Species identification of <Emphasis Type="Italic">Pinctada imbricata</Emphasis> using intergenic spacer of nuclear ribosomal RNA genes and mitochondrial 16S ribosomal RNA gene regions

ABSTRACT:   For pearl production, pearl oyster seeds from foreign pearl oysters as well as hybrids between native and such foreign pearl oysters are produced in Japanese hatcheries. However, it is very difficult to identify these pearl oysters and hybrids based on morphological measurements. Thus, a molecular identification method for distinguishing Atlantic pearl oysters Pinctada imbricata from the Indian-Pacific pearl oyster group including P. martensii and P. fucata , was developed. The polymerase chain reaction (PCR) products of the partial intergenic spacer (IGS) of nuclear ribosomal RNA (rRNA) genes exhibited length polymorphism between P. imbricata (590 bp) and the other two species (427 bp). The restriction fragment length polymorphism analysis of the PCR products (PCR-RFLP) cleaved with Mse  I observed in the IGS of nuclear rRNA genes also gave different profiles between P. imbricata and the other two species. The difference in PCR-RFLP using Alu  I was also detected in the mitochondrial 16S rRNA gene regions between P. imbricata and the other two species. Thus, the method developed enables the distinction of P. imbricata from P. martensii and P. fucata .     

Identification of razor clams <Emphasis Type="Italic">Ensis arcuatus</Emphasis> and <Emphasis Type="Italic">Ensis siliqua</Emphasis> by PCR-RFLP analysis of ITS1 region

ABSTRACT:   Ensis arcuatus and Ensis siliqua are the most economically important species of razor clams in the European Union. Due to similarities between their shell morphology, and the differing retail value, these species are often misidentified. Therefore, it is necessary to develop an appropriate protocol to allow accurate differentiation between these species of razor clam in order to protect consumer rights, avoid commercial fraud (whether intentional or unintentional), and to enforce labeling and safety regulations. With the aim of developing a rapid and reliable method of differentiation, individuals of E. arcuatus and of E. siliqua were examined by polymerase chain reaction restriction fragment polymorphism (PCR–RFLP) using the internal transcribed spacer region 1 (ITS1). A species-specific restriction endonuclease pattern was found with the enzyme Ksp I for both species, allowing their exact identification. Thus, this work provides a simple, reliable and rapid protocol for accurate discrimination between E. arcuatus and E. siliqua , which proves useful for traceability and enabling the enforcement of labeling regulations.     

Evaluation of three phytoplankton species as food for the pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis>

In the pearl cultivation farms of the Ehime Prefecture, Japan, mass mortalities of the pearl oyster Pinctada fucata have occurred since 1994. The occurrences of mass mortality roughly coincided with a shift of the dominant phytoplankton from Skeletonema and Chaetoceros to Chaetoceros and Nitzschia all of which belong to Bacillariophyceae. Hence, we evaluated Nitzschia, together with Chaetoceros and Isocrysis, as food for the oyster. Wet weights, lengths, widths, glycogen contents, and growth rates in terms of wet weight of the oysters in all the feeding treatments were significantly higher than those in the non-feeding treatment. The highest glycogen content (2.34%) and growth rate (2.21 g month⁻¹) were found in the Chaetoceros treatment. Growth rate in the Isocrysis treatment (1.63 g month⁻¹) was also high, although glycogen content in this treatment (0.41%) was low. In the Nitzschia treatment, growth rate of the oyster (0.94 g month⁻¹) was the lowest and glycogen content (0.83%) was also low relative to that in the Chaetoceros treatment. Chlorophyll a concentration in fecal pellets was lowest in the Nitzschia treatment (<2.7 μg mg⁻¹), suggesting more complete digestion of Nitzschia by the oyster. Thus, Nitzschia was edible and digestible but not assimilated by P. fucata. We propose the following scenario for the relationship between Nitzschia dominance and mass mortality. When Nitzschia dominates in a culture area, the physiological condition of P. fucata deteriorates due to low assimilation of Nitzschia by the oyster, followed by susceptibility of the oyster to infection by agents lethal to the oyster.     

