ZHOU Ji-Shu, LIAN Qing-An, YU Er-Meng, XIE Jun, JI Hong and YU Hai-Bo

(1. College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China; 2. Key Laboratory of Tropical &Subtropical Fishery Resource Application & Cultivation, Ministry of Agriculture, Pearl River Fisheries Research Institute of CAFS, Guangzhou 510380, China)

Abstract: To investigate the influence of artificial substrates (AS) on the growth performance of fish in pond poly-culture systems, a feeding experiment was conducted in six individual earthen ponds, which were divided into two groups. The ponds with AS in the water were labeled as the “AS group” and that without the use of AS were labeled as the “control group” (three ponds per group). A total of 3867 common carp [Cyprinus carpio, (310±11) g], being artificial-feeds feeding fish, and 370 bighead carp (Aristichthys nobilis) and silver carp [Hypophthalmichthys molitrix, (810±15) g], being plankton filter feeding fish, were divided into six groups and cultured in the ponds respectively. The common carp in each pond were fed with commercial particle feeds three times a day and the feeding period was 62 days. Water quality, concentration of plankton and the bacterial community presenting in the water and sediment were monitored during the experiment. At the end of the experiment, all fish were harvested and the growth of the fish and feed efficiency ratio were determined. Results showed that the weight gain rate and feed efficiency ratio of common carp in AS group was significantly higher than that of the control group. Final body weights of bighead carp and silver carp in the AS group were significantly lower than that of the controls. Water transparency and diversity indices of the bacterial community in the AS group were significantly higher than that of the control (P＜0.05) and the concentration of plankton in the AS group was lower than that of the control. Results showed that the application of AS in poly-culture ponds was beneficial for the growth of common carp, being artificial-feeds feeding fish,while it was not beneficial for the growth of bighead carp and silver carp, being plankton filter feeding fish.

Key words: Common carp; Bighead carp; Silver carp; Water quality parameters; Bacteria community

Artificial substrates (AS), made of high-density polymers, with high surface adhesive force[1]and having been reported to promote the growth of microbes and algae[2], could be used as food for fish[3]and might improve the purification of water via bacterial metabolism[4—8]. The use of AS has been reported to improve the yield of shrimp[1,9,10], fresh water crayfish[11,12], Japanese eel[13], and Rohu, catla and kalbaush[14—16]in intensive ponds but not in extensive ponds or poly-culture ponds.

Poly-culture systems are a traditional aquaculture system in China and include a mixed culture of different kinds of fish, such as artificial-feeds feeding fish, which eat food by swallowing it, and filter feeding fish, which eat food by filtering water through their gill rakes. Common carp (Cyprinus carpio) is a type of artificial-feeds feeding fish and bighead carp(Aristichthys nobilis) and silver carp (Hypophthalmichthys molitrix) are filter feeding fish that mainly feed on plankton. These three types of fish are mainly cultured in poly-culture ponds in China, and the effects of AS on the growth of these fish in poly-culture ponds remains unclear and poorly studied. Therefore, in the present study common carp, and bighead carp and silver carp, were divided into two groups and cultured in six earthen ponds, where AS was only used in three ponds. After 62 days of feeding, the growth of the fish, water quality, concentration of plankton and bacterial communities in the water and sediments were monitored to show the effect of AS.

1 Materials and methods

1.1 Fish feeding and management of ponds in polyculture system

In total, 3867 common carp (310±11) gand 370 silver carp and bighead carp (810±15) g, purchased from a farm of Ankang (Shaanxi province, China),were randomly divided into 6 groups and cultured in six earthen ponds (220 m2each, triplicate ponds per group) respectively. The ponds with the use of AS (1 m width×10 m length) placed vertically in the water were the AS group (Fig. 1). The ponds without the use of AS were control groups.

Common carp in each pond were fed with the same amount of commercial feeds by hand 3 times a day (09:00, 13:30 and 17:00). Commercial feeds was purchased from Huaqing feed factory (Shaaxi province, China) and contained 418 g/kg crude protein, 305 g/kg crude fiber and 143 g/kg ash. The feeding period was 62 days, from 27thAugust to 27thOctober. No water-exchange occurred between the ponds during the whole experiment.

Fig. 1 Pictorial representation of AS placement

1.2 Sampling procedure

At 0, 11, 18, 26, 33, 42, 54, 58 and 62 days after the beginning of the experiment, water from five sampling points (four corners and one middle) in each pond were sampled by water bottle at a depth of 0.5 m and mixed well respectively. During the experimental period a sample of water and sediment from five sampling points of each pond (four corners and one middle) were sampled on Sep. 6 (10 days feeding),Oct. 6 (36 days feeding) and Nov. 6 (62 days feeding and 4 days more starvation for the low water temperature) and mixed well. Sediment samples were taken with a foot bottom sampler. At the termination of the experiment, all of the fish were harvested and weighed.

