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\begin{document}

\title{A comparison of digestive strategies for fishes with different feeding
habits: digestive enzyme activities, intestinal morphology and gut
microbiota}



\author[1]{Fang Jiao}%
\author[2]{Lei Zhang}%
\author[3]{Samwel Mchele Limbu}%
\author[2]{hua Rong}%
\affil[1]{South China Agricultural University}%
\affil[2]{Xiangyang Polytechnic}%
\affil[3]{University of Dar es Salaam}%


\vspace{-1em}



  
  \date{May 17, 2024}


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\selectlanguage{english}
\begin{abstract}
Feeding habit determines the digestive tract structure and intestinal
microflora. However, the relationship among feeding habit, digestive
physiology intestinal, and microbial diversity of omnivorous,
herbivorous, filter-feeder and carnivorous fish reared in the same pond
has not been compared. This study compared the digestive enzyme
activities, intestinal morphology and intestinal microflora of
omnivorous (Carassius auratus), herbivorous (Ctenopharyngodon idellus),
carnivorous (Siniperca chuatsi) and filter-feeder (Shizothorax grahami)
and predicted the potential functions of specific microflora on
different nutrients. Twelve intestine samples were collected from each
of the four fishes from Dianchi Lake. The composition and diversity of
microbial communities were determined by using high throughput
sequencing of 16S rDNA. The results showed that the filter-feeder fish
had significantly higher protease but lower amylase activities in the
intestine than herbivorous. The carnivorous fish intestine had more
microvilli branches and complex structures than other fish species in
the order carnivorous \textgreater{} herbivorous \textgreater{}
filter-feeder \textgreater{} omnivorous. The diversity of intestinal
microflora was higher in omnivorous and followed the order omnivorous
\textgreater{} herbivorous \textgreater{} filter-feeder \textgreater{}
carnivorous. Acinetobacter species and Bacteroides species were the most
dominant flora in carnivorous and herbivorous fish, respectively.
Acinetobacter johnsonii, Acinetobacter lwoffii and Pseudomonas stutzeri
might help the host to digest protein, while Bacteroidetes species may
help the host to digest cellulose. Taken together, feeding habit
determines the digestive enzyme activities, intestinal tissue morphology
and differential colonization of fish intestinal flora. The knowledge
obtained is useful in designing appropriate approaches for feed
formulation and feeding practices in for fish.%
\end{abstract}%



\sloppy


\textbf{A comparison of digestive strategies for fishes with different
feeding habits:digestive enzyme activities,} \textbf{intestinal
morphology and gut microbiota}

Fang Jiao\textsuperscript{1}, Lei Zhang\textsuperscript{2}, Samwel
Mchele Limbu\textsuperscript{3}, Hua Rong\textsuperscript{2,*}

\textsuperscript{1}College of Marine Sciences, South China Agricultural
University, Guangzhou 510642, China

\textsuperscript{2}Xiangyang Polytechnic, Xiangyang, 441050, China

\textsuperscript{3}Department of Aquaculture Technology, School of
Aquatic Sciences and Fisheries Technology, University of Dar es Salaam,
P. O. Box 60091, Dar es Salaam, Tanzania

*Corresponding author.

\emph{E-mail address} : \emph{huarong@scau.edu.cn} (H. Rong)

\textbf{Abstract}

Feeding habit determines the digestive tract structure and intestinal
microflora. However, the relationship among feeding habit, digestive
physiology intestinal, and microbial diversity of omnivorous,
herbivorous, filter-feeder and carnivorous fish reared in the same pond
has not been compared. This study compared the digestive enzyme
activities, intestinal morphology and intestinal microflora of
omnivorous (\emph{Carassius auratus} ), herbivorous
(\emph{Ctenopharyngodon idellus} ), carnivorous (\emph{Siniperca
chuatsi} ) and filter-feeder (\emph{Shizothorax grahami} ) and predicted
the potential functions of specific microflora on different nutrients.
Twelve intestine samples were collected from each of the four fishes
from Dianchi Lake. The composition and diversity of microbial
communities were determined by using high throughput sequencing of 16S
rDNA. The results showed that the filter-feeder fish had significantly
higher protease but lower amylase activities in the intestine than
herbivorous. The carnivorous fish intestine had more microvilli branches
and complex structures than other fish species in the order carnivorous
\textgreater{} herbivorous \textgreater{} filter-feeder \textgreater{}
omnivorous. The diversity of intestinal microflora was higher in
omnivorous and followed the order omnivorous \textgreater{} herbivorous
\textgreater{} filter-feeder \textgreater{} carnivorous.
\emph{Acinetobacter} species and \emph{Bacteroides} species were the
most dominant flora in carnivorous and herbivorous fish, respectively.
\emph{Acinetobacter johnsonii} , \emph{Acinetobacter lwoffii} and
\emph{Pseudomonas stutzeri} might help the host to digest protein, while
\emph{Bacteroidetes} species may help the host to digest cellulose.
Taken together, feeding habit determines the digestive enzyme
activities, intestinal tissue morphology and differential colonization
of fish intestinal flora. The knowledge obtained is useful in designing
appropriate approaches for feed formulation and feeding practices in for
fish.

