Eel Intestine Research Articles

SUPPLEMENTAL DESCRIPTION OF CABALLEROTREMA ANNULATUM (DIESING, 1850) OSTROWSKI DE NÚÑEZ AND SATTMANN, 2002 (DIGENEA: CABALLEROTREMATIDAE) FROM A NEW HOST (ELECTROPHORUS CF. VARII) AND LOCALITY (AMAZON RIVER, COLOMBIA) WITH PHYLOGENETIC ANALYSIS AND EMENDED GENERIC DIAGNOSIS.

Herein, we provide a supplemental description of Caballerotrema annulatum (Diesing, 1850) Ostrowski de Núñez and Sattmann, 2002 (Digenea: Caballerotrematidae Tkach, Kudlai, and Kostadinova, 2016) based on specimens collected from the intestine of an electric eel, Electrophorus cf. varii (Gymnotiformes: Gymnotidae) captured in the Amazon River (Colombia). This caballerotrematid can be differentiated from its congeners by the following combination of morphological features: body surface spines forming contiguous transverse rows, concentric (wrapping dorso-ventrally around body), distributing into posterior body half (vs. restricted to anterior body half in Caballerotrema brasiliensePrudhoe, 1960; indeterminate for Caballerotrema aruanenseThatcher, 1980 and Caballerotrema piscicola [Stunkard, 1960] Kostadinova and Gibson, 2001); head collar lacking projections (vs. having them in C. brasiliense, C. aruanense, and C. piscicola), narrow (head collar more narrow than maximum body width vs. the head collar being obviously wider than the body in C. brasiliense, C. aruanense, and C. piscicola); corner spines clustered (vs. corner spines distributing as 2 separated pairs in C. brasiliense, C. aruanense, and C. piscicola); pharynx approximately at level of the corner spines (vs. pharynx far anterior to corner spines in C. brasiliense, C. aruanense, and C. piscicola); and testes ovoid and nonoverlapping (C. aruanense; vs. sinuous and overlapping in C. brasiliense and C. piscicola). Based on our results, we revise the diagnosis of CaballerotremaPrudhoe, 1960 to include features associated with the shape and distribution of body surface spines, orientation and position of head collar spines, cirrus sac, seminal vesicle, oviduct, Laurer's canal, oötype, vitellarium, and transverse vitelline ducts. We performed Bayesian inference analyses using the partial large subunit ribosomal (28S) DNA gene. Our 28S sequence of C. annulatum was recovered sister to that of Caballerotrema sp. (which is the only other caballerotrematid sequence available in GenBank) from an arapaima, Arapaima gigas (Schinz, 1822) (Osteoglossiformes: Arapaimidae) in the Peruvian Amazon. Our sequence of C. annulatum comprises the only caballerotrematid sequenced tethered to a morphological description and a voucher specimen in a lending museum. The present study is a new host record and new locality record for C. annulatum. The phylogeny comprises the most resolved and taxon-rich evolutionary hypothesis for Echinostomatoidea published to date.

Effects of compound plant extracts on growth performance, antioxidant capacity, and histomorphology of liver and intestine of rice field eel (Monopterus albus)

AbstractCompound plant extracts (CPE) are beneficial for aquatic animals on growth performance and antioxidant capacity. A 56‐day experiment was conducted to investigate its positive effect on rice field eel. The fish were fed a commercial diet and supplementing CPE (mainly containing eucommia polysaccharides, reducing sugar) at 0, 0.8, 1.6, 2.4, and 3.2 g/kg. Results showed that dietary CPE significantly increased weight gain and specific growth rate (p < 0.05). Liver lipid content in 1.6 and 3.2 g/kg groups was significantly lower than in 0.8 and 2.4 g/kg groups (p < 0.05). Muscle lipid content in 0.8, 2.4, and 3.2 g/kg CPE groups was significantly lower than in control (p < 0.05). Meanwhile, intestinal digestive enzymatic activities in the 3.2 g/kg CPE group were the highest in comparison to all other groups (p < 0.05). Dietary CPE enhanced the antioxidant activities of serum and intestine (p < 0.05). Compared with the control, intestinal fold thickness in CPE groups was significantly increased (p < 0.05). Furthermore, dietary CPE reduced the degree of liver steatosis and the number of lipid droplet vacuoles. This study indicated that dietary CPE was beneficial to growth performance, antioxidant capacity, liver, and intestinal histology. Supplementation with 1.6–3.2 g/kg CPE is optimal for this eel species.

