Influence of timing of fishing on trophic levels and diets of typical fish and invertebrate species in the Bohai Strait over a single year based on carbon and nitrogen isotope analysis

Ecological Indicators 70 (2016) 348–356

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Influence of timing of fishing on trophic levels and diets of typical fish and invertebrate species in the Bohai Strait over a single year based on carbon and nitrogen isotope analysis Pei Qu a,b , Min Pang a , Qixiang Wang c , Zhao Li d , Chengyue Liu b , Zhipeng Zhang b , Xuexi Tang b,∗ a

The First Institute of Oceanography, State Oceanic Administration, Qingdao 266061, China College of Marine Life, Ocean University of China, Qingdao 266003, China c Marine Biology Institute of Shandong Province, Qingdao 266104, China d China National Environmental Monitoring Centre, Beijing 100029, China b

a r t i c l e

i n f o

Article history: Received 27 November 2015 Received in revised form 12 June 2016 Accepted 15 June 2016 Keywords: Bohai Strait Fishing periods Predation Stable isotopes Trophic levels

a b s t r a c t Fishing is the most widespread human exploitation of marine resources, which has an annual cyclical influence on aquatic species in Chinese offshore waters. This study used carbon and nitrogen isotopic ratios as tracers to reveal the changes in trophic level and dietary composition of offshore organisms during four cruises in March, June, August and November 2014. The results indicated that the trophic levels of fishes declined during two fishing periods, from March (average trophic level = 3.36) to June (3.01), and from August (2.99) to November (2.57), while most invertebrates did not show this trend. The self-restoring ability of this ecosystem was reflected in the trophic level changes after the closed fishing season (from June 1 to September 1). The trophic levels of fishes remained stable, and some species even recovered such as Enchelyopus elongates (trophic level increased from 2.84 in June to 2.86 in August), Cryptocentrus filifer (from 3.10 to 3.12), and Ernogrammus hexagrammus (from 2.91 to 2.96). According to the trophic results, we selected the invertebrates Octopus minor and Asterias amurensis from the top trophic levels for dietary composition analysis. The composition of their diets changed significantly after fishing periods, and the proportions of some smaller and “non-commercial” species increased, such as Notoacmea schrenckii and Chlorostoma rustica. After the closed fishing season, the larger and “commercial” species contributed a greater proportion to their diet composition. These results indicated that the closed fishing season should be prolonged to give the ecosystem enough time to restore itself and further halt the trend of this fishery towards environment deterioration. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Analyses of stable carbon (13 C/12 C) and nitrogen (15 N/14 N) isotope ratios in organisms and food sources can be used as tracers to study nutritional relationships among species in ecological studies (Michener and Lajtha, 2007). Nitrogen stable isotope values (␦15 N) of a consumer change with changes in its trophic position through isotopic fractionation or discrimination during metabolic processes (Fry and Sherr, 1984; Fry, 2006). Controlled laboratory experiments show that the ␦15 N values of animals are significantly positively correlated with the prey that they consume (Post, 2002; Fry, 2006;

∗ Corresponding author. Present address: 5 Yushan Road, 266003 Qingdao, Shandong Province, China. E-mail address: [email protected] (X. Tang). http://dx.doi.org/10.1016/j.ecolind.2016.06.029 1470-160X/© 2016 Elsevier Ltd. All rights reserved.

Sweeting et al., 2007; Caut et al., 2009). ␦15 N values can act as timeintegrated indicators of trophic position (Wada et al., 1991; Vander Zanden and Rasmussen, 2001). The average increases of ␦15 N values per trophic level have been investigated in previous studies. The per-trophic-level fractionation values most commonly used ranged from 2 to 4‰ (Minagawa and Wada, 1984; Post, 2002; Sweeting et al., 2007; Caut et al., 2009). These fractionation values were used to build isotope mixing models for the assessment of the trophic structure of food chains involving multiple food sources (Phillips and Koch, 2002; Deehr et al., 2014). Many laboratory and field studies have found that carbon isotopic compositions in animals can reflect their dietary sources (Vander Zanden and Rasmussen, 2001; Fry, 2006; Michener and Lajtha, 2007), and that stable carbon isotope (␦13 C) values are indicative of food source origins (Cherel et al., 2010). However, indi-

