Influence of Herba Houttuyniae added to fodder on the morphological structure of the intestinal tract, the digestive enzymes, the intestinal flora, and immune function of koi carp (Cyprinus carpio) infected with Aeromonas veronii.

This study investigated whether adding Herba Houttuyniae to feed can improve intestinal function and prevent diseases for koi carp (Cyprinus carpio) infected with Aeromonas veronii. There was a total of 168 koi carp with an average body length of (9.43 ± 0.99) cm and an average body weight of (26.00 ± 11.40) g. The K group was the control group fed with basal feed, while the C group was fed with feed with a H. houttuyniae content of six per thousand. After 14 days of feeding, the fish were fasted for a day and then intraperitoneally injected with A. veronii for artificial infection, injection dose is 0.2 mL, and the concentration is 1 × 107 CFU/mL. Samples were collected from the two groups on days 0, 1, 2, and 4. The fold height, intestinal villus width, and muscle layer thickness in the gut of the koi carp were measured. In addition, on day 4, the activities of trypsin, α-amylase, and lipase in the gut were determined, and the intestinal flora of the carp in both groups was tested. The results showed that on the second and fourth days of sampling, the fold height and muscle layer thickness in the C group were significantly higher than those in the K group (P < 0.05). The villus width in the C group was slightly higher than that in the K group, but the difference was not significant (P > 0.05). Microscopic observation revealed that the intestinal structure of the carp in the C4 (day 4 in C group) group was more intact than that in the K4 (day 4 in K group) group. Moreover, the activities of trypsin, α-amylase, and lipase in the foregut and midgut in the C4 group were higher than those in the K4 group (P < 0.05). The activities of trypsin and α-amylase in the hindgut in the C4 group were higher than those in the K4 group (P < 0.05). Furthermore, beneficial bacteria, especially those in the genus Cetobacterium, were more abundant in the intestinal tract of the carp in the C4 group compared to the K group. In addition, comparisons and tests of IL-4 and IL-10 in the intestines of the fish in both groups demonstrated that the H. houttuyniae added to feed enhanced the immune function of the fish intestines after bacterial attack. In conclusion, for koi carp infected with A.veronii, adding H. houttuyniae to their feed not only improves the activity of digestive enzymes and the morphological structure of the intestine but also optimizes the beneficial intestinal microbiota, thereby protecting the intestinal tract.

The Circadian Rhythm of the Behavior and Gut Microbiota in Dybowski's Frogs (Rana dybowskii) during the Autumn Migration Period.

Many amphibian behaviors and physiological functions adapt to daily environmental changes through variations in circadian rhythms. However, these adaptations have yet to be reported in Dybowski's frog (Rana dybowskii). We aimed to elucidate the dynamic changes in the behavior and gut microbiota of R. dybowskii within a 24 h cycle during their migration to hibernation sites. Thus, we monitored their behavior at 4 h intervals and collected samples for microbiome analysis. We found that the juvenile frogs arrived at hibernation sites earlier than the adults. Among the adults, the male frogs arrived earlier. The richness and diversity of the gut microbiota in the adult R. dybowskii were lowest at 14:00. At 6:00, the differences between the males and females were most significant. At 18:00, there was an increase in the activity of Bacteroides, Coprobacillus, Ruminococcus, and Dorea in the intestinal tracts of the male frogs, whereas in the intestinal tract of the female frogs, there was an increase in the activity of Pseudoramibacter_Eubacterium, Desulfovibrio, Anaerotruncus, and PW3. This indicated diurnal rhythmic variations in the gut microbiota and significant sex-based differences in the microbial activity at different time points. Our findings contribute to the understanding of the circadian rhythm of R. dybowskii and provide crucial insights into improving breeding strategies.

Replacing Dietary Fish Meal with Defatted Black Soldier Fly (Hermetia illucens) Larvae Meal Affected Growth, Digestive Physiology and Muscle Quality of Tongue Sole (Cynoglossus semilaevis).

For solving the global shortage of fish meal (FM) supplies from fisheries, the black soldier fly (Hermetia illucens) has become a new protein alternative in aquatic feeds. The present study investigated the effects of dietary inclusion of defatted H. illucens larvae meal (DBLM) on growth, serum biochemical parameters, digestive function, and muscle quality of tongue sole (Cynoglossus semilaevis). The feeding experiment consisted of five experimental diets: a control diet based on FM protein (H0) and four DBLM diets, substituting 25% (H25), 50% (H50), 75% (H75), and 100% (H100) of FM. C. semilaevis (initial weight 563.48 ± 22.81 g) were randomly allocated over five treatments in quadruplicate. After 65 days of feeding, the weight gain rate (WGR), specific growth rate (SGR), and protein efficiency ratio (PER) were significantly higher in H0 and H25 groups with less feed conversion ratio (FCR) and feed intake (FI). The concentrations of serum ALT, TG, T-CHO, ALB, and GLO and their ratio (i.e., A/G) in the H25 group were also significantly higher than those in the other DBLM diet-feeding groups. The digestive enzyme activities first increased (from 25% to 75%) and then decreased (from 75%) with the increased level of DBLM in diets. Meanwhile, there were significant improvements in the thickness of the intestinal longitudinal muscle (LM), circular muscle (CM), columnar epithelium (CE), and lamina propria (LP) in H25 C. semilaevis compared to the control group (p < 0.05). The fish from the other DBLM diets groups presented significant reductions in the thicknesses of LM, CM, CE, and LP, as well as the length of microvilli (ML) in a dose-dependent manner (p < 0.05). However, the substitution of FM increased up to 50% would result in intestinal structural damage. Moreover, the proximate compositions, antioxidant and water holding capacity, and muscular structures of C. semilaevis fillets were all significantly affected after substituting 25% FM with DBLM (p < 0.05). Except for the dry matter, moisture, ash, crude fat, and protein contents were significantly higher in H25 C. semilaevis muscles. The SOD activity in the H0 group was significantly lower than that in the H25 group. The CAT activity in C. semilaevis muscles prominently reduced along with the increase in DBLM content in feeding diets (p < 0.05). The water holding capacity of C. semilaevis fillets was best in the H25 group. In summary, the optimum proportion of DBLM with FM for feeding C. semilaevis may be around 25%.

