Fisheries science JSFS , tập 77, số 6, 2011 11

Fish Sci (2011) 77:891–901 DOI 10.1007/s12562-011-0400-5 ORIGINAL ARTICLE Fisheries Hydroacoustic survey of fish density, spatial distribution, and behavior upstream and downstream of the Changzhou Dam on the Pearl River, China Xichang Tan • Myounghee Kang • Jiangping Tao Xinhui Li • Daoming Huang • Received: March 2011 / Accepted: 18 August 2011 / Published online: 22 September 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract Hydroacoustic surveys were conducted to understand the relationship between fish density, spatial distribution, and behavior upstream and downstream of the Changzhou Dam on the Pearl River, China, and the condition (open/closed) of the spillways When the spillways were open on 24 June 2010, numerous fish were observed to be densely distributed in the forebay upstream of the dam, with an average fish density was 0.22 fish m-3 When the spillways were closed on 25 June 2010, the fish upstream of the dam dispersed, and the average fish density decreased to 0.007 fish m-3 Prior to operating the spillways on 24 May 2010, the average fish density downstream of the dam was 0.28 fish m-3; in comparison, on 26 June, immediately following closure of the spillways, the average fish density downstream of the dam was 0.08 fish m-3 Fish were more active on June 24 than on 25 June: they swam faster and their positions in the water column varied greatly On 26 June, fish did not to swim as freely in the water column as those measured on 24 May Based on these observations, we conclude that a large number of fish are able to swim to the upstream side of the dam while the spillways are open X Tan Á X Li Pearl River Fishery Research Institute, Chinese Academic of Fishery Science, 510380 Guangzhou, China M Kang (&) Myriax Software Pty Ltd, 110 Murray Street, Hobart, Tasmania 7000, Australia e-mail: size100@hotmail.com J Tao Á D Huang Institute of Hydroecology, Ministry of Water Resources & Chinese Academy of Science, 430079 Wuhan, China Keywords Hydroacoustic survey Á Fish density Á Fish spatial distribution Á Fish behavior Á Pearl River Á Changzhou Dam Introduction The Pearl River is the largest river in southern China, with three major tributaries—the Xi Jiang River, the Bei Jiang River, and the Dong Jiang River—that all flow into the South China Sea The Pearl River is 2,218 km long with 450,000 km2 of catchment areas and 3.3 1011 m3 of mean annual discharge The Pearl River exhibits a seasonal flow, with a high flow during the summer and a low flow in the spring and winter The water temperature in the river vanes between approximately 10 and 35°C, depending on the season At one time the Pearl River was home to 321 fish species, with approximately 208 of these species being freshwater and approximately 113 being estuary species The construction of the Changzhou Dam began in 2003 and was completed in 2006 The dam spans the mainstream of Xi Jiang River, close to Wu Zhou City, Guangxi Zhuang Autonomous Region, China It stretches 3.47 km across the Xi Jiang River and is 34.6 m high When operating at full capacity, it can generate 630 MW from 15 turbines The dam has two powerhouses and 41 spillways; however, only 33 spillways are currently functional due to operational problems During flooding events, when the discharge reaches 22,000 m3 s-1, the spillways of the Changzhou Dam are open to release the floodwater The Changzhou Dam is the first dam on the mainstream of the Xi Jiang River—i.e., it is nearest to the South China Sea Downstream of the dam is a habitat that is extremely important in the life cycles of many commercially valuable fish species that are native to the Pearl River However, fish distribution 123 892 and behavior above and below the dam have not been investigated despite the geographical importance Available information comes only from interviews of local fishermen and surveys of their catches [1] A great concern among ecologists is that the dam obstructs fish migration Commercially important fish species, such as black carp Mylopharyngodon piceu, grass carp Ctenopharyngodon idellus, silver carp Hypophthalmichthys molitrix, and bighead carp Aristichthys nobilis, should be able to swim upstream of the Changzhou for spawning, and their larvae should be able to float downstream to the estuary for early development It can be assumed that fish can swim upward when the spillways are open during the flood period However, due to having to take into account the fluctuations of water level upstream and downstream of the dam, the spillways were open for only about 10 days per year in the period 2007–2009 This raises the question of whether such short periods of time are sufficient to enable the majority of fish to migrate upward while the spillways are open? There is only one fish pass and this has not been properly functional since it was built This fish pass is managed by a private company, and no regulations are in place to allow the company to run the fish pass In addition, a recent investigation on fish migration around the Gezhouba Dam on the Yangtze River concluded that ship locks can not be used as channels for fish migration [2] Fish migration toward upstream regions through spillways is an extremely important issue in fisheries science in general and, more specifically, in dam management in the river basin Therefore, it is exceedingly important to collect data on fish density and their spatial distribution as well as their behavior both upstream and downstream of the dam in the light of the condition of the spillways Here, the phrase ‘‘condition of the spillways’’ refers to the state of whether the spillways are open or closed The development and improvement of scientific acoustic instruments and new monitoring techniques during the last decade now enable researchers to monitor fish migration both efficiently and non-obtrusively [3, 4] Acoustic techniques can also be used in a practical context, such as for monitoring ecosystems in deep lakes or reservoirs, where traditional shallow-water netting techniques (e.g., gill nets, traps net, and seining) are difficult to employ, enabling a precise characterization of fish spatial distribution and furthering our understanding of fish behavior Although hydroacoustic studies have been performed in the reservoir, these mostly focused on fish abundance and distribution; little