Disruption of intestinal structure, tight junction complex, immune response and microbiota after chronic exposure to copper in swamp eel (Monopterus albus)

As an essential micronutrient, copper is crucial in aquatic organisms' growth and development. Numerous studies have consistently reported that excessive intake of copper can have harmful effects on organisms. However, there are limited studies on the impact of copper on the intestine of the swamp eel (Monopterus albus). This study aimed to investigate the changes of intestinal histopathology, tight junction complex, immune response, and microbiota in swamp eel treated with 0 mg/L Cu2+, 0.05 mg/L Cu2+, and 0.10 mg/L Cu2+ for 56 d. Intestinal histopathology showed major changes such as the increased number of erythrocytes and goblet cells in the lamina propria, and separation of the lamina propria. The expression of genes involved in tight junction complex (ZO-1, Claudin-3, Claudin-12 and Claudin-15) was significantly changed. In addition, copper exposure significantly increased the mRNA levels of TLR3, TLR7, TLR8, NF-κB, I-κB, TNF-α and IL-8, especially in 0.10 mg/L Cu2+ group. In contrast, the relative expression level of anti-inflammatory cytokine TGF-β was significantly decreased after exposure to copper. Analysis of the intestinal microbiome showed the intestinal microbiota of swamp eels in the control and copper exposure groups were dominated by Firmicutes and Proteobacteria at the phylum level. Notably, copper exposure changed the diversity of the intestinal microbiota and decreased the relative abundance of Firmicutes and Proteobacteria in the intestine of swamp eel. Collectively, this study demonstrates that chronic copper exposure induces intestinal pathologic changes and inflammatory response, disrupts the intestinal microbial diversity and microbiota composition, and decreases intestinal barrier function in swamp eel, which enhances our understanding of copper-induced intestinal toxicity in fish.

Molecular mechanisms underlying guanylin-induced transcellular Cl− secretion into the intestinal lumen of seawater-acclimated eels

Guanylin (GN) stimulates Cl− secretion into the intestinal lumen of seawater-acclimated eels, but the molecular mechanisms of transepithelial Cl− transport are still unknown. In Ussing chamber experiments, we confirmed that mucosal application of eel GN reversed intestinal serosa-negative potential difference, indicating Cl− secretion. Serosal application of DNDS or mucosal application of DPC inhibited the GN effect, but serosal application of bumetanide had no effect. Removal of HCO3– from the serosal fluid also inhibited the GN effect. In intestinal sac experiments, mucosal GN stimulated luminal secretion of both Cl− and Na+, which was blocked by serosal DNDS. These results suggest that Cl− is taken up at the serosal side by DNDS-sensitive anion exchanger (AE) coupled with Na+–HCO3– cotransporter (NBC) but not by Na+-K+-2Cl− cotransporter 1 (NKCC1), and Cl− is secreted by unknown DPC-sensitive Cl− channel (ClC) at the mucosal side. The transcriptomic analysis combined with qPCR showed low expression of NKCC1 gene and no upregulation of the gene after seawater transfer, while high expression of ClC2 gene and upregulation after seawater transfer. In addition, SO42− transporters (apical Slc26a3/6 and basolateral Slc26a1) are also candidates for transcellular Cl− secretion in exchange of luminal SO42. Na+ secretion could occur through a paracellular route, as Na+-leaky claudin15 was highly expressed and upregulated after seawater transfer. High local Na+ concentration in the lateral interspace produced by Na+/K+-ATPase (NKA) coupled with K+ channels (Kir5.1b) seems to facilitate the paracellular transport. In situ hybridization confirmed the expression of the candidate genes in the epithelial enterocytes. Together with our previous results, we suggest that GN stimulates basolateral NBCela/AE2 and apical ClC2 to increase transcellular Cl− secretion in seawater eel intestine, which differs from the involvement of apical CFTR and basolateral NKCC1 as suggested in mammals and other teleosts.

