Environ Sci Pollut Res DOI 10.1007/s11356-017-0153-5
RESEARCH ARTICLE
Factors affecting palatability of four submerged macrophytes for grass carp Ctenopharyngodon idella Jian Sun 1,2 & Long Wang 1,2 & Lin Ma 1 & Fenli Min 1,2 & Tao Huang 1,2 & Yi Zhang 1 & Zhenbin Wu 1 & Feng He 1
Received: 15 May 2017 / Accepted: 7 September 2017 # Springer-Verlag GmbH Germany 2017
Abstract Grass carp can weaken the growth and reproductive capacity of submerged macrophytes by consuming valuable tissues, but factors affecting palatability of submerged macrophytes for grass carp rarely are considered. In this study, relative consumption rate of grass carp with regard to submerged macrophytes was in the following order: Hydrilla verticillata > Vallisneria natans > Ceratophyllum demersum > Myriophyllum spicatum. Firmness of macrophytes was in the following order: M. spicatum > C. demersum > H. verticillata = V. natans, whereas shear force was M. spicatum > C. demersum > H. verticillata > V. natans. After crude extracts of M. spicatum were combined with H. verticillata, grass carp fed on fewer macrophyte pellets that contained more plant secondary metabolites (PSMs). This indicated that structure and PSMs affected palatability of macrophytes. PSMs do not contribute to reduction in palatability through inhibition of intestinal proteinases activity, but they can cause a decrease in the abundance of Exiguobacterium, Acinetobacter-yielding proteases, lipases, and cellulose activity, which in turn can weaken the metabolic capacity of grass carp and adversely affect their growth. Thus, the disadvantages to the growth and development of grass carp Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-017-0153-5) contains supplementary material, which is available to authorized users. * Feng He [email protected] 1
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
caused by PSMs may drive grass carp to feed on palatable submerged macrophytes with lower PSMs. Keywords Palatability . Grass carp . Submerged macrophytes . Plant structure . PSMs . Intestinal microbiota
Introduction As the main primary producers, submerged macrophytes play vital roles in freshwater ecosystems (Wang et al. 2014). They maintain ecological integrity and stability, reduce turbidity from sediments, and increase the diversity of the physical habitat (Genkai-Kato and Carpenter 2005; Bolduc et al. 2016). However, the growth and resultant biomass of macrophytes are influenced by many factors, including nutrients, light, competition from other plants, and herbivorous fishes (Burks et al. 2006). Several studies have demonstrated that herbivory has a great influence on macrophytes, especially when exotic herbivores are introduced. Common carp Cyprinus carpio, crayfish Procambarus clarkii and Orconectes rusticus, and snail Pomacea canaliculata can dramatically change freshwater habitats by reducing macrophyte abundance from consumption and destruction (Lodge et al. 1994; Wong et al. 2010; van der Wal et al. 2013; Bajer et al. 2016). Similar to common carp and crayfish, grass carp Ctenopharyngodon idella, well known for their high feeding rate, generally consume 25 to 100% of their body mass of macrophytes per day. This can have a large influence on macrophytes through weakening their growth and reproductive capacity and influencing their community succession (Dorenbosch and Bakker 2012). However, not all macrophytes are favored by grass carp as their palatability varies (Parker and Hay 2005).
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To date, there have been many studies on the factors determining the palatability of aquatic plants for grazers. Many of these reports suggest that the presence of deterrent secondary compounds in plants can result in the low feeding rates of herbivores. Kapuscinski et al. (2014) found that soluble phenolic compounds negatively affected palatability for rudd Scardinius erythrophthalmus. In addition, plants with high chemical deterrents exhibited low vulnerability to amphipod Ampithoe longimana and isopod Idotea chelipes (Taylor et al. 2003; Martínez-Crego et al. 2016). Herbivores are normally nitrogen-limited and prefer plants with a high protein content (Cruz-Rivera and Hay 2003; Sanchez and Trexler 2016). In addition to secondary compounds and protein content, structural determinants of plants play an important role in palatability for herbivores (Elger and Willby 2003). Palatability of macrophytes for the snail Lymnaea stagnalis was negatively related to dry matter content (DMC), and the talitrid amphipod Orchestoidea tuberculata preferred fresh Macrocystis integrifolia over Lessonia nigrescens, because Lessonia nigrescens has firmer physical features (Elger and Lemoine 2005; Duarte et al. 2014). Although Parker et al. (2006) have reported that plant secondary metabolites were associated with herbivory of grass carp, we know little about the mechanisms of palatability of submerged macrophytes for grass carp. Some studies have focused on the relationship between traits of plants and their palatability for herbivores. However, these studies have examined how those traits interact to affect fitness of herbivores as it relates to survivorship, growth, and reproduction over a relatively long period of time (Duarte et al. 2014; Tao et al. 2014). Nevertheless, the physiological reactions such as proteinase activity in aquatic herbivores due to different traits of plants, which may affect feeding intake, have been barely explored. It is well known that gut microbiota of fish can influence biological processes, including digestion (Giatsis et al. 2015; Liu et al. 2016). However, there is a lack of research on the relationship between palatability of macrophytes and fish intestinal microbiota affected by macrophyte traits. Taken together, further investigations are needed to elucidate the mechanism of palatability of submerged macrophytes for grass carp and to provide useful information to develop strategies for the restoration of submerged macrophytes in ecosystems containing herbivorous fishes. We here measured the relative consumption rate (RCR) of grass carp with regard to four submerged macrophytes: Hydrilla verticillata (H. verticillata), Vallisneria natans (V. natans), Ceratophyllum demersum (C. demersum), and Myriophyllum spicatum (M. spicatum). Each submerged macrophyte was further analyzed for physical, nutritional, and chemical traits to assess whether any of these parameters were correlated with palatability for grass carp. Additionally, we investigated the effect of crude extracts from the least palatable plant (M. spicatum) on the activity of intestinal proteinases and the gut bacterial community.