1.3 Growth of fish

The growth of fish was determined by the following formulae.

Weight gain rate=(Final body weight–Initial body weight)/Initial body weight;

Feed efficiency ratio, (%)=(Final total weight–Initial total weight)/Feed intake×100.

1.4 Determination of water quality parameters

Temperature, pH and transparency of the water were determined by a thermometer, pH probe (Shanghai Rex, Shanghai Yidian Company, Shanghai,China) and transparency scale (JCT-8 Plug transparency, Beijing dameida technology co., LTD, Beijing,China), respectively. Dissolved oxygen, ammonia, nitrate, nitrite and total phosphorus were determined by Winkler’s method, Na’s reagent method, Diazo nitrogen colorimetric method and Phosphorus molybdenum blue method, respectively[17].

1.5 Determination of plankton concentration in pond water

Firstly, a 1000 mL water sample was fixed with 15 mL Lugol’s iodine, then water were sedated for 48h and the upper layer was deleted and down lay with 30 mL water was kept and mixed well, then 0.1 mL down layer water was put in a 0.1 mL plankton counting chamber to count the total number of phytoplankton and zooplankton under a microscope. The total number of plankton in each water sample was counted twice.

1.6 Determination of microbes in water and sediment by method of PCR-DGGE

Total genomic DNA was extracted respectively from 100 mL water and 250 mg sediment samples using the DNA Kit (Omega America) according to the manufacturers’ instructions. A region of the 16S rRNA gene was targeted with bacterial primers 338f-GC (5′-CGCCCGCCGCGCCCCGCGCCCGGCC CGCCGCCCCCGCCCC-3′ and 518 r (5′-ATTACCG CGGCTGCTGG-3′) and the primers were ordered from Fuheng Biology Company (Shanghai, China).The PCR was run with 30s at 94℃, 30s at 47℃(0.5℃ increase after each cycle), 1min at 72℃×9 and 30s at 94℃, 30s at 52℃ and 1min at 72℃×19. For analysis, 6% polyacrylamide gels were used with a denaturant gradient of 30%—60% composed of deionized formamide and urea. Gels were run 1× TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid, 1 mmol/L EDTA pH 8) at 60℃ and 60 V for 16h. Bands were visualized by staining with 1× SYBR?Gold and illuminated on a UV-trans-illuminator (STUV-10,Nanjing Shuntai Technology Co., Ltd, Nanjing,China) under a SYBR?gold filter.

The electrophoretic bands in the gel were extracted and the DNA in the band was purified using the QIAquick Gel Extraction Kit (Shanghai Sangon Biological Engineering Co., Ltd, Shanghai, China) and then amplified. The digital information of DGGE and microbe DNA sequence was obtained from Quantity One software. A phylogenetic tree of primary microbes was constructed using the neighbor - joining(NJ) method[18]with MEGA 4.1 software.

Tab. 1 Effect of AS on growth and feed efficiency ratios of fish in ponds

1.7 Data analysis

Data are presented as mean±SD. The results were analyzed by one-way ANOVA, followed byT-tests using SPSS 11.5 software (SPSS, Chicago, IL,USA) and results were regarded as significant atP＜0.05.

2 Result

2.1 The effect of AS on growth of fish

Weight gain and feed efficiency ratios for common carp in the AS group were higher than controls(P＜0.05, Tab. 1). The final mean weight of bighead carp and silver carp in AS group were significantly lower than that in the control group (P＜0.05, Tab. 1).

2.2 The effect of AS on variation of water quality

Water temperature and pH, dissolved oxygen,ammonia, nitrate, nitrite and total phosphorus of water in the AS group and control groups were not significantly different (Tab. 2 and Fig. 2A-H), while water transparency in the AS group was significantly higher than the control group (Tab. 2 and Fig. 2I).

2.3 The effect of AS on concentration of plankton

The concentration of phytoplankton was not affected by usage of AS, while the concentration of zooplankton was significantly decreased by AS (P＜0.05,Tab. 3).

2.4 The effect of AS on similarity indices of bacterial communities

In Sep., Oct. and Nov., the similarity indices of bacterial communities in the water of AS and control groups were 100%, 57.3% and 55.3% and 31.5%,34.5% and 32.2%, respectively (Fig. 3), indicating a significantly higher similarity index in the AS groups than control groups (P＜0.05).