Evaluation of dried porcine solubles in diets of Rice field eel (Monopterus albus)

Porcine solubles, originating from porcine mucosal tissues, is a by-product of heparin production. The nutritional value and functionality of a carrier-free, spray-dried porcine soluble (DPS) product was evaluated in 56-day experiment with juvenile rice field eel Monopterus albus (initial weight: 15.5 ± 0.19 g) fed diets with optimal (42%) and a low level (22%) of fishmeal (FM) inclusion. Six isonitrogenous and isolipidic experimental diets were formulated with two levels of FM (22% and 42%) and three levels of DPS (0%, 2.5% and 5%). Growth performance, humoral immunity, digestive enzyme activities, intestinal histology and expression of intestinal tight junction protein-related mRNA were analyzed at the end of the trial. Similar to FM, inclusion of 5% DPS significantly enhanced growth rate and reduced feed conversion ratio of rice field eel, although no significant interaction between FM and DPS on growth performance were observed. Positive influences of DPS on fish health and immunity were seen in that inclusion of DPS significantly enhanced serum complement (C3, C4), immunoglobulin M (IgM) levels and lysozyme activities, and altered total cholesterol and triglyeride contents in the serum of rice field eel. In regard to gut health, villus height, goblet cell quantity and expression of ZO-1 transcripts significantly (P < 0.05) increased with DPS inclusion in fish fed low-FM diets but was not observed in fish fed diets containing the optimal level of fishmeal. Expression of ZO-2 and CL-12 transcripts in intestine also was significantly (P < 0.05) elevated by DPS inclusion in fish fed diets containing low or optimal FM. Moreover, FM and DPS showed significant (P < 0.05) interactions in the expression of inflammatory cytokines mRNA in the intestine of rice field eel with 5% DPS inclusion significantly (P < 0.05) promoting the expression of Tgfb2 and IκBe mRNA, while repressing IL-1β, IL-8, IL-12 and IFNγ expression in fish fed diets containing low FM. This study is the first to show functionality of DPS supplementation at 5% of diet in any aquatic species. Based on these results, DPS supplementation should be considered as an effective, innovative and sustainable strategy to improve health, as well as reduce intestinal inflammation and maintain normal intestine structure of rice field eel fed low-FM diets.

Systematic evaluation of the gut microbiome of swamp eel (Monopterus albus) by 16S rRNA gene sequencing.

BackgroundThe swamp eel (Monopterus albus) is a commercially important farmed species in China. The dysbiosis and homeostasis of gut microbiota has been suggested to be associated with the swamp eel’s disease pathogenesis and food digestion. Although the contributions of gut microbiome in fish growth and health has been increasingly recognized, little is known about the microbial community in the intestine of the swamp eel (Monopterus albus).MethodsThe intestinal microbiomes of the five distinct gut sections (midgut content and mucosa, hindgut content and mucosa, and stools) of swamp eel were compared using Illumina MiSeq sequencing of the bacterial 16S rRNA gene sequence and statistical analysis.ResultsThe results showed that the number of observed OTUs in the intestine decreased proximally to distally. Principal coordinate analysis revealed significant separations among samples from different gut sections. There were 54 core OTUs shared by all gut sections and 36 of these core OTUs varied significantly in their abundances. Additionally, we discovered 66 section-specific enriched KEGG pathways. These section-specific enriched microbial taxa (e.g., Bacillus, Lactobacillus) and potential function capacities (e.g., amino acid metabolism, carbohydrate metabolism) might play vital roles in nutrient metabolism, immune modulation and host-microbe interactions of the swamp eel.ConclusionsOur results showed that microbial diversity, composition and function capacity varied substantially across different gut sections. The gut section-specific enriched core microbial taxa and function capacities may perform important roles in swamp eel’s nutrient metabolism, immune modulation, and host-microbe interactions. This study should provide insights into the gut microbiome of the swamp eel.

Molecular mechanisms for intestinal HCO3- secretion and its regulation by guanylin in seawater-acclimated eels.