Genetic comparison between torafugu <Emphasis Type="Italic">Takifugu rubripes</Emphasis> and its closely related species karasu <Emphasis Type="Italic">Takifugu chinensis</Emphasis>

Abstract:   Sequence analyses of mitochondrial (mt) and nuclear genes were performed for genetic comparison between two Takifugu pufferfish species: torafugu T. rubripes and karasu T.  chinensis . With a sequence coverage of 20% in mtDNA, 640, 308, 344, 522 and 697 bp encoding mt 16S ribosomal RNA (rRNA), adenosine triphosphatase 6 ( ATPase 6 ), nicotinamide adenine dinucleotide dehydrogenase subunit 4 ( ND4 ), ND5 and cytochrome b (cyt b ), respectively, among 24 wild torafugu, 24 wild karasu and six hybrid-like samples, 15% of the torafugu identified by external color patterns showed nucleotide sequences consistent with karasu. Meanwhile, sequences of 60% karasu were consistent with those registered for torafugu (AJ421455). As for the hybrid-like samples, two possessed karasu-specific sequences in some base positions while torafugu-specific sequences in others. The remaining hybrid-like samples possessed torafugu-specific sequences. On the other hand, the mt control region did not show such type of consistency. Analysis of nuclear melanocortin receptor genes ( MC1R , MC4R ) among 54 samples showed 99–100% inter- and intraspecific sequence identity. Partial nuclear 18S  rRNA, complete internal transcribed spacer 1 ( ITS1 ), partial 5.8S  rRNA and ITS2 genes showed similar levels of identity, indicating a very low level of variation in their respective gene fragments between the two Takifugu species.     

Complete mitochondrial DNA sequence of the Japanese flying fish <Emphasis Type="Italic">Cypselurus hiraii</Emphasis>

ABSTRACT:   In this study, to develop a technique that enables authentication of processed seafood, the complete nucleotide sequence of the mitochondrial genome for the Japanese flying fish Cypselurus hiraii was determined. Three segments spanning the entire genome were amplified using polymerase chain reaction, and products were subsequently used as templates for direct sequencing with 60 primers. The genome (16 528 base pairs) was found to contain the same 37 genes (two ribosomal RNA, 22 transfer RNA, and 13 protein-coding genes) as those found in other vertebrate mitochondrial genomes, with the gene order being identical to that typical of vertebrates. A major non-coding region between the tRNA^Pro and tRNA^Phe genes (868 base pairs) appears to be the control (D-loop) region, as it has several conserved blocks characteristic of control regions.     

Detection of hybrids between masu salmon <Emphasis Type="Italic">Oncorhynchus masou masou</Emphasis> and amago salmon <Emphasis Type="Italic">O. m. ishikawae</Emphasis> occurred in the Jinzu River using a random amplified polymorphic DNA technique

Identification of masu salmon Oncorhynchus masou masou and amago salmon O. m. ishikawae was accomplished using a random amplified polymorphic DNA (RAPD) technique. Of 80 primers screened, three primers amplified subspecies-specific fragments (OPA11-1095 ^*, OPB5-1618^* and OPD5-2038 ^*). Based on fragment patterns, 33 hybrids were detected among 150 upstream-migrating individuals in the Jinzu River, central Japan, a long-time habitat of masu salmon, to which amago salmon were recently introduced. All of the individuals examined in the 2000–2002 cohorts were identified as F ₁ and F _x as well as pure masu and amago forms. These results indicated continuing hybridization, the genetic pollution of the native masu salmon population by amago salmon possibly being a serious problem in the Jinzu River. Both standard length and body weight in F ₁ hybrids tended to be less than in pure masu salmon. However, F _x hybrids showed similar body sizes to masu salmon.     

Exploration of the antibacterial proteins in pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis> induced by bacterial inoculation

The aim of this research was to characterize immune-related antibacterial substances from pearl oyster Pinctada fucata induced by bacterial invasion. Bacteria inoculation was performed by injecting 0.1 ml of 1.0 × 10¹² colony-forming units/ml Vibrio parahaemolyticus into adductor muscle. Acidic extracts were prepared with 0.1% trifluoroacetic acid from different tissues after 8 h of injection, and antibacterial activity against V. parahaemolyticus was determined via the microdilution broth method. The acidic extracts from gills of inoculated oysters (AEg) showed stronger antibacterial activity than those from non-inoculated ones. Based on this result, antibacterial proteins were purified from AEg via two-step gel filtration chromatography, followed by high-performance liquid chromatography using a TSkgel G3000 column. Protein components were analyzed by both sodium dodecyl sulfate and native polyacrylamide gel electrophoresis. As a result, two antibacterial proteins, APg-1 (with a molecular mass of approximately 210 kDa) and APg-2 (of approximately 30 kDa), were obtained from AEg. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis and partial amino acid sequences revealed that these proteins might be novel antibacterial proteins. These results indicate that antibacterial proteins are potentially upregulated in the gill of pearl oysters or released therefrom for defense against bacterial invasion.     

