研究揭示气呼吸泥鳅兼具消化与呼吸功能的消化道

These authors contributed equally to this work.

Received 2024 Apr 11; Revised 2024 May 17; Accepted 2024 May 22; Collection date 2024 Jun.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Fish digestive tracts always show remarkable morphological differences depending on their functions. The weatherloach, Misgurnus anguillicaudatus, is a facultative air-breathing teleost fish, which employs its partial digestive tract as an accessory respiratory organ. The specificity of its digestive tract structure and function is unclear. To clarify how the digestive tract of the weatherloach serves a dual function of digestion and respiration simultaneously, the histological structures of its digestive tract, the passage of digesta and air passing through its intestine and the rate of intestinal evacuation have been studied. The study also explored the structural features of the different regions of the weatherloach intestine and how these structures coordinate digestion and respiration. This provides basic data for the coordination between digestion and respiration in the weatherloach. The results showed that the weatherloach intestine is mainly divided into five functional regions with specific structures. The process of gastrointestinal air breathing can not only aid respiration, but also promotes the excretion of the contents of the intestine. This study can support further exploration of the physiological mechanisms of gastrointestinal air breathing in fish.

To clarify how the digestive tract of the weatherloach, Misgurnus anguillicaudatus, serves a dual function of digestion and respiration simultaneously, the histological structures of its digestive tract, the passage of digesta and air passing through its intestine and the rate of intestinal evacuation have been studied. The results indicate that the digestive tract is divided into five functional regions, i.e., esophagus, anterior intestine, middle intestine, posterior intestine and rectum. The diverse intestinal structures have the specialized function of coordinating digestion and respiration. An X-ray barium meal examination showed in the normal breathing state, the contents of the intestine are diffusely semifluid, and air is distributed as bubbles in the dorsal intestine 2 h after feeding. After 5 h, the contents accumulated in the mid and posterior intestine, and gas flowed above the contents as bundles. After 8 h, the intestinal food was basically evacuated. In the intestinal air-breathing restricted group, the contents of the intestine remained diffuse, and a large number of digesta entered and remained in the rectum after 5 h. After the inhibition was relieved, the contents of the rectum were rapidly discharged. Measurement of the intestinal evacuation rate in the intestine showed that the evacuation of the intestinal contents lagged behind that of the normal group in the air-breathing restricted group. Compared to the normal state and inhibited GAB (gastrointestinal air breathing), we could deduce that GAB could promote the movement of the intestine.

Keywords: air-breathing, digestive tract, histology, barium meal, evacuation rate, Misgurnus anguillicaudatus

Fish digestive tracts always show remarkable morphological differences. Although the digestive tract appears to be a unified organ, it has been divided into a collection of related segments based on their function, location and internal anatomy. All these segments possess different functional attributes and team up to provide the functionality of the whole gut [1].

The weatherloach, M. anguillicaudatus, is a facultative air-breathing teleost fish, which employs its partial digestive tract as an accessory respiratory organ. The digestive tract is distinguished by a relatively short length and small volume. The ratio of intestinal length to body length is notably low, which is considerably below that observed in fish belonging to the Callichthyidae, which also exhibit intestinal breathing [1,2]. During gastrointestinal air breathing (GAB), they need to inhale through the mouth, then transport the gas to the posterior intestine for gas exchange and finally expel the exhaust gas through the anus [3]. Almost all studies of M. anguillicaudatus have focused on the respiratory region and physiological aspects of gas exchange. The posterior intestine in M. anguillicaudatus is highly modified, being thin-walled, air-filled and highly vascularized [4]. These modifications make it unsuitable for digestive functions, and it is generally found to be devoid of digesta in this region. The digesta can rapidly pass the respiratory region under the pressure from the anterior regions [5], and at times the air is excreted under contraction. But how this is done is not clear since the anterior intestine is always full of food that would seriously hamper the passage of airflow. Zhang et al. proposed that it is the disorder between the digestion and breathing that leads to a high mortality in the aquaculture [6].

It is likely that the contractions of muscles in the middle spiral rotation compress digesta into a narrow string before entering the posterior respiratory region, according to McMahon’s research [3]. Actually, the mucosa and muscle layer in the spiral region are very undeveloped [7]. Additionally, there exists a phenomenon that the digesta in this region are coated gradually by a layer of the mucus sac [5]. Little is known about the specific structures and functions of other regions.

A barium meal is a diagnostic procedure which uses X-ray imaging to detect the abnormalities in the esophagus, stomach and small intestine [8]. The gastrointestinal tract, like other soft tissue structures, does not show up clearly enough on plain radiographs for diagnostic purposes. The barium salt is radiopaque and easily distinguishable on radiographs [9]. The barium meal experiment is capable of providing a visual representation of the intestinal structure and the relative positioning of the contents and gas, which is an invaluable tool for investigating intestinal peristalsis in the weatherloach.