Methods Study organisms During spring time (March 2016), grass carp (mean ± SD, weight 80 ± 5 g; total length 19 ± 0.5 cm) were purchased from the Xiantao County Fishery, Hubei Province, China (30° 27′ N, 113° 52′ E) and reared in laboratory conditions for 1 week before experiments. All animal experiments were approved by the Institute of Hydrobiology, Chinese Academy of Sciences (Approval ID: IHB 2013724) and were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals. Four species of submerged macrophytes (H. verticillata, V. natans, C. demersum, and M. spicatum) were collected from East Lake, Wuhan City, China (30° 53′ N, 114° 38′ E). Before the experiment, running tap water was used to flush away marl and periphyton coverage on the macrophytes. Feeding assays Feeding assays were performed in aquaria (100 cm × 50 cm × 50 cm), in which the flow rate of recirculating water of 26 °C was maintained at 25 L min −1 and the water was filtered through biofilters to remove fish feces and to reduce the ammonia concentration. To avoid stressful conditions caused by low oxygen concentrations, aeration was maintained at a constant level in aquaria. In each aquarium, there were five grass carp that have been fed pond food for 2 days of acclimatization (Dorenbosch and Bakker 2011). Before feeding assays, grass carp had been starved for 24 h. During the experiments, grass carp were permitted to feed for 2 h and there was no mortality of grass carp. Feeding assay with fresh submerged macrophytes Each of four species of fresh submerged macrophytes (100 g) was added to six aquaria, with six replicates for each species for a total of 24 aquaria. Given autogenic mass change in the macrophytes, the paired control in other six aquaria contained water and macrophytes without grass carp. After removing excess water with a paper towel, we weighed macrophyte biomass at the beginning and the end of the experiment. The percent RCR was calculated as [H0 × Cf/C0 ‐ Hf]/W × 100%, where H0 and Hf were the wet masses of macrophytes exposed to grass carp before and after the experiment, respectively; C0 and Cf were the wet masses of macrophytes from the paired control before and after experiment, respectively; W was the mass of grass carp (Wong et al. 2010). Feeding assay with reconstituted macrophytes To control for plant structure, each species of fresh macrophytes was lyophilized at − 70 °C for 24 h to a constant weight
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and then finely ground to a homogeneous consistency by sieving through a 149-μm mesh screen. First, heating agar and distilled water were put in a microwave oven until they melt, then the resulting plant material from each species was added to the mixture and stirred evenly to a final ratio of 1 g plant material: 5 mL water: 0.5 g agar. This agar-based food was formed into a uniform cylinder of 2 mm diameter using a mold. After it solidified, we cut the cylinder into pellets of 7 mm size. In assays using the artificial pellets, each of the four types of pellets was added to six aquaria, with six replicates for each type for a total of 24 aquaria. The number of pellets provided to the grass carp before experiment was counted as N0 and the number of pellets remaining after the experiment was Nf. RCR was then calculated and expressed as a percentage of each type of pellet using the formula (Nf ‐ N0) × G/W × 100%, where G was the weight of each pellet; W was the mass of grass carp (Miller and Provenza 2007).
measured at 620 nm after dissolving dried macrophytes with 60% mass/volume sulfuric acid and adding 2% mass/volume anthranone. Soluble protein content of macrophytes was determined at 595 nm after grinding the plant material and using Coomassie Brilliant Blue G-250 to react. Lysine was measured at 500 nm after degreasing dried ground macrophytes with 60–90 °C petroleum ether for 8 h and addition of ninhydrin. The amount of tannins was estimated using permanganate titration methods, where the solution turned bright golden yellow after slow addition of 0.1 mol L−1 KMnO4. The concentration of flavonoids was analyzed at 510 nm after addition of 5% mass/volume NaNO2, 10% mass/volume Al(NO3)3, and 4% mass/volume NaOH into the 70% ethanol extract. All analyses for the same individual macrophytes were performed in triplicates except for firmness and shear force, which were conducted in quintuplicates.
Feeding assay with PSMs
Crude extract was obtained from M. spicatum using the same method as described above. We then prepared five concentrations of PSMs solutions (0, 18, 180, 1800, 18,000 μg flavonoids·L−1). The solutions were stored at 4 °C until enzyme assays were conducted.
According to the results of two feeding assays above, very little M. spicatum was consumed either in the form of fresh plant or reconstituted pellets; therefore, we added its crude extracts into H. verticillata (the most palatable plant) to assess whether PSMs influenced palatability for grass carp in the third assay. For M. spicatum, five different masses (0, 1.25, 2.5, 3.75, and 5 g) of lyophilized and ground plant powder were extracted in 70% ethanol three times for 24 h each, followed by further extraction with petroleum ether thrice (exhibiting a boiling range of 30–60 °C) to remove chlorophyll and lipid. These crude extracts were concentrated by rotary evaporation and then we got five concentrations of crude extracts named 0 PSMs, 0.25 PSMs, 0.50 PSMs, 0.75 PSMs, and 1.00 PSMs. These crude extracts were added to 5 g of H. verticillata powder separately. Five different food pellets were prepared and RCR was calculated for each type of pellet as described above. Measurement of plant traits To understand the palatability of submerged macrophytes for grass carp, we measured three aspects of their traits: physical properties (firmness, shear force, dry matter content (DMC), ash, and fibrin), nutritional contents (soluble protein and lysine) and chemical contents (tannins and flavonoids). All trait measurements were performed on randomly chosen plant samples. Firmness and shear force of four macrophytes were examined at 2 cm from the top of each plant, using a texture analyzer (TA. XT Plus). DMC was determined following Elger and Lemoine (2005). Ash content was assessed according to the methods described by Qiu and Kwong (2009). Fibrin was
Effects of PSMs on the activity of intestinal proteinases
Effects on protease activity Intestinal tracts were dissected from five grass carp and divided into three sections (foregut, midgut, and hindgut). After removing the gut contents by ice-cold stoke-physiological saline solution, the tissues were homogenized at a ratio of 1 g tissue: 9 mL phosphate buffer saline (PBS, 0.1 mol/mL, pH 8.0). After centrifugation at 12,000 r min−1 for 20 min at 4 °C, three kinds of intestinal crude enzymes were obtained and incubated with the five PSM concentrations separately for 10 min at 37 °C, followed by addition of 0.5% casein to react at 37 °C for 20 min. Then, 10% trichloroacetic acid was added to terminate the reaction, and optical density (OD) was measured at 680 nm after the addition of 0.55 mol L−1 Na2CO3 and Folin-Phenol to react at 37 °C for 15 min. The unit of protease activity in the fish intestine is described as unit per gram proteases. Briefly, 1 U g−1 prot is expressed as 1 g proteases hydrolyzing casein to generate 1 μg tyrosine at 37 °C for 1 min. All analyses were performed in triplicates. Effects on trypsin activity Trypsin activity in the foregut, midgut, and hindgut of grass carp was determined by spectrophotometry using kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The unit of enzyme activity was expressed as unit·per milligram proteases based on the manufacturer’s recommendations, which was expressed as the equivalent enzyme activity
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required to generate an optical density (OD) change of 0.003 at pH 8.0 and 37 °C. All analyses were performed in triplicates. Effects of PSMs on the bacterial community in grass carp intestine. Two treatments were used: one containing six fish that were fed food pellets containing H. verticillata only (HV) and another containing six fish that were fed food pellets containing H. verticillata with crude extracts from M. spicatum at a concentration of 1.00 PSMs (HVP). The food pellets were prepared in the same way as described above. The fish were fed twice a day (09:00, 15:00 h) with 30 g food pellets to last 40 days in the two aquaria, which were filled with water of 26 °C from the same source that was changed once a day. At the end of the experiment, four fish from each aquarium were chosen randomly and their percentage weight gain (PWG) was calculated as [(Wf ‐ W0)/ W0] × 100%, where W0 and Wf were the masses of grass carp before and after the experiment, respectively (Wang et al. 2015). The intestinal contents were then collected from each fish and stored in a refrigerator using the same method as described by Wu et al. (2012). DNA extraction, PCR, and high-throughput 16S rRNA pyrosequencing of each sample were performed following the methods described by Borsodi et al. (2017).