The intestine of marine teleosts secretes HCO3- into the lumen and precipitates Ca2+ and Mg2+ in the imbibed seawater as carbonates to decrease luminal fluid osmolality and facilitate water absorption. However, the hormonal regulation of HCO3- secretion is largely unknown. Here, mucosally added guanylin (GN) increased HCO3- secretion, measured by pH-stat, across isolated seawater-acclimated eel intestine bathed in saline at pH7.4 (5% CO2). The effect of GN on HCO3- secretion was slower than that on the short-circuit current, and the time course of the GN effect was similar to that of bumetanide. Mucosal bumetanide and serosal 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) inhibited the GN effect, suggesting an involvement of apical Na+-K+-2Cl- cotransporter (NKCC2) and basolateral Cl-/HCO3- exchanger (AE)/Na+-HCO3- cotransporter (NBC) in the GN effect. As mucosal DNDS failed to inhibit the GN effect, apical DNDS-sensitive AE may not be involved. To identify molecular species of transporters involved in the GN effect, we performed RNA-seq analyses followed by quantitative real-time PCR after transfer of eels to seawater. Among the genes upregulated after seawater transfer, AE genes (draa, b, and pat1a, c) on the apical membrane, and NBC genes (nbce1a, n1, n2a) and an AE gene (sat-1) on the basolateral membrane were candidates involved in HCO3- secretion. Judging from the slow effect of GN, we suggest that GN inhibits NKCC2b on the apical membrane and decreases cytosolic Cl- and Na+, which then activates apical DNDS-insensitive DRAs and basolateral DNDS-sensitive NBCs to enhance transcellular HCO3- flux across the intestinal epithelia of seawater-acclimated eels.

Disturbance in the homeostasis of intestinal microbiota by a high-fat diet in the rice field eel (Monopterus albus)

Intestinal microbiota plays crucial roles in nutrient uptake and contributes to obesity in aquatic animals, although the association between intestinal microbiota and obesity remains unclear. In this study, high-throughput sequencing was employed to determine the composition, interspecific interactions, and potential function of the microbial community in the intestine of rice field eel (Monopterus albus) fed with basal diet (Control group, 6.30% lipid) or high-fat diet (HF group, 12.11% lipid). The high-fat diet decreased species richness, accompanied by a significant reduction in the diversity of intestinal microbiota, and significantly increased the relative abundance of Fusobacteriia (classified as Cetobacterium), concurrent with a significant reduction in Clostridia (classified as Clostridium) in the eel. However, the eels in both groups shared the same dominant gut microbiota, comprising Clostridia, Fusobacteriia, and Gammaproteobacteria. Molecular ecological network analysis described the complex inter-species interactions within the intestinal bacterial community, which in both groups was predominated by positive interactions. The dominant intestinal microbiota was found to be the major component of the ecological network and could play vital roles, for instance, as connectors, which have a major contribution in maintaining the stability of the bacterial community. The high-fat diet disturbed the intestinal microbiota homeostasis through the deterioration of the ecological network by reducing the average connectivity, negative interactions, and the number of connectors. Analysis of predicted functions (PICRUSt) showed that the high-fat diet significantly increased some potential functions of intestinal microbiota involved in lipid, amino acid, carbohydrate, and energy metabolism, in accordance with metabolic changes observed in terms of low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, cholesterol, and glucose in the serum. These results suggested that the change in intestinal microbiota may contribute to the development of diet-induced fat deposition in eels.

Histochemical and immunohistochemical characterization of rodlet cells in the intestine of two teleosts, Anguilla anguilla and Cyprinus carpio.

Rodlet cells (RC) are characterized by a distinctive cell cortex and conspicuous inclusions named "rodlets." These cells are particularly abundant and large in size in intestine of eels. Histochemical, immunohistochemical and ultrastructural investigations were carried out on European eel Anguilla anguilla and Common carp Cyprinus carpio from Northern Italy. Eight biotinylated lectins were used to probe for specific carbohydrate residues in deparaffinized, hydrated intestinal sections of eel and carp. Five antibodies were tested on intestinal sections of both fish species: inducible nitric oxide synthase (i-NOS), leu-enkephalin, lysozyme, serotonin and tumour necrosis factor-α. Lectin histochemistry revealed rodlet cells (RCs) of the eel intestine to react with two of the eight lectins tested, specifically Concanavalin A (ConA) and Sambucus Nigra Agglutinin (SNA). This contrasted to lectin staining of RCs in the intestine of common carp, where four of the eight lectins showed a positive reaction; Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), SNA and ConA. RCs in eel and carp intestine were immunoreactive with antibodies to lysozyme and i-NOS. The occurrence of the inflammatory peptides lysozyme and i-NOS in RCs of the eel and common carp poses in favour that these cells are involved in the mechanism of defence against pathogens.