Characterization of <Emphasis Type="Italic">kiss2</Emphasis> and <Emphasis Type="Italic">kissr2</Emphasis> genes and the regulation of kisspeptin on the HPG axis in <Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>

Reproduction allows organisms to produce offspring. Animals shift from immature juveniles into mature adults and become capable of sexual reproduction during puberty, which culminates in the first spermiation and sperm hydration or ovulation. Reproduction is closely related to the precise control of the hypothalamic–pituitary–gonadal (HPG) axis. Kisspeptin peptides are considered as the important regulator of HPG axis in mammalian. However, the current understanding of kisspeptin in flatfish is not comprehensive. In this study, we cloned and analyzed the kiss2 and kissr2 genes in Cynoglossus semilaevis. Interesting alternative splicing in the 5′-untranslated regions (UTR) of the Cskissr2 gene was found. The expression profiles of Cskiss2 and Cskissr2 showed relative high messenger RNA (mRNA) levels at the late gastrula stage during embryonic development, at total length = 40 mm during early gonadal differentiation, and in the brains and gonads of all investigated tissues. These results suggested that the kisspeptin system participated in embryogenesis and in the regulation of gonadal differentiation and development. Considering that the control and regulatory mechanisms of kisspeptin in the central reproductive axis are still unclear, we documented that the intramuscular injection of kisspeptin caused different sGnRH and cGnRH mRNA levels in a dose- and tissue-dependent manner. The mRNA expressions of FSH and LH were stimulated in the ovary and were inhibited in the testis under the kisspeptin treatments. These results provided foundations for understanding the roles of kisspeptin in the neuroendocrine system in fish. The manipulation of the kisspeptin system may provide new opportunities to control the gonadal development and even reproduction in fish.     

A baseline study on lipid and fatty acid composition in the pearl oyster, <Emphasis Type="Italic">Pinctada fucata</Emphasis>

The pearl oyster Pinctada fucata is cultured for pearl production in China, but its germplasm is degenerated. Thus, lipid and fatty acids were investigated in P. fucata based on different tissues, body sizes and condition indexes using GC–MS to provide a lipidomic baseline for evaluation of breeding variety in terms of physiology. The major fatty acids over all samples included saturated (C16:0 and C18:0), monounsaturated [C16:1(n-7), C18:1(n-7) and C18:1(n-9)], and polyunsaturated fatty acids [arachidonic acid: C20:4(n-6), EPA: C20:5(n-3) and DHA: C22:6(n-3)]. Relative percentages of lipid contents of the mantle and visceral mass were, respectively, 40.75 and 42.03 %, significantly higher than adductor muscle (14.48 %) and gill (17.15 %). Fatty acid analysis showed that there were 15 types of fatty acids in adductor muscle, 17 types in mantle and gill, respectively, and 19 types in visceral mass. Meanwhile, total fatty acids, SFA, MUFA and PUFA were the highest in visceral mass (P < 0.05). Yet the lipid contents and fatty acid compositions showed no significant differences between body sizes or condition indexes based on visceral mass. The observations above suggested that visceral mass and mantle are the major tissues for P. fucata to store lipids and thus are important candidate tissues for lipidomic study.     

