Antibiotic accumulation, growth performance, physiologic status and intestinal ora diversication of Nile tilapia (Oreochromis niloticus) feed by diets supplemented with different dose of sulfamethoxazole

Sulfamethoxazole (SMZ) is an antibiotic used globally to treat sh disease in aquaculture, but the effects of exposure to legal aquaculture doses of SMZ in sh are still unclear. To comprehensively investigate the effects of exposure to legal doses of sulfamethoxazole (SMZ) in Nile tilapia (Oreochromis niloticus), sh were exposed to diets supplemented with different doses of SMZ (blank group, normal feed; LS, 0.67 g/kg; MS, 6.67 g/kg and HS, 33.33 g/kg) for 4 weeks. General SMZ accumulation, growth performance, physiologic status, intestinal and hepatic health were systemically evaluated. in changed


Background
Antibiotics poor absorption after medication, and overdose, coupled with their bioactivity and persistent behavior in global environment that has caused severe ecological sustainability and health risks [1][2][3], especially in sh, which also plays a vital role in human food safety. In aquaculture, sh cannot avoid exposure to antibiotics because of legal dietary medication to prevent and cure diseases [4,5].
Sulfamethoxazole (SMZ) is bacteriostatic antibiotics used for treatments of bacterial diseases in cultured sh species, which blocks dihydrofolate intermediate production, thereby restricts the normal bacterial folic acid synthesis [4,6]. The SMZ is commonly administered at 100 to 200 mg/kg sh body weight per day for 5 days [7], depending on sh species, infection and country-speci c legal requirements. Despite these legal directives, the antibiotics is used for long periods in aquaculture production [4], sometimes on daily basis [8]. Previous studies indicated that, SMZ is poorly absorbed in the guts of animals after medication, subsequently are excreted in urine and feces, either unchanged or modi ed into metabolites, which are transported into surface waters through runoff and subsurface drainage systems [9]. SMZ have been detected in freshwater at concentrations ranging from 259.60 ng/L to 385.00 ng/L [10]. These concentrations pose risks on aquatic species, raising global public concern on human health upon sh consumption [6,11]. However, no study has thoroughly evaluated the potential effects of using legal doses of SMZ in sh.
In this work, in order to deeply explore the differences of sh and the aquaculture environment, with the different SMZ addition set according to the aquaculture recommended doses. For this purpose, we exposed Nile tilapia (Oreochromis niloticus), a global economic, cultured and consumed species, to dietary treatments containing SMZ for 8 weeks (4 weeks with antibiotics fed and 4 weeks with normal fed, SCT 1084-2006 recommended the withdrawal period was 30 days). The present work aimed to explore the systemic effects of legal SMZ doses on antibiotic accumulation, general body functions, hepatic health and intestinal ora in sh. It provides a theoretical basis for the scienti c use of SMZ in the aquaculture industry.

Methods
Antibiotics and sh diet exposure Sulfamethoxazole (SMZ, purity w > 98.0%) were purchased from ANPEL Laboratory Technologies Co., Ltd., Shanghai, China. About 400 O. niloticus ngerlings were supplied by shery breeding base, the experiment was carried out in the ecological room of the Freshwater Fisheries Research Center of the Chinese Academy of Fishery Sciences. After acclimatization, all tested ngerlings were randomly distributed into 12 sterile 500-litre tanks (30 sh per tank) lled with dechlorinated water to a volume of 400 L per tank.
Twenty-four hours before the experiment, the total initial mean weights of O. niloticus were determined as 16.33 ± 0.50 g for all groups. In the experiment, refer to SCT 1084-2006 "Regulations for the use of sulfamethoxazole in aquaculture", considering the uctuation of SMZ use in actual production, O. niloticus were exposed to three concentrations in feeds supplemented with the low-dose (LS, 20 mg/(kg·d)) and medium-dose (MS, SCT 1084-2006 recommended The therapeutic dose of 200 mg/(kg·d)) and high dose (HS, 5 times the normal dose, 1000 mg/(kg·d)). Convert it to the SMZ content in the feed as 0.67, 6.67 and 33.33 g/kg, respectively. A control tank was included in which O. niloticus were reared and treated similarly but deprived of antibiotics, each group set 3 parallel, total of 12 aquaculture tanks. All O. niloticus were hand-fed twice daily at 9:00 h and 15:00 h and at 3% of their average body weight per day for 8 weeks (4 weeks with antibiotics fed and 4 weeks with blank fed). The entire breeding experiment does not change the water in order to maintain relatively stable concentrations as designed. The weight of individual O. niloticus was recorded weekly and the feed rations were adjusted accordingly.