research has been carried out to date on the influence of the functional aspects of the dam [4] The results of these studies suggested that fish behavior at two dams was different according to the use of the turbine units In China, 123 Fish Sci (2011) 77:891–901 hydroacoustic surveys have been widely used for fish resource research in inland waters, such as species identification of the Chinese sturgeon Acipenser sinensis [5], fish distribution in the forebay of the Three Gorge Reservoir [6], and spatial and temporal distribution of the naked carp Gymnocypris przewalskii in Qinghai Lake [7] However, in the Pearl River, a hydroacoustic survey has only been performed as a means to observe the spawning aggregation of black Guangdong bream Megalobrama hoffmanni in the two spawning grounds [8] Therefore, it is imperative to apply the hydroacoustic method for monitoring fishery and fish ecology in the Pearl River, especially around the Changzhou Dam We have therefore undertaken a hydroacoustic survey with the aim of acquiring reliable information on the density, spatial distribution, and behavior of fish both upstream and downstream of the Changzhou Dam We also sampled using a gill net The aim of the study was to gain an understanding of the density, spatial distribution, and behavior of fish upstream and downstream of the Changzhou Dam in relation to the condition of the spillways in order to provide valuable information for (1) the effective management and operation of spillways in terms of fish migration, (2) scientific assessment of the dam as an obstacle to fish migration and (3) the development of a methodology for estimating fish resources in the Pearl River Materials and methods Study area The chosen research region upstream of the Changzhou Dam was km in length, 1.7–3 km in width, and had an average depth of 12.4 m; the region chosen for study downstream of the dam was 22 km in length, approximately 1.5 km in width, and had an average depth of 14.6 m (Fig 1) A downstream fish conservation zone, established in December 2009, and its core area are indicated in Fig Two spillways, with 33 gates, are located across two branches of the river Each gate is 16 m high and 16.6 m wide During the period when the hydroacoustic survey was being conducted in the downstream region (23–25 May 2010), the amount of discharge was low (11,200 m3 s-1) When the second survey was carried out in both the upstream and downstream of the dam to understand the dynamic changes in the fish population during 24–26 June 2010, the discharge was relatively high (22,400 m3 s-1) The data on water levels upstream and downstream of the dam were obtained from the Wu Zhou Channel Authority’s website (http://wzhd.gxgh.cn) in the Guangxi Zhuang Autonomous Region Fish Sci (2011) 77:891–901 893 Fig Map of river system in the upstream and downstream of the Changzhou Dam in the Pearl River, China U Upstream study area, D downstream study area, x spillway locations, a fish conservation zone, b center of the fish conservation zone Fish sampling From 19 to 24 May 2010, 23 gillnettings with various mesh sizes (5–8 cm) were carried out to sample fish downstream of the Changzhou Dam All catches were identified for species, each fish was measured (body length, in millimeters) and weighed (grams) stored during daylight hours because of the danger involved in navigating at night The acoustic surveys were carried out following a zigzagging route, and the degree of coverage was calculated for each survey using the Aglen’s formula [9] The coverage degree in this study was in the range of 7.1–16.2, which exceeds the level recommended by Aglen [9] Hydroacoustic surveys Data analysis Two hydroacoustic surveys were conducted in two separate time periods The first survey started downstream of the area marked D in Fig on 23 May, proceeded upstream, reaching area D on 24 May, and then moved back downstream on 25 May The second hydroacoustic survey started upstream of the area marked U in Fig on the first day, on 24 June The survey then proceeded downstream to area U in Fig on 25 June, and then was conducted in area marked D in Fig on 26 June A calibrated Simrad EY60 portable echosounder (Horten, Norway) and a circular split-beam 120 kHz transducer (7° of nominal angle) were used in the surveys The pulse duration was 64 ls and the pulse interval was 128 ls The transducer was mounted on the fore-port side of the ship at the depth of 0.5 m below the water surface The EY60 echosounder was connected to a laptop computer running the ER60 software in Windows XP in order to store data, and the computer was also linked to a Garmin GPS (Olathe, USA) A 25-m vessel was utilized for the survey, and the cruising speed was approximately 7–8 knots The total recorded acoustic data was about 11 GB, which included 537 sections, and each section is approximately 300 m in length All survey data were The raw data files of the EY60 sounder were directly added into Echoview (ver 4.90), which is a fisheries acoustics data analysis software (Myriax, Hobart, Australia) The volume backscattering strength (SV) echograms were presented with a 20 log R time varied gain (TVG), where R is the range to a target from the transducer The target strength (TS) echograms were displayed with a 40 log R TVG The angular position (angle) echograms were also shown There was considerable noise at the level of the water surface due to the movement of the vessel Therefore, a straight line, either 0.8 or m deep, was drawn to exclude any data samples considered as water surface noise or plankton-like organisms However, some noise echoes appeared up to the middle of the water column These echoes were manually selected and defined as bad data, which means that they were removed from further data analysis The river bottom line was defined using the Best bottom candidate algorithm in Echoview [10] and was manually edited when it was necessary Accordingly, any data above the water surface line, below the river bottom line, and regions defined as bad data were eliminated from the data analysis 123 894 Fish Sci (2011) 77:891–901 To detect fish tracks for evaluating fish density and behavior in studies like the one presented here, a single target detection echogram should be created since the fish track technique should be performed on a single target detection echogram There are several single target