Takifugu Obscurus Research Articles

Effect of cadmium exposure on expression of antioxidant gene transcripts in the river pufferfish, Takifugu obscurus (Tetraodontiformes)

Cadmium (Cd) is a non-essential toxic heavy metal with the potential to induce oxidative stress. Cd toxicity and its capacity for accumulation in aquatic habitats have earned its recognition as a pollutant of immediate and widespread concern. To obtain a better understanding of oxidative stress-associated gene expression in different tissues, six antioxidant genes such as catalase ( CAT), glutathione reductase ( GR), glutathione peroxidase 1a ( GPx1a), glutathione peroxidase 1b ( GPx1b), Cu/Zn superoxide dismutase ( Cu/Zn-SOD), and Mn superoxide dismutase ( Mn-SOD) were cloned and fully sequenced in the river pufferfish, Takifugu obscurus. On tissue specific mRNA expression, the liver showed the highest expression when compared to other tissues, even though each antioxidant gene showed different modes of expression patterns in the examined tissues. Of the various antioxidant genes, GR was the most highly expressed in the liver, followed by CAT, GPx1, and Cu/Zn-SOD. For the time-course experiment, all the antioxidant genes were significantly induced over time except for Cu/Zn-SOD in the liver, and there was a 5-fold induction in hepatic GR, CAT, and Mn-SOD mRNA compared to the control. These findings indicate that the liver of T. obscurus has a robust antioxidant system. In addition, these results suggest that Cd exposure modulates the expression of antioxidant genes, and would indicate that the antioxidant genes would be a relevant biomarker of trace metal pollution such as Cd exposure in T. obscurus.

A novel TTX-producing Aeromonas isolated from the ovary of Takifugu obscurus

Puffer fish ( Takifugu obscurus) from the Yangtze River of China were screened for tetrodotoxin-producing bacteria. An Aeromonas strain was isolated from the ovary of the puffer fish and was shown to produce tetrodotoxin; this strain was denoted Ne-1. The identity of tetrodotoxin produced by strain Ne-1 was confirmed by mouse bioassay, high performance liquid chromatography coupled with mass chromatography (LC-MS), and ELISA. Strain Ne-1 was characterized morphologically, biochemically, and by 16S rDNA phylogenetic analysis; these analyses suggested that strain Ne-1 is closely related to Aeromonas molluscorum. Given that strain Ne-1 was isolated from the ovary of T. obscurus, we propose that the TTX-producing Aeromonas sp. is a parasite or symbiotic bacterium rather than a sample contaminant. Collectively, our studies suggest that Aeromonas sp. strain Ne-1 produces tetrodotoxin in T. obscurus.

Effects of salinity on survival and Na+/K+ ATPase activity of obscure puffer Takifugu obscurus embryos

Effects of salinity on survival and Na+/K+ ATPase activity of obscure puffer Takifugu obscurus embryos

Identification of renal transporters involved in sulfate excretion in marine teleost fish

Sulfate (SO(4)(2-)) is the second most abundant anion in seawater (SW), and excretion of excess SO(4)(2-) from ingested SW is essential for marine fish to survive. Marine teleosts excrete SO(4)(2-) via the urine produced in the kidney. The SO(4)(2-) transporter that secretes and concentrates SO(4)(2-) in the urine has not previously been identified. Here, we have identified and characterized candidates for the long-sought transporters. Using sequences from the fugu database, we have cloned cDNA fragments of all transporters belonging to the Slc13 and Slc26 families from mefugu (Takifugu obscurus). We compared Slc13 and Slc26 mRNA expression in the kidney between freshwater (FW) and SW mefugu. Among 14 clones examined, the expression of a Slc26a6 paralog (mfSlc26a6A) was the most upregulated (30-fold) in the kidney of SW mefugu. Electrophysiological analyses of Xenopus oocytes expressing mfSlc26a6A, mfSlc26a6B, and mouse Slc26a6 (mSlc26a6) demonstrated that all transporters mediate electrogenic Cl(-)/SO(4)(2-), Cl(-)/oxalate(2-), and Cl(-)/nHCO(3)(-) exchanges and electroneutral Cl(-)/formate(-) exchange. Two-electrode voltage-clamp experiments demonstrated that the SO(4)(2-)-elicited currents of mfSlc26a6A is quite large (approximately 35 microA at +60 mV) and 50- to 200-fold higher than those of mfSlc26a6B and mSlc26a6. Conversely, the currents elicited by oxalate and HCO(3)(-) are almost identical among mfSlc26a6A, mfSlc26a6B, and mSlc26a6. Kinetic analysis revealed that mfSlc26a6A has the highest SO(4)(2-) affinity as well as capacity. Immunohistochemical analyses demonstrated that mfSlc26a6A localizes to the apical (brush-border) region of the proximal tubules. Together, these findings suggest that mfSlc26a6A is the most likely candidate for the major apical SO(4)(2-) transporter that mediates SO(4)(2-) secretion in the kidney of marine teleosts.