1.50% for M. spicatum to 7.29% for H. verticillata. There were significant differences in the RCR among macrophyte treatments (one-way ANOVA, d.f. = 3, F = 104.237, P < 0.001). The order of RCR of the four macrophyte species was H. verticillata > V. natans > C. demersum > M. spicatum (Fig. 1a). Combined with the results in Fig. 2 that the order of the firmness of each species was M. spicatum > C. demersum > H. verticillata = V. natans and the order of shear force was M. spicatum > C. demersum > H. verticillata > V. natans, this indicated that an increase in firmness and shear force can
Data analysis All statistical analyses were performed using SPSS IBM Statistics version 23, Illinois, USA. RCR of each feeding assay, physical/nutritional/chemical traits of submerged macrophytes, and the proteinases activity of each gut were assessed the differences (P < 0.05) by using one-way ANOVA, followed by the Tukey post hoc tests to identify homogenous groups. PWG of grass carp and microorganisms of HV and HVP at genus level were compared using independent samples t test, respectively. For sequence analysis of gut microbiota, the Shannon index was determined on the base of the calculated operational taxonomic units (OTUs) by Mothur ver. 1.30.1 (Giatsis et al. 2015). The sequences gained were phylogenetically classified to the phylum and genus levels at 97% similarity for the community composition analysis. MEGA 6.0 was used to depict a phylogenetic tree with the neighbor-joining algorithm. The representative sequences from each OTU were subjected to the RDP-II Classifier of the Ribosomal Database Project (RDP) and the National Center for Biotechnology Information (NCBI) BLAST for taxonomic analysis.
Results and discussion Feeding assays When grass carp were offered four different species of fresh submerged macrophytes for 2 h, their RCR ranged from
Fig. 1 Mean (± SD) relative consumption rate (RCR) of submerged macrophytes by grass carp: a fresh submerged macrophytes, b reconstituted macrophytes, c crude chemical extracts from Myriophyllum spicatum. Different letters (a, b, and c) above each bar represent statistical differences between species based on RCR
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decrease RCR of grass carp, and macrophyte selection by grass carp can be determined, at least in part, by the structure of submerged macrophytes.
The RCR of reconstituted macrophytes by grass carp differed among species, but there was no significant difference between H. verticillata, V. natans, and C. demersum (one-way
Fig. 3 Effects of plant secondary metabolites (PSMs) on protease activity in the gut of grass carp: a foregut, b midgut, c hindgut. Data are presented as mean ± SD. Different letters (a, b, and c) above each bar represent statistical differences between different concentrations of PSMs
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Fig. 4 Effects of plant secondary metabolites (PSMs) on trypsin activity in the gut of grass carp: a foregut, b midgut, and c hindgut. Data are presented as mean ± SD. Different letters (a and b) above each bar represent statistical differences between different concentrations of PSMs
ANOVA, d.f. = 2, F = 3.732, P = 0.089) (Fig. 1b). Compared with RCR of fresh macrophytes, RCR was less for reconstituted macrophytes (Fig. 1a, b). This suggested that the lyopholization-grinding process may degrade cells, causing secondary metabolites to be released more readily, leading to decreased intake by grass carp. M. spicatum was the least palatable when presented either as fresh or reconstituted plant matter. When the crude extracts from M. spicatum were combined with the most palatable H. verticillata, the RCR of grass carp decreased while the dose of PSMs increased (one-way ANOVA, d.f. = 4, F = 107.646, P < 0.001). After adding 0.5 PSMs or more, the feeding rates were as little as 0 (Fig. 1c). These results suggested that PSMs have a significant effect on reducing the RCR of grass carp, presumably due to recognition by olfaction or gustation. PSMs of M. spicatum were determined to be quantitative defenses and dose dependent (Hay 2016). PSMs contain many chemical deterrents such as tannins and flavonoids (Table S1). Furthermore, some reports have demonstrated that herbivores exhibit low preference for plants with relatively high levels of secondary metabolites (Parker et al. 2006; Dorenbosch and Bakker 2011; Goodman and Hay 2013; O’brien and Scheibling 2016).
Effects on trypsin activity In the foregut, trypsin activity was significantly improved at concentrations of 180, 1800, and 18,000 μg L−1 PSMs compared to the concentrations of 0 and 18 μg L−1 (one-way ANOVA, d.f. = 4, F = 10.765, P = 0.001). In the midgut, there was a significant difference only at the concentration of 18,000 μg L − 1 PSMs (one-way ANOVA, d.f. = 4, F = 4.423, P = 0.026). However, in the hindgut, trypsin activity was not affected by different concentrations of PSMs (oneway ANOVA, d.f. = 4, F = 0.807, P = 0.548). (Fig. 4). The results above suggest that PSMs of M. spicatum can improve the activity of intestinal proteinases, which is a contrast to research showing that tannins, phenolics, and some specific monoterpenes can limit protein digestibility in terrestrial organisms intestine (Lowry et al. 1996; Niho et al. 2001; Dearing et al. 2005b; Degabriel et al. 2009; Kohl et al. 2015;). Additionaly, Mouradov and Spangenberg (2014) demonstrated that flavonoids inhibit activity of Table 1 Number of sequences, OTUs number, and Shannon index of microbial communities in grass carp intestine Sample
Number of sequences
OTUs number
Shannon index
HV1 HV2 HV3 HV4 HVP1 HVP2 HVP3 HVP4
14,358 15,089 15,528 12,245 3382 4455 3471 3736
887 768 655 716 429 409 448 389
4.02 3.65 3.14 3.94 3.62 3.69 3.90 3.26
Effects of PSMs on the activity of intestinal proteinases Effects on protease activity The protease activity in the foregut, midgut, and hindgut of grass carp responded similarly to the increase in concentration of PSMs. Protease activity significantly increased at concentrations of 1800 and 18,000 μg L−1 (foregut: P < 0.001; midgut: P < 0.001; hindgut: P < 0.001). However, there was no difference at 0, 18, or 180 μg L−1 (Fig. 3). These results demonstrated that low levels of PSMs did not affect protease activity, and that high levels can contribute to improving the activity.