detection methods in Echoview, each based on different echosounder algorithms Among these, the single target detection split beam method [11] used by the algorithm from the Simrad EK60 echosounder was assessed to be appropriate to detect single targets using split beam data from the EY60 echosounder The parameters used in single target detection split beam method were: TS threshold = -70 dB; pulse length determination level = -6 dB; minimum normalized pulse length = 0.4; maximum normalized pulse length = 1.4; maximum beam compensation = 10 dB; maximum standard deviation of minor-axis angles = 0.6; maximum standard deviation of major-axis angles = 0.6 The fish track technique is generally used to identify groups of single targets which show a pattern of systematic movement The targets grouped into a fish track are assumed to have been generated by a single object moving through space Echoview’s a–b Fish Tracker algorithm implements a fixed coefficient filtering method, as presented in Blackman [12] The parameters used for fish tracking in this study are shown in Table Output from the fish track technique was exported in the comma-separated values format to investigate fish density and fish behavior The fish tracks were extracted with a focus on four separate days of acoustic data: 24 June, 25 June, 24 May and 26 June 2010, since the condition of spillways are unique In the subsection Fish behavior in the Results section, fish tracks extracted from the acoustic data on 24 June 2010 are called ‘‘24 June fish’’; those from the acoustic data on 25 June 2010, ‘‘25 June fish’’; those from data on 24 May, ‘‘24 May fish’’; those from data on 26 June ‘‘26 June fish’’ The spatial distribution of fish upstream and downstream of the dam was described using Surfer software Table Setting parameters for detecting fish tracks using acoustic data from the 200-kHz echosounder Results Gillnet sampling A total of 642 fish were caught by gillnetting, and the identify of 35 fish species was confirmed The dominant species in the catch were barbel chub Squaliobarbus curriculus (23.7%), mud carp Cirrhina molitorella (16.9%), black Guangdong bream (12.5%), white amur bream Parabramis pekinensis (7.1%), and common carp Cyprinus carpio (4.9%) These five species made up 82.4% of the total biomass of the catch in the gill nets The majority of the catch had body lengths ranging from 9.2 to 58 cm Spillways operation The difference of water levels between the study regions upstream and downstream of Changzhou Dam during January through October 2010 is shown in Fig Few floods occurred due to the unusual drought in the spring of 2010 in the Pearl River area Therefore, the spillways were opened only for days—from 21 to 24 June 2010 Within that time frame, information on the density, spatial distribution, and behavior of fish were compiled and later examined with reference to the condition of the spillways The acoustic data collected on 24 June was used since the spillways were open on that date For comparison purposes, acoustic data collected on 25 June, when the spillways were closed, were also used Both data sets were collected upstream of the dam Two more acoustic data sets were derived from the study area downstream of the dam on 24 May and 26 June, respectively The first of these dates precedes by a long time the opening date of the spillways, and the second date is immediately after the spillways were closed Category Parameter Value Tracking detection Alpha for major and minor axes and range 0.7 Beta for major and minor axes and range 0.5 Target gates Exclusion distance for major and minor axes and range 2m Weights Major axis 20% Minor axis 20% Range 60% Minimum number of single targets Minimum number of pings Maximum gap between single targets Track acceptance 123 (ver 8; Golden Software, Golden, USA) with a minimum curvature gridding method Fish Sci (2011) 77:891–901 895 Fig Difference in water levels between the study areas upstream and downstream of Changzhou Dam between January and October 2010 Fish density and their spatial distribution Echogram analysis showed that fish communities chiefly consisted of many individual fish Figure shows an example of numerous single fish detected as fish tracks The three-dimensional fish track (Fig 3c) is an example of the orientation of the fish track in the beam On 24 June 2010, the average fish density upstream of the Changzhou Dam was 0.22 fish m-3, with a maximum density of 0.71 fish m-3 A large number of fish found upstream were distributed adjacent to the dam When the spillways of the dam were closed on 25 June 2010, the fish quickly dispersed, and the average fish density decreased to 0.007 fish m-3, with a maximum value of 0.03 fish m-3 On 24 May 2010, average fish density downstream of the Changzhou Dam was 0.28 fish m-3, with a maximum density of 1.57 fish m-3 A large number of fish were distributed in the fish conservation area On 26 June 2010, average fish density downstream of the Changzhou Dam was 0.08 fish m-3, with a maximum density of 0.27 fish m-3 Comparison of the fish densities upstream and downstream of the dam demonstrated that the downstream fish density on 26 June was nearly tenfold higher than the upstream fish density upstream on 25 June 2010 The summary of the average fish density upstream and downstream of the dam based on the condition of the spillways (open/closed) is shown in Table The fish communities upstream of the dam were not evenly distributed, as shown in Fig On 24 June 2010, during the time the spillways were open, few fish were observed in an open-water area (black arrow in Fig 4a) On 25 June 2010, when the spillways were closed, most fish had shifted (right side of the figure, gray arrow in Fig Fish tracks in the Changzhou Dam on 25 June 2010 a SV echogram, b single target detection echogram, c three-dimensional (3D) single targets in the beam from a fish track circled in black in b The region on the top of SV and single target echograms is defined as a bad region and excluded from further analysis The line at the bottom of the figure indicates the river bottom Note that weak signals in the SV echogram not appear on the single target echogram Fig 4b) A small number of fish were present in front of the spillways In comparison with the fish density on 24 June, on 25 June nearly all fish had disappeared during the one night upstream of the dam