Expression profiles of seven glutathione S-transferase ( GST) genes in cadmium-exposed river pufferfish ( Takifugu obscurus)

Glutathione S-transferase (GST; EC 2.5.1.18) plays a critical role in detoxification pathways. In this study, we report cloning and expression of seven genes of the GST family of the pufferfish Takifugu obscurus together with mRNA tissue distribution pattern and time-course of expression in response to exposure to cadmium. At basal levels of tissue expression, GST- Mu is highly expressed in liver compared with other tissues. When fish were exposed to cadmium (5 mg/L for 96 h), expression of GST- MAPEG, GST-Mu, GST-Omega, and GST- Zeta was greatly increased, whereas GST- Alpha and GST- Kappa genes showed no significant response. These findings suggest that gene expression of a number of GST isoforms in T. obscurus is modulated in response to exposure to cadmium. We propose GST- Mu, GST-Theta, and GST-Zeta as candidate biomarkers for heavy metal exposure in this fish.

Nutritional condition determined using RNA/DNA ratios of the river pufferfish Takifugu obscurus under different salinities

We measured dry weight and nucleic acid (RNA/DNA) contents of larvae and juveniles of the anadromous river pufferfish Takifugu obscurus for 2 aquaculture salinity series in order to evaluate the effect of different rates of salinity change on fish growth and to determine appropriate rearing conditions for one of the most valuable aquaculture species. In addition, the effect of salinity during starvation was evaluated for 3 stages of development (larvae, juveniles and young fish). Analyses were performed at 1 to 2 d intervals over a period of 1 to 60 d after hatching (DAH). Differ- ences in growth rates of the 2 groups were observed from 36 DAH until the end of the experiment. Specifically, the group in which salinity was increased by 1 ± 0.5 psu d -1 grew faster than the group in which salinity was increased by 3 ± 0.5 psu d -1 . Significant differences in RNA/DNA ratios between both groups occurred from 30 DAH, with juvenile fish of the 1 psu d -1 group showing a higher ratio. Under starving conditions, the RNA content per individual decreased against a roughly constant DNA content. Significant differences in the RNA/DNA ratios were found between fed and starved larvae and juvenile fish. Consequently, we verified that RNA/DNA ratios could be used as an index of nutritional condition with respect to the effects of salinity change and recommend that maintaining stable salinity would be helpful in the aquaculture of anadromous river pufferfish.

Molecular cloning, phylogenetic analysis and expression of a MAPEG superfamily gene from the pufferfish Takifugu obscurus

The microsomal glutathione S-transferases (MGSTs) of membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) superfamily play an important role in xenobiotics detoxification. Compared to mammals, there is limited information on MAPEGS from fish. We cloned a full length of cDNA sequence of a MGST gene from the river pufferfish ( Takifugu obscurus), studied its phylogenetic relationship, and measured its expression in different tissues and in liver of fish exposed to cadmium. Phylogenetic analysis revealed that the identified gene encoded for MGST3. Liver showed the highest expression of MGST3 transcripts. When MSGT expression was compared with the expression of other GSTs ( GST-Alpha, GST-Mu and GST-Theta), a similar pattern of highest expression was observed in the liver. Upon Cd exposure (5 ppm) for 96 h, the highest expression of MGST was observed at 24 h. GST-Mu also showed highest expression at 24 h. These findings indicate that MGSTs may be playing a role in detoxification of xenobiotics or free radicals generated by Cd-induced oxidative stress in fish.