\textbf{KEYWORDS} feeding habit; intestinal microbiota; digestive
enzymes; 16S rDNA

\textbf{1 \textbar{} INTRODUCTION}

The feeding habits of fish are reflected by their digestive organ,
mainly the intestine. Scholars generally classify fish feeding habits as
herbivorous, carnivorous, omnivorous and filter-feeders according to
feeding method and food content. The intestine tract is the main site
for digestion and nutritional uptake, which has been regarded as a key
organ in fish nutrition (Kumar et al., 2005; Wang et al., 2018; Zhou et
al., 2021). The fish digestive enzymes activities are closely related to
the diet consumed and the ability of fish to digest and absorb different
nutrients (Bakke et al., 2010; Liu et al., 2021). Evidently, previous
studies found herbivorous fish such as Roho labeo (\emph{Labeo rohita} )
and Japanese eel (\emph{Anguilla japonica} ) had stronger amylase
activity compared to carnivorous fish such as Great white catfish
(\emph{Wallago attu} ) (Agrawal et al., 1975) and rainbow trout
(\emph{Oncorhynchus mykiss} ) (Hidalgo et al., 1999). Therefore, the
influence of feeding habits on digestive enzymes activities is beyond
doubt.

The feeding habits of fish also affect digestive tract structure and
intestinal microorganisms (Li et al., 2019; Meng et al., 2014; Valdes et
al., 2018). Interference with intestinal morphology such as muscularis
thickness and villi width affect nutrients absorption and intestinal
microbiota (Limbu et al., 2018). Fish gut microbiota contribute to
digestion and affect gastrointestinal tract development and overall
growth of fish (Clements et al., 2014; Ghanbari et al., 2015). However,
feeding habits (Larsen et al., 2014; Meng \& Nie, 2019; Roeselers et
al., 2011), which determines the feed composition consumed (Benson et
al., 2010; Spor et al., 2011; Sullam et al., 2012) has been reported to
shape microbial communities in fish (Larsen et al., 2014; Meng \& Nie,
2019; Roeselers et al., 2011). Accordingly, diet has been reported as a
dominant source of variation in the microbiota composition of rainbow
trout (Desai et al., 2012; Ingerslev et al., 2014). The disruption in
intestinal microbiota induced by feeding habit via diet usually affect
digestive functions of the host through disturbance in bacterial
digestive enzyme production (Ghanbari et al., 2015). Certain gut
microbiota such as the cellulolytic enzyme-producing bacterial
community, which were isolated from the intestinal tract of herbivorous
fish species are known to metabolize a remarkable variety of substrates
(Li et al., 2016) improving growth performance. Therefore, several
studies have explored the manipulation of gut microbiota through diet to
improve fish growth (Fan et al., 2021; Li et al., 2019; Pan et al.,
2021). However, studies exploring the relationship between feeding
habits, digestive enzyme activity, intestinal structure and gut
microbial composition in fish are currently limited. Such a knowledge
gap limits our knowledge on designing appropriate approaches for feed
formulation and feeding practices in aquaculture.

China is currently the largest producer and consumer of cultured fish
(FAO, 2022), including Grass carp (\emph{Ctenopharyngodon idellus} ), a
herbivorous fish (Liu et al., 2017) and mandarin fish (\emph{Siniperca
chuatsi} ), a carnivorous fish (Shen et al., 2021). Aquaculture
production in China also include species such as Dianchi high-back
crucian carp (\emph{Carassius auratus} ), an omnivorous fish (Shi et
al., 2017) and Kunming Schizothoracin (\emph{Schizothorax grahami} ), a
filter-feeding fish, which are endemic to Yunnan, China. These species
are the main economic fish produced in Yunnan because of their
nutritional value (Zheng et al., 2016). To ensure continued production
of these species, knowledge on the influence of feeding habits on
digestive enzymes, intestinal morphology and microbiota composition is
needed for effective feed formulation. The present study compared the
relationship between feeding habits, and digestive enzyme, intestinal
morphology and intestinal microbiota of \emph{C. idella, S. chuatsi}
,\emph{C. auratus} and \emph{S. grahami} as herbivorous, carnivorous,
omnivorous and filter-feeding fish representative species, respectively.
The results obtained provide a scientific basis for development of
appropriate formulation of compound fish feeds.