Duplicated CFTR isoforms in eels diverged in regulatory structures and osmoregulatory functions

Two cystic fibrosis transmembrane conductance regulator (CFTR) isoforms, CFTRa and CFTRb, were cloned in Japanese eel and their structures and functions were studied in different osmoregulatory tissues in freshwater (FW) and seawater (SW) eels. Molecular phylogenetic results suggested that the CFTR duplication in eels occurred independently of the duplication event in salmonid. CFTRa was expressed in the intestine and kidney and downregulated in both tissues in SW eels, while CFTRb was specifically expressed in the gill and greatly upregulated in SW eels. Structurally, the CFTR isoforms are similar in most functional domains except the regulatory R domain, where the R domain of CFTRa is similar to that of human CFTR but the R domain of CFTRb is unique in having high intrinsic negative charges and fewer phosphorylation sites, suggesting divergence of isoforms in terms of gating properties and hormonal regulation. Immunohistochemical results showed that CFTR was localized on the apical regions of SW ionocytes, suggesting a Cl− secretory role as in other teleosts. In intestine and kidney, however, immunoreactive CFTR was mostly found in the cytosolic vesicles in FW eels, indicating that Cl− channel activity could be low at basal conditions, but could be rapidly increased by membrane insertion of the stored channels. Guanylin (GN), a known hormone that increases CFTR activity in mammalian intestine, failed to redistribute CFTR and to affect its expression in eel intestine. The results suggested that GN-independent CFTR regulation is present in eel intestine and kidney.

Guanylin activates Cl(-) secretion into the lumen of seawater eel intestine via apical Cl(-) channel under simulated in vivo conditions.

Guanylin (GN) action on seawater eel intestine was examined under simulated in vivo conditions, where isotonic luminal fluid has low NaCl and high MgSO4 (MgSO4 Ringer). In Ussing chamber, MgSO4 Ringer induced serosa-negative potential difference (PD) even after bumetanide treatment, which is due to the higher paracellular Na(+) permeability over Cl(-), as confirmed by the replacement by MgCl2 (no Cl(-) gradient) or Na2SO4 Ringer (no Na(+) gradient). Luminal GN reversed serosa-negative PD, probably by enhancing Cl(-) secretion into the lumen, as the GN effect was blocked by apical Cl(-) channel blockers [diphenylamine-2-carboxylic acid (DPC), 5-nitro-2-(3-phenylpropylamino) benzoic acid, glibenclamide but not cystic fibrosis transmembrane regulator (CFTR)inh-172] or replacement of luminal fluid by MgCl2 Ringer. The blockers' effect was undetectable when normal Ringer was on both sides. In the sac preparation, NaCl secretion occurred into the lumen (Na(+) > Cl(-)), and GN further enhanced Cl(-) secretion (Cl(-) > Na(+)), resulting in water secretion. These GN effects were also blocked by DPC. Quantitative analyses showed that isotonic NaCl is absorbed when luminal fluid is normal Ringer, but, when luminal fluid is MgSO4 Ringer, hypertonic NaCl, almost equivalent to seawater, is secreted into the lumen after GN. These results indicate that GN stimulates the secretion of hypertonic NaCl into the lumen of seawater eel intestine, like rectal gland of marine elasmobranchs, to get rid of excess NaCl although marine teleost intestine is thought to have only absorptive-type cells with a unique Na-K-Cl cotransport system. The secreted NaCl may activate the cotransport system and further help absorb water in the final segment of seawater eel intestine.

Cucullanus egyptae sp. nov. (Nematoda, Cucullanidae) infecting the European eel Anguilla anguilla in Egypt. Morphological and molecular phylogenetic studies.