HV and HVP represent the treatments of fish feeding food pellets containing H. verticillata only and food pellets containing H. verticillata with crude extracts from M. spicatum at a concentration of 1.00 PSMs, respectively. OTUs is abbreviated as operational taxonomic units
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proteinases. The reason for this may be that the inhibition caused by these metabolites was much less than the improvement from other molecules that were not detected or tested for. Effects of PSMs on the bacterial community in grass carp intestine To identify the bacterial communities from eight intestinal content samples, more than 3300 sequences were obtained for each sample. After downstream analyses, 4701 OTUs were obtained at a 97% sequence identity threshold. In each sample, 389 to 887 OTUs were observed. The sequence information and diversity index (Shannon) of the samples are shown in Table 1. Hierarchical clustering analysis was used to show the similarity in the bacterial community structure among the eight intestinal content samples (Fig. 5). There were two clear clusters Fig. 5 Taxonomic composition of bacterial 16S rRNA gene reads at a phylum level and b genus level from eight intestinal content samples
observed: one group consisting of samples collected from HV and another one clustered with samples collected from HVP, which indicated a clear distinction in the bacterial community structure between HV and HVP. To compare the phylogenetic differences in the compositions of the bacterial community structures of intestinal content in HV and HVP, relative bacterial community abundance was characterized at the phylum and genus levels (Fig. 5). At the phylum level, there were 36 phyla classified for each and the major phyla of the samples were uniform, but their relative abundances differed (Fig. 5a). For example, Proteobacteria, detected as the dominant phylum, were less abundant in HV (60.75–67.24%) than in HVP (67.86–73.58%). However, Firmicutes (19.93–24.74 vs. 17.99–19.75%) and Planctomycetes (1.89–3.97 vs. 0.91–1.86%) were more abundant in HV as compared to HVP. At the genus level, the prevalent species in HV were Exiguobacterium (17.04–22.10%),
Environ Sci Pollut Res Fig. 6 Comparison analysis of microorganisms between two treatments (HV vs. HVP) at genus level. HV and HVP represent the treatments of fish feeding food pellets containing H. verticillata only and food pellets containing H. verticillata with crude extracts from M. spicatum at a concentration of 1.00 PSMs, respectively
Acinetobacter (6.39–7.65%), Methylocystis (3.63–7.06%), and Blastopirellula (0.42–1.00%). Yet, Exiguobacterium (13.20– 16.07%), Acinetobacter (4.09–5.68%), Methylocystis (0.65– 1.58%), and Hydrogenophaga (1.12–1.59%) were dominant in HVP (Fig. 5b). A comparison analysis of microorganisms with a significant difference between the two treatments (HV vs. HVP) at the genus level showed that the two major species, Exiguobacterium and Acinetobacter, were lower in HVP (t test, for Exiguobacterium, d.f. = 6, F = 2.793, P = 0.007; for Acinetobacter, d.f. = 6, F = 0.150, P = 0.004) (Fig. 6), suggesting that PSMs have negative effects on the abundance of bacterial community in the grass carp intestine. As protease-producing, lipase-producing, and cellulaseproducing bacteria, Acinetobacter in the digestive tracts of fishes can yield protease, lipase, and cellulose activity (Jiang et al. 2011; Ray et al. 2012). Similar to Acinetobacter, the genus Exiguobacterium can secrete alkaline protease (Vishnivetskaya et al. 2009). Thus, gut microbiota play an essential role in the digestion of food and could be affected by multiple factors, such as diet (Liu et al. 2016). Therefore, PSMs can lessen enzymatic activity in the intestines by decreasing the abundance of Acinetobacter and Exiguobacterium, which in turn may weaken the metabolic capacity of grass carp and cause series of adverse effects on growth. After feeding for 40 days, the PWG of grass carp in HVP was 1.67% and in HV was 5.45%. The PWG in HVP was significantly lower than that in HV (t test, d.f. = 5.412, F = 5.731, P = 0.012). Thus, the disadvantageous feedback signal for the growth and development of grass carp induced by PSMs may motivate grass carp to feed on the palatable submerged macrophytes with lower PSMs.
Conclusions This study was undertaken to explore the palatability mechanisms in grass carp for submerged macrophytes. The results
demonstrated that the order of palatability of the four species of fresh submerged macrophytes species was H. verticillata > V. natans > C. demersum > M. spicatum. These results demonstrated that not only the structure of macrophytes but also PSMs played roles in affecting palatability for grass carp. Higher firmness and shear force can decrease the RCR of grass carp. And higher content of PSMs can reduce palatability of macrophytes by significantly decreasing the abundance of Exiguobacterium, Acinetobacter-yielding protease, lipase, and cellulose activity, but not by weakening the activity of intestinal proteinases.
Acknowledgements This work was financially supported by the National Science and Technology Support Program of the 12th FiveYear Plan, China (No. 2012BAJ21B03-04), the National Natural Science Foundation of China (51178452), the Major Science and Technology Program for Water Pollution Control and Treatment of China of the 12th Five-Year Plan (No. 2012ZX07101007-005), the Knowledge Innovation Program of the Chinese Academy of Sciences and the Hubei Province Science Foundation for Youths (2014CFB282). We thank Drs. Yafen Wang, Qiaohong Zhou, Biyun Liu, Enrong Xiao, Dong Xu, Junmei Wu, and Ms. Liping Zhang for the experimental help (Institute of Hydrobiology, Chinese Academy of Sciences).