The difference in distributions between 24 and 25 June (as shown in Fig 4a, b) may be due to the condition of the spillways Regarding the fish spatial distribution downstream of the dam, the fish communities were not uniformly distributed (Fig 5) On 24 May 2010, a higher density was found between 111.31°300 E and 111.37°480 E along the river, with a great number of fish being detected in the fish conservation zone (Fig 5a) On 26 June 2010, most of the fish were distributed rather evenly downstream, although an area of dense distribution was found further down from the dam (Fig 5b) Fish behavior A variety of fish behavioral descriptors, such as TS, depth, depth change, tortuosity, vertical direction, and speed, were used to understand fish behavior in the study areas upstream and downstream of the dam The behavioral descriptors are explained in Table Here, TS is not technically a behavioral descriptor However, it can be a factor of body length since the body length of fish can be estimated from TS and two other species-specific constants 123 896 Fish Sci (2011) 77:891–901 Table Average fish density upstream and downstream of the Changzhou Dam based on the condition of the spillways (open/closed) Upstream Downstream -3 Date Average density (fish m ) Spillways Date Average density (fish m-3) Spillways 24 June 2010 0.022 Open 24 May 2010 0.28 Before opening 25 June 2010 0.007 Closed 26 June 2010 0.08 After closing Fig Fish spatial distribution upstream of Changzhou Dam on 24 June 2010 when the dam spillways were open (a) and on 25 June 2010 when the spillways were closed (b) This location is the same as that marked U in Fig Note that different density (fish m-3) scales Fig Fish spatial distribution downstream of Changzhou Dam on 24 May 2010, which is prior to the opening of the spillways (a), and on 26 June 2010, which is after the spillways have been closed (b) This location is as that marked D in Fig Note that different density (fish m-3) scales are used [13] Therefore, TS is also included in these descriptors Fish behavior was compared based on the condition of the spillways (open/closed), such as fish behavior on 24 June versus that on 25 June fish, and fish behavior on 24 May 123 are used The vertical bar in dark gray on the right side of b indicates the dam Arrows indicate the area where the spillways are being operated versus that on 26 June (Figs 6, 7) Figure compares fish behavior on 24 June and 25 June in a box plot The TS of the 24 June fish was larger than that of the 25 June fish For example, 50% of the 24 June fish (the first quartile through the third quartile) had the TS range of -60.0 and 51.1 dB, although that of the 25 June fish had a range of 66.6 to –60.3 dB The depth of the 24 June fish was slightly shallower than that of the 25 June fish; however, the maximum depth of the 24 June fish was deeper than that of the 25 June fish The depth change of the 24 June fish was somewhat larger than that of the 25 June fish These results could lead to the interpretation that the 24 June fish swam more dynamically in a vertical direction The tortuosity of the 24 June fish school was marginally larger than that of the 25 June fish school The middle half of the 24 June fish had a tortuosity range of 1.03–1.6 and that of the 25 June fish had a range of 1.01–1.28 In terms of vertical direction, up to the third quartile of the 24 June fish had a minus sign (i.e., -1.44), which meant that 75% of the fish headed down In comparison, half of the 25 June fish swam in a mixed direction (some fish heading up and other fish heading down) Fifty percent of the 24 June fish swam approximately m s-1 faster than the 25 June fish To summarize, in comparison to the 25 June fish, the 24 June fish had larger TS, were distributed at a shallower water depth, showed relatively large changes in water depth, were swimming more dynamically, were heading slightly Fish Sci (2011) 77:891–901 Table Delineation of each behavioral descriptor 897 Name (unit) Definition TS (dB re m-2) Mean TS value of the targets in the track Depth (m) Average depth over all targets in the track Depth change (m) The depth of the first target minus the depth of the last target in a track The depth change with a plus sign means that the fish swims toward the water surface, while the depth change with a minus sign shows that the fish swims toward the river bottom The sum of the distances between adjacent targets in a track (that is, the total distance traveled) divided by the straight line distance between the first and last targets in a track Tortuosity TS, Target strength Vertical direction It is calculated from a line drawn from the first to the last target in a track The vertical direction of 0° describes a direction parallel to the X–Y (horizontal) plane (i.e., no vertical direction), -90° describes a downward direction, and 90° an upward direction Speed (m s-1) The accumulated distance between targets divided by the total time Fig Box plot of various behavioral descriptors of the 24 June fish and 25 June fish Fifty percent of the samples (box) are within the first quartile (bottom of the box) and the third quartile (top of the box) The minimum and maximum values (bars) are shown However, the upper inner fence is replaced with the maximum value for target strength (TS), tortuosity, and speed Each descriptor is precisely explained in Table down, and were swimming relatively fast It can be presumed that the reason why 24 June fish were more actively moving around compared to 25 June fish was that a great number of fish had migrated from the downstream area while the spillways were open Therefore, the fish were acting more vigorously because the fish which were already upstream would be stimulated by the presence of new fish and fish newly arrived from the downstream area would be in a new environment A comparison of fish behavior on 24 May and 26 June are shown in Fig Half of the 24 May fish had a TS range of –66.70 and –60.79 dB, and the 26 June fish had a TS range of –65.86 to –58.85 dB Fifty percent of the 24 May fish were distributed at a depth approximately 1.9 m shallower than that of the 26 June fish Both groups of fish did not show a significant depth change, although the range of the depth change in the 26 June fish was larger than that in the 25 May fish The tortuosity of the 24 May fish was 123 898 Fish Sci (2011) 77:891–901 