Cloning and characterization of <Emphasis Type="Italic">Bax1</Emphasis> and <Emphasis Type="Italic">Bax2</Emphasis> genes of <Emphasis Type="Italic">Ctenopharyngodon idellus</Emphasis> and evaluation of transcript expression in response to grass carp reovirus infection

Multidomain proapoptotic Bcl-2-associated X (Bax) protein is an essential effector responsible for mitochondrial outer membrane permeabilization, resulting in cell death via apoptosis. In this study, two Bax genes of grass carp (Ctenopharyngodon idellus), designated as CiBax1 and CiBax2, were isolated and analyzed. The obtained CiBax1 cDNA is 2058 bp long, with a 579 bp open reading frame (ORF) coding a protein of 192 amino acid residues. The full-length cDNA of CiBax2 is 1161 bp, with a 618 bp ORF coding 205 amino acids. Both CiBax1 and CiBax2 are typical members of Bcl-2 family containing conserved Bcl and C-terminal domains, and they share conserved synteny with zebrafish Bax genes despite the grass carp Bax mapping to different linkage groups. Phylogenetic analysis showed that CiBax1 was clustered with Bax from most teleost fish, and CiBax2 was close to Bax2 from teleost fish but far separated from that of Salmo salar. Quantitative real-time PCR analysis revealed broad expression of CiBax1 and CiBax2 in tissues from healthy grass carp, but the relative expression level differed. The mRNA expression of CiBax1 and CiBax2 was both upregulated significantly and peaked in all examined tissues at days 5 or 6 post-infection with grass carp reovirus. Subcellular localization indicated that CiBax1 protein was localized in both nucleus and cytosol, while CiBax2 protein only in cytosol. Moreover, CiBax2, but not CiBax1 was colocalized with mitochondrion under normal condition. Taken together, the findings would be helpful for further understanding of the function of Bax in teleost fish.     

Comparison of larval thermal maxima between <Emphasis Type="Italic">Fundulus heteroclitus</Emphasis> and <Emphasis Type="Italic">F. grandis</Emphasis>

Fundulus heteroclitus and F. grandis are resident salt marsh fishes that overlap in distribution over a narrow range in northeastern Florida. The objective of the present study was to examine whether the limits of the species’ ranges could be explained by differences in thermal tolerance. Two populations of each species were collected and then spawned in the laboratory, and 9-day-old larvae were used for critical thermal maxima trials. Mean LOE temperatures of larvae ranged from 43.04 to 43.65°C and showed little difference between species. Therefore, differences in high temperatures experienced cannot account for the differences of the distributions of the two species. Condition-specific competition may play a greater role in determining the observed range of the two species.     

Identification of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> and <Emphasis Type="Italic">Undaria undarioides</Emphasis> Laminariales,Phaeophyceae using mitochondrial 23S ribosomal DNA sequences

ABSTRACT:   Mitochondrial 23S ribosomal (r) DNA were sequenced from two Undaria species. The 23S rDNA from seven Undaria pinnatifida individuals were 2707 bp in length, whereas the 23S rDNA from four Undaria undarioides individuals were 2708–2709 bp in length. We found 15–20 nucleotide substitutions and 1–2 gaps between U. pinnatifida (the major haplotype) and U. undarioides . Based on the differences in sequences, we carried out PCR/RFLP analyses to distinguish between U. pinnatifida and U. undarioides , which showed different PCR/RFLP patterns upon Hin fI digestion. Sequence differences and PCR/RFLP analyses of mt 23S rDNA are useful for species identification of Undaria species .     

Comparison of behavioral responses to a novel environment between three teleosts,bluegill <Emphasis Type="Italic">Lepomis macrochirus</Emphasis>, crucian carp <Emphasis Type="Italic">Carassius langsdorfii</Emphasis>, and goldfish <Emphasis Type="Italic">Carassius auratus</Emphasis>

To investigate the emotional reactivity of fish in a novel environment, the swimway test was developed. The swimway apparatus consists of a shaded start chamber and an open, illuminated swimway. Fish were first introduced and habituated to the start chamber. A door partitioning the start chamber from the swimway was then opened, and behavioral responses of the fish in the apparatus were measured. By using the swimway test, behavioral responses to a novel environment of bluegill Lepomis macrochirus, crucian carp Carassius langsdorfii, and goldfish Carassius auratus were quantified and compared. The emotional reactivities in blue gill were found to be the lowest and crucian carp the highest, indicating bluegill are relatively active or ‘bold’, and that the crucian carp are relatively passive or ‘shy’, in a novel environment. It is suggested that the swimway test is applicable to examning inter-species differences in relative emotional reactivity or boldness in a simplified novel situation.     