A total of 80 specimens of the European eel Anguilla anguilla were collected during the period from February 2013 to March 2014 at the coast of the Gulf of Suez (Red Sea, Egypt). A new species of parasitic nematodes was recovered and described as Cucullanus egyptae. It was found in the eel's intestine with a prevalence of 68.7%. The morphology of the recovered parasite was studied by light and scanning electron microscopy. The adult worms had a wide cephalic extremity with a slit-like oral aperture being surrounded by a cuticular ring and delimited internally by a row of small teeth in addition to four sub-median cephalic papillae and one pair of lateral amphids. Body measurements showed that the male worms were smaller than females measuring 7.5-8.9 mm (8.3 ± 0.2) in length and 1.6-1.9 mm (1.8 ± 0.1) in width. Females measured 12.9-13.5 mm (13.1 ± 0.2) in length and 2.9-3.2 mm (3.1 ± 0.1) in width. The posterior end of the males is provided with ten pairs of caudal papillae and two long spicules which are slightly sclerotized, equal in size measuring 0.59-0.65 mm (0.62 ± 0.01) in length. Comparing the present worms with other species of the genus Cucullanus, several similarities were observed. However, peculiar new characteristics such as the precloacal sucker (especially the spicula length), the arrangement and the distribution of the post-cloacal papillae, and the position of the excretory pore make it reasonable to describe a new species. The sequence data of the small subunit (SSU) ribosomal DNA (rDNA) obtained from the present nematode supported its taxonomic position within the genus Cucullanus. The new species is closely related to the first clade of Spirurina and even more closely related to Cucullanus dodsworthi as a sister taxon with a high percentage of identity. The sequence of the recorded SSU rDNA of this parasite is deposited in the GenBank with the accession no. KF681520. It is proposed to name the new species C. egyptae as the first representative of Cucullanidae in Egypt with a new host record.

Mechanisms of guanylin action on water and ion absorption at different regions of seawater eel intestine.

Guanylin (GN) inhibited water absorption and short-circuit current (Isc) in seawater eel intestine. Similar inhibition was observed after bumetanide, and the effect of bumetanide was abolished by GN or vice versa, suggesting that both act on the same target, Na(+)-K(+)-2Cl(-) cotransporter (NKCC), which is a key player for the Na(+)-K(+)-Cl(-) transport system responsible for water absorption in marine teleost intestine. However, effect of GN was always greater than that of bumetanide: 10% greater in middle intestine (MI) and 40% in posterior intestine (PI) for Isc, and 25% greater in MI and 34% in PI for water absorption. After treatment with GN, Isc decreased to zero, but 20-30% water absorption still remained. The remainder may be due to the Cl(-)/HCO3 (-) exchanger and Na(+)-Cl(-) cotransporter (NCC), since inhibitors for these transporters almost nullified the remaining water absorption. Quantitative PCR analysis revealed the presence of major proteins involved in water absorption; the NKCC2β and AQP1 genes whose expression was markedly upregulated after seawater acclimation. The SLC26A6 (anion exchanger) and NCCβ genes were also expressed in small amounts. Consistent with the inhibitors' effect, expression of NKCC2β was MI > PI, and that of NCCβ was MI << PI. The present study showed that GN not only inhibits the bumetanide-sensitive Na(+)-K(+)-Cl(-) transport system governed by NKCC2β, but also regulates unknown ion transporters different from GN-insensitive SLC26A6 and NCC. A candidate is cystic fibrosis transmembrane conductance regulator Cl(-) channel, as demonstrated in mammals, but its expression is low in eel intestine, and its role may be minor, as indicated by the small effect of its inhibitors.

Intestinal expression of peptide transporter 1 (PEPT1) at different life stages of Japanese eel, Anguilla japonica

The expression of peptide transporter 1 (PEPT1) was investigated at the different life stages of Japanese eel, Anguilla japonica. The cDNA encoding Japanese eel PEPT1 was cloned and sequenced. The hydrophilicity plot analysis of its deduced amino acid sequence showed high similarities with topological features of known PEPT1 molecules in other species. Tissue distribution analysis confirmed that PEPT1 mRNA was detected specifically in the anterior and posterior intestines of adult eel. In eel larvae at 13days post hatching (dph), PEPT1 mRNA expression was mainly detected in the intestinal tract regions. The trypsinogen mRNA was only detected in the gastric region including the pancreas. Intense immunoreaction for PEPT1 was observed in the apical membrane of the intestinal epithelial cells of both larval and adult eel. These results indicated that PEPT1 was an intestine-specific transporter, which was localized at the luminal side of the epithelial cells, suggesting that di/tri-peptide absorption via PEPT1 takes place in the eel intestine. According to the ontogenetic analyses by quantitative PCR, PEPT1 and trypsinogen mRNA expressions were simultaneously increased at 5-7 dph. It is thus assumed that nutrient absorption systems in the intestinal tracts of larvae become functional at this age.