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Lowry JB, McSweeney CS, Palmer B (1996) Changing perceptions of the effect of plant phenolics on nutrient supply in the ruminant. Crop Pasture Sci 47:829–842 Martínez-Crego B, Arteaga P, Tomas F, Santos R (2016) The role of seagrass traits in mediating zostera noltei vulnerability to mesograzers. PLoS One 11:1–19 Miller SA, Provenza FD (2007) Mechanisms of resistance of freshwater macrophytes to herbivory by invasive juvenile common carp. Freshw Biol 52:39–49 Mouradov A, Spangenberg G (2014) Flavonoids: a metabolic network mediating plants adaptation to their real estate. Front Plant Sci 5:1–16 Niho N, Shibutani M, Tamura T, Toyoda K, Uneyama C, Takahashi N, Hirose M (2001) Subchronic toxicity study of gallic acid by oral administration in F344 rats. Food Chem Toxicol 39:1063–1070 O’brien JM, Scheibling RE (2016) Nipped in the bud: mesograzer feeding preference contributes to kelp decline. Ecology 97:1873–1886 Parker JD, Hay ME (2005) Biotic resistance to plant invasions? Native herbivores prefer non-native plants. Ecol Lett 8:959–967 Parker JD, Collins DO, Kubanek J, Sullards MC, Bostwick D, Hay ME (2006) Chemical defenses promote persistence of the aquatic plant Micranthemum umbrosum. J Chem Ecol 32:815–833 Qiu JW, Kwong KL (2009) Effects of macrophytes on feeding and lifehistory traits of the invasive apple snail Pomacea canaliculata. Freshw Biol 54:1720–1730 Ray AK, Ghosh K, Ringø E (2012) Enzyme-producing bacteria isolated from fish gut: a review. Aquac Nutr 18:465–492 Sanchez JL, Trexler JC (2016) The adaptive evolution of herbivory in freshwater systems. Ecosphere 7:1–15 Tao L, Berns AR, Hunter MD (2014) Why does a good thing become too much? Interactions between foliar nutrients and toxins determine performance of an insect herbivore. Funct Ecol 28:190–196 Taylor RB, Lindquist N, Kubanek J, Hay ME (2003) Intraspecific variation in palatability and defensive chemistry of brown seaweeds: effects on herbivore fitness. Oecologia 136:412–423 Vishnivetskaya TA, Kathariou S, Tiedje JM (2009) The Exiguobacterium genus: biodiversity and biogeography. Extremophiles 13:541–555 van der Wal JEM, Dorenbosch M, Immers AK, Forteza CV, Geurts JJM, Peeters ETHM, Koese B, Bakker ES (2013) Invasive crayfish threaten the development of submerged macrophytes in lake restoration. PLoS One 8:1–11 Wang HJ, Wang HZ, Liang XM, Wu SK (2014) Total phosphorus thresholds for regime shifts are nearly equal in subtropical and temperate shallow lakes with moderate depths and areas. Freshw Biol 59: 1659–1671 Wang B, Liu Y, Feng L, Jiang WD, Kuang SY, Jiang J, Li SH, Tang L, Zhou XQ (2015) Effects of dietary arginine supplementation on growth performance, flesh quality, muscle antioxidant capacity and antioxidant-related signalling molecule expression in young grass carp (Ctenopharyngodon idella). Food Chem 167C:91–99 Wong PK, Liang Y, Liu NY, Qiu JW (2010) Palatability of macrophytes to the invasive freshwater snail Pomacea canaliculata: differential effects of multiple plant traits. Freshw Biol 55:2023–2031 Wu S, Wang G, Angert ER, Wang W, Li W, Zou H (2012) Composition, diversity, and origin of the bacterial community in grass carp intestine. PLoS One 7:1–11
RESEARCH ARTICLE
Factors affecting palatability of four submerged macrophytes for grass carp Ctenopharyngodon idella Jian Sun 1,2 & Long Wang 1,2 & Lin Ma 1 & Fenli Min 1,2 & Tao Huang 1,2 & Yi Zhang 1 & Zhenbin Wu 1 & Feng He 1
Received: 15 May 2017 / Accepted: 7 September 2017 # Springer-Verlag GmbH Germany 2017
Abstract Grass carp can weaken the growth and reproductive capacity of submerged macrophytes by consuming valuable tissues, but factors affecting palatability of submerged macrophytes for grass carp rarely are considered. In this study, relative consumption rate of grass carp with regard to submerged macrophytes was in the following order: Hydrilla verticillata > Vallisneria natans > Ceratophyllum demersum > Myriophyllum spicatum. Firmness of macrophytes was in the following order: M. spicatum > C. demersum > H. verticillata = V. natans, whereas shear force was M. spicatum > C. demersum > H. verticillata > V. natans. After crude extracts of M. spicatum were combined with H. verticillata, grass carp fed on fewer macrophyte pellets that contained more plant secondary metabolites (PSMs). This indicated that structure and PSMs affected palatability of macrophytes. PSMs do not contribute to reduction in palatability through inhibition of intestinal proteinases activity, but they can cause a decrease in the abundance of Exiguobacterium, Acinetobacter-yielding proteases, lipases, and cellulose activity, which in turn can weaken the metabolic capacity of grass carp and adversely affect their growth. Thus, the disadvantages to the growth and development of grass carp Responsible editor: Philippe Garrigues Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11356-017-0153-5) contains supplementary material, which is available to authorized users. * Feng He [email protected] 1
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
caused by PSMs may drive grass carp to feed on palatable submerged macrophytes with lower PSMs. Keywords Palatability . Grass carp . Submerged macrophytes . Plant structure . PSMs . Intestinal microbiota
Introduction As the main primary producers, submerged macrophytes play vital roles in freshwater ecosystems (Wang et al. 2014). They maintain ecological integrity and stability, reduce turbidity from sediments, and increase the diversity of the physical habitat (Genkai-Kato and Carpenter 2005; Bolduc et al. 2016). However, the growth and resultant biomass of macrophytes are influenced by many factors, including nutrients, light, competition from other plants, and herbivorous fishes (Burks et al. 2006). Several studies have demonstrated that herbivory has a great influence on macrophytes, especially when exotic herbivores are introduced. Common carp Cyprinus carpio, crayfish Procambarus clarkii and Orconectes rusticus, and snail Pomacea canaliculata can dramatically change freshwater habitats by reducing macrophyte abundance from consumption and destruction (Lodge et al. 1994; Wong et al. 2010; van der Wal et al. 2013; Bajer et al. 2016). Similar to common carp and crayfish, grass carp Ctenopharyngodon idella, well known for their high feeding rate, generally consume 25 to 100% of their body mass of macrophytes per day. This can have a large influence on macrophytes through weakening their growth and reproductive capacity and influencing their community succession (Dorenbosch and Bakker 2012). However, not all macrophytes are favored by grass carp as their palatability varies (Parker and Hay 2005).
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To date, there have been many studies on the factors determining the palatability of aquatic plants for grazers. Many of these reports suggest that the presence of deterrent secondary compounds in plants can result in the low feeding rates of herbivores. Kapuscinski et al. (2014) found that soluble phenolic compounds negatively affected palatability for rudd Scardinius erythrophthalmus. In addition, plants with high chemical deterrents exhibited low vulnerability to amphipod Ampithoe longimana and isopod Idotea chelipes (Taylor et al. 2003; Martínez-Crego et al. 2016). Herbivores are normally nitrogen-limited and prefer plants with a high protein content (Cruz-Rivera and Hay 2003; Sanchez and Trexler 2016). In addition to secondary compounds and protein content, structural determinants of plants play an important role in palatability for herbivores (Elger and Willby 2003). Palatability of macrophytes for the snail Lymnaea stagnalis was negatively related to dry matter content (DMC), and the talitrid amphipod Orchestoidea tuberculata preferred fresh Macrocystis integrifolia over Lessonia nigrescens, because Lessonia nigrescens has firmer physical features (Elger and Lemoine 2005; Duarte et al. 2014). Although Parker et al. (2006) have reported that plant secondary metabolites were associated with herbivory of grass carp, we know little about the mechanisms of palatability of submerged macrophytes for grass carp. Some studies have focused on the relationship between traits of plants and their palatability for herbivores. However, these studies have examined how those traits interact to affect fitness of herbivores as it relates to survivorship, growth, and reproduction over a relatively long period of time (Duarte et al. 2014; Tao et al. 2014). Nevertheless, the physiological reactions such as proteinase activity in aquatic herbivores due to different traits of plants, which may affect feeding intake, have been barely explored. It is well known that gut microbiota of fish can influence biological processes, including digestion (Giatsis et al. 2015; Liu et al. 2016). However, there is a lack of research on the relationship between palatability of macrophytes and fish intestinal microbiota affected by macrophyte traits. Taken together, further investigations are needed to elucidate the mechanism of palatability of submerged macrophytes for grass carp and to provide useful information to develop strategies for the restoration of submerged macrophytes in ecosystems containing herbivorous fishes. We here measured the relative consumption rate (RCR) of grass carp with regard to four submerged macrophytes: Hydrilla verticillata (H. verticillata), Vallisneria natans (V. natans), Ceratophyllum demersum (C. demersum), and Myriophyllum spicatum (M. spicatum). Each submerged macrophyte was further analyzed for physical, nutritional, and chemical traits to assess whether any of these parameters were correlated with palatability for grass carp. Additionally, we investigated the effect of crude extracts from the least palatable plant (M. spicatum) on the activity of intestinal proteinases and the gut bacterial community.