In contrast to some other GAB fish (Callichthyidae), M. anguillicaudatus is a gastric fish and has a simpler and shorter gut. The GAB process of the weatherloach has been described [3]. It was found that there may be a mechanism for coordination between digestion and respiration. In order to clarify the coordination between digestion and air breathing, this study was conducted to investigate the digesta and ventilation following barium meal ingestion in conjunction with X-ray examination, determine the rate of intestinal evacuation and also examine the histological structures of the intestinal tract using light microscopy (LM) and scanning electron microscopy (SEM). The findings contribute to the establishment of a theoretical framework for the healthy breathing process of the weatherloach.

Two hundred adult weatherloaches, M. anguillicaudatus (the average body weight and body length were 14.22 ± 4.27 g and 11.74 ± 1.60 cm, respectively), were bought from the Baishazhou Aquatic Products Market and raised in the Aquaculture Base of the College of Fisheries, Huazhong Agricultural University in Wuhan, Hubei Province, China. All the fish were acclimated at 20 °C~26 °C and DO > 6 mg/L prior to experimental use. After 2 weeks of temporary rearing to acclimatize the fish to the experimental conditions, all the fish being investigated were starved for 24 h. All the experimental procedures involving fish were approved by the institution’s animal care and use committee of the Huazhong Agricultural University, China.

Ten adult fish (five for LM and five for SEM) were euthanized using tricainemethanesulfonate (MS-222, 100 mg L−1). After dissecting them, the specimens’ intestinal tracts were picked out and divided into smaller samples (0.5–1.0 mm) based on the external morphology. The samples for LM were fixed in Bouin’s fluid, dehydrated in a series of alcohols (70%, 80%, 90%, 95% and 100%), and finally embedded in paraffin blocks. The whole digestive tract was sectioned at 5–7 μm and stained with hematoxylin and eosin. Histological sections were observed and photographed via microscopy (Axio Imager A2, Zeiss, Jena, Germany). For SEM analysis, the samples were cut into an appropriate size (2 × 2 mm2), fixed in glutaraldehyde and dehydrated, dried in a critical point drier, mounted on aluminum stubs, vacuum coated with gold and then examined via SEM (JSM-6390 LV, Tokyo, Japan) operating between 10 and 30 kV.

A pre-experiment showed that the proper addition of barium sulfate (BaSO4) to feed had little effect on the appetite of M. anguillicaudatus, and the digesta took 7–8 h to pass through the GI tract. The experiments were conducted at 20 °C~26 °C and in a DO > 6 mg/L environment. Based on this result, thirty loaches in the present study were divided into two groups (the normal group and intestinal air-breathing restricted group) and cultured in the same plastic tank, which was divided into two parts using mesh. The intestinal air-breathing restricted group was covered by mesh under water to prevent air breathing at the surface. The fish were fed with excess barium meal (eel powder feed and BaSO4 were mixed in the ratio of 2 to 1). The remaining feed was cleaned up half an hour after feeding. In the control group, three fish were randomly sampled at 0 h, 2 h, 5 h, 8 h post-feeding. In the restricted group, the first sampling was at 8 h post-feeding. After that, the mesh was removed, and the fish came to the surface to take their first breath of air. The second and third samplings were performed after the first air breath and half an hour later, respectively. The sampled fish were narcotized and photographed under an X-ray generator. The operating parameters of the X-ray generator were 40.00 KVP, 12.50 ms and 2.50 mAs (the optimal parameter setting, balancing the lowest radiation dose with the highest imaging quality, was achieved through rigorous verification processes).

The remaining fish were divided into two groups under the same conditions. Ten fish (five in each group) were randomly sampled every hour from 1 to 7 h after feeding. The fish were weighed and killed under anesthesia. The contents were extracted from the bulbous section (called the digestive intestine), intermediate spiral section (called the spiral intestine) and rectum section of the intestine (called the respiratory intestine) and weighed.

Evaluations of the mucous cell and erythrocyte were made using LM on randomly selected 100 μm lengths of mucosal epithelium from the consecutive five whole-mount preparations [10]. The relative blood capillary density was reflected by the number of erythrocytes. The digestive tract was composed of four layers: mucosa, submucosa, muscularis and serosa. The general four layers were measured using WCIF ImageJ (Win 64-bit). The proportion of the contents of each intestinal segment to the net weight was also calculated. Excel 2016 and SPSS 19.0 software packages were used to perform the statistical analysis. All the statistics were analyzed via one-way analysis of variance and represented by the mean ± standard deviation. The different letters in the same column show the significant difference at the 5% level in the tables.

The digestive tract of M. anguillicaudatus is a short and straight tube (Figure 1). The ratio of the length of the gut to the body length was 0.52. From a gross anatomical view, the anterior inflated portion is separated by an intermediate spiral section from the posterior respiratory zone. The histological studies allowed a more precise identification of the digestive tract into five different regions, namely esophagus, anterior intestine, middle intestine, posterior intestine and rectum. All these regions ar