SMZ residual determination
The O. niloticus muscles, intestinal contents and aquaculture water were collected at the beginning of the experiment, 4th, 6th and 8th weeks, the aquaculture sediment was collected at the end of the 8th week, the analysis of antibiotics concentrations was performed following [12,13].

Growth performance
In the 8th week of the experiment, the O. niloticus were collected and their individual weights determined for weight gain and nal weight estimations. The amount of feed and the weight of O. niloticus were used to calculate feed coe cient as the ratio of total wet weight gained by O. niloticus to total amount of feed fed in each group.

Liver morphology and detection
Randomly select 3 sh in each tank, MS-222 anesthesia was used to sample the sh and take the liver with dissection. The liver were washed with normal saline and xed with 2.5% glutaraldehyde. The sections were stained according to the routine preparation procedures of transmission electron microscopy ultrathin section samples, which were to be observed, analyzed and photographed [14,15]. Meanwhile, a reagent test kit was used to determine the triglyceride content (TG) of liver tissue.

Short-chain fatty acid determination
In the 4th week of the experiment, a 100 mg of intestinal contents were collected from each breeding barrel for SCFAs, 100 µL of 15% phosphoric acid, 50 µL of 50 µg/mL of internal standard (isohexanoic acid) solution and 100 µL of ether was added to homogenize for 1 min, centrifuge at 4℃, 12000 rpm for 10 min, the supernatant was analyzed by GC-MS, the detail instrument conditions was performed following [16] .

Intestinal ora diversity determination
In the 4th week of the experiment, the intestinal contents was quickly frozen in liquid nitrogen and stored at -80 °C for DNA extraction and 16 s rRNA (V3 + V4) high-throughput sequencing. After extracting the total DNA of the sample, primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') were designed according to the conserved region, and sequencing adapters were added to the ends of the primers to perform PCR ampli cation and the products puri cation, quanti cation, and homogenization are performed to form a sequencing library. The built library is rst subjected to library quality inspection, and the quali ed library is sequenced with lllumina HiSeq 2500. Filtered the sequencing data, and fed back the effective sequences after splicing. UCLUST (Edgar, 2002) in QIIME [17] software was used to cluster Tags at a similarity level of 97%, and obtain Operational Taxonomic Units (OTU), based on Silva (bacteria). Taxonomy database provides taxonomic annotations to OTU; Sobs, Ace, Chao1, Shannon, and Simpson indexes were used to compare the biological diversity within different treatment samples; PCoA analysis was used to compare the difference in the composition and structure of the biological community between different groups.

Statistical analysis
Isanger Shengxinyun was used to analyze and plots differences in bacterial ora structure. SPSS 25.0 software was used for signi cant difference analysis (P < 0.05 indicates signi cant difference), all of the data are expressed as mean ± SE (n = 3). Graphpad Prism 5.0, Adobe Illustrator and Origin 81 software ware used for mapping.

SMZ residual characteristics
In this study, the food intake of sh performed well, the feed coe cient at 4th week showed no signi cant difference. Various nutrients in the feed were digested and absorbed by the sh intestinal epithelial cells [3,18]. The feed ingredients also contained amount of SMZ (0.019 µg/kg). In NS group, the SMZ residual in sh muscles was below the maximum limited value (MRLs value, 100 µg/kg) stipulated in the 235 announcement of the Ministry of Agriculture (Fig. 1a). And in other groups, the SMZ residual in sh muscles and intestinal contents reached the highest value in the 4th week, and the residual dose of SMZ in each group was closely related to the feed exposure (Fig. 1a, b). After returning to normal feed in the 6th -8th week, the residual SMZ in the sh intestinal contents were still at a high level, which may release into environment through sh excretion activity.
The SMZ residual in the aquaculture system mainly comes from the excretion of sh and the release of SMZ from the unconsumed bait [19]. The SMZ concentration in NS group were in the range of 0.14-1.31 µg/L. And in other groups, the SMZ concentration increased rapidly from the start of aquaculture and reached a maximum in the 6th week, and then decreased rapidly, and the growth rate in the rst 4 weeks was higher than that in the 4th to 6th weeks. At the 8th week, there was no difference in the residual of SMZ in the aquaculture waters of each group (Fig. 1c). These results shows that with SMZ feed, the SMZ residual in the aquaculture water will increase due to the presence of SMZ; after stopping SMZ feed, the content of SMZ in the aquaculture water environment will decrease rapidly. The sediment located at the bottom of the culture system, and there is a large amount of sh excrement and bait accumulation [20]. And low oxygen and low temperature condition at bottom was not conducive to the degradation of SMZ [21]. Therefore, the SMZ residues are still at a high level at the end of aquaculture period. The SMZ residues in the sediments of each treatment group were also positively correlated with the amount of feed antibiotics.