Methods Study organisms During spring time (March 2016), grass carp (mean ± SD, weight 80 ± 5 g; total length 19 ± 0.5 cm) were purchased from the Xiantao County Fishery, Hubei Province, China (30° 27′ N, 113° 52′ E) and reared in laboratory conditions for 1 week before experiments. All animal experiments were approved by the Institute of Hydrobiology, Chinese Academy of Sciences (Approval ID: IHB 2013724) and were conducted in accordance with the Guiding Principles for the Care and Use of Laboratory Animals. Four species of submerged macrophytes (H. verticillata, V. natans, C. demersum, and M. spicatum) were collected from East Lake, Wuhan City, China (30° 53′ N, 114° 38′ E). Before the experiment, running tap water was used to flush away marl and periphyton coverage on the macrophytes. Feeding assays Feeding assays were performed in aquaria (100 cm × 50 cm × 50 cm), in which the flow rate of recirculating water of 26 °C was maintained at 25 L min −1 and the water was filtered through biofilters to remove fish feces and to reduce the ammonia concentration. To avoid stressful conditions caused by low oxygen concentrations, aeration was maintained at a constant level in aquaria. In each aquarium, there were five grass carp that have been fed pond food for 2 days of acclimatization (Dorenbosch and Bakker 2011). Before feeding assays, grass carp had been starved for 24 h. During the experiments, grass carp were permitted to feed for 2 h and there was no mortality of grass carp. Feeding assay with fresh submerged macrophytes Each of four species of fresh submerged macrophytes (100 g) was added to six aquaria, with six replicates for each species for a total of 24 aquaria. Given autogenic mass change in the macrophytes, the paired control in other six aquaria contained water and macrophytes without grass carp. After removing excess water with a paper towel, we weighed macrophyte biomass at the beginning and the end of the experiment. The percent RCR was calculated as [H0 × Cf/C0 ‐ Hf]/W × 100%, where H0 and Hf were the wet masses of macrophytes exposed to grass carp before and after the experiment, respectively; C0 and Cf were the wet masses of macrophytes from the paired control before and after experiment, respectively; W was the mass of grass carp (Wong et al. 2010). Feeding assay with reconstituted macrophytes To control for plant structure, each species of fresh macrophytes was lyophilized at − 70 °C for 24 h to a constant weight
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and then finely ground to a homogeneous consistency by sieving through a 149-μm mesh screen. First, heating agar and distilled water were put in a microwave oven until they melt, then the resulting plant material from each species was added to the mixture and stirred evenly to a final ratio of 1 g plant material: 5 mL water: 0.5 g agar. This agar-based food was formed into a uniform cylinder of 2 mm diameter using a mold. After it solidified, we cut the cylinder into pellets of 7 mm size. In assays using the artificial pellets, each of the four types of pellets was added to six aquaria, with six replicates for each type for a total of 24 aquaria. The number of pellets provided to the grass carp before experiment was counted as N0 and the number of pellets remaining after the experiment was Nf. RCR was then calculated and expressed as a percentage of each type of pellet using the formula (Nf ‐ N0) × G/W × 100%, where G was the weight of each pellet; W was the mass of grass carp (Miller and Provenza 2007).
measured at 620 nm after dissolving dried macrophytes with 60% mass/volume sulfuric acid and adding 2% mass/volume anthranone. Soluble protein content of macrophytes was determined at 595 nm after grinding the plant material and using Coomassie Brilliant Blue G-250 to react. Lysine was measured at 500 nm after degreasing dried ground macrophytes with 60–90 °C petroleum ether for 8 h and addition of ninhydrin. The amount of tannins was estimated using permanganate titration methods, where the solution turned bright golden yellow after slow addition of 0.1 mol L−1 KMnO4. The concentration of flavonoids was analyzed at 510 nm after addition of 5% mass/volume NaNO2, 10% mass/volume Al(NO3)3, and 4% mass/volume NaOH into the 70% ethanol extract. All analyses for the same individual macrophytes were performed in triplicates except for firmness and shear force, which were conducted in quintuplicates.
Feeding assay with PSMs
Crude extract was obtained from M. spicatum using the same method as described above. We then prepared five concentrations of PSMs solutions (0, 18, 180, 1800, 18,000 μg flavonoids·L−1). The solutions were stored at 4 °C until enzyme assays were conducted.