Comparison between α-LNA and DHA in early developmental stages ofTakifugu obscurusandTakifugu rubripes

Rearing experiments were conducted to investigate the essential fatty acid requirements in the early developmental stages of river puffer Takifugu obscurus and tiger puffer T. rubripes using two n−3 series unsaturated fatty acids, α-linolenic acid (18∶3n−3, α-LNA) and docosahexaenoic acid (22∶6n−3, DHA), under salinity of 30 and 18.5–20.3°C. River and tiger puffer larvae used in this study were 15 and 14 days old after hatching, and their average body weights were 30.1 and 20.8 mg, respectively. The results on fatty acid requirements of these two species were evaluated from fish growth, survival, fatty acid composition of the fish body and activity test results. The DHA groups of both river and tiger puffer exhibited better survival and weight gain. However, there was no difference in the mean final body weights of river puffer between two dietary groups. Also, the DHA group of tiger puffer showed better results in the recovery test from anesthetic condition than that obtained in the LNA group. In an examination of the fatty acid compositions of the whole body, the LNA group containing no dietary DHA resulted in 0.5% DHA in tiger puffer and 1.1% DHA in river puffer. These results suggest that α-LNA from Artemia converted to eicosapentaenoic acid (20∶5n−3, EPA) and to DHA successively by their fatty acid metabolism. Symptoms following essential fatty acid deficiency were not observed in any experimental groups. As river puffer did not represent a significant difference in the dietary effects between α-LNA and DHA treatment groups, its essential fatty acid requirement was assumed to be somewhat closer to that of the freshwater fishes in comparison with that of marine fishes, including tiger puffer.

Differences in reproductive strategies between obscure pufferTakifugu obscurusand ocellated pufferTakifugu ocellatusduring their spawning migration

Summary Reproductive strategies were compared between obscure puffer Takifugu obscurus and ocellated puffer Takifugu ocellatus captured in waters near Yangzhong Island in the lower reaches of the Yangtze River during the spawning migration season from February to June in two consecutive years (1995 and 1996). Results showed that obscure puffer and ocellated puffer have adopted different reproductive strategies, including different spawning times, different size at maturity, and different number and size of oocytes, resulting in two different larval sizes. In detail, the timing of the spawning migration and status of gonad development of obscure puffer was about 1 month earlier than that of ocellated puffer; the obscure puffer was obviously longer and heavier than ocellated puffer in both mature male and female fish; mean GSI of obscure puffer females (15.8%) was significantly higher than that of ocellated puffer females (14.6%); the average diameter of ocellated puffer eggs (1.49 ± 0.12 mm) was significantly larger than that of the obscure puffer (1.22 ± 0.08 mm); and obscure puffer females (320.8 oocytes mg−1 somatic wet weight) had significantly higher relative fecundity than ocellated puffer females (125.2 oocytes mg−1 somatic wet weight). These differences in reproductive strategies between two closely related species of the Takifugu genus indicate that both obscure puffer and ocellated puffer fit the r/K dichotomy. Obscure puffer shows K-selected characters with maturity at relatively large size and r-selected characters with relatively many and small offspring, whereas ocellated puffer shows r-selected characters with maturity at a relatively small size and K-selected characters with relatively few and large offspring.

Functional Characterization of Pufferfish Slc26a6A and Slc26a6B

Euryhaline pufferfish (Takifugu obscurus, mefugu) can live in freshwater (FW) with ion concentration less than 1/100 of its body fluid or acclimate to seawater (SW) which has more than 3 times the ion concentration of its body fluid. However, the body‐fluid ion concentrations are maintained stable by the function of epithelial cells in the gill, intestine, and kidney which transport ions directionally and selectively. To understand the mechanisms of the fish body‐fluid homeostasis at the molecular level, there are several essentials: identification of transporters expressed in the epithelia, determination of transporter localization, and characterization of transporter activities. Analyses of various transporter mRNA levels in mefugu tissues identified increases for Slc26a6A and Slc26a6B in the intestine and the kidney of SW‐acclimated mefugu. To study their functions for SW acclimation, we subcloned the cDNAs into a Xenopus expression vector and assayed their functions using microelectrodes in oocytes.The electrophysiological analyses of the oocytes revealed that (a) Slc26a6A and Slc26a6B are electrogenic Cl−/nHCO3− exchangers; (b) Slc26a6A and Slc26a6B are also exchangers of Cl−/formate, Cl−/oxalate, and Cl−/SO42−; and (c) the slope of I–V relationship of Slc26a6A in the presence of SO42− is 70 times higher than that of Slc26a6B. These results suggest significant roles for Slc26a6A and Slc26a6B in intestinal HCO3− secretion and a role for Slc26a6A in renal SO42− excretion during SW acclimation.This work was supported by MEXT Japan, NIH (EY017732) and CFF grant (Romero‐06G0).

Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish

Marine teleost fish precipitate divalent cations as carbonate deposits in their intestine to minimize the potential for excessive calcium entry and to stimulate water absorption by removing osmolyte ions. This carbonate deposit formation therefore plays an important role in osmoregulation in the seawater environment and also requires controlled secretion of HCO3− to match the amount of calcium entering the intestinal lumen. Despite this physiological importance, the process of carbonate precipitate formation has not been characterized at the molecular level. We first isolated 24 candidate cDNA clones representing all possible membrane proteins capable of transporting HCO3− from the euryhaline pufferfish, mefugu Takifugu obscurus based on the DNA sequence information on the T. rubripes genome, then performed Northern blot analyses to select clones highly expressed in the intestine of seawater mefugu, and finally determined the cellular locations by immunohistochemistry. Based on these observations, we proposed a model for HCO3− excretion across the intestinal epithelium of marine teleost fish that includes a basolateral NBCe1 (Na+/bicarbonate cotransporter) and apical Slc26a6 (Cl−/HCO3− exchangers).

Isolation and characterization of polymorphic microsatellite loci from a dinucleotide-enriched genomic library of obscure puffer (Takifugu obscurus) and cross-species amplification

Obscure puffer (Takifugu obscurus) is an anadromous fish species in China. Here, we reported 10 polymorphic microsatellite loci isolated from a dinucleotide-enriched genomic library of T. obscurus. The number of alleles, observed and expected heterozygosity per locus in 30 individuals ranged from four to 10, from 0.57 to 0.86 and from 0.68 to 0.90, respectively. Three loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in T. obscurus.

Cloning of a river pufferfish (Takifugu obscurus) metallothionein cDNA and study of its induction profile in cadmium-exposed fish

We report here the full-length cDNA sequence of metallothionein (MT) gene from an anadromous river pufferfish, Takifugu obscurus (order: Tetradotiformes; family: Tetradontidae). Phylogenetic relationship analysis revealed that the identified MT has high sequence similarity with many Perciformes fish species. The tissue distribution and concentration- and time-dependent expression of MT mRNA were studied in fish exposed to cadmium. Liver showed the highest level of MT gene expression followed by other tissues (brain, gill and kidney) in response to cadmium exposure. Muscle showed a weak expression response of MT gene. Time-course study revealed highest early phase (at 6h) expression in the brain and late persistence of induction in the intestine. MT mRNA expression showed a concentration-dependent expression in all the tissues. However, induction in brain and liver occurred at much lower concentrations as compared to other tissues. Our results demonstrate that MT in T. obscurus is induced by cadmium exposure which indicates that it plays a functionally conserved function of metal detoxification.

Identification of intestinal bicarbonate transporters involved in formation of carbonate precipitates to stimulate water absorption in marine teleost fish