Fig Box plot of various behavioral descriptors of the 24 May fish and 26 June fish Fifty percent of the samples (box) are within the first quartile (bottom of the box) and the third quartile (top of the box) The minimum and maximum values (bars) are shown However, the upper inner fence is replaced with the maximum value for TS, tortuosity, and speed Each descriptor is precisely explained in Table slightly larger than that of the 26 June fish; for example, the third quartile was 1.94 compared to the 1.31 of the 26 June fish The 24 May fish had a tendency to swim upwards since the first through to the third quartile of the vertical direction had all plus signs, that is 2.6–10.1° On the other hand, the middle half of the 26 June fish had a narrow range of -2.6 and 1.4° in the first and third quartiles The swimming speed of the 26 June fish was approximately 1.8 m s-1 faster than that of the 24 May fish In summary, compared to the 24 May fish, the 26 June fish had larger TS values, they were distributed at a deeper depth, their movement did not vary along the depth axis, and they swam relatively straighter and faster Based on these results, it can be assumed that after the spillways had closed, the 26 June fish preferred to stay at a one level in relatively deep water Discussion In general, spillways operate to maximize the storage of water upstream The overriding rule when operating spillways is that the safety of the dam is paramount, and the rate of outflow must not exceed the rate of inflow during the rising stage of an incoming flood [14] Little consideration 123 given to the fish community during the operation of spillways In this study, density, spatial distribution, and the behavior of fish communities upstream and downstream of the Changzhou Dam were discovered to have altered, depending on the operation of spillways In particular, the difference in fish density owing to the operation of spillways demonstrated that the dam—or the poor operation of the spillways—created a discontinuity that shifts fish communities from downstream to upstream An nonfunctional fish pass also contributed to the dam being an obstruction for fish migration The fish pass must operate as designed and remain fully operational at all times A blocked or damaged fish pass is simply a waste of precious resources Ours observations demonstrated that most fish arriving upstream of the dam between 21 and 24 June remained in front of the spillways after having swum through them Based on the fish density difference between 24 and 25 June, the number of fish that swam upstream can be estimated to be about 4.5 million fish This implies that 43 fish migrated through the spillways every second during the period 21–24 June 2010 Using similar reasoning, on 24 May 2010, the fish biomass can be loosely estimated as 134.9 million fish, indicating that only 10.7% of fish downstream of the dam were able to migrate up through the Fish Sci (2011) 77:891–901 spillways during the flood period These estimations suggest that the operating days and the operating frequency of the spillways must be increased for the purpose of fish resource protection and ecology management, since only one operation per year may not be adequate for fish migration in the Pearl River A detailed understanding of fish behavior in dam environments is of great importance; for example, mechanical structures designed to divert or guide juvenile migrant salmon at dams often work less effectively than expected This likely results from a lack of understanding of fish behavior [15] Biologically substantiated techniques for studying fish behavior in the dam environment require a deeper knowledge of fish behavior in a broad ecological context For example, the most appropriate selection method for a specific location, such as a fish conservation area, should take into account the spatial fish distribution and fish behavior This report is the first to provide information on fish behavior using the acoustic method Little information on fish behaviors in the study area is currently available Therefore, it is nearly impossible to directly compare our results with other research However, Our study may contribute valuable information on the complexity of the ecological systems for sustainable dam management and initiate further study We found the spatial fish distribution downstream of the dam to be within the range of the fish conservation region However, the coverage of survey area should be extended farther downstream in order for ecologists to acquire a larger picture of spatial distribution throughout the entire downstream area of the Pearl River For mobile organisms such as fish, micro- (response to local hydrodynamic conditions) and macro- (life-cycle features) movement and migrations are critical for maintaining viable populations [16–18] The life cycle of each and every fish species can be considered to be a sequence of residential and migratory phases Thus, the operation of spillways should consider the spatial and temporal patterns of fish distribution and dispersal and migratory activity in terms of fish behavior Our results show that the key to successfully operating the spillways and the fish pass lies in a better understanding of fish behavior and their response to hydrodynamic conditions at obstructions Although our results only show fish micromovement around the dam, they can be used as a foundation for the comprehensive study on macro-movement and migrations Ongoing monitoring should be mandatory to increase knowledge on how to improve the ecological status of the regions upstream and downstream of the dam Therefore, information (fish density, spatial distribution and behavior) from monitoring should be collected for a relatively long time period during regular time intervals Two methods, namely, catch statistics and fish larvae sampling, have been used to estimate fish resources in the 899 Pearl River However, these may not provide quantitatively representative results from fish communities in a targeted area We have demonstrated that a hydroacoustic survey can provide valuable information on fish density, fish spatial distribution, and fish behavior upstream and downstream of the Changzhou Dam This kind of information is very difficult to obtain using conventional methods Accordingly, the hydroacoustic method provides a simple, yet reliable way to