O. niloticus growth performance
Growth performance is one of the most important aspects to sh farmers because it affects production and economic bene t in the aquaculture. LS and MS feeding group promoted growth of O. niloticus by reducind feed coe cient in LS and MS group (Fig. 2c). This result most likely occurred because the appropriate amount SMZ improved the activity of intestinal epithelial cells and promoted the digestion and absorption of nutrients [22,23]. Antibiotics reduced the negative effects of anti-nutritional factors in feed. This nding agrees with those obtained in previous studies on an appropriate amount of antibiotics can promote the growth of farmed aquatic products [23][24][25]. For the exposure of O. niloticus to SMZ feed signi cantly retarded growth performance and weight gain in HS group (P < 0.05; Fig. 2a, b, Table 1). This result is in line with excessive antibiotics may inhibit the growth of farmed aquatic products [26]. This nding may account for the excessive SMZ produced toxicity and impaired normal life activities of O. niloticus.
The liver is an accumulation organ for sh body fat. The fat in the sh liver mainly comes from the conversion and synthesis of excess protein, carbohydrates and absorption of fat in the feed [27]. The O. niloticus exposed to SMZ had fatter liver and higher viscera than control sh with the increased number of fat particles (P < 0.05; Fig. 3). These results indicated that, exposured to LS and MS concentration of SMZ have positive effects on sh growth performance, feed coe cient, except for HS diet. Exposure of O. niloticus to SMZ also increased the content of TG in liver (P < 0.05; Fig. 2d). These nding agree with those obtained in previous studies on several animals including mice and rainbow trout [28,29], which showed that penicillin and erythromycin exposure greatly improved the fat accumulation may be limited sh energy metabolism and growth performance, this is most likely the reasons why the weight gain of O. niloticus in this experiment is LS, MS > NS > HS.

The SCFAs contents
SCFAs are the products of intestinal ora metabolizing and fermenting nutrients in feed. The contents of 7 SCFAs in the intestinal contents of O. niloticus in each treatment group were quantitatively analyzed. Only acetic acid, propionic acid, butyric acid, isovaleric acid, valeric acid and caproic acid were detected among all samples, isobutyric acid was not detected. The total content of 6 SCFAs in the intestinal contents of tilapia is between 45.11 µg/g − 68.92 µg/g (Fig. 4a). And the percentage content of SCFAs in all samples are mainly acetic acid (accounting for 90.45%-94.44%), propionic acid (2.88%-4.38%), butyric acid (0.76%-1.49%), isovaleric acid (0.31%-0.89%), valeric acid (0.33%-0.42%) and caproic acid (0.65%-3.15%), respectively (Fig. 4b). In animal intestines, Bacteroides mainly produces acetic acid and propionic acid and Phylum Firmicutes can produce butyric acid [30], while acetic acid and propionic acid not only provide energy for cells but also participate in energy metabolism in the liver [31], and butyric acid can regenerate mucosal cells in the intestine [32,33]. The results of SCFAs showed that LS and MS diet would increase the content of SCFAs, and the main increase was propionic acid and butyric acid; HS group would reduce the content of SCFAs, and the main reduction was acetic acid. The content of SCFAs in the intestinal of O. niloticus in the SMZ treatment group was: LS, MS > NS > HS. These explanation supported the results that proper SMZ was fed to promote growth and excessive SMZ inhibited growth.