\textbf{2 \textbar{} MATERIALS AND METHODS}

\textbf{2.1 \textbar{} Fish sampling}

Ten individual fish for each species (\emph{Ctenopharyngodon idellus}
,\emph{Siniperca chuatsi} , \emph{Carassius auratus}
and\emph{Schizothorax grahami} ) were caught by trolling boats in the
Dianchi Lake, Kunming, Yunan, China. The fish were transported live in
plastic bags provided with dissolved oxygen by car to the Aquaculture
Laboratory of Yunnan Agricultural University, where they were euthanized
by immersing them into 40 mg/L eugenol (Shanghai Reagent, China). The
average weights of the sampled fish were determined by using a weighing
scale as 1323.60 \selectlanguage{ngerman}± 40.20 g for \emph{C. idellus} , 471.10 ± 23.94 for
\emph{S. grahami} , 841.30 ± 34.54 g for \emph{S. chuatsi} and 350.4 ±
25.98 g for \emph{C. auratus} .

\textbf{2.2 \textbar{} Determination of digestive enzymes activities}

Three fish for each species were carefully dissected and intestine and
hepatopancreas were sampled and transferred into an Eppendorf tube and
immediately placed into liquid nitrogen. The tubes containing the
samples were stored at \selectlanguage{english}-80 degC until needed for analysis of enzymes
activities. On analysis day, the hepatopancreas and intestine samples
were weighed and mixed with 9 times phosphate buffer saline (PBS) (w:v =
1:9), then homogenized by using an electric homogenizer (Ningbo Scientz
Biotechnology) in ice bath for 15 s. The resulting homogenate was
carefully pipetted and centrifuged at 12,000 rpm at 4 degC for 20 min.
Finally, the liquid supernatant was removed for digestive enzymes
analysis. The digestive enzymes activities including pepsin, trypsin,
lipase and amylase, and total protein concentration of hepatopancreas
and intestine were determined by using specific commercial kits (Nanjing
Jiancheng Bioengineering Institute, Jiangsu, China) based on
instructions from the manufacturer.

\textbf{2.3 \textbar{} Intestinal morphology analysis}

Three fish tissues from midgut for each feeding habit was collected and
prepared for intestinal morphology analysis as described previously
(Limbu et al., 2018). The tissues were fixed with 4\% paraformaldehyde
for 24 h, then dehydrated with 75\% absolute ethanol. The tissues were
then transferred into xylene twice for transparent, immersed into
paraffin wax three times, embedded and cooled. The intestine tissues
were sliced transversely into pieces with approximately 5 to 6 \selectlanguage{greek}µ\selectlanguage{english}m, dried
and stained by using hematoxylin and eosin (H \& E). The slides were
finally examined by using electronic biological microscope KOPPACE at
40x to 1600x (Kopace Technology Co., LTD, Shenzhen, China). Villi height
(VH), villi width (VW) and muscularis thickness (MT) were measured from
at least 30 segments for each feeding habit by using Case Viewer
software. Villi height index (VHI), villi width index (VWI) and
muscularis thickness index (MTI) were calculated as VH, VW and MT
divided by individual fish body weight\textsuperscript{1/3}.

\textbf{2.4 \textbar{} DNA extraction and high-throughput sequencing
analysis of intestinal microbiota}

We sterilized the scalpels, tweezers, and scissors by heating at 180 \selectlanguage{ngerman}°C
for 2h before using them for DNA extraction. We also wiped the fish
surface, the desktop and instruments used by using 75\% alcohol to
disinfect them. Afterwards, we collected the gut contents of the
remaining four fish and placed them into sterile tubes under sterile
conditions. The tubes containing the samples were immediately placed
into liquid nitrogen and then stored at \selectlanguage{english}-80 degC until DNA extraction. The
four intestine samples for herbivorous fish\emph{C. idellus} were
abbreviated as HE, filter feeder \emph{S. chuatsi} as FI, omnivorous
\emph{C. auratus} as OM and carnivorous \emph{S. grahami} as CA for
convenient reporting. These samples were subjected to DNA extraction
using the PowerFood Microbial DNA isolation kit (QIAGEN Srl, Milan,
Italy) following manufacturer's instructions. The quality and quantity
of the DNA were checked by using gel electrophoresis and a Qubit 4
Fluorometer (Thermo Fisher Scientific, USA). Primers 341F:
ACTCCTACGGGAGGCAGCAG and 806R: GGACTACHVGGGTATCTAAT were used to
generate the PCR amplicons for the 16S rRNA gene V3 - V4 region on
Illumina sequencing platform (HiSeq\selectlanguage{english} 2500, Beijing igeneCode Biotech
Co., Ltd., Beijing, China).

\textbf{2.5 \textbar{} Bioinformatics analysis}

To obtain high-quality clean reads, raw reads were demultiplexed and
filtered for quality based on the methods developed by Fadrosh et al.
(2014). Cleaned tags were obtained by FASTP (Chen et al., 2018).
Sequences were assigned to operational taxonomic units (OTUs) based on a
similarity cut-off of 97\% by using UPARSE (v7.0.1090) (Edgar, 2013).
The representative sequences from each OTU were then taxonomically
classified using the Ribosomal Database Project (RDP) Classifier (v2.2)
with confidence threshold of 80\%.