Structure and possible function of <Emphasis Type="Italic">N</Emphasis>-glycans of an invertebrate C-type lectin from the acorn barnacle <Emphasis Type="Italic">megabalanus rosa</Emphasis>

ABSTRACT:   A C-type lectin (BRA-2) isolated from the acorn barnacle Megabalanus rosa , which was a glycoprotein having an N -linked sugar chain, was deglycosylated by N -glycopeptidase F. The structure of the released sugar chains was determined by a 2-D mapping method after derivatization with a fluorescent reagent, 2-aminopyridine, to be Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc and Manα1-6(GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc. The structures were confirmed by matrix-assisted laser desorption ionization mass spectrometry and a comparison with authentic sugar chains by high-pressure liquid chromatography. Various properties of BRA-2 were examined before and after deglycosylation. The susceptibility of BRA-2 to protease digestion was increased by deglycosylation. However, the inhibitory activity toward calcium carbonate crystallization as well as the hemagglutinating activity of deglycosylated BRA-2 was significantly decreased. These results suggest that the sugar chains of BRA-2 are important to both its structural stability and its function.     

Random sequencing of genomic DNA of <Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis>

ABSTRACT:   Random sequencing of the genomic DNA of Photobacterium damselae subsp. piscicida was conducted. The sequences were assembled into 930 contigs. The total length of these contigs accounts for 37.5% of the genome of P. damselae subsp. piscicida. The contigs contain 2055 open reading frames (ORFs) that have homology with genes in the GenBank database. Furthermore, some of the ORFs have homology with reported virulence related genes, and are classified as encoding colonization factors, exotoxin, and lipopolysaccharide (LPS). Thirty-seven ORFs have homology with colonization factors. In those colonization factors, 27, three, and four ORFs have homology with polar flagellar-related genes, capsule-related genes, and others (accessory colonization factors, superoxide dismutase (Cu-Zn), MshA biogenesis protein, and heme receptor genes), respectively. Five ORFs have homology with exotoxin-related genes (2 hemolysin, phospholipase, hyaluronidase, lysophospholipase L2 genes). Further, six ORFs have homology with LPS-related genes.     

Complete nuclear ribosomal DNA sequence analysis of Pacific abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>

Pacific abalone Haliotis discus hannai (Haliotidae, Gastropoda) is an economically important shellfish species in northern China. The complete nuclear ribosomal DNA (nrDNA) of Pacific abalone was amplified, sequenced, and analyzed. The length of the nrDNA was determined to be around 10.7 kb, and to contain, in order, small subunit ribosomal RNA (nrSSU) genes (1871 bp), internal transcribed spacer (ITS, 759–762 bp), large subunit ribosomal RNA (nrLSU) gene (3411 bp), and an intergenic spacer (IGS, 4624–4654 bp). The SSU and LSU regions were almost identical in different individuals, and show little variation from those of other abalone species. The two different variations of the ITS2 region were presented, and this phenomenon also existed in other species. A phylogeny tree was constructed, based on ITS region sequence datasets, to determine the evolutionary relationships of abalones. Abalones have two major subclades, mainly distributed in the North Pacific, Europe and Australia. The IGS region of the nrDNA was sequenced and analyzed for the first time. Several repeat fragments were present upstream of the sequence, and were significantly different between individuals (93.86% sequence identity). The complete nrDNA sequence will be useful for the classification, identification, phylogeny, germplasm management, and breeding of this shellfish.     

Immune response of sea bass <Emphasis Type="Italic">Dicentrarchus labrax</Emphasis> to <Emphasis Type="Italic">Tenacibaculum maritimum</Emphasis> antigens

ABSTRACT:   Seawater fishes are affected by a pathology commonly called 'myxobacteriosis', caused by Tenacibaculum maritimum (formerly Flexibacter maritimus ). The disease is characterized by fin erosion and necrotic ulcers of skin and muscle, and by low but constant mortality in cultured marine fish; in Italy is one of the most important and widely spread diseases affecting sea bass Dicentrarchus labrax , gilthead seabream Sparus aurata , sharp-snouted bream Diplodus puntazzo , white bream Diplodus sargus , and six-tooted bream Dentex dentex . In order to obtain an effective vaccine against the disease, formalin killed cells (FKC), extracellular products (ECPs) and crude lipopolysaccharide (LPS) preparations were obtained from the T. maritimum strain SPVId and injected intraperitoneally twice into the sea bass. The fish immune response to the preparations was studied: agglutinating antibody titer and in vitro phagocytosis were determined after the first and second injection in order to evaluate whether the preparations are immunogenic or not and if the booster effect took place. The results show that FKC and LPS preparations increased the antibody titer after the first injection when compared to the control sea bass. Moreover, all the preparations stimulated a secondary (booster) response. In vitro phagocytosis of the total blood was significantly higher for all the preparations when compared to the controls, but the crude LPS immunized sea bass showed the highest activity.     