A Novel Phospholipase C‐X‐domain (PLCXD) containing protein exhibits differential gene expression in the European eel ( Anguilla anguilla ) intestine following “silvering”

Adult European eels undergo a metamorphosis where sexually immature freshwater “yellow” eels, exhibit anatomical and physiological adaptations resulting in the development of sexually maturing and migratory “silver” eels. Microarray studies investigating gene expression during this stage of development identified one cDNA with homology to the mammalian phospholipase C X-domain (PLCXD) containing gene family, which exhibited loss of intestinal expression following silvering. The eel PLCXD gene, like PLCXD proteins from all other species, contains only the X domain of the normal X and Y domains that form the catalytic active site of other PLCs. To date the functional roles of these proteins are unknown. Real time PCR analysis validated the original microarray data and showed a 60-fold increase in the expression of PLCXD mRNA in the intestine of yellow compared to silver eels. Following sequencing of the full length cDNA antibodies were developed for characterisation of protein expression. Our data suggest that PLCXD expression is essential for maintenance of the normal absorptive function of the intestine of the yellow eel but is not required in the silver eel when the fish stops eating and the role of the GI tract is limited only to osmoregulation.

Expression of aquaporins 3, 8 and 10 in the intestines of freshwater- and seawater-acclimated Japanese eels Anguilla japonica

Euryhaline teleosts possess excellent adaptability to a wide range of environmental salinities. Although intestinal water absorption is important for seawater (SW) adaptation, little is known about the molecular mechanisms of the water-transporting pathway in the intestine. We have cloned three homologs of the mammalian aquaporins (AQPs) 3, 8 and 10 from the intestine of the SW-acclimated Japanese eel. The deduced amino-acid sequences shared 47–98% homology with other known respective AQP isoforms. Topology prediction of the identified sequences showed six membrane-spanning domains, intracellular N- and C-terminal tails, and two NPA (asparagine–proline–alanine) motifs in the second and fifth connecting loops, all of which are highly conserved among known AQPs. Reverse transcription PCR analysis revealed that AQP3 was expressed in various tissues, whereas the expression of AQP 8 and 10 mRNAs was detected predominantly in the intestine. The expression levels of AQPs 1, 3, 8 and 10 in the anterior and posterior intestines and the rectum were determined by real-time quantitative PCR and compared for FW (freshwater)- and SW-acclimated eels. AQP1 expression levels in the posterior intestine and rectum were significantly higher in the SW-acclimated eel than in the FW-acclimated fish. AQP3 expression in the SW-acclimated eel was only higher in the rectum than that in the FW-acclimated eel. Expression levels of AQPs 8 and 10 in the intestinal segments tended to be higher in the SW-acclimated eel than in the FW-acclimated eel. These results showed that intestinal AQP expression is closely related to SW adaptation, suggesting the presence of a water-absorbing mechanism associated with multiple AQP isoforms in the intestinal tract.

Role of BK Channels in the Apoptotic Volume Decrease in Native Eel Intestinal Cells

High conductance Ca⁺-activated K⁺ channels (BK channels) have previously been demonstrated in the eel intestine. They are specifically activated following a hypotonic stress and sustain Regulatory Volume Decrease (RVD). The aim of the present work was to address the possible role of these channels in the Apoptotic Volume Decrease (AVD) of isolated eel enterocytes, and the possible interaction between BK channels and the progression of apoptosis. The detection of apoptosis was performed by confocal microscopy and annexin V and propidium iodide labelling; cell volume changes were monitored by video imaging. Within a few hours after isolation, enterocytes underwent anoikis (apoptosis induced by detachment from the extracellular matrix). They showed an early normotonic volume decrease (AVD) preceding the appearance of annexin V positivity. AVD occurred in correspondence with an increase in the [Ca²⁺]_i, measured with Fura-2. When the cells were resuspended in high K⁺ solution or treated with iberiotoxin, AVD was completely abolished. In addition, treatment with high K⁺ or iberiotoxin significantly inhibited apoptosis progression. It was demonstrated for the first time in native enterocytes that BK channels, which are involved in RVD in these cells, plays also a crucial role in the AVD process and in the progression of apoptosis.