\textbf{2.6 \textbar{} Prediction of microbiome functions by using
bioinformatics analysis}

We predicted the gut microbiome functions by using Phylogenetic
Investigation of Communities by Reconstruction of Unobserved States
(PICRUSt) to elucidate the physiological features and metabolism
capability during the process of dietary conversion. To compare the
functional categories of microbiota among the four groups (HE, FI, OM
and CA) by PICRUSt analyses, functional profile heatmaps based on 423
categories (KEGG level-3) were constructed, which showed marked
differences among feeding habits.

\textbf{2.7 \textbar{} Statistical analyses}

All the data for enzymes activities and intestinal morphology were
tested for normality and homogeneity of variances by using Shapiro-Wilk
and Levene's tests, respectively. Afterwards, one-way analysis of
variance (ANOVA) was used to test for statistical differences on the
data for enzymes activities and intestinal morphology among the four
feeding habits. Tukey multiple comparisons test was used to compare for
significant differences among the four feeding habits when ANOVA
indicated statistical differences. The analysis of enzymes activities
and intestinal morphology data was conducted by using SPSS 20.0 (SPSS,
Inc., Chicago, IL, USA). Results with \emph{P} \selectlanguage{english}[?] 0.05 were considered
significant different. The results obtained are expressed as mean +-
standard error of the mean (SEM).

The differences in bacterial phylotype distribution was assessed by
using principal component analysis (PCA). The alpha-diversity indices
(the abundance coverage-based Estimator - ACE, Chao1, Shannon and
Simpson indices) were generated by using Mothur v1.31.2
(http://www.mothur.org/wiki/Calculators). Abundance of microbiota was
analyzed by using ACE and Chao1 indices while microbiota species
diversity was assessed by using Shannon and Simpson indices. Linear
discriminant analysis effect size (LEfSe) was analyzed by using the R
statistical package (v3.1.1).

\textbf{3 RESULTS}

\textbf{3.1 \textbar{} Comparative analysis of digestive enzymes
activity}

The feeding habits affected differently the digestive enzymes activities
of the four fish species in the hepatopancreas and intestine
(\emph{p}\textless{} .05; Table 1). The carnivorous fish (\emph{S.
chuatsi} ) had higher trypsin and pancrelipase activities in the
hepatopancreas and enteropeptidase in the intestine than herbivorous
(\emph{C. idella} ), omnivorous (\emph{C. auratus} ) and the
filter-feeder fish (\emph{S. grahami} ) (\emph{p} \textless{} .05).
Moreover, the filter-feeder fish had higher trypsin and pancrelipase
activities in the hepatopancreas but lower entero-amylase in the
intestine than the herbivorous and omnivorous fishes (\emph{p}
\textless{} .05). The omnivorous fish had significantly higher
enteropeptidase activity in the intestine than herbivorous and
filter-feeder fishes (\emph{p} \textless{} .05). However, the
herbivorous and omnivorous fish species had no significant difference in
trypsin and pancrelipase activities in the hapatopancreas (\emph{p}
\textgreater{} .05). Similarly, the herbivorous and filter-feeder fishes
had no significant difference in enteropeptidase activity in the
intestine (\emph{p} \textgreater{} .05).

Interestingly, the carnivorous fish (\emph{S. chuatsi} ) had
significantly lower amylopsin activity in the hapatopancreas and
entero-amylase in the intestine than the herbivorous, omnivorous and
filter-feeder fishes (\emph{p} \textless{} .05). Similarly, the
omnivorous fish had significantly lower amylopsin activity than
herbivorous and filter feeding fishes in the hepatopancreas
(\emph{p}\textless{} .05). The herbivorous and filter feeding fishes had
no significant difference in amylopsin activity in the hepatopancreas
(\emph{p} \textgreater{} .05). Equally, herbivorous and filter-feeder
fishes had no significant difference in enteropeptidase in the intestine
(\emph{p} \textgreater{} .05). The herbivorous and omnivorous had
significantly higher intestinal lipase activity than filter feeder and
carnivorous fish species (\emph{p} \textless{} .05). Similarly, the
carnivorous fish had significantly higher intestinal lipase than the
filter-feeder fish (\emph{p} \textless{} .05). The filter-feeder fish
had significantly lower entero-amylase activity than herbivorous and
omnivorous fishes in the intestine (\emph{p} \textless{} .05). However,
the herbivorous and omnivorous fish species had no significant
differences in intestinal lipase and entero-amylase activities
(\emph{p}\textgreater{} .05).