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vidual animals have several primary food sources, which make it difficult to find a single solution to the proportional contributions of multiple food sources. (Phillips, 2012) built a computer program “IsoSource” to solve this problem. The program has been successfully applied to food web studies of bears (Ben-David and Flaherty, 2012), fish (Sarà et al., 2004), shrimp (Burford and Lorenzen, 2004), and humans (Newsome et al., 2004). In the northern China Sea, although both ␦15 N and ␦13 C values have been studied to investigate trophic levels and food source composition, isotope based trophodynamic assessment has been limited. The Bohai Sea was once the most important fishing region in the northern China Sea, and provided the main spawning grounds, feeding grounds and habitats for a variety of fishery biological resources (Xu et al., 2010, 2011). In recent years, the development of the Chinese Bohai Sea Economic Zone placed extra pressure on the biological fishery resources and their ecosystems, and continued overfishing, marine reclamation and pollution resulted in the loss of biodiversity and ongoing deterioration in habitat quality (Jin, 2004; Nie and Tao, 2010; Xu et al., 2011 Zheng and You, 2014). The quality and quantity of biological resources in the area has declined sharply, reducing high quality resources, increasing inferior ones and selecting for smaller sizes in fishery species as their age at sexual maturity fell (Jin, 2004; Xu et al., 2010; Ribeiro et al., 2013; Durán et al., 2013; Zheng and You, 2014). Furthermore, some traditional fishery species with larger body sizes and higher trophic levels have gradually disappeared from the Bohai Sea, such as Trichiurus haumel and Cynoglossus semilaevis (Shan et al., 2012). Because the adverse consequences of overfishing appear over long periods of time, they are always ignored in favor of immediate, temporary benefits. According to previous research on the changes in local fishery resources (Jin, 2004; Nie and Tao, 2010; Xu et al., 2011; Zheng and You, 2014), we predicted that the trophic level and diets of local species may be affected by fishing activities. The objectives of the present study were therefore to analyze the influence of the timing of fishing on common species’ trophic levels (Minagawa and Wada, 1984; Post, 2002; Sweeting et al., 2007; Caut et al., 2009) and predation (Vander Zanden and Rasmussen, 2001; Fry, 2006; Michener and Lajtha, 2007) over a single year using nitrogen and carbon isotopic ratios of common species around Xiaoheishan Island in the Bohai Sea during four specific periods in 2014, in order to reveal the changes in trophic levels and predation rates caused by periodic fishing activity. 2. Methods 2.1. Sampling area The Bohai Strait lies between the Liaodong and Shandong Peninsulas, and is the sole channel connecting the Bohai Sea and the northern Yellow Sea. It has clear seasonal characteristics typical of a continental monsoon climate. Winter starts in November and the seawater temperature drops considerably. Deep winter follows from December to March. June to August comprises the summer period, and April to May and September to November are transitional periods. The sampling stations in this study were located in the coastal waters off Xiaoheishan Island in the Bohai Strait (Fig. 1) at longitude 120◦ 38 00 E to 120◦ 40 00 E and latitude 37◦ 57 30 E to 37◦ 58 30 E. 2.2. Sampling periods Our study focused on the influence of fishing activities on the fishery biological resources over a single year, so the choice of sampling periods was important. Because stable isotopic ratios link

349

Fig. 1. Sampling area and stations (S1, S2, S3).

the assimilatory accumulation of consumers during the preceding period, we chose sampling periods of 18–20 March, 1–3 June, 29–31 August and 17–19 November, marking the beginning and end of fishing activities in the Bohai Sea. • Fishing activities were lower from November to March due to lower seawater temperatures, and samples collected in March reflect the variation of trophic relationships during this winter period. • A fishing moratorium forbidding fishing activity was in force during the summer period from June to August. Samples collected in August reflected the trophic relationship variation during this period. • During spring (from April to June) and autumn (from September to November), fishing activity was at a peak and biological resources were most affected by human activity. Samples collected in June and November reflected the variation in these two periods, respectively. 2.3. Sample preparation and stable isotope analysis Samples were taken using trapping cages as stationary fishing devices with 20 jointed metal square frameworks with two ends fixed to stones on the seabed. The sampling period extended over four tidal cycles (two days). After onboard identification, the specimens were photographed and then frozen at −20 ◦ C until stable isotope analysis. Zooplankton samples were collected from vertical hauls using a 70 ␮m mesh plankton net, followed by sieving through a 500 ␮m mesh. Specific pretreatment processes were required according to the heterogeneity of particular samples. Whether samples were treated in whole or in part was dependent on the feeding habits of consumers. Since prey at lower trophic levels are often ingested as whole organisms, the entire bodies of benthic invertebrates were used for stable isotope analyses, excluding inorganic carbon material such as carbonate shells and skeletons (Pinnegar and Polunin, 1999; Pitt et al., 2009); carbonate was removed from the samples by hydrochloric acid treatment. In contrast, tissues such as the muscle of higher level prey had undergone some digestion and so reflected longer term information regarding food selection (Pitt et al., 2009; McIntyre and Flecker, 2006). Consequently, the white dorsal muscles of fishes obtained through dissection were used for further isotope analyses. Since acidification not only removes carbonate evidence but can also distort ␦15 N values in food sources (Pinnegar and Polunin, 1999; Kanaya et al., 2007), all of the samples collected were divided into two portions for separate carbon and nitrogen stable isotope analyses. One part was treated with hydrochloric acid (1 mol/L) to remove calcium carbonate for ␦13 C analysis, while the other,