Marine teleost fish precipitate divalent cations as carbonate deposits in the intestine to minimize the potential for excessive Ca2+ entry and to stimulate water absorption by reducing luminal osmotic pressure. This carbonate deposit formation, therefore, helps maintain osmoregulation in the seawater (SW) environment and requires controlled secretion of HCO3(-) to match the amount of Ca2+ entering the intestinal lumen. Despite its physiological importance, the process of HCO3(-) secretion has not been characterized at the molecular level. We analyzed the expression of two families of HCO3(-) transporters, Slc4 and Slc26, in fresh-water- and SW-acclimated euryhaline pufferfish, mefugu (Takifugu obscurus), and obtained the following candidate clones: NBCe1 (an Na+-HCO3(-) cotransporter) and Slc26a6A and Slc26a6B (putative Cl(-)/HCO3(-) exchangers). Heterologous expression in Xenopus oocytes showed that Slc26a6A and Slc26a6B have potent HCO3(-)-transporting activity as electrogenic Cl(-)/nHCO3(-) exchangers, whereas mefugu NBCe1 functions as an electrogenic Na+-nHCO3(-) cotransporter. Expression of NBCe1 and Slc26a6A was highly induced in the intestine in SW and expression of Slc26a6B was high in the intestine in SW and fresh water, suggesting their involvement in HCO3(-) secretion and carbonate precipitate formation. Immunohistochemistry showed staining on the apical (Slc26a6A and Slc26a6B) and basolateral (NBCe1) membranes of the intestinal epithelial cells in SW. We therefore propose a mechanism for HCO3(-) transport across the intestinal epithelial cells of marine fish that includes basolateral HCO3(-) uptake (NBCe1) and apical HCO3(-) secretion (Slc26a6A and Slc26a6B).

Molecular cloning and β-naphthoflavone-induced expression of a cytochrome P450 1A ( CYP1A) gene from an anadromous river pufferfish, Takifugu obscurus

In recent years, there has been a decline in the wild populations of river pufferfish, Takifugu obscurus. Besides overexploitation for commercial purposes, environmental pollution is believed to have contributed to its decline. However, almost no information exists about genes involved in metabolism of xenobiotics by this species. Nevertheless, there is interest in fugu fishes, since they possess the smallest genome among vertebrates. We cloned and characterized the full-length cDNA sequence of a cytochrome P450 1A ( CYP1A) gene from T. obscurus. Phylogenic relationship of T. obscurus CYP1A was also compared to other fish species. The tissue distribution and time-dependant induction of CYP1A mRNA were studied by real-time PCR in T. obscurus exposed to an aryl hydrocarbon receptor (Ahr) agonist, β-naphthoflavone (BNF). The greatest basal expression in livers of control as well as BNF-treated individuals. However, brain, gill, gonad, intestine, and kidney also expressed CYP1A. Muscles expressed the least CYP1A. The results of the time-course study revealed induction in brain and gills after 6 h and at 12 h in most tissues. Except for gills, all other organs retained induced expression of CYP1A mRNA up to 96 h.

Effect of Timing of First Feeding on Survival and Growth of Obscure Puffer (Takifugu obscurus) Larvae

ABSTRACT The effect of timing of the first feeding (2, 3, 4, 5, 6, 7, and 8 days post-hatch, dph), on survival and on growth of laboratory-reared obscure puffer (Takifugu obscurur) larvae was examined. Larvae began to feed exogenously at 2–3 dph (2.96±0.05 mm in total length) at 24 °C. Survival of larvae initially fed at 2, 3, 4, and 5 dph was similar (73.2–78.8%), while survival decreased sharply for those larvae whose first feeding was at 6 dph (22.1%), and no larvae were able to survive when food was not offered until 7 and 8 dph. The feeding incidence of larvae fed for the first time at 2, 3.4, and 5 dph reached 100% in 24 h, whereas the feeding incidence decreased gradually in the larvae fed for the first time at 6 and 7 dph, and no feeding occurred in the larvae introduced to food at 8 dph. According to the feeding incidence, the point of no return occurred between days 6 and 7 after hatching. After 18 dph, no statistically significant differences in mean total length were detected among the larvae fed for the first time at 2, 3, 4, and 5 dph, while the larvae fed for the first time at 6 dph were significantly smaller. To ensure a successful transition from endogenous to exogenous feeding, timing of first feeding of obscure puffer larvae must be before 5 dph.

Headless splice variant acting as dominant negative calcitonin receptor

Calcitonin receptor (CTR) is a member of class B G protein-coupled receptor (GPCR) superfamily. We have recently identified the CTR gene and its two transcriptional isoforms, mfCTR and mfCTRΔN, in mefugu (mf) ( Takifugu obscurus). Here we characterized the mfCTRΔN that lacks hormone-binding extracellular N-terminal domain. Strong expression in the liver and weak but broad tissue distribution of its mRNA, revealed by Northern analysis, suggested physiological significance of this headless splice variant. Biochemical and immunocytochemical analyses revealed that it acts as a naturally occurring dominant negative isoform by forming a heterodimer with normal CTR. The headless mfCTRΔN characterized here is the first case of N-terminally truncated dominant negative form of GPCR, and immunocytochemistry used for detecting the heterodimer formation may be useful as a novel method for analyzing membrane protein interaction in a living cell.