assess the impact of the dam on fish migration This method can also be used to estimate fish resources relatively quickly and easily This study was the first trial of the hydroacoustic method to estimate fish resources in the Pearl River and to demonstrate the effect of operating spillways on fish migration However, the method does not directly provide data on species composition, which requires a ground-truth method In our study, gillnetting was utilized as the ground-truth method The standardized sampling of fish in the majority of freshwater environments is done with gillnets [19] However, gillnetting can only be performed easily in pelagic areas; thus, results from gillnetting most likely are not representative of the entire fish community of that area In freshwater, fishery science trawling is even less frequently applied as it is associated with a relatively high sampling effort and requires sophisticated equipment Trawl net sampling has less selectivity than gillnet sampling, which means that the former method can be utilized to catch representative fish samples more reliably than the latter method Thus, combined surveys of hydroacoustics and trawling methods can complement each other by balancing their individual drawbacks; the hydroacoustics method provides data on absolute fish density and the trawling method provides data on species composition and length distribution In multispecies circumstances, where information on species contributions and detailed size spectra are needed, the combination of hydroacoustics with trawl catches is the favored approach, although manpower and finances remain major constraints The Pearl River has been seriously overfished, and fish communities require protection, especially with respect to spawning and growth [20] Certain fish species, such as the black carp, grass carp, silver carp, and bighead carp, urgently need to migrate upstream to finish spawning (unpublished information provided by fisheries scientists) Hereby, the enhanced reliability of the combination approach can provide essential data on Pearl River populations that will enable researchers to obtain an ecological understanding of these species and, consequently, to protect them This combined method is flexible enough to be conducted with other observation and experimental systems In the distant future it would be ideal to take into account complicated factors such as biological (food availability, predation risk and competition), environmental (oxygen concentration and water temperature) and 123 1070 increase AR gene expression [3] AR inhibitors have been reported to inhibit AGE formation [4, 5], suggesting that the polyol pathway and AGE formation are interconnected to promote diabetic complications AR, a member of the aldo–keto reductase family, is a nicotinamide adenine dinucleotide phosphate (NADPH)dependent oxidoreductase and a pivotal enzyme in the polyol pathway Under diabetic conditions, AR catalyzes the reduction of aldo sugars to sugar alcohols, such as glucose to sorbitol, followed by intracellular accumulation of sorbitol and corresponding metabolites due to their poor passage across membranes, resulting in the onset of diabetic complications [6–8] AGEs are also formed through the reactions between reducing sugars and proteins Under chronic hyperglycemia, carbonyl or ketone groups of sugars can react nonenzymatically with the amino groups of proteins, giving rise to a Schiff base and Amadori product These products can then undergo rearrangement and eventually dehydration, condensation, fragmentation, and oxidation, followed by the irreversible formation of the AGEs AGEs can also be cross-linked with long-lived proteins, such as serum albumin, lens crystalline, and collagen, leading to the accumulation of AGEs in tissues and subsequent protein modification The formation and accumulation of AGEs have been implicated in the progression of diabetic complications [9] Thus, both AR inhibitors (ARIs) and AGE formation inhibitors (AGEIs) can serve as important targets for the prevention and treatment of diabetic complications Many potent synthetic ARIs and AGEIs have been studied in vitro and in vivo Among these, aminoguanidine, an AGEI, reacts with the Amadori product to prevent cross-linking with proteins [10] However, the use of aminoguanidine to treat diabetic human patients and rats has yielded side effects, including pernicious-like anemia, nausea, headache, and higher rates of pancreatic and renal-neoplastic tumors [11] Hence, several pharmaceutical companies and various researchers have focused upon the search for potent and safe ARIs and AGEIs from nature [12, 13] We have therefore focused our work on the therapeutic and preventive effects of Laminaria japonica against diabetic complications The sea tangle, Laminaria japonica Areschoung, is a brown alga that belongs to the family Laminariaceae It is widely distributed throughout the coastal waters of Korea, Japan, China, and France and has long been used as a common seafood as well as traditional medicine to promote maternal health and treat diabetes mellitus [14] It possesses several biological activities, including scavenging activity against free radicals [15], antimutagenic activity [16], hypotensive activity [17], inhibition of lipid peroxidation and down-regulation of blood glucose in diabetic rats [18], inhibitory effects on oxidative stress and xanthine oxidase activity in streptozotocin-induced diabetic rat liver and protective effects against high glucose-induced oxidative stress in 123 Fish Sci (2011) 77:1069–1079 human umbilical vein endothelial cells [19, 20] However, the inhibitory activities of L japonica on AGE formation and rat lens AR (RLAR) have not been investigated Therefore, the aim of the study reported here was to evaluate the effects of L japonica and two porphyrin derivatives from L japonica against diabetic complications based on the results of in vitro assays measuring the inhibitory activities of L japonica and these two porphyrin derivatives on RLAR and AGE formation To evaluate the relationship between the chemical structure and inhibitory activity of the porphyrin derivatives on both RLAR and AGE formation, we used four commercially available porphyrin derivatives and two active porphyrin derivatives, including pheophorbide a (1) and pheophytin a (2) from L japonica Furthermore, to determine