\textbf{3.2 \textbar{} Intestinal tissue morphology}

The VH, VW and MT differed significantly among the four feeding habits
of the fish species (Figure 1; \emph{p} \textless{} .05). The intestinal
microvilli of carnivorous fish had many branches and complex structures.
The order of microvilli complexity was carnivorous \textgreater{}
herbivorous \textgreater{} filter-feeder \textgreater{} omnivorous.
Herbivorous fish had significantly higher VH and VHI than filter-feeder,
carnivorous and omnivorous fish species (\emph{p} \textless{} .05).
Similarly, carnivorous fish had significantly higher VH and VHI than
filter-feeder and omnivorous fish species (\emph{p} \textless{} .05).
Likewise, filter-feeder fish had significantly higher VH and VHI than
omnivorous fish species (\emph{p} \textless{} .05). On the contrary,
carnivorous fish had significantly higher VW, MT, VWI and MTI than
herbivorous, filter-feeder and omnivorous fish species (\emph{p}
\textless{} .05). Similarly, herbivorous fish had significantly higher
VW, MT, VWI and MTI than filter-feeder and omnivorous fish species
(\emph{p} \textless{} .05). Likewise, omnivorous fish had significantly
higher MT, VWI and MTI than filter-feeder fish species (\emph{p}
\textless{} .05). However, filter-feeder and omnivorous fish species had
no significant difference in VW (\emph{p} \textgreater{} .05).

\textbf{3.3 \textbar{} Microbial complexity of fish gut flora}

A total of 2,300 OTU were obtained for all the four fishes. A total of
223 OTUs were shared by all species (9.7\%), while 332 (223 + 42 + 47 +
20) OTUs (14.4\%) were shared by herbivorous and carnivorous (Figure 2).
The herbivorous fish had relatively higher number of unique OTUs (467)
equivalent to 20.3\% followed by omnivorous fish (251 OTUs) representing
10.9\%, while filter-feeder fish had 113 OTUs equivalent to 4.9\% and
carnivorous fish had only 43 OTUs making up 1.9\%.

\textbf{3.4 \textbar{} Microbiota abundance and diversity}

The carnivorous species had significantly lower number of microbiota
species than omnivorous species (Figure 3A; \emph{p} \textless{} .05).
However, carnivorous, herbivorous and filter-feeder had no significant
number of microbiota species (\emph{p} \textgreater{} .05). Similarly,
carnivorous and filter-feeder fish species had no significant difference
in number of microbiota species (\emph{p} \textgreater{} .05). The
carnivorous species also had significantly lower abundance as reflected
by Chao1 (Figure 3B) and ACE (Figure 3C) than omnivorous and herbivorous
fish species (\emph{p} \textless{} .05). The filter-feeder species also
had significantly lower Chao1 than omnivorous species (\emph{p}
\textless{} .05). However, omnivorous, herbivorous and filter-feeder
species had statistically no difference in Chao1 while herbivorous and
filter-feeder species had no significant difference in ACE (\emph{p}
\textgreater{} .05). Similarly, carnivorous and filter-feeder fish
species had no significant difference in in Chao1 and ACE (\emph{p}
\textgreater{} .05). The carnivorous fish had significantly lower
Shannon diversity index (Figure 3D), but higher Simpson's diversity
index (Figure 3E). However, omnivorous, herbivorous and filter-feeder
species had no significant difference in Shannon diversity index and
Simpson's diversity index (\emph{p} \textgreater{} .05). Similarly,
carnivorous, herbivorous and filter-feeder fish species had no
significant difference in Shannon diversity index and Simpson's
diversity index (\emph{p} \textgreater{} .05). The community diversity
of the four fish followed the order omnivorous \textgreater{}
herbivorous \textgreater{} filter-feeder \textgreater{} carnivorous.

\textbf{3.5 \textbar{} Abundance and composition of microbiota at phyla,
genera and species levels}

A total of 37 phyla were obtained from all fish species (Supplementary
Figure 1S). We then selected the most abundant phyla with above 5\%. We
obtained nine phyla classified as Proteobacteria, Firmicutes,
Bacteroidetes and Actinobacteria phyla with relatively high abundance,
representing 66.60\%, 9.82\%, 9.04\% and 5.18\%, respectively (Figure
4). Herbivorous fish species had significantly lower abundance of
Proteobacteria phylum than carnivorous and filter-feeding species
(\emph{p} \textless{} .05). On the contrary, herbivorous fish species
had significantly higher abundance of Firmicutes and Bacteroides phyla
than omnivorous, filter-feeder and carnivorous species
(\emph{p}\textless{} .05). The omnivorous fish species had significantly
higher abundance of \emph{Verrucomicrobia} phylum than omnivorous,
filter-feeder and herbivorous species (\emph{p} \textless{} .05).

A total of 324 bacterial genera were obtained. We then removed
unidentified bacterial genera, and others and selected only those with
more than 0.5\% abundance. The results on composition showed 45
bacterial genera were obtained (Supplementary Figure 2S). The four fish
species with different feeding habits had distinct microbiota
composition at genera for the nine phyla. Carnivorous and filter-feeder
species were dominated with \emph{Limnobacter} species (25.05\% and
23.56\%) and \emph{Pseudomonas} species (26.07\% and 8.16\%),
respectively. The microbiota of omnivorous species was mainly composed
of \emph{Rhodobacter} species (14.99\%), \emph{Zymomonas} species
(10.63\%), \emph{Clavibacter} species (8.57\%) and
\emph{Luteolibacter}species (6.68\%). The herbivorous fish species was
mainly composed of\emph{Bacteroides} species (22.56\%) and
\emph{Citrobacter} species (9.43\%).