Gossypol affects ion transport in the isolated intestine of the seawater adapted eel, Anguilla anguilla

Cottonseed ( Gossypium sp.) meals are protein rich and inexpensive, but the presence of the polyphenolic dialdehyde, gossypol, is responsible of many toxic effects in animals including fishes. Recently an effect on the transepithelial ion transport in rat colon has been demonstrated. In this study we investigated the effect of gossypol on the transepithelial electrical parameters of the isolated intestine of seawater adapted eel, Anguilla anguilla, by employing a Ussing chamber technique. We showed that the addition of gossypol to the perfusion media reduced short circuit current ( I sc), a measure of Cl − active absorption in this tissue, and increased tissue conductance ( g t). The observation that the effect of gossypol on both I sc and g t was modified by the pretreatment with TFP, a calmodulin inhibitor, suggests that the substance acts via a Ca 2+ calmodulin pathway and excludes the possibility that the observed effects were due to a cytotoxic action. In addition, experiments performed in the presence of verapamil suggest that the polyphenolic pigment increases Ca 2+ influx. It is likely that gossypol stimulates a basolateral quinine sensitive K + conductance producing a K + flux in absorptive direction that explains the reduction of I sc. In addition dilution potential experiments showed that the polyphenolic aldehyde increases the anion conductance of the paracellular pathway. In conclusion our study suggests that gossypol alters ion transport in eel intestine by acting on both transcellular and paracellular pathways. Since the intestine is an important organ for maintaining the water and ion balance in seawater adapted fish, it is conceivable that gossypol could impair the ability of the animals to adapt to the environment.

Regulation of Ion Transport in Eel Intestine by the Homologous Guanylin Family of Peptides

Since the gene expression of guanylin peptides and their receptors, guanylyl cyclase Cs, is enhanced in the intestine of seawater (SW)-adapted eels compared with fresh water (FW)-adapted fish, the guanylin family may play an important role in SW adaptation in eels. The present study analyzed the effect of three homologous guanylin peptides, guanylin, uroguanylin and renoguanylin, on ion movement through the eel intestine, and examined the target of guanylin action using Ussing chambers. The middle and posterior parts of the intestine, where water and ion absorption occurs actively in SW eels, exhibited serosa-negative transepithelial potential, while the anterior intestine was serosa-positive. Mucosal application of each guanylin in the middle or posterior intestine reduced the short-circuit current (Isc) dose dependently and reversed it at high doses, and reduced electric tissue resistance. The effects were greater in the middle intestine than in the posterior intestine. All three guanylins showed similar potency in the middle segment, but guanylin was more potent in the posterior segment. 8-bromo cGMP mimicked the effect of guanylins. The intestinal response to guanylin was smaller in FW eels. The mucosal presence of NPPB utilized as a CFTR blocker, but not of other inhibitors of the channels/transporters localized on the luminal surface in SW fish intestine, inhibited the guanylin-induced decrease in Isc. In eels, therefore, the guanylin family may be involved in osmoregulation by the intestine by binding to the receptors and activating CFTR-like channels on the mucosal side through cGMP production, perhaps resulting in Cl(-) and HCO3(-) secretion into the lumen.

Histological and ultrastructural study of the digestive tract of rice field eel, Monopterus albus

Summary The histological characteristics of the digestive tract and the ultrastructure of mucosal cells of the stomach and intestine of rice field eel, Monopterus albus, are described to provide a basis for future studies on its digestive physiology. The digestive tract of the rice field eel is a long and coiled tube composed of four layers: mucosa, lamina propria-submucosa, muscularis and serosa. The pharynx and oesophagus mucosa is lined with a stratified epithelium. The stomach includes the cardiac and pyloric portions and the fundus. Many gastric pits are formed by invaginations of the mucosal layer and tubular gastric glands formed by the columnar cells in the fundus. The intestine is separated from the stomach by a loop valve and divided into a proximal portion and a distal portion. The proximal intestinal epithelium consists of columnar cells with microvilli towards the lumen and goblet cells. The enterocytes are joined at the apical surface by the junctional complex, including the evident desmosomas. Numerous lysosomes and some vesicles are evident in the upper cytoplasm of the cells, and a moderate amount of endoplasmic reticulum and lysosomes are scattered in the supranuclear cytoplasm. The epithelium becomes progressively thicker and the folds containing large numbers of goblet cells are fewer and shorter in the distal portion of the intestine. At the ultrastuctural level, the columnar cells of the tubular gastric glands have numerous clear vacuoles and channels. A moderate amount of pepsinogen granules are present in the stomach. The enterocytes of the intestinal mucosa display a moderate amount of endoplasmic reticulum and lysosomes, and long and regular microvilli.