the optimized extraction conditions of the two porphyrin derivatives from L japonica, quantitative analysis of these compounds in different extracts of L japonica, including MeOH, CH2Cl2, and acetone extracts, was performed simultaneously in a high-performance liquid chromatography–ultraviolet (HPLC-UV) detector system Materials and methods General experimental procedures The 1H- and 13C-nuclear magnetic resonance (NMR) spectra were determined using a JEOL JNM ECP-400 spectrometer (JEOL, Tokyo, Japan) at 400 mHz for 1H and 100 mHz for 13 C Column chromatography was conducted using silica (Si) gel 60 (70–230 mesh; Merck, Darmstadt, Germany), Diaion HP20 (250–850 lm; Sigma, St Louis, MO), and Lichroprep RP-18 (40–63 lm; Merck) All thin-layer chromatography (TLC) runs were conducted on precoated Merck Kieselgel 60 F254 plates (20 20 cm, thickness 0.25 mm; Merck) and RP-18 F254s plates (5 10 cm; Merck), using 50% H2SO4 as the spray reagent Chemicals and reagents Bovine serum albumin (BSA), aminoguanidine hydrochloride, D-(-)-fructose, D-(?)-glucose, NADPH, quercetin, DL-glyceraldehyde dimer, dimethyl sulfoxide (DMSO), chlorophyll a, protoporphyrin IX, hemin, and hematin were purchased from Sigma Sodium azide was purchased from Junsei Chemical Co (Tokyo, Japan) All solvents used were purchased from Merck and DUKSAN Pure Chemical Co (AnSan City, Korea), unless stated otherwise Plant materials Laminaria japonica Areschoung was collected from Gi-Jang, Kyeong Nam Province, Korea, in May 2009 Fish Sci (2011) 77:1069–1079 A voucher specimen (no 20090501) was deposited in one author’s laboratory (J.S Choi) Extraction, fractionation, and isolation Laminaria japonica powder (5.50 kg) was refluxed with methanol (MeOH) for h (5 l) The total filtrate was then concentrated to dryness in vacuo at 40°C in order to obtain the MeOH extract (990.85 g) This extract was suspended in distilled water and successively partitioned with methylene chloride (CH2Cl2), ethyl acetate (EtOAc), and n-butanol (n-BuOH) to yield the CH2Cl2 (100.05 g), EtOAc (1.60 g), and n-BuOH (36.52 g) fractions, respectively, as well as an aqueous residue (515.02 g) (Fig 1) The CH2Cl2 fraction (46.0 g) was first chromatographed through a Si gel column (8 80 cm), using a mixed solvent of n-hexane and EtOAc (n-hexane:EtOAc, 20:1 ? 0:1 gradient) to obtain 18 subfractions (F01–F18) Fraction (F09, 2.3 g), which exhibited higher activities than the other subfractions in both the RLAR and AGE formation assays (data are not shown), was subjected to chromatography on a Diaion HP20 column with the ethanol (F0901), MeOH (F0902– F0903), and acetone (F0904) subfractions Repeated column chromatography of F0904 conducted with a solvent mixture of n-hexane and acetone (n-hexane:acetone, 10:1 ? 0:1 gradient) resulted in the isolation of pheophorbide a (compound 1, 15.0 mg) Pheophytin a (compound 2, 95.0 mg) was purified from F090403 on an RP-18 column and eluted with a solvent mixture of CH2Cl2, MeOH, and H2O (CH2Cl2:MeOH:H2O = 8:22:1) All isolated compounds (1 and 2) were characterized and identified by spectroscopic methods, including 1H-NMR (400 MHz) and 13C-NMR (100 MHz), as well as by comparison with published data [21–24] 1071 Pheophorbide a (compound 1) Dark-green amorphous solid; 1H-NMR (CDCl3, 400 MHz): d 9.42 (s, 10-H), 9.25 (s, 5-H), 8.53 (s, 20-H), 7.90 (dd, J = 18.0, 12.0 Hz, 31-H), 6.13 (d, J = 11.0 Hz, 32-H), 6.23 (s, 132-H), 4.19 (brd, J = 8.0 Hz, 17-H), 4.43 (q, J = 9.0 Hz, 18-H), 3.63 (q, J = 9.0 Hz, 81-CH2), 3.55 (s, 121-Me), 3.34 (s, 21-Me), 3.13 (s, 71-Me), 3.85 (s, 132CO2Me), 2.58 (m, 171-CH2), 2.24 (m, 172-CH2), 1.63 (t, J = 8.0 Hz, 82-Me), 1.81 (d, J = 7.0 Hz, 181-Me), 0.00 (NH); 13C-NMR (CDCl3, 100 MHz): 11.1 (C-71), 12.0 (C-21), 12.0 (C-121), 17.3 (C-82), 19.3 (C-81), 23.0 (C-181), 29.5 (C-172), 30.7 (C-17 1), 50.0 (C-18), 50.9 (C-17), 52.8 (C-132, CO2Me), 64.6 (C-132), 93.0 (C-20), 97.4 (C-5), 104.3 (C-10), 105.0 (C-15), 122.7 (C-32), 128.8 (C-13), 128.9 (C-31), 128.9 (C-12), 131.8 (C-2), 136.0 (C-3), 136.2 (C-7), 136.4 (C-4), 137.8 (C-11), 142.0 (C-1), 145.1 (C-8), 149.5 (C-9), 150.8 (C-14), 155.5 (C-6), 161.0 (C-16), 169.6 (C-132, CO2), 172.0 (C-19), 177.8 (C-172, CO2), 189.6 (C-131) Pheophytin a (compound 2) Dark-green amorphous solid; 1H-NMR (CDCl3, 400 MHz): d 9.34 (s, 10-H), 9.16 (s, 5-H), 8.52 (s, 20-H), 7.82 (dd, J = 18.0, 11.6 Hz, 31-H), 6.19 (dd, J = 11.6, 1.6 Hz, 32-CH2), 6.28 (s, 132-H), 4.23 (brd, J = 9.0 Hz, 17-H), 4.49 (q, J = 8.0 Hz, 18-H), 3.49 (q, J = 9.0 Hz, 81CH2), 3.90 (s, 121-Me), 3.33 (s, 21-Me), 3.04 (s, 71-Me), 3.64 (s, 132-OMe), 2.50–2.64 (m, 171-C), 2.21–2.36 (m, 172-C), 1.49 (t, J = 8.0 Hz, 82-Me), 1.82 (d, J = 8.0 Hz, 181-Me) Phytyl group: 5.16 (t, J = 6.5 Hz, P2-H), 4.44 (dd, J = 12.9, 6.5 Hz, P1-H), 1.88 (m, P4-C), 1.59 (s, P17Me), 0.9–1.3 (m, P5 * P14-C), 0.85 (m, P18, 19-CH3), 0.79 (P16, 20-CH3); 13C-NMR (CDCl3, 100 MHz): 11.0 (C-71), 12.0 (C-21), 12.0 (C-121), 16.3 (C-P17), 17.3 (C-82), 19.2 (C-81), 22.6 (C-P20), 22.7 (C-P16), 23.1 (C-181), 24.4 (C-P15), 24.7 (C-P9), 24.9 (C-P5), 27.9 (C-P13), 29.8 (C-172), 31.2 (C-17 1), 32.6 (C-P11), 32.7 (C-P7), 36.6 (C-P14), 37.2 (C-P12), 37.3 (C-P10), 37.3 (C-P8), 39.3 (C-P6), 39.8 (C-P4) 50.1 (C-18), 51.1 (C-17), 52.8 (C-132, OMe), 61.5 (C-P1), 64.7 (C-132), 93.0 (C-20), 97.3 (C-5), 104.2 (C-10), 105.2 (C-15), 117.7 (C-P2), 122.6 (C-32), 128.8 (C-13), 128.8 (C-31), 128.9 (C-12), 131.8 (C-2), 135.9 (C-7), 136.0 (C-3), 136.3 (C-4), 137.8 (C-11), 141.9 (C-1), 142.8 (C-P3), 145.0 (C-8), 150.8 (C-9), 149.6 (C-14), 155.4 (C-6), 161.2 (C-16), 172.9 (C-132, OH), 172.1 (C-19), 172.9 (C-172, CO2), 189.6 (C-131) Assay for RLAR inhibitory activity Fig Extraction and fractionation of Laminaria japonica The Guidelines for Care and Use of Laboratory Animals, as approved by Pukyong National University, were 123 1072 Fish Sci (2011) 77:1069–1079 followed in all experiments A rat lens homogenate was prepared according to the modified method of Hayman and Kinoshita [25] Briefly, the lenses were removed from the eyes of Sprague-Dawley rats (weight 250–280 g; Samtako BioKorea, Gyeonggi-do, South Korea) The lenses were homogenized in sodium phosphate buffer (pH 6.2) prepared from sodium phosphate dibasic (Na2HPO4ÁH2O, 0.66 g) and sodium phosphate monobasic (NaH2PO4ÁH2O, 1.27 g) in 100 ml of double distilled H2O The supernatant was obtained by centrifugation of the homogenate