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non-acidified, part was used directly for ␦15 N analysis. All samples were cut into small pieces, oven dried for at least 48 h at 80 ◦ C until constant weight was achieved, and then homogenized into a fine powder using a mortar and pestle. After pretreatment, carbon and nitrogen isotope ratios were determined by continuous flow isotope ratio mass spectrometry (CF-IRMS) using a Thermo Delta VTM isotope ratio mass spectrometer. Stable isotopic ratios were expressed in standard ␦-unit notation (␦13 C and ␦15 N), defined as follows: ı13 C(‰) = ( ı15 N(‰) = (

13 C/12 C

sample 13 C/12 C VPDB

15 N/14 N

sample 15 N/14 N air

− 1) × 1000 − 1) × 1000

(1)

(2)

where 13 C/12 Csample and 15 N/14 Nsample are the ratios of heavy isotopes to light isotopes in the samples, 13 C/12 CVPDB represents the Vienna Pee Dee Belemnite (VPDB) standard for 13 C, and 15 N/14 Nair represents atmospheric N2 for 15 N, respectively. 2.4. Trophic level and diet analyses Because nitrogen isotopic ratios in a predator can be enriched in a predictable way as the food chain moves up the trophic ladder (Smit et al., 2005), the fractionation of nitrogen stable isotope tends to result in isotopic differences between trophic levels. The trophic level can therefore be calculated according to the traditional model (Cresson et al., 2014; Valls et al., 2014) as follows: (3) TL = 2 + (␦15 Nconsumer − ␦15 Nreference )/␦15 NTEF where TL is the trophic level, ␦15 Nconsumer is the nitrogen isotopic ratio of consumers, ␦15 Nreference is the nitrogen isotopic ratio of the marine primary consumers (zooplankton), and ␦15 NTEF is the trophic enrichment factor. Because the primary consumers were at the bottom of the trophic ladder, they were assumed to occupy the 2nd trophic level in this study. For this model, two key parameters, ␦15 NTEF and ␦15 Nreference , mainly determined the uncertainty. ␦15 NTEF determined the relative distance between two adjacent trophic levels in the TL model, and changes with territory and species (Post, 2002; Sweeting et al., 2007; Akin and Winemiller, 2008; Caut et al., 2009; Plass-Johnson et al., 2013). Caut et al. (2009) summarized estimates of ␦15 NTEF variations based on 66 publications and found that the ␦15 NTEF value should be determined specifically for a local area. Therefore, we adopted the ␦15 NTEF value of 2.5‰ that had been reported in a previous study on neighboring waters (Cai et al., 2005). This value also approximates the average ␦15 NTEF of fish and invertebrates calculated in the research of Caut et al. (2009). ␦15 Nreference was the other key parameter in TL calculation using the traditional model formula; this determines the threshold of the trophic structure. Generally, in this formula, the nitrogen isotope ratios of primary consumers such as zooplankton (Post, 2002; Fernández de Puelles et al., 2014) and bivalves (Cai et al., 2001; Vander Zanden and Rasmussen, 2001) were considered as the ␦15 Nreference . Diets were analyzed using IsoSource (Phillips and Gregg, 2003), which has already been applied to other food web studies successfully (Ben-David and Flaherty, 2012; Sarà et al., 2004; Burford and Lorenzen, 2004; Newsome et al., 2004). When the number of food sources in a food web is small, isotope mixing models can provide unique solutions regarding their contributions to the diets of consumers. The IsoSource model can provide upper and lower limits for the contribution of each source, and all possible solutions for each source contribution (0%–100%) can be examined in small increments (1%–2%). The frequency and range of potential source contributions can then be represented with histograms. According to the series of reports by Phillips (2012), Phillips et al. (2005),

Fig. 2. The ␦15 N (‰) change of samples in water off Xiaoheishan Island.

and Newsome et al. (2004), the IsoSource model can be mainly applied to determine the distribution of all feasible solutions, and the uncertainty derived from this model is that it generates a set of solutions rather than a unique value. When we further calculated the mathematical expectations of each given food source using formula (4), the uncertainty in this process was ineluctable. Therefore, to control the model’s uncertainty and to avoid the misrepresenting results, the distribution of feasible solutions should also be considered.