The delimitation of intestine segments of koi carp (Cyprinus carpio var. koi) based on histological features.

In this study, the delimitation of intestine segments of koi carp (Cyprinus carpio var. koi) was conducted using a histological approach with the measurements of height of mucosa folds (HF), width of mucosa folds (WF), thickness of muscularis (TM) and cross-sectional area (CSA). According to the change trends for these four parameters, the intestine of the koi carp was divided into anterior intestine, middle intestine and posterior intestine. The locations of the three intestine segments were defined, and their ratios along the entire intestine were accounted for 23.84 ± 1.18%, 46.77 ± 2.29% and 29.39 ± 1.65%, respectively. The anterior intestine had a significantly higher HF, compared with the middle (p < .01) or posterior intestines (p < .01). The muscularis became thin gradually from the anterior intestine to posterior part. TM was significantly different among the anterior, middle and posterior intestines (p < .01). The anterior intestine had a significantly higher CSA, compared with the middle (p < .01) and posterior intestines (p < .01), and the latter two segments had similar CSA values (p > .05). The procedure of the delimitation of the koi carp intestine segments can offer useful information for future studies on other fish species. The presented results are meaningful for studies on differential functions of the different intestine segments in fish.

Impact of DNA extraction methods on the observed microbial communities from the intestinal flora of the penaeid shrimp Litopenaeus vannamei.

Two DNA extraction methods, the Zirmil-beating cell disruption method (ZBC) and the QIAamp fast DNA stool mini kit (QIA), were used to extract DNA from the intestinal flora of the penaeid shrimp Litopenaeus vannamei, and their microbial communities were analyzed using 16S rDNA high-throughput sequencing. Results were obtained in terms of the number of reads, alpha diversity indexes, beta diversity indexes and taxonomic composition. The alpha diversity indexes of the community, according to the ZBC method, were higher than those according to the QIA method. Furthermore, results from the three samples using the ZBC method were less consistent than those where the QIA method was used. Further, using the latter method led to substantive clustering. It is suggested that the QIA method is more stable and repeatable than the ZBC method. Although the two extraction methods shared the major abundant microflora based on 16S rDNA high-throughput sequencing, bias associated with diversity analysis indexes and certain species was observed.

Effects of Bacillus subtilis on the growth, colony maintenance, and attached bacterial community composition of colonial cyanobacteria.

In freshwater aquaculture ponds, application of algicidal Bacillus is a promising way in the control of cyanobacterial blooms. To best understand Bacillus algicidal characters and mechanisms in the field, different-sized colonial cyanobacteria were isolated from an aquaculture pond, and the effects of B. subtilis on their growth, colony maintenance, and colony-attached bacterial community composition were investigated. The results showed that B. subtilis could inhibit the growth of colonial cyanobacteria. Bigger-sized colonies isolated from the field could spontaneously disintegrate into smaller-sized colonies in the laboratory. Algicidal B. subtilis could accelerate the disintegration of colonies and decrease colony size. B. subtilis not only decreased the colony-attached bacterial community diversity but also changed its composition. B. subtilis increased the relative abundances of some attached bacterial genera, including Pseudomonas, Shewanella, Bacillus, Shinella, Rhizobium, and Ensifer. These bacteria with algicidal, microcystin-degrading, and flocculating activities might be an important contributor to algicidal effects of B. subtilis on colonial cyanobacteria.

Isolation, Identification and Characteristics of Aeromonas veronii From Diseased Crucian Carp (Carassius auratus gibelio).

Aeromonas species often cause disease in farmed fish. In the present study, dominant bacteria were isolated from diseased crucian carp (Carassius auratus gibelio). Based on this, a bacterial isolate was tentatively named CFJY-623. This isolate was identified as Aeromonas veronii based on analysis of its morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. Six virulence genes related to pathogenicity including aerolysin, cytotonic enterotoxins, elastase, glycerophospholipid: cholesterol acyltransferase, lipase, and serine protease were identified in this A. veronii isolate. The median lethal dosage (LD50) of the CFJY-623 isolate for crucian carp was determined as 1.31 × 107 CFU/mL. Artificial experimental infection showed that the CFJY-623 isolate caused considerable histological lesions in the fish, including tissue cell degeneration, necrosis, and inflammatory cell infiltrating. Drug sensitivity testing showed that the isolate was susceptible to aminoglycosides, carbapenemes, and nitrofurans. Exploring its growing features showed that this isolate exhibited a high level of environmental adaptability. These results provided a scientific basis for the identification of A. veronii and treatment for fish infected by this pathogen.

A modified method for genomic DNA extraction from the fish intestinal microflora.

A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280 ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.