We obtained 14 genera with significant differences in abundance of
microbiota (Table 3). The carnivorous fish species had significant
higher \emph{Acinetobacter} species than the other three fish species
(\emph{p} \textless{} .05). The omnivorous fish species had higher
abundance of \emph{Anaerospora} , \emph{Arenimonas}
,\emph{Dechloromonas} , \emph{Deefgea} , \emph{Luteolibacter}
and\emph{Zymomonas} genera than herbivorous and carnivorous fish species
(\emph{p} \textless{} .05). The herbivorous fish species had
significantly higher \emph{Bacteroides} genus than omnivorous,
carnivorous and filter feeder fish species (\emph{p} \textless{}
.05).\emph{Escherichia} , \emph{Limnobacter} and \emph{Mycoplana} genera
were abundant flora of filter-feeder and carnivorous fish species,
while\emph{Pseudomonas} genus was abundant flora of filter-feeder and
omnivorous fish species.

A total of 2048 species of bacteria were obtained. \emph{Pseudomonas
stutzeri} , \emph{Escherichia coli} and \emph{Acinetobacter lwoffii}
were the most abundant species (Figure 5). The \emph{Acinetobacter
johnsonii} , \emph{Acinetobacter lwoffii} , \emph{Escherichia coli}
and\emph{Pseudomonas stutzeri} had significant different abundance among
omnivorous, herbivorous, filter-feeder and carnivorous fish species
(\emph{p} \textless{} .05). The carnivorous fish species had
significantly higher \emph{Acinetobacter johnsonii} ,
\emph{Acinetobacter lwoffii} and \emph{Pseudomonas stutzeri} than the
other three fish species (\emph{p} \textless{} .05). The filter-feeder
and carnivorous fish species had significantly higher \emph{Escherichia
coli} than omnivorous and herbivorous fish species.

\textbf{3.6 \textbar{} LEfSe analysis of significantly enriched
microbial communities}

Linear discriminant analysis effect size (LEfSe) was used to
characterize enriched microbial communities (Figure 6A). There were 48
differences among the four fish species with different feeding habits,
classified from phylum to genus. The Proteobacteria phylum was common to
all the four fish species. The omnivorous species had significantly
higher enriched gut microbiota species than the other three species.
Indeed, omnivorous species had significantly enriched Fusobacteria,
Actinobacteria and Verrucomicrobia phyla compared to carnivorous,
herbivorous and filter-feeder fish species. The most enriched bacteria
in the gut of the four fish species followed the trend omnivorous (25)
\textgreater{} carnivorous (10) \textgreater{} filter-feeder (8)
\textgreater{} herbivorous (5) (Figure 6B). The omnivorous species
enriched \emph{Fusobacteriales, Fusobacteriaceae, Fusobacteriia,
Fusobacteria, Dechloromonas, Saprospirales, Saprospirae,
Chitinophagaceae, Paucibacter, Microbacteriaceae, Luteolibacter,
Clavibacter, Actinomycetales, Actinobacteria, Actinobacteria, Zymomonas,
Verrucomicrobiales, Verrucomicrobiae, Verrucomicrobia,
Verrucomicrobiaceae, Rhodobacter, Rhodobacterales}
and\emph{Rhodobacteraceae} . The herbivorous fish species
enriched\emph{Aeromonadales, Aeromonadaceae, Lachnospiraceae,
Clostridia} and\emph{Clostridiales} . Moreover, carnivorous fish species
enriched\emph{Acinetobacter, Moraxellaceae, Hyphomonadaceae, Mycoplana,
Pseudomonas, Pseudomonadaceae, Pseudomonadales, Caulobacteraceae,
Caulobacterales} and \emph{Proteobacteria} . The filter-feeder fish
enriched \emph{Anaerolineae, Neisseriales, Neisseriaceae, Escherichia,
Comamonadaceae, Burkholderiales, Betaproteobacteria}
and\emph{Limnobacter} .

\textbf{3.7 \textbar{} Predicted gut microflora functions}

A total of 423 metabolism pathways (Kyoto Encyclopedia of Genes and
Genomes, KEGG level-3) were constructed. The four fish species showed
marked differences in functional profile (Supplementary Figure 3S). The
microbial functions among the four fishes with different feeding habits
showed that, 39 pathways related to digestion were identified, including
those associated with carbohydrate, protein and amino acid, energy and
lipid metabolism (Figure 7). Of all the pathways identified, 27 pathways
were significantly changed (Figure 7). The herbivorous fish species had
higher carbohydrate metabolism than carnivorous fish species
(\emph{p}\textless{} .05). Moreover, herbivorous fish species increased
pathways related to carbohydrate metabolism {[}i.e. glycolysis III (from
glucose), galactose degradation I (Leloir pathway), superpathway of
D-glucarate and D-galactarate degradation, reductive TCA cycle I and
incomplete reductive TCA cycle{]} than in carnivorous fish species.
Interestingly, carnivorous had more enriched protein and amino acid
metabolism pathways {[}superpathway of ornithine degradation,
superpathway of L-arginine and L-ornithine degradation and L-arginine
degradation II (AST pathway){]} and lipid metabolism (fatty acid
salvage) than herbivorous and omnivorous fish species.