100%

E (x) =

xf (x) dx

(4)

0

where E(x) is the plausible contribution of the given food source and f(x) is the percent frequency of x contribution. We used ␦13 C data to determine the primary food sources of each predator. 2.5. Statistical analysis Graphics were generated using ArcGIS software v.10.2.2 (ESRI, Redlands, CA, USA) and Microsoft Excel 2010 (Microsoft Corporation, Redmond, WA, USA). All statistical analyses were performed using the statistics package SPSS v.17.0 (SPSS Inc., Chicago, IL, USA). Calculated results were compared using the t-test and analysis of variance. 3. Results 3.1. Stable isotope analyses Samples of 34 species were collected in four cruises in 2014. We divided the samples into two main categories: 18 species of fishes and 16 species of invertebrates. Some species, such as Sebastes schlegelii, Enchelyopus elongates, Charybdis japonica, and Asterias amurensis, were collected on all four cruises, while some, like Scombermorus niphonius, Chelon affinis, Oratosquilla oratoria, and Alpheus japonicas were not (Table 1). Therefore, we focused on the species captured in all four cruises, which could better indicate the influence of fishing periods (Tables 2 and 4). The ␦15 N of fishes collected in the study area were significantly higher than in invertebrates (t-test, t34 = 7.176, p < 0.01). The averages of fish and invertebrate ␦15 N values in each cruise also show a similar result (Fig. 2). In March, the average ␦15 N of fishes and invertebrates were 13.68‰ and 10.61‰, respectively. The averages of fish and invertebrate ␦15 N were 11.84‰ and 11.35‰ in June, 13.10‰ and 11.15‰ in August, and 11.88‰

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Table 1 Sampling of fishes and invertebrates in 2014 (March, June, August, November, “*” denotes collected in four cruises). Fishes

* * * * * *

Invertebrates Species

Phylum

Cryptocentrus filifer Enchelyopus elongatus Enchelyopus fangi Ernogrammus hexagrammus Hexagrammos otakii Sebastes schlegelii Pleuronichthys yokohamae Eupleurogrammus muticus Acanthopagrus schlegelii Thamnaconus septentrionalis Platycephalus indicus Clupanodon punctatus Liparis tanakae Hemiramphus sajori Raja pulchra Chelon affinis Sebastiscus marmoratus Scombermorus niphonius

Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata Chordata

* * * * * * * * * * * *

Species/family

Phylum

Chlorostoma rustica Mytilus galloprovincialis Mya arenaria Notoacmea schrenckii Ostreidae Octopus minor Actiniidae Apostichopus japonicas Anthocidaris crassispina Asterias amurensis Charybdis japonica Oregonia gracilis Oratosquilla oratoria Alpheus japonicus Aplysiidae Rapana venosa

Mollusca Mollusca Mollusca Mollusca Mollusca Mollusca Cnidaria Echinodermata Echinodermata Echinodermata Arthropoda Arthropoda Arthropoda Arthropoda Mollusca Mollusca

Table 2 Sampling number (n), ␦15 N and ␦13 C (mean values ± SD) of six species fishes in March, June, August and November in 2014. Species

Cryptocentrus filifer Enchelyopus elongatus Enchelyopus fangi Ernogrammus hexagrammus Hexagrammos otakii Sebastes schlegelii

March

June

August

November

n

␦15 N (‰)

␦13 C (‰)

n

␦15 N (‰)

␦13 C (‰)

n

␦15 N (‰)

␦13 C (‰)

n

␦15 N (‰)

␦13 C (‰)

5 7 3 3 16 2

14.46 ± 0.70 12.87 ± 1.27 13.62 ± 0.72 13.82 ± 0.72 15.27 ± 1.05 12.94 ± 1.07

−18.25 ± 0.24 −17.55 ± 0.81 −15.36 ± 0.04 −18.01 ± 0.21 −17.55 ± 0.41 −19.16 ± 0.28

3 3 7 2 10 15

13.17 ± 0.98 12.52 ± 1.15 12.44 ± 1.00 12.70 ± 0.64 13.63 ±1.20 13.26 ± 1.15

−18.13 ± 0.27 −18.01 ± 0.64 −19.19 ± 0.41 −18.59 ± 0.33 −19.59 ± 0.35 −18.99 ± 0.32