According to the results of two feeding assays above, very little M. spicatum was consumed either in the form of fresh plant or reconstituted pellets; therefore, we added its crude extracts into H. verticillata (the most palatable plant) to assess whether PSMs influenced palatability for grass carp in the third assay. For M. spicatum, five different masses (0, 1.25, 2.5, 3.75, and 5 g) of lyophilized and ground plant powder were extracted in 70% ethanol three times for 24 h each, followed by further extraction with petroleum ether thrice (exhibiting a boiling range of 30–60 °C) to remove chlorophyll and lipid. These crude extracts were concentrated by rotary evaporation and then we got five concentrations of crude extracts named 0 PSMs, 0.25 PSMs, 0.50 PSMs, 0.75 PSMs, and 1.00 PSMs. These crude extracts were added to 5 g of H. verticillata powder separately. Five different food pellets were prepared and RCR was calculated for each type of pellet as described above. Measurement of plant traits To understand the palatability of submerged macrophytes for grass carp, we measured three aspects of their traits: physical properties (firmness, shear force, dry matter content (DMC), ash, and fibrin), nutritional contents (soluble protein and lysine) and chemical contents (tannins and flavonoids). All trait measurements were performed on randomly chosen plant samples. Firmness and shear force of four macrophytes were examined at 2 cm from the top of each plant, using a texture analyzer (TA. XT Plus). DMC was determined following Elger and Lemoine (2005). Ash content was assessed according to the methods described by Qiu and Kwong (2009). Fibrin was
Effects of PSMs on the activity of intestinal proteinases
Effects on protease activity Intestinal tracts were dissected from five grass carp and divided into three sections (foregut, midgut, and hindgut). After removing the gut contents by ice-cold stoke-physiological saline solution, the tissues were homogenized at a ratio of 1 g tissue: 9 mL phosphate buffer saline (PBS, 0.1 mol/mL, pH 8.0). After centrifugation at 12,000 r min−1 for 20 min at 4 °C, three kinds of intestinal crude enzymes were obtained and incubated with the five PSM concentrations separately for 10 min at 37 °C, followed by addition of 0.5% casein to react at 37 °C for 20 min. Then, 10% trichloroacetic acid was added to terminate the reaction, and optical density (OD) was measured at 680 nm after the addition of 0.55 mol L−1 Na2CO3 and Folin-Phenol to react at 37 °C for 15 min. The unit of protease activity in the fish intestine is described as unit per gram proteases. Briefly, 1 U g−1 prot is expressed as 1 g proteases hydrolyzing casein to generate 1 μg tyrosine at 37 °C for 1 min. All analyses were performed in triplicates. Effects on trypsin activity Trypsin activity in the foregut, midgut, and hindgut of grass carp was determined by spectrophotometry using kits from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The unit of enzyme activity was expressed as unit·per milligram proteases based on the manufacturer’s recommendations, which was expressed as the equivalent enzyme activity
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required to generate an optical density (OD) change of 0.003 at pH 8.0 and 37 °C. All analyses were performed in triplicates. Effects of PSMs on the bacterial community in grass carp intestine. Two treatments were used: one containing six fish that were fed food pellets containing H. verticillata only (HV) and another containing six fish that were fed food pellets containing H. verticillata with crude extracts from M. spicatum at a concentration of 1.00 PSMs (HVP). The food pellets were prepared in the same way as described above. The fish were fed twice a day (09:00, 15:00 h) with 30 g food pellets to last 40 days in the two aquaria, which were filled with water of 26 °C from the same source that was changed once a day. At the end of the experiment, four fish from each aquarium were chosen randomly and their percentage weight gain (PWG) was calculated as [(Wf ‐ W0)/ W0] × 100%, where W0 and Wf were the masses of grass carp before and after the experiment, respectively (Wang et al. 2015). The intestinal contents were then collected from each fish and stored in a refrigerator using the same method as described by Wu et al. (2012). DNA extraction, PCR, and high-throughput 16S rRNA pyrosequencing of each sample were performed following the methods described by Borsodi et al. (2017).
1.50% for M. spicatum to 7.29% for H. verticillata. There were significant differences in the RCR among macrophyte treatments (one-way ANOVA, d.f. = 3, F = 104.237, P < 0.001). The order of RCR of the four macrophyte species was H. verticillata > V. natans > C. demersum > M. spicatum (Fig. 1a). Combined with the results in Fig. 2 that the order of the firmness of each species was M. spicatum > C. demersum > H. verticillata = V. natans and the order of shear force was M. spicatum > C. demersum > H. verticillata > V. natans, this indicated that an increase in firmness and shear force can
Data analysis All statistical analyses were performed using SPSS IBM Statistics version 23, Illinois, USA. RCR of each feeding assay, physical/nutritional/chemical traits of submerged macrophytes, and the proteinases activity of each gut were assessed the differences (P < 0.05) by using one-way ANOVA, followed by the Tukey post hoc tests to identify homogenous groups. PWG of grass carp and microorganisms of HV and HVP at genus level were compared using independent samples t test, respectively. For sequence analysis of gut microbiota, the Shannon index was determined on the base of the calculated operational taxonomic units (OTUs) by Mothur ver. 1.30.1 (Giatsis et al. 2015). The sequences gained were phylogenetically classified to the phylum and genus levels at 97% similarity for the community composition analysis. MEGA 6.0 was used to depict a phylogenetic tree with the neighbor-joining algorithm. The representative sequences from each OTU were subjected to the RDP-II Classifier of the Ribosomal Database Project (RDP) and the National Center for Biotechnology Information (NCBI) BLAST for taxonomic analysis.
Results and discussion Feeding assays When grass carp were offered four different species of fresh submerged macrophytes for 2 h, their RCR ranged from
Fig. 1 Mean (± SD) relative consumption rate (RCR) of submerged macrophytes by grass carp: a fresh submerged macrophytes, b reconstituted macrophytes, c crude chemical extracts from Myriophyllum spicatum. Different letters (a, b, and c) above each bar represent statistical differences between species based on RCR
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decrease RCR of grass carp, and macrophyte selection by grass carp can be determined, at least in part, by the structure of submerged macrophytes.
The RCR of reconstituted macrophytes by grass carp differed among species, but there was no significant difference between H. verticillata, V. natans, and C. demersum (one-way
Fig. 3 Effects of plant secondary metabolites (PSMs) on protease activity in the gut of grass carp: a foregut, b midgut, c hindgut. Data are presented as mean ± SD. Different letters (a, b, and c) above each bar represent statistical differences between different concentrations of PSMs
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Fig. 4 Effects of plant secondary metabolites (PSMs) on trypsin activity in the gut of grass carp: a foregut, b midgut, and c hindgut. Data are presented as mean ± SD. Different letters (a and b) above each bar represent statistical differences between different concentrations of PSMs
ANOVA, d.f. = 2, F = 3.732, P = 0.089) (Fig. 1b). Compared with RCR of fresh macrophytes, RCR was less for reconstituted macrophytes (Fig. 1a, b). This suggested that the lyopholization-grinding process may degrade cells, causing secondary metabolites to be released more readily, leading to decreased intake by grass carp. M. spicatum was the least palatable when presented either as fresh or reconstituted plant matter. When the crude extracts from M. spicatum were combined with the most palatable H. verticillata, the RCR of grass carp decreased while the dose of PSMs increased (one-way ANOVA, d.f. = 4, F = 107.646, P < 0.001). After adding 0.5 PSMs or more, the feeding rates were as little as 0 (Fig. 1c). These results suggested that PSMs have a significant effect on reducing the RCR of grass carp, presumably due to recognition by olfaction or gustation. PSMs of M. spicatum were determined to be quantitative defenses and dose dependent (Hay 2016). PSMs contain many chemical deterrents such as tannins and flavonoids (Table S1). Furthermore, some reports have demonstrated that herbivores exhibit low preference for plants with relatively high levels of secondary metabolites (Parker et al. 2006; Dorenbosch and Bakker 2011; Goodman and Hay 2013; O’brien and Scheibling 2016).