Characterization of <Emphasis Type="Italic">pax3a</Emphasis> and <Emphasis Type="Italic">pax3b</Emphasis> genes in artificially induced polyploid and gynogenetic olive flounder (<Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>) during embryogenesis

Although chromosome set manipulation techniques including polyploidy induction and gynogentic induction in flatfish are becoming increasingly mature, there exists a poor understanding of their effects on embryonic development. PAX3 plays crucial roles during embryonic myogenesis and neurogenesis. In olive flounder (Paralichthys olivaceus), there are two duplicated pax3 genes (pax3a, pax3b), and both of them are expressed in the brain and muscle regions with some subtle regional differences. We utilized pax3a and pax3b as indicators to preliminarily investigate whether chromosome set manipulation affects embryonic neurogenesis and myogenesis using whole-mount in situ hybridization. In the polyploid induction groups, 94 % of embryos in the triploid induction group had normal pax3a/3b expression patterns; however, 45 % of embryos in the tetraploid induction group showed abnormal pax3a/3b expression patterns from the tailbud formation stage to the hatching stage. Therefore, the artificial induction of triploidy and tetraploidy had a small or a moderate effect on flounder embryonic myogenesis and neurogenesis, respectively. In the gynogenetic induction groups, 87 % of embryos in the meiogynogenetic diploid induction group showed normal pax3a/3b expression patterns. However, almost 100 % of embryos in the gynogenetic haploid induction group and 63 % of embryos in the mitogynogenetic diploid induction group showed abnormal pax3a/3b expression patterns. Therefore, the induction of gynogenetic haploidy and mitogynogenetic diploidy had large effects on flounder embryonic myogenesis and neurogenesis. In conclusion, the differential expression of pax3a and pax3b may provide new insights for consideration of fish chromosome set manipulation.     

Effect of combination feeding of <Emphasis Type="Italic">Nannochloropsis</Emphasis> and freshwater <Emphasis Type="Italic">Chlorella</Emphasis> on the fatty acid composition of rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> in a continuous culture

ABSTRACT:   A continuous culture of rotifer was conducted to investigate the effect of combination feeding of both a high density of Nannochloropsis oculata (N) and condensed freshwater Chlorella (FC) on the fatty acid composition of L-type rotifer Brachionus plicatilis in a continuous culture system. The algal feeding of the rotifers was carried out in three successive steps: N-feeding → N+FC-feeding → FC-feeding. The culture was conducted at 24°C and 25–27 psu in a 2000 mL bottle with 50% of water exchanged daily. The combination N+FC-feeding was effective in increasing rotifer density. The rotifers fed on N+FC (N+FC-R) had more non-polar lipids than polar ones, similar to those on N (N-R), opposite to the rotifers fed on FC (FC-R). N+FC-R contained higher levels of 16:2, 18:2n-6 (linoleic acid [LA]) and 20:2n-6, but lower levels of 18:1, 20:4n-6 (arachidonic acid), 20:5n-3 (eicosapentaenoic acid [EPA]) and 22:5n-3 (docosapentaenoic acid [DPA]) compared with N-R. Whereas N+FC-R contained higher levels of 16:1n-7, EPA and DPA, but lower levels of 16:2 and LA compared with FC-R. N+FC-R had more DPA in polar lipids than in non-polar ones. The Σn-6/Σn-3 ratio in N+FC-R was 0.9–1.0, significantly different from those in N-R (0.4) and FC-R (6.6–8.4). Therefore, it is inferred that the fatty acid profile of the N+FC-R cultured in a continuous culture system was affected by both N and FC. Also, the combination N+FC-feeding may be effective in manipulating the Σn-6/Σn-3 ratio in continuously cultured rotifers.