23 3 3 3 39 11

13.21 ± 0.80 12.56 ± 0.72 12.42 ± 1.50 12.82 ± 0.77 12.85 ± 0.72 13.53 ± 1.20

−18.75± 0.40 −20.06 ± 0.55 −17.11 ± 0.06 −18.11 ± 0.33 −19.96 ± 0.55 −19.37 ± 0.21

4 12 3 3 2 5

11.78 ± 0.90 11.66 ± 1.00 11.82 ± 1.01 11.48 ± 0.89 12.49 ± 0.70 11.87 ± 0.71

−17.79 ± 0.17 −15.94 ± 0.68 −17.87 ± 0.17 −18.60 ± 0.23 −18.83 ± 0.21 −18.03 ± 0.12

and 10.86‰ in November, respectively. This result indicated the tendency of the trophic levels of most fishes to be higher than invertebrates. Of particular importance, however, was that, as the t-test results of each cruise indicated, the ␦15 N of fishes were significantly higher than invertebrates in March (t = 4.008, p < 0.01) and August (t = 2.413, p < 0.05), but not in June (t = 1.565, p = 0.146) and November (t = 1.935, p = 0.089), which mark the times of reduced intensity of fishing activity. In March, the ␦15 N of fishes ranged from 12.87‰ to 15.72‰, while the ␦15 N of invertebrates ranged from 9.13‰ to 12.28‰. In June, the ␦15 N of fishes ranged from 11.66‰ to 13.63‰ and the ␦15 N of invertebrates ranged from 8.56‰ to 14.72‰, although it is worth noting that the ␦15 N of some invertebrates, such as O. oratoria, were even higher than in fishes. In August, the ␦15 N of fishes ranged from 12.56‰ to 13.53‰ and the ␦15 N of invertebrates ranged from 7.35‰ to 12.86‰. In November, the ␦15 N of fishes ranged from 10.84‰ to 13.06‰ and the ␦15 N of invertebrates ranged from 10.86‰ to 11.45‰. The ␦15 N of fishes showed a significant trend and values declined from March to June, rose slightly in August, and then continued to decline in November (Fig. 2). The two periods of decline matched the corresponding timing of fishing activity. Nevertheless, invertebrates did not show a similar trend and their highest ␦15 N values appeared in June and August (Fig. 2). Compared to fishes, the trophic levels of invertebrates were less influenced by fishing activity, and invertebrates possibly even multiplied as a result of lower predation pressure from fishes. The ␦13 C values of both categories of samples ranged from −21.95‰ to −15.36‰, indicating their original sources. In contrast to ␦15 N, the ␦13 C values showed no significant trend during the year under study (Fig. 3). The averages for fishes and invertebrates were −18.04‰ and −19.05‰ in March, −18.55‰ and −19.38‰ in June, −18.68‰ and −19.53‰ in August, −17.54‰ and −17.52‰

Fig. 3. The ␦13 C (‰) change of samples in water off Xiaoheishan Island.

in November, respectively. We could not discern any trend due to the influence of fishing activity based purely on the ␦13 C values, so further study of the predation relationships based on ␦13 C is needed. 3.2. Trophic level changes Trophic levels of samples were calculated based on ␦15 N, using the primary consumers set as a reference at the second trophic level. In our study, typical fishes of six species, including Hexagrammos otakii, E. elongates, Enchelyopus fangi, Cryptocentrus filifer, S. schlegelii, and Ernogrammus hexagrammus, were captured dur-

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Table 3 The average trophic levels of invertebrates in a year. No.

Species

Average (␦15 N)

SD (␦15 N)

Trophic level

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Mytilus galloprovincialis Ostreidae Apostichopus japonicus Notoacmea schrenckii Mya arenaria Chlorostoma rustica Rapana venosa Actiniidae Anthocidaris crassispina Alpheus japonicus Aplysiidae Oregonia gracilis Asterias amurensis Charybdis japoncia Octopus minor Oratosquilla oratoria

9.22 9.47 9.56 9.76 10.50 10.56 10.57 10.67 10.77 11.67 12.22 12.52 12.89 13.04 13.43 13.93