Effects on trypsin activity In the foregut, trypsin activity was significantly improved at concentrations of 180, 1800, and 18,000 μg L−1 PSMs compared to the concentrations of 0 and 18 μg L−1 (one-way ANOVA, d.f. = 4, F = 10.765, P = 0.001). In the midgut, there was a significant difference only at the concentration of 18,000 μg L − 1 PSMs (one-way ANOVA, d.f. = 4, F = 4.423, P = 0.026). However, in the hindgut, trypsin activity was not affected by different concentrations of PSMs (oneway ANOVA, d.f. = 4, F = 0.807, P = 0.548). (Fig. 4). The results above suggest that PSMs of M. spicatum can improve the activity of intestinal proteinases, which is a contrast to research showing that tannins, phenolics, and some specific monoterpenes can limit protein digestibility in terrestrial organisms intestine (Lowry et al. 1996; Niho et al. 2001; Dearing et al. 2005b; Degabriel et al. 2009; Kohl et al. 2015;). Additionaly, Mouradov and Spangenberg (2014) demonstrated that flavonoids inhibit activity of Table 1 Number of sequences, OTUs number, and Shannon index of microbial communities in grass carp intestine Sample
Number of sequences
OTUs number
Shannon index
HV1 HV2 HV3 HV4 HVP1 HVP2 HVP3 HVP4
14,358 15,089 15,528 12,245 3382 4455 3471 3736
887 768 655 716 429 409 448 389
4.02 3.65 3.14 3.94 3.62 3.69 3.90 3.26
Effects of PSMs on the activity of intestinal proteinases Effects on protease activity The protease activity in the foregut, midgut, and hindgut of grass carp responded similarly to the increase in concentration of PSMs. Protease activity significantly increased at concentrations of 1800 and 18,000 μg L−1 (foregut: P < 0.001; midgut: P < 0.001; hindgut: P < 0.001). However, there was no difference at 0, 18, or 180 μg L−1 (Fig. 3). These results demonstrated that low levels of PSMs did not affect protease activity, and that high levels can contribute to improving the activity.
HV and HVP represent the treatments of fish feeding food pellets containing H. verticillata only and food pellets containing H. verticillata with crude extracts from M. spicatum at a concentration of 1.00 PSMs, respectively. OTUs is abbreviated as operational taxonomic units
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proteinases. The reason for this may be that the inhibition caused by these metabolites was much less than the improvement from other molecules that were not detected or tested for. Effects of PSMs on the bacterial community in grass carp intestine To identify the bacterial communities from eight intestinal content samples, more than 3300 sequences were obtained for each sample. After downstream analyses, 4701 OTUs were obtained at a 97% sequence identity threshold. In each sample, 389 to 887 OTUs were observed. The sequence information and diversity index (Shannon) of the samples are shown in Table 1. Hierarchical clustering analysis was used to show the similarity in the bacterial community structure among the eight intestinal content samples (Fig. 5). There were two clear clusters Fig. 5 Taxonomic composition of bacterial 16S rRNA gene reads at a phylum level and b genus level from eight intestinal content samples
observed: one group consisting of samples collected from HV and another one clustered with samples collected from HVP, which indicated a clear distinction in the bacterial community structure between HV and HVP. To compare the phylogenetic differences in the compositions of the bacterial community structures of intestinal content in HV and HVP, relative bacterial community abundance was characterized at the phylum and genus levels (Fig. 5). At the phylum level, there were 36 phyla classified for each and the major phyla of the samples were uniform, but their relative abundances differed (Fig. 5a). For example, Proteobacteria, detected as the dominant phylum, were less abundant in HV (60.75–67.24%) than in HVP (67.86–73.58%). However, Firmicutes (19.93–24.74 vs. 17.99–19.75%) and Planctomycetes (1.89–3.97 vs. 0.91–1.86%) were more abundant in HV as compared to HVP. At the genus level, the prevalent species in HV were Exiguobacterium (17.04–22.10%),
Environ Sci Pollut Res Fig. 6 Comparison analysis of microorganisms between two treatments (HV vs. HVP) at genus level. HV and HVP represent the treatments of fish feeding food pellets containing H. verticillata only and food pellets containing H. verticillata with crude extracts from M. spicatum at a concentration of 1.00 PSMs, respectively
Acinetobacter (6.39–7.65%), Methylocystis (3.63–7.06%), and Blastopirellula (0.42–1.00%). Yet, Exiguobacterium (13.20– 16.07%), Acinetobacter (4.09–5.68%), Methylocystis (0.65– 1.58%), and Hydrogenophaga (1.12–1.59%) were dominant in HVP (Fig. 5b). A comparison analysis of microorganisms with a significant difference between the two treatments (HV vs. HVP) at the genus level showed that the two major species, Exiguobacterium and Acinetobacter, were lower in HVP (t test, for Exiguobacterium, d.f. = 6, F = 2.793, P = 0.007; for Acinetobacter, d.f. = 6, F = 0.150, P = 0.004) (Fig. 6), suggesting that PSMs have negative effects on the abundance of bacterial community in the grass carp intestine. As protease-producing, lipase-producing, and cellulaseproducing bacteria, Acinetobacter in the digestive tracts of fishes can yield protease, lipase, and cellulose activity (Jiang et al. 2011; Ray et al. 2012). Similar to Acinetobacter, the genus Exiguobacterium can secrete alkaline protease (Vishnivetskaya et al. 2009). Thus, gut microbiota play an essential role in the digestion of food and could be affected by multiple factors, such as diet (Liu et al. 2016). Therefore, PSMs can lessen enzymatic activity in the intestines by decreasing the abundance of Acinetobacter and Exiguobacterium, which in turn may weaken the metabolic capacity of grass carp and cause series of adverse effects on growth. After feeding for 40 days, the PWG of grass carp in HVP was 1.67% and in HV was 5.45%. The PWG in HVP was significantly lower than that in HV (t test, d.f. = 5.412, F = 5.731, P = 0.012). Thus, the disadvantageous feedback signal for the growth and development of grass carp induced by PSMs may motivate grass carp to feed on the palatable submerged macrophytes with lower PSMs.
Conclusions This study was undertaken to explore the palatability mechanisms in grass carp for submerged macrophytes. The results
demonstrated that the order of palatability of the four species of fresh submerged macrophytes species was H. verticillata > V. natans > C. demersum > M. spicatum. These results demonstrated that not only the structure of macrophytes but also PSMs played roles in affecting palatability for grass carp. Higher firmness and shear force can decrease the RCR of grass carp. And higher content of PSMs can reduce palatability of macrophytes by significantly decreasing the abundance of Exiguobacterium, Acinetobacter-yielding protease, lipase, and cellulose activity, but not by weakening the activity of intestinal proteinases.
Acknowledgements This work was financially supported by the National Science and Technology Support Program of the 12th FiveYear Plan, China (No. 2012BAJ21B03-04), the National Natural Science Foundation of China (51178452), the Major Science and Technology Program for Water Pollution Control and Treatment of China of the 12th Five-Year Plan (No. 2012ZX07101007-005), the Knowledge Innovation Program of the Chinese Academy of Sciences and the Hubei Province Science Foundation for Youths (2014CFB282). We thank Drs. Yafen Wang, Qiaohong Zhou, Biyun Liu, Enrong Xiao, Dong Xu, Junmei Wu, and Ms. Liping Zhang for the experimental help (Institute of Hydrobiology, Chinese Academy of Sciences).
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