In Sep., Oct. and Nov., similarity indices of bacterial communities in the sediment of AS and control groups were 100%, 61.9%, 38.4% and 54.2%, 49.9%,38.3%, showing no significant differences between the two groups (P＞0.05, Fig. 4).

Tab. 2 Effects of artificial substrates on water quality parameters in ponds

Fig. 2 Variation of water quality parameters during the experimental periodValues are mean±SD (n=3). The * on each bar indicates significant differences between the two groups (P＜0.05)

2.5 The identification of bacteria by PCR-DGGE

Seven bacterial phyla were identified, where the most prevalent bacteria in water and sediment wereProteobacteriaand the second most prevalent bacteria wereBacteroidetes,FirmicutesandActinobac-

Tab. 3 The effect of AS on the concentration of phytoplankton and zooplankton

Note: Values are mean±SD (n=3). The*indicates a significant difference between the two groups (P＜0.05)teria, with the least prevalent bacteria beingChloroflexi,ThermosandCyanobacteria(Tab. 4). A phylogenetic tree of the 54 main bacteria was constructed using the DNA sequences of these bacteria(Fig. 5).

Fig. 3 The effect of AS on similarity indices of bacterial communities in water of pondsThe number under each line labeled 1, 2, 3 and 4, 5, 6 means the water respectively sampled in AS group and in control group on 6 Sep. (10 days feeding), 6 Oct. (36 days feeding) and 6 Nov. (62 days feeding and 4 days more starvation for the low water temperature). The percent under each line labeled 1—6 means the similarity indices (%) compared with line 1

Fig. 4 The effect of AS on similarity indices of bacterial communities in sediments from the pondsThe number under each line labeled 1, 2, 3 and 4, 5, 6 means the sediments respectively sampled in AS group and in control group on 6 Sep.(10 days feeding), 6 Oct. (36 days feeding) and 6 Nov. (62 days feeding and 4 days more starvation for the low water temperature). The percent under each line labeled 1—6 means the similarity indices (%) compared with line 1

3 Discussion

The present results showed that the application of AS in the ponds significantly improved the growth of common carp (Tab. 1), which was in agreement with previous reports that the application of AS improved the growth ofFarfantepenaeus brasiliensis[11],Litopenaeus vannamei[19,20],Penaeus monodon[21],Macrobrachium rosenbergii[22],Litopenaeus van-namei,Labeo rohita[23],Catla catla[15]andAnguilla japonica[13]. The present results showed that water transparency in the AS group was significantly higher than that of the control group (Tab. 2), indicating that clean water and plentiful food were suitable for the growth of common carp.

Fig. 5 Phylogenetic tree of the 54 main bacteria was constructed by the NJ method. The bacteria mainly belong to the phyla of Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria

The higher water transparency in the present study probably meant that the amount of plankton in the AS group was lower, which was in agreement with our present result that the concentration of zoo-luted source water by simulated river bioreactor with different carriers [J].Environmental Science, 2010, 31(11):plankton in the AS ponds was significantly lower than that in control ponds (Tab. 3). Plankton are live food for bighead carp and silver carp, which are filter feeding fish, and the combination of higher water transparency and lower concentration of live food for bighead carp and silver carp in the AS group probably resulted in significantly lower growth of these filter feeding fish in the present study (Tab. 1).

The surface of the non-woven gauze artificial substrates are known to absorb organic debris from the water and provide an available support for the growth of many bacteria, such asBacillusspp,Acinetobacterspp,Pseudomonasspp,Micrococcusspp,Zoogloea itzigsohn monocytogenesspp and also various algae species. These bacteria and algae had been reported to take up nitrogen, phosphorus and degraded nitrate from the water, and subsequently increase oxygen levels due to algal photosynthesis[13,24],which resulted in improved water quality. Our results supported these previous findings, where water quality parameters, especially water transparency and dissolved oxygen, were improved by application of AS in ponds (Tab. 2). This was in accordance with previous results that water quality was improved by the use of AS in the culture of Pacific white shrimp[7,19,25],pink shrimp[11], juvenile tiger shrimp[21], Rohu and catla[15], freshwater prawn[22], freshwater crayfish[12],rainbow trout[26]and grass carp[17].

4 Conclusion

The application of AS in a pond poly-culture system improved the growth of common carp, but did not promote the growth of bighead and silver carp.

Acknowledgement:

We would like to thank Magnus ?sli (NMBU in Norway) and Yang Li (Northwest A&F University)for language revision.