0.41 0.42 0.43 0.44 0.47 0.48 0.45 0.48 0.49 1.85 1.45 1.75 1.31 0.78 0.56 1.76

1.12 1.22 1.26 1.33 1.63 1.65 1.66 1.70 1.74 2.10 2.32 2.44 2.59 2.65 2.80 3.00

three species of invertebrates appeared at high trophic levels of 2.59–2.80: O. minor, C. japonica, and A. amurensis, which were captured on all four cruises, and were selected for the analysis of their dietary composition. Their ␦15 N and ␦13 C values are shown in Table 4. Nine species at lower levels were identified as their potential food sources. Their contributions to predator diets in each fishing period were analyzed based on ␦13 C using the IsoSource procedure. Fig. 6 shows the dietary changes of O. minor (the contributing proportions of nine potential food sources), which can be divided into two Groups. Group One contained “non-commercial” species, such as Chlorostoma rustica, N. schrenckii, and Oregonia gracilis, and Group Two was composed of species with greater fishery value, including Ostreidae, Rapana venosa, Mytilus galloprovincialis, Apostichopus japonicas, C. japonica, and Mya arenaria. Each time period reflected the corresponding condition of ingested prey during the previous period. For example, O. minor collected in March revealed the assimilation from the previous December to that time, and O. minor sampled in June revealed the situation from March to June. According to the IsoSource analysis, the contribution of the nine species to the O. minor diet in March ranged from 8% to 14%. O. minor primarily preyed on species in Group One. Each species in that group provided 14% of the diet and thus 42% of the total of all the nine species. In June, the contribution of the nine resource species broadened and ranged from 6% to 21%. However, the top three contributors were still from Group One and their proportions increased significantly to 21%, 17%, and 16%, respectively. The total contribution to the diet reached about 54%. Correspondingly, the proportion of other food sources was reduced, e.g. R. venosa fell from 10% to 7%. In August, the diet of O. minor changed significantly, not only reflecting in the narrower range of proportions but also in the main primary food sources. The range of contributions narrowed to 4% (between 9% and 13%). Meanwhile, the primary food sources changed. C. japonica and M. arenaria became the main dietary contributors and their proportion increased to 12% and 13%, respectively. In November, the range of proportions widened again to 6%–16%. Group One reverted to the original top three contributors, accounting for 16%, 16%, and 15%, respectively. In summary for O. minor, Group One comprised the primary food sources and accounted for high proportions in three periods, but were not economically important fishery species. Only after the closed fishing season (from June to August), did the feeding intensity on each food source equalize and the contribution of prime fishery species such as C. japonica and M. arenaria increase. The dietary changes of A. amurensis showed a similar trend to O. minor, with the changes in response to fishing activities being more obvious (Fig. 7). The food sources were also divided into two groups on a similar basis to O. minor. The “non-commercial” Group One species comprised C. rustica, N. schrenckii and Actiniidae, while Group Two was composed of the more valuable species Ostreidae, R. venosa, M. galloprovincialis, A. japonicas, Anthocidaris crassispina, and M. arenaria. In March, the contribution of each food source to the diet of A. amurensis ranged from 6% to 13%. Each species in Group Two contributed more than 12%, and the total contribution of Group Two accounted for 77% of the diet. In June, the contribution of each

ing the four cruises in the study area and analyzed to reveal the changes in their trophic levels at each period. Their trophic levels ranged from 2.42 to 3.94 over the year. The trophic levels of invertebrates, including Octopus minor, A. amurensis, O. oratoria, and C. japonica, ranged from 1.83 to 3.72 during the year. As shown in Fig. 4, the average trophic levels of fishes showed a downward trend during the year, especially from March (3.36) to June (3.01), and from August (2.99) to November (2.57). The trophic level changes of six fishes were then compared in each period (Fig. 4). Sebastes schlegelii showed the highest trophic level with 3.94 in March, which then fell steadily to 3.29 in June, and 2.97 in November. The other four fish species also showed downward trends, except H. otakii whose trophic level was 3.01 in March, rising to 3.14 in June and again to 3.24 in August. Nevertheless, it is notable that, from June to August, the trophic levels changed little, or even rebounded, as in E. elongates (2.84 in June to 2.86 in August), C. filifer (3.10 in June to 3.12 in August) and E. hexagrammus (2.91 in June–2.96 in August). The trophic levels of invertebrates showed a different trend over the year (Fig. 5), consistent with ␦15 N values. C. japonica showed a higher trophic level in March (2.94) and August (3.01), then declined in June (2.30) and November (2.56), coinciding with the timing of fishing activity. But other invertebrates’ trophic levels showed no such trend. The trophic level of A. amurensis even exhibited a fluctuating trend compared to C. japonica. The trophic level of O. oratoria showed a declining trend except in March. The trophic level of O. minor varied between 2.5 and 3.5. 3.3. Dietary changes of invertebrate predators The average trophic levels of invertebrates (Table 3) were calculated based on ␦15 N using the trophic level Eq. (3). Seven species were above the second trophic level, while the other nine species were below the second trophic level, including six species of shellfish. The invertebrate predators were selected according to two rules. First, they should be captured on all cruises, and, second, they should occupy the top trophic levels. As shown in the results,