Intestinal ora diversity
The intestinal ora of O. niloticus has been established 20-60 days after hatching [34]. 16 s highthroughput sequencing was used to obtain bacterial data for each treatment group. Alpha diversity index analysis showed that all the community richness index (Sobs, Ace and Chao1) were decreased with the increase of SMZ dose, and the Sobs index in HS group was signi cantly lower than that of NS group (P < 0.05). As for community evenness index (Shannon and Simpson), Shannon index diminished with increasing dose of SMZ, and Simpson index increased with increasing dose of SMZ. These results suggested that exposure to SMZ impair species abundance, biological abundance and intestinal ora biodiversity of O. niloticus (Table. 2). SMZ reduces the biodiversity of the intestinal ora, which was consistent with the results of other antibiotic feeding and breeding experiments [35]. PCoA was used to explore the similarity or difference of sample community composition. Based on the sample OUT level, the binary-jaccard was used to calculate the distance between the samples and to explore the in uence of SMZ on the structure of the intestinal ora. The SMZ group (LS, MS, and HS) were signi cantly separated from the NS group (Fig. 5), the distance with NS group increased with the increase of the SMZ dose. These results indicated that the structure of intestines ora were altered in O. niloticus treated with SMZ feed.

The intestinal ora composition and function
Based on the annotation of OUT, the in uence of SMZ on the intestinal ora composition and function from the perspective of community phylogeny was clari ed, the species consumption of each group in the classi cation level (Phylum, class, and Genus) were shown in Fig. 6.
The main dominant species in the tested group were Fusobacteria, Proteobacteria, Cyanobacteria and Actinobacteria at the phylum level, which were basically consistent with the results of He's study [36], which indicating that SMZ reduced the biological diversity and changed the structure of the tilapia intestinal ora, and effected the number and proportion of dominant bacteria, but the main dominant bacteria at the phylum level was not changed. Fusobacteria in MS and HS group were much higher than NS and LS group, Proteobacteria in NS group was highest. Cyanobacteria in LS and MS group were higher than NS and HS group (Fig. 6a). These results implying that SMZ would affect the relative abundance of major dominant species.
The main dominant species were Fusobacteriia, Alphaproteobacteria, Cyanobacteria and Unclassi ed-pproteobacteria at the class level (Fig. 6b), and were Cetobacterium in Fusobacteria and norank-ccyanobacteria in Cyanobacteria at the genus level (Fig. 6c). These results showed that the abundance of Fusobacteria was positively correlated with the exposure dose. Species composition analysis showed that SMZ can inhibit the growth of Fusobacterium in the intestine of O. niloticus. Fusobacterium have obvious advantages in all samples, which may be related to their phylogenetic and phenotypic advantages [37]. Proper SMZ promotes its growth, which may be related to the large number of Fusobacterium and the characteristics of broad-spectrum antibacterial SMZ. The Cyanobacteria was main distributed in the aquaculture water, and the abundance was related to the nutrient content of the input (mainly feed) and the eutrophication of the aquaculture environment [38]. The Cyanobacteria in all tested tilapia intestine came from the same species; Actinomycetes are an important component of the freshwater plankton bacterial community [39], the Actinomycetes in the intestine of tilapia were also from the same species. This is most likely related to the culture environment (feed nutrition, SMZ and temperature).The intestinal ora is the most important group to maintain the intestinal microenvironment. The greater the number and uniformity of the intestinal ora, the stronger the resistance of the intestinal ora, micro ecology is also more stable [40]. Obviously, the addition of SMZ changed the balance of intestinal ora in O. niloticus.

Conclusion
The sh exposed to SMZ will cause the accumulation of antibiotics in organs and aquaculture environment. The LS and MS doses of SMZ will promote the growth (weight gain) of tilapia, except for HS group. This effect is that SMZ reduces the consumption of feed by reducing the biological diversity of the intestinal ora and induces intestinal ora produces more SCFAs, which in turn promotes the growth of O. niloticus; while HS group severely reduces the intestinal ora and affects the production of SCFAs, and also causes abnormal accumulation of fat in viscera, thereby inhibiting the growth of O. niloticus. Therefore, in the process of aquaculture, antibiotics should be used scienti cally and controlled strictly.  Tables   Due to technical limitations, table 1 is only available