\textbf{4 \textbar{} DISCUSSION}

Feeding habit reflects directly the digestive ability of fish to
different nutrient components. The ability of fish to digest and utilize
different nutrients in feed is affected by the structure of digestive
tract, the secretion of digestive enzymes and the composition and
diversity of intestinal microbes. This study explored the relationship
among feeding habits of omnivorous, carnivorous, herbivorous and
filter-feeder fish species and digestive physiology, intestinal
morphology, and intestinal microbial diversity. We also predicted the
functions of microbiome from the four fishes. We found clear differences
in the enzymes activities of the four fish species depending on their
feeding habits. Evidently, the carnivorous fish (\emph{S. chuatsi} ) had
higher trypsin and pancrelipase activities in the hepatopancreas and
enteropeptidase in the intestine than herbivorous (\emph{C. idella} ),
omnivorous (\emph{C. auratus} ) and the filter-feeder fish (\emph{S.
grahami} ). The variations in digestive enzymes are caused by the
different feeding habits (Xu et al., 2011). Accordingly, the activities
of trypsin, intestinal protease and pancreatic lipase were roughly
carnivorous \textgreater{} omnivorous \textgreater{} herbivorous,
reflecting the feeding on animal materials with high protein and lipid
requiring secretions of related enzymes to digest them. Similarly, Liu
et al. (2014) reported higher protease activity in carnivorous fish than
omnivorous and herbivorous fish.

However, the intestinal lipase activity in this study was higher in
herbivorous and omnivorous fish species than filter-feeder and
carnivorous fish species. Contrary, Parrizas et al. (1994) reported
higher lipase activity in carnivorous fish than herbivorous and
omnivorous fish. The higher lipase activity in stomachless fish is
probably due to the relationship between intestinal tissue structure and
digestive enzymes (Pan et al., 1996). In our study, the mandarin fish
(\emph{S. chuatsi} ) represents a carnivorous fish with a stomach, grass
carp (\emph{C. idellus} ) and Dianchi high-back crucian carp (\emph{C.
auratus} ) are typical herbivorous and omnivorous species, respectively,
without stomach and, Kunming Schizothoracin (\emph{S. grahami} ) is a
filter-feeder fish with an enlarged sac between eosophagus and
intestinal tract, which can secrete digestive fluid and perform some
stomach functions. Therefore, the higher intestinal lipase activity in
the stomachless fish was due to secretions of the enzyme from the
intestine, which performs some functions of the stomach. Interestingly,
this study found higher pancreatic amylase and intestinal amylase
activities in herbivorous than carnivorous species. Similarly, Li et al.
(2012) found higher amylase in herbivorous than omnivorous and Liu and
Zhang (2001) reported higher amylase activity in omnivorous fish than
carnivorous fish. The higher amylase activity in herbivorous fish is
related to the utilization of carbohydrates in the feed. Similarly,
omnivorous fish can use higher carbohydrate levels than carnivorous fish
(Li et al., 2015a).

This study found significant differences in intestinal villi height,
villi width and muscularis thickness among the four fish. These
variations are due to the morphological and structural characteristics
of the fish gut reflected by the different feeding habits (Liu \& Zhang,
2001; Zeng \& Ye, 1998). The intestinal microvilli of carnivorous fish
had many branches and complex structures. The complexity of the
microvilli (villi length and villi width) and muscularis thickness, was
in the order carnivorous \textgreater{} herbivorous \textgreater{}
omnivorous. The intestinal structure accommodates, transports and
digests feed and absorbs nutrients. The height, width and complexity of
intestinal microvilli increase the surface area for digestion and
absorption of digested nutrients (Sun et al., 2019). The muscularis
thickness is composed of smooth muscle, which promotes the movement of
food in the intestine through rhythmic relaxation and contraction. The
thickness of the muscle layer directly reflects the contraction and
peristalsis ability of the intestine. Accordingly, strengthening
intestinal contraction and peristalsis is an effective means to increase
feed digestion and reduces chyme circulation (Bian et al., 2021).
Generally, carnivorous fish have shorter intestines (Day et al., 2014).
Accordingly, carnivorous increase the complexity of the intestinal
structure to reduce circulation rate of chyme and enhance absorption of
nutrients. Therefore, the complexity of the intestinal structure is
adapted to the feeding habits of fish so as to achieve fully absorption
of nutrients.