Table 4 Sampling number (n), ␦15 N and ␦13 C (mean values ± SD) of four invertebrate predators in March, June, August and November in 2014. Species

Octopus minor Asterias amurensis Oratosquilla oratoria Charybdis japonica

March

June

August

November

n

␦ N (‰)

␦ C (‰)

n

␦ N (‰)

␦ C (‰)

n

␦ N (‰)

␦ C (‰)

n

␦15 N (‰)

␦13 C (‰)

2 12 0 3

11.98 ± 0.73 10.01 ± 1.15 – 12.78 ± 1.82

−17.74 ± 0.13 −19.41 ± 0.94 – −17.45 ± 0.57

9 5 9 49

13.09 ± 0.38 12.93 ± 0.39 14.72 ± 0.51 11.16 ± 1.12

−17.32 ± 0.55 −18.53 ± 0.53 −19.20 ± 0.34 −19.49 ± 0.91

2 6 2 35

12.69 ± 1.53 10.90 ± 1.14 12.86 ± 0.73 12.94 ± 1.42

−18.02 ± 0.15 −19.10 ± 0.78 −18.85 ± 0.31 −17.69 ± 0.85

9 2 2 2

11.95 ± 1.19 13.77 ± 1.20 11.20 ± 1.45 11.81 ± 1.06

−17.31 ± 0.22 −17.87 ± 0.24 −18.30 ± 0.31 −17.91 ± 0.44

15

13

15

13

15

13

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Fig. 4. The trophic level changes of fishes in coastal water off Xiaoheishan Island. Left: the variation of fish trophic levels statisticed by “SigmaPlot”; Right: the specific comparison of six fishes in each period.

Fig. 5. The change of trophic levels of invertebrates in coastal water off Xiaoheishan Island. Left: the variation of invertebrate trophic levels statisticed by “SigmaPlot”; Right: the specific comparison of four invertebrate predators (trophic level > 2.5) in each period.

Fig. 6. The diet change of O. minor. Left: the contribution change of nine potential food sources; Right: the proportions of two groups, Group One containing worthless species was in orange and Group Two containing species of greater values was in blue.

Fig. 7. The diet change of A. amurensis. Left: the contribution change of nine potential food sources; Right: the proportions of two groups, Group one containing worthless species was in orange and Group two containing species of greater values was in blue.

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food source ranged from 7% to 15%. The average contributions of Group One and Two were 14% and 9%, respectively. Compared with March, Group One became more important and the total contribution increased to 43%. In August, Group Two contributed 71% of the total and became the primary food source again. In November, the total contribution of Group One increased significantly to 59%, and became the primary source once more, with C. rustica, N. schrenckii, and Actiniidae accounting for 25%, 19% and 14% of the diet, respectively. This variation in diet was mainly related to the periods of fishing and confirmed the indirect influence of fishing activity.

