Park, Min Kyun 123 Fish Sci (2011) 77:159–160 Podrabsky, Jason Pol, Michael Saito, Hiroaki Saitoh, Kenji Sakakura, Yoshitaka Sano, Motohiko Saruwatari, Toshiro Satoh, Keisuke Satoh, Shuichi Satomi, Masataka Satuito, Cyril Sawada, Kohichi Sawabe, Tomoo Sekino, Masashi Senoo, Shigeharu Seoka, Manabu Shimizu, Akio Shimizu, Munetaka Shimizu, Susumu Shimose, Tamaki Shimura, Tsuyoshi Shiode, Daisuke Shirakihara, Kunio Shirakihara, Miki Shoji, Jun Shoji, Takayuki Subramoniam, Thanumalayaperumal Sugita, Tsuyoshi Suyama, Satoshi Suzuki, Nobuaki Suzuki, Nobuhiro Suzuki, Tohru Tagawa, Masatomo Tahara, Daisuke Takada, Takenori Takada, Yoshitake Takagi, Motohiro Takahashi, Akiyoshi Takahashi, Motomitsu Takahashi, Tetsumi Takami, Hideki Takao, Yoshimi Takasuka, Akinori Takatsu, Tetsuya Tanaka, Eiji Tanaka, Hideki Tanaka, Hiroyuki Tanaka, Ryusuke Tanaka, Sho Tanaka, Yohsuke Tanaka, Yuji Taniguchi, Yoshinori Todo, Takashi Tokai, Tadashi Tokuda, Masaharu Tominaga, Osamu Tomiyama, Takeshi Tsuboi, Junichi Tsutsumi, Hiroaki Ueda, Hiroshi Ueda, Yuji Uehara, Shinji Umino, Tetsuya Uthicke, Sven Vizziano, Denise Wada, Katsuhiko Wada, Toshihiro Watanabe, Satoshi Watanabe, Yoshiro Won, Nam-Il Yagi, Nobuyuki Yamada, Hideaki Yamaguchi, Atsuko Yamamoto, Tamiji Yamamura, Orio Yamashita, Michiaki Yamashita, Yuho Yamazaki, Koji Yambe, Hidenobu Yasuda, Tohya Yatsuya, Kousuke Yokoyama, Hiroshi Yokoyama, Hisashi Yokoyama, Saichiro Yokoyama, Takehiko Yokoyama, Yoshihiro Yoneda, Chie Yoneda, Michio Yoshida, Terutoyo Yoshimatsu, Takao Yoshinaga, Ikuo Yoshinaga, Tatsuki Yumoto, Isao [...]... 0 .15 Exploitable biomass (e) (b) 0.50 0 .10 0.05 0.00 50 55 60 65 70 75 8 0 8 5 90 9 5 0 0 05 10 1 9 1 9 19 19 19 19 19 19 1 9 19 20 2 0 20 14 0 13 0 12 0 11 0 10 0 90 80 70 60 50 40 30 20 10 0 19 Biomass 95% CI BMSY 5 0 9 5 5 9 6 0 9 65 9 7 0 9 75 9 8 0 9 85 9 9 0 9 9 5 0 0 0 0 0 5 0 1 0 2 2 1 2 1 1 1 1 1 1 1 1 Year Year 220 Exploitable biomass 200 18 0 (f) Biomass 95% CI 0.30 0.25 Harvest Rate 95% CI 16 0... that Fish Sci (2 011 ) 77:23–34 33 (d) 220 200 Biomass 95% CI 0.50 Harvest Rate 95% CI 0.45 0.40 18 0 16 0 Harvest rate Exploitable biomass (a) 240 14 0 12 0 10 0 80 0.30 0.25 HMSY 0.20 0 .15 BMSY 60 40 0.35 0 .10 0.05 20 0 50 5 5 60 65 70 75 80 85 90 9 5 00 0 5 1 0 1 9 19 1 9 1 9 1 9 1 9 19 1 9 19 19 20 20 20 0.00 5 0 5 5 60 6 5 7 0 7 5 8 0 8 5 9 0 9 5 0 0 0 5 10 1 9 1 9 1 9 19 19 1 9 19 19 1 9 1 9 2 0 2 0 2... rate (c) 14 0 12 0 10 0 80 BMSY 60 40 0.20 0 .15 HMSY 0 .10 0.05 20 0 50 55 60 65 70 75 8 0 85 9 0 9 5 0 0 0 5 10 1 9 19 1 9 1 9 1 9 1 9 1 9 1 9 1 9 19 2 0 20 20 Year 0.00 19 5 0 9 5 5 96 0 9 65 9 70 97 5 9 80 9 85 99 0 9 95 0 00 0 0 5 0 10 2 2 2 1 1 1 1 1 1 1 1 1 Year Fig 6 Trends in exploitable biomass (10 00 t) and exploitation rate of North Pacific swordfish under the single-stock scenario, 19 51 2006:... 0.30 0.25 0.20 0 .15 0 .10 0.00 0.3 0.2 0.0 50 955 960 965 970 975 980 985 990 995 000 005 010 2 1 1 1 1 1 2 1 2 1 1 1 19 19 Year 0.45 50 955 960 965 970 975 980 985 990 995 000 005 010 1 1 1 1 1 1 1 1 2 2 1 2 Year (b) 0.6 Observed Predicted 0.40 0.5 0.35 Swordfish CPUE Swordfish CPUE 0.4 0 .1 0.05 (b) Observed Predicted 0.5 0.35 Swordfish CPUE Swordfish CPUE 31 0.30 0.25 0.20 0 .15 0 .10 Observed Predicted... 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Biol Bull 18 3:233–2 41 11 Kim YK, Tsutsui N, Kawazoe I, Okumura T, Kaneko T, Aida K (2005) Localization... 39:527–537 15 6 Gunamalai V, Kirubagaran R, Subramoniam T (2006) Vertebrate steroids and the control of female reproduction in two Fish Sci (2 011 ) 77 :1 21 157 15 8 15 9 16 0 16 1 16 2 decapod crustaceans, Emerita asiatica and Macrobrachium rosenbergii Curr Sci 90 :11 9 12 3 Yano I (19 85) Induced ovarian maturation and spawning in greasyback shrimp, Metapenaeus ensis, by progesterone Aquaculture 47:223–229 Yano I (19 87)... hawaii.edu/PFRP/sctb15/papers/BBRG-3.pdf; accessed 24 Sept 2 010 ) In two subsequent studies, Wang et al [ 2, 3] applied a similar age- and length-structured modeling 12 3 24 Fish Sci (2 011 ) 77:23–34 Catch (t, live-weight) 30000 25000 20000 Japan Chinese-Taipei Korea Mexico United States 15 000 10 000 5000 0 50 955 960 965 970 975 980 985 990 995 000 005 1 1 1 1 1 1 1 1 2 2 1 19 Year Fig 1 Swordfish landings... shallow—set 1 Hawaii longline shallow—set 2 Single-stock scenario 0 .12 0 0.334 0.265 0.2 01 Two-stock scenario subarea 1 0 .12 0 0.400 0.352 0.204 Two-stock scenario subarea 2 0.267 0.340 – – CPUE Catch per unit effort 12 3 30 Fish Sci (2 011 ) 77:23–34 (a) 0.55 0.50 Swordfish CPUE 0.45 Observed Predicted 0.40 0.35 0.30 0.25 0.20 0 .15 0 .10 0.05 0.00 50 55 60 65 70 75 80 85 90 95 00 05 10 19 19 19 19 19 19 19 19 19 19 ... 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