The Gastric Phenotype in the Cypriniform Loaches: A Case of Reinvention?

<div><p>The stomach, which is characterized by acid peptic digestion in vertebrates, has been lost secondarily multiple times in the evolution of the teleost fishes. The Cypriniformes are largely seen as an agastric order; however, within the superfamily Cobitoidea, the closely related sister groups Nemacheilidae and Balitoridae have been identified as gastric families. The presence of these most recently diverged gastric families in an otherwise agastric clade indicates that either multiple (>2–3) loss events occurred with the Cyprinidae, Catostomidae and Cobitidae, or that gastric reinvention arose in a recent ancestor of the Nemacheilidae/Balitoridae sister clade. In the present study, the foregut regions of Cobitidae, Nemacheilidae/Balitoridae and the ancestral Botiidae family members were examined for the presence of gastric glands and gastric proton pump (Atp4a) α subunit expression by histology and immunohistochemistry respectively. <i>Atp4a</i> gene expression was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR). Gastric glands expressing apical H⁺/K⁺-ATPase α subunit and isolated partial sequences of <i>atp4a</i>, identified using degenerate primers showing clear orthology to other vertebrate <i>atp4a</i> sequences, were detected in representative species from Nemacheilidae/ Balitoridae and Botiidae, but not Cobitidae (<i>Misgurnus anguillicaudatus</i>). In summary, we provide evidence for an uninterrupted gastric evolutionary lineage in the Cobitoidea, making it highly improbable that the stomach was reinvented in the Nemacheilidae/Balitoridae clade consistent with Dollo’s principle. These results also indicate that the gastric trait may be present elsewhere in the Cobitoidea.</p></div

<i>Beaufortia kweichowensis</i> (Balitoridae) immunohistochemistry.

<p>Double immunohistochemical localization of HKα1/NKα1 (green; αR1 antibody) and NKCC (red; T4 antibody) in <i>B</i>. <i>kweichowensis</i> (<b>a,b</b>) cardiac stomach, (<b>c</b>) proximal intestine and (<b>d</b>) kidney (upper) and distal intestine (lower). Asterisk indicate gut lumen. Scale bar (a,c,d) 100 μm (b) 25 μm.</p

<i>Nemacheilus angorae</i> (Nemacheilidae) anterior gastrointestinal tract immunohistochemistry.

<p>Double immunofluoresent localization of HKα1 (<b>a,b,c</b>; green; C2 antibody) and NKAα1 (<b>d,e,f</b>; red; α5 antibody) in <i>N</i>. <i>angorae</i> esophagus (<b>a,d,g</b>), cardiac stomach (<b>b,e,h</b>) and proximal intestine (<b>c,f,i</b>). IHC images are overlaid with corresponding DAPI (<b>g,h,i</b>) and DIC (<b>j,k,l</b>). Scale bar 100 μm.</p

Molecular phylogenetic analysis of the three loach sequences.

<p>Phylogenetic tree obtained using the Maximum Likelihood method based on the JTT matrix-based model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163696#pone.0163696.ref045" target="_blank">45</a>]. The percentage of trees in which the associated taxa clustered together is shown next to the branches.</p

Summary of histology for the presence/absence of gastric glands and immunofluorescence (IF) microscopy results for HKα1 expression.

<p>Summary of histology for the presence/absence of gastric glands and immunofluorescence (IF) microscopy results for HKα1 expression.</p

Histology of <i>Misgurnus anguillicaudatus</i> (Cobitinae) anterior intestine indicating the absence of gastric glands.

<p>The intestine is thrown into longitudinal folds and is covered by a columnar epithelium regularly interspersed with goblet cells. H&E staining viewed with differential interference contrast technique. Gut lumen (*). Scale bars (a) 500, (b) 100, (c) 25 μm.</p

<i>Botia histrionica</i> (Botiidae) stomach and intestine immunohistochemistry.

<p>Immunohistochemical staining of golden zebra loach cardiac (a,b) and pyloric (c) stomach and intestine (d). Sections are probed with the HKα1/NKα1 antibody (αR1; green a,c,d) and HKα1 antibody (C2; green; b) and and colabeled with NKCC (T4; red) and NKα1 (α5; red) and respectively. Sections are counter stained with the nuclear stain DAPI (blue) and over laid with the DIC image for orientation. Scale bar 100μm.</p

<i>Chromobotia macracanthus</i> (Botiidae) stomach and intestine.

<p>(a,b) Demonstration of acinar-type gastric glands expressing apical H⁺/K⁺-ATPase (HKα1; green) as determined by immunofluorescence microscopy in <i>Chromobotia macracanthus</i> (Botiidae). In the gastric glands Na⁺:K⁺:2Cl^- cotransporter (red) has a basolateral distribuition. c) PAS staining (magenta) indicates the presence of a protective mucous secreting epithelium in the stomach. (d; bottom) The intestinal epithelium shows the presence of typical intestinal enterocytes with basolateral Na⁺/K⁺-ATPase (green) and brush border Na⁺:K⁺:2Cl^- cotransporter (red) staining, while the pyloric region of the stomach (d; top) shows no such staining. Scale bar (a,c,d) 100 μm (b) 50 μm.</p

A simplified schematic of the phylogenetic relationships in the Cypriniformes (modified from Slechtova <i>et</i>. <i>al</i>. (2007) [22] and Wilson and Castro (2010) [12]).

<p>The dashed lines indicate families in which the absence of a stomach (-) is observed (Cobitidae and Cyprinidae/Catostomidae). The crown families Nemacheilidae/Balitoridae are known to be gastric (+). It is unknown if the stomachless condition exists in Villantellidae (?) and the presence of the stomach in Botiidae requires confirmation (+?) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163696#pone.0163696.ref012" target="_blank">12</a>].</p