4. Discussion 4.1. Trophic level changes The two periods of trophic level decline, from March to June, and from August to November, coincided with the timing of fishing activity. In the closed fishing season (from June to August), the trophic level of fish remained largely unchanged, some species even showed the ability to recover. Fishes are the primary fishery resource in the Bohai Sea (Chang et al., 2014; Zheng and You, 2014) and are strong influenced by local fishing activity, which was reflected in the changes in their trophic levels. There were two aspects of the primary influences from fishing activities. One was the direct capture of larger individual fish and the other was the indirect influence on their food sources. The results from Akin and Winemiller (2008) and Romanuk et al. (2011) previously suggested that trophic level and body size should be positively correlated. Therefore, the direct influence of fishing activity causes fishes to reach sexual maturity at smaller body sizes (Arreguín-Sánchez and Ruiz-Barreiro, 2014; Micheli et al., 2014) and the loss of larger individuals resulted in a population of predominantly smaller individuals. On the other hand, fishing activity might have reduced the supply of good quality food sources, so that fishes began to prey on other, low trophic level food sources instead, and even on some primary producers such as algae (Boström et al., 2014; Churchill et al., 2014). We concluded that both direct and indirect influences of fishing activity could affect fish trophic levels, with direct capture being dominant, because its influence on food sources constitutes a long-term effect (Klarner et al., 2013; Cresson et al., 2014). As reported previously (Xu et al., 2010; Valls et al., 2014), trophic levels are also associated with the quality of fishery resources. If this downward trend continued, both the quality of the fishery and the local ecosystem would become degraded in the long term as a result of overfishing (Reich et al., 2012; Micheli et al., 2014). This would break the rules guiding sustainable fishery development and ecosystem conservation. Furthermore, our results also demonstrated the positive effect of a closed fishing season: compared with June, most of the fish trophic levels did not decline in August, and some species even rose, such as E. elongates, C. filifer, and E. hexagrammus. Therefore, lengthening the closed fishing season was an effective approach for the preservation of fishery resources and the fishery ecosystem. The trophic levels of invertebrates showed a different trend over the year (Fig. 5), consistent with ␦15 N values. C. japonica exhibited a high trophic level in March (2.94) and August (3.01), which then decreased in June (2.30) and November (2.56), coinciding with the timing of fishing activity. However, the trophic levels of other invertebrates showed no comparable trend. The trophic level of A. amurensis even exhibited a fluctuating trend compared with C. japonica. The trophic level of O. oratoria showed a declining trend except in March, while that of O. minor varied between 2.5 and 3.5. C. japonica is a primary economic fishery species in local coastal waters (Xu et al., 2013; Sudo et al., 2008), and its trophic levels

were directly influenced by fishing activity. The trophic level of C. japonica clearly declined as a result of fishing, especially in June and November.

4.2. Dietary composition changes As previously reported (Chang and Kim, 2003; Zheng et al., 2014), O. minor preferred crabs and shellfish as its main food. However, our study showed that in the coastal waters off Xiaoheishan Island O. minor primarily preyed on C. rustica, Oregonia gracilis, and Notoacmea schrenckii; smaller sized species with a lower economic value. However, while the contribution of fishery species such as C. japonica and M. arenaria increased to 12% in August after the closed fishing period, it was still less than the contribution of the three primary food sources. This unusual phenomenon might also be related to the dietary changes caused by fishing activity from March to June, and from August to November. Contrary to many other invertebrates, A. amurensis was typical of species with better predatory capability but lower economic value (Beddingfield et al., 1993; Ross et al., 2003, 2006; Wong and Barbeau, 2005) and it was scarcely affected by direct fishing activity except regarding its diet. A. amurensis has sometimes been considered harmful due to its preference for larger shellfish with higher nutritional value as prey (Ross et al., 2003, 2006). However, in our study, smaller shellfish like C. rustica and N. schrenckii were the predominant diet species for A. amurensis during the fishing periods from March to June and from August to November. This abnormal phenomenon indicated the short supply of high quality food sources during the fishing seasons and also reflected the indirect effects of fishing activity. We also analyzed the diet of C. japonica, but it did not show a similar trend to either A. amurensis or O. minor. The diet of C. japonica was clearly directly influenced by capture rather than fishing activity. The analyses of diet of both A. amurensis and O. minor indicated the effect of fishing activity on the predation behavior of higher level invertebrates. They preyed mainly on certain fishery species, with a more balanced diet during the closed fishing season, but during the fishing season focused on food sources with smaller size and lower economic values, such as N. schrenckii, C. rustica and Oregonia gracilis. The timing of fishing compelled some predators to change their ecological strategies. Some invertebrates with a high trophic level changed the composition of their diets by substituting “noncommercial” low nutritional value food sources, such as C. rustica and N. schrenckii, as the dominant prey in order to survive the fishing season when high quality food sources were in short supply. In summary, stable nitrogen and carbon isotopic ratios revealed trophic level and dietary composition changes in typical fish and invertebrate species in the Bohai Strait over a single year. This study showed that the changes were related to fishing activities. To further distinguish between the overfishing and seasonal signal, another program in a marine protected area (119◦ 05 E to 119◦ 31 E; 37◦ 35 N to 37◦ 57 N) in the Bohai Sea acting as a “control” zone with no fishing activity has now been established. This will provide more useful information and lead to more robust conclusions.

Acknowledgements This work was supported by the Public Science and Technology Research Funds Project of Ocean (grant No. 201305009-4, 201305030), the NSFC-Shandong Joint Funds Project “Marine Ecology and Environmental Sciences” (grant No. U1406403), the National Natural Science Foundation of China (grant No. 41476091), and the Qingdao Municipal Science and Technology Plan Project

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(grant No. 13-1-4-234-jch). Pei Qu benefited from a post-doctoral grant provided by the Qingdao Government.

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