Fish calcitonin receptor has novel features

Calcitonin (CT), a 32-amino acid peptide, was initially isolated from fish. Fish CT has higher affinity to mammalian CT receptor (CTR), and has activity on calcium homeostasis. Therefore, fish CT has been used as a drug for the treatment of human bone diseases. However, the physiological roles of CT in fish as well as the characteristics of the fish CTR have not been clarified. Here, we cloned and characterized CTR from mefugu ( Takifugu obscurus). Full-length cDNA sequencing revealed that mfCTR (mf, mefugu) consists of N-terminal four tandem putative hormone-binding domains (HBDs). Database mining showed that the multiple HBD-containing CTR is a common feature for some other fishes. Detailed pharmacological studies revealed that mfCTR generated cAMP in response to (1) fish CT, (2) calcitonin gene-related peptide (CGRP) in combinations with receptor activity-modifying proteins (mfRAMPs) 1 and 4, and (3) amylin in combinations with mfRAMPs 1−5. Unlike mammalian CTR, mfCTR showed dual affinity sites. Corresponding EC 50 values of those are in close proximity of the in vivo concentration of CT in fish. Analyses of the deletion mutants of mfCTR demonstrated that only the nearmost HBD to the first transmembrane region is functional to the ligands. Although, fish CT has higher affinity to the human CTR, human CT did not bind to the mfCTR. This is the first report that demonstrates the structure and property of fish receptor for CT, CGRP, and amylin. Fish CTR is the first example that has multiple HBD-like sequences.

Cloning and polymorphism analysis of intron 2 of the GH gene in Takifugu

Size and nucleotide polymorphisms of intron 2 of the growth hormone (GH) gene was studied in 82 individuals from Takifugu obscurus, Takifugu rubripes and Takifugu niphobles with PAGE, SSCP and cloning. Results showed that there were nine length variants (A~I) in these fishes, ranging from 271351 bp, with a frequency of variation of 24.22%. Sequence analysis of the 9 length variants showed the average percentages of the four bases (ATG and C) were 17.15, 20.77, 37.38 and 24.70, respectively, and the difference between GC content (45.47) and AT content (54.53) was not remarkable. Length variation was mainly due to the variable number of microsatellite TCTG repeats (N=20 approximately 40). Further sequence comparison revealed 4 substitution sites: a (C-->A) transversion at nucleotide 103 and 3 transitions 83(C-->T), 101(A-->G) and 296(G-->A), respectively. Phylogenetic analysis of these length variants placed DD and II into one group first, which was subsequently joined by GG and AA, BB and CC, EE and HH were then clustered together, FF was joined last. Thus, intraspecific variations were much smaller than interspecific variations for the intron 2 of the GH gene.

Analysis of the composition of the bacterial community in puffer fish Takifugu obscurus

The food chain has been postulated as one pathway for tetrodotoxin (TTX)-producing bacteria to enter into fish. However, the background composition of the bacterial community in puffer fish is unclear. Using 16S rDNA PCR denaturing gradient gel electrophoresis (PCR-DGGE) technology, we investigated the composition of bacterial communities in the skin, intestines, and TTX-accumulating organs (ovary, liver) of the striped puffer fish Takifugu obscurus. A total of 38 species of different culture-independent bacteria were isolated and classified according to phylogenetic analysis. Of these species, those belonging to the class γ-Proteobacteria dominated the microbial community in the puffer fish, while others belonged to the group of low DNA G + C content, Cytophaga– Flavobacterium– Bacteroides (CFB) group, α-Proteobacteria, β-Proteobacteria, ε-Proteobacteria and Spirochaetales. Gram positive bacteria of the group of low DNA G + C content were seen as the dominant component in the intestine, while γ-Proteobacteria was the dominant group in the skin or TTX-accumulating organs. The TTX-accumulating organs were found to contain bacteria from all TTX-producing genera reported previously. We also observed large differences in the bacterial assemblages in the intestines of fish fed natural and artificial diets.