The intestinal microflora of vertebrates plays an important role in host
nutrition, (Liu et al., 2016; Valdes et al., 2018). Many studies have
shown that dietary feeding habits (Miyake et al., 2015; Zhou et al.,
2021) and host species (Li et al., 2019; Youngblut et al., 2019) are the
main factors affecting the gut microbiota of fish. This study also found
that the diversity of fish gut microbiota was significantly affected by
feeding habits and host species. The diversity of gut microbiota species
and abundance determine the stability of the host gut microbiota
associated with host nutrition (Kuang et al., 2020). The community
diversity of the four fish species followed the order omnivorous
\textgreater{} herbivorous \textgreater{} filter-feeder \textgreater{}
carnivorous. Contrary, Li et al. (2015b) reported higher bacterial
diversity in the gut of the filter-feeding than herbivorous. It has been
reported that the higher the Shannon index, the better the stability of
the bacterial community and the better the digestion of nutrients (Zhang
et al., 2019).

The results of this study showed Proteobacteria and Firmicutes phyla as
typical dominant flora in the gut of the four fish species.
Proteobacteria and Firmicutes are two phyla typical dominant flora in
the intestine of many fish, such as \emph{Oncorhynchus mykiss}(Ingerslev
et al., 2014), \emph{Nibea coibor} and \emph{Nibea diacanthus} (Li et
al., 2019), \emph{Megalobrama terminalis} (Liu et al., 2021),
\emph{Micropterus salmoides} (Zhou et al., 2021) and\emph{Symphysodon
haraldi} (Zhang et al., 2021). The four fish species had variations in
abundant of microbiota species. The different symbiotic bacteria carried
by fish species may be caused by the selective enrichment of different
microorganisms due to variations in feeding and host species. A previous
study indicated that during evolution, hosts tend to acquire suitable
environmental bacteria by recognizing adhesion mechanisms on the cell
surface (McFall-Ngai, 2015). In addition, the differential enrichment of
specific flora under different feeding conditions may also adapt to the
function of the flora, for example, \emph{Bacillus} species and
\emph{Cetacea} species are potential candidates for probiotics (Larsen
et al., 2014),\emph{Pseudomonas} species produces vitamin
B\textsubscript{12}, and\emph{Fusobacterium} species produces butyrate
(Zhou et al., 2019). Accordingly, the carnivorous gut was dominated by
\emph{A. johnsonii} ,\emph{A. luckii} and \emph{P. stutzeri} bacteria
species, which may contribute to the digestion of proteins, while
\emph{Bacteroides}species were dominant in herbivorous fish gut may help
the host to digest cellulose. The presence of these microbiome in the
fish species are useful in host nutrition. Indeed, the intestinal
microbes of the carnivorous (\emph{S. chuatsi} ) showed higher protein
digestion potential and lower carbohydrate digestion potential, while
gut microbes of herbivorous fish (\emph{C. idellus} ) showed lower
protein and high carbohydrate digestion, consistent with their feeding
habits.

\textbf{5 \textbar{} CONCLUSION}

Taken together, the digestive enzymes activities, intestinal morphology,
and intestinal microbiome composition and diversity of fish are
significantly affected by feeding habits. Accordingly, carnivorous fish
possess higher trypsin and lipase activities related to their higher
feeding habit on protein and lipid. On the contrary, herbivorous fish
species utilizes plant materials related to the higher amylase enzyme
activity. The intestinal microvilli of carnivorous fish had many
branches and complex structures to increase surface area for digestion
and absorption of digested nutrients as an adaption to the short
intestine. The feeding habits led to various adaptations of microbiota
related to the selective colonization for various biological functions.
The results provide an understanding of the different digestive
strategies of omnivorous, carnivorous, herbivorous and filter-feeder
fish to improve feed formulation for better feed utilization and
digestibility in order to enhance nutrient absorption for promoting
growth performance.

\textbf{AUTHOR CONTRIBUTION}

Conceived and designed the experiments: Hua Rong; Performed the
experiments: Fang Jiao, Lei Zhang; Analyzed the data: Fang Jiao, Lei
Zhang; Wrote the paper: Fang Jiao, Hua Rong; Review and editing: Hua
Rong and Samwel Mchele Limbu. All authors read the manuscript and
approved it in its final version.

\textbf{CONFLICT OF INTEREST}

Authors declare that they have no conflicts of interest.



\textbf{DATA AVAILABILITY STATEMENT}

The data that support the findings of this study are openly available in
{[}Rong, Hua (2022), ``Analysis of intestinal microbes in fish'',
Mendeley Data, V1, doi: 10.17632/5zmmwbfhhv.1{]}

\textbf{ACKNOWLEDGEMENTS}

We are grateful to Beijing igeneCode Biotech Co., Ltd and all laboratory
members for technical advice and valuable help during the feeding trial,
sample collection and data analysis.

\textbf{FUNDING INFORMATION}

This study was financially supported by Guangdong Province Basic and
Applied Basic Research Fund---Provincial and Municipal Joint Fund
(2021A1515110179), Basic Research Program of Yunnan Province
(202201AT070251), Agricultural Basic Research Joint Special Project of
Yunnan Province (2018FG001-044).

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\textbf{Hosted file}

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\textbf{Hosted file}

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