at 10,000 rpm at 4°C for 20 and frozen until use (-20°C) A crude AR homogenate, with a specific activity of 6.5 U/mg, was used in the evaluations of enzyme inhibition Reaction solutions consisted of 620 ll of 100 mM sodium phosphate buffer (pH 6.2), 90 ll of AR homogenate, 90 ll of 1.6 mM NADPH, 9.0 ll of the samples (final concentration 100 lg/ml for the extracts and fractions, and 1.0–100 lg/ml for the compounds, dissolved in 100% DMSO) or 9.0 ll of 100% DMSO, and 90 ll of 50 mM of DL-glyceraldehyde as the substrate AR activity was determined by measuring the decrease in NADPH absorption at 340 nm over a 4-min period on a Ultrospec 2100pro UV/Visible spectrophotometer with SWIFT II Applications software (Amersham Biosciences, Piscataway, NJ) Quercetin, a well-known ARI, was used as a reference The inhibition percentage (%) was calculated using the following equation: Inhibition percentageð%Þ ¼ ½1 À ðDA sample= ÀDA blank= minÞ =ðDA control= ÀDA blank= minފ  100; where DA sample/min represents the reduction of absorbance for with the test sample and substrate, respectively, and DA control/min represents the same, but with 100% DMSO instead of the sample The IC50 of RLAR inhibitory activity were calculated from the logdose inhibition curve within the test concentrations All IC50 values are expressed as the mean ± standard error of the mean Inhibition of AGE formation Inhibition of AGE formation was determined according to the modified method of Vinson and Howard [26] To prepare the AGE reaction solution, 10 mg/ml BSA in 50 mM sodium phosphate buffer (pH 7.4), with 0.02% sodium azide to prevent bacterial growth, was added to 0.2 M fructose and 0.2 M glucose The reaction mixture (950 ll) was then mixed with various concentrations of the samples (50 ll; final concentration: 50–1,200 lg/ml for the extracts and fractions, and 12.5–200 lg/ml for the compounds) dissolved in 10% DMSO After incubation at 37°C for 123 days, the fluorescence intensity of the glucose–BSA reaction products was determined using a spectrofluorometric detector (FLx800 microplate fluorescence reader; Bio-Tek Instruments, Winooski, VT), with excitation and emission wavelengths at 350 and 450 nm, respectively The percentage inhibition of AGE formation was determined from a graphical plot of the data and expressed as the mean ± SEM (triplicate experiments) The nucleophilic hydrazine compound aminoguanidine was used as a reference in the AGE assay HPLC quantitative analyses of and Reversed-phase HPLC was performed in a JASCO HPLC model 1580 system (Tokyo, Japan) equipped with a UV/Vis detector at 410 nm and a column thermostat set at 25°C The Borwin chromatographic software system (JMBS Developments, Le Fontanil, France) was used for HPLC data analysis Chromatographic separation was accomplished on a ODS-Hypersil column (4.6 250 mm, lm; Thermo Scientific, Waltham, MA) The A and B mobile phases consisted of MeOH:0.5 M ammonium acetate (8:2, solvent A) and MeOH:acetone (7:3, solvent B) The gradient elution was performed at a flow rate of 0.8 ml/min as follows: 0–5 min, 75% A and 25% B; 5–10 min, 50% A and 50% B; 10–15 min, 25% A and 75% B; 15–35 min, 0% A and 100% B; 35–45 min, 0% A and 100% B; 45–50 min, 100% A and 0% B; 50–60 min, 100% A and 0% B The injection volume was 20 ll, and the CH2Cl2, MeOH, and acetone extracts of L japonica were dissolved in 100% acetone at concentrations of 1.0 mg/ml, then filtered through a centrifugal filter device (0.45 lm; Millipore, Bedford, MA) The stock solutions (1.0 mg/ml) of compounds and isolated from L japonica were prepared individually in acetone and different concentrations (compound 1: 31.25–1.95 lg/ml, compound 2: 125.0–7.8 lg/ml) of these were loaded onto an HPLC for preparation of the calibration curves In order to determine the optimized solvent condition, the relative quantities of the two compounds in the CH2Cl2, MeOH, and acetone extract were expressed as contents (mg/g) calculated from calibration curves Each crude extract was prepared by extraction of the sample (each 100 g) with the respective solvent (each 500 ml) by reflux for h The yield of the CH2Cl2, MeOH, and acetone extracts was 15.4, 16.6, and 20.0%, respectively Statistics The statistical significance was analyzed via a one-way analysis of variance (ANOVA) and Student’s t test (Systat, Evanston, IL) and considered at P \ 0.01 All results are presented as the mean ± SEM Fish Sci (2011) 77:1069–1079 1073 Results Inhibitory effects of the MeOH extract and fractions from L japonica on RLAR and AGE formation To evaluate the effects of L japonica on diabetic complications, we measured the inhibitory activities of the MeOH extract and its fractions on both the formation of both RLAR and AGE formation The RLAR inhibitory activity of the MeOH extract and fractions, observed at concentrations of 100.0 lg/ml, are listed in Table The MeOH extract showed promising inhibitory activities against RLAR, with an inhibition percentage of 27.44 ± 1.13% at 100 lg/ml Among the several fractions tested, the inhibition percentage of the EtOAc fraction (50.83%) was the most significant, followed by that of the CH2Cl2 and n-BuOH fractions (30.24 and 31.85%, respectively) The aqueous fraction also showed marginal inhibitory activity (inhibition percentage of 19.11% at 100 lg/ml) In the AGE assay, the MeOH extract exhibited detectable inhibitory activity on AGE formation, with an IC50 of 1,206.08 ± 21.29 lg/ml Among the several fractions tested, the EtOAc and CH2Cl2 fractions manifested potent AGE formation inhibitory activity, with an IC50 of 150.32 ± 2.84 and 167.65 ± 1.76 lg/ml, respectively Conversely, the n-BuOH and aqueous fractions showed no inhibitory activity toward AGE formation up to 1600.0 lg/ml Although the CH2Cl2 fraction was less active than the Table In vitro activities of the methanolic extract and its fractions from Laminaria japonica on RLAR and AGE formation Samples RLARa Inhibition (%) AGEb IC50 (lg/ml) 1,206.08 ± 21.29 MeOH extract 27.44 ± 1.13 CH2Cl2 fraction 30.24 ± 0.36 167.65 ± 1.76 EtOAc fraction 50.83 ± 0.12 150.32 ± 2.84 n-BuOH fraction 31.85 ± 1.01 [1,600 H2O fraction 19.11 ± 1.34 [1,600 Quercetinc 65.89 ± 0.95 Aminoguanidined 86.77 ± 0.62 RLAR Rat lens aldose reductase, AGE advanced glycation end products Values are expressed as the mean ± standard error of the mean (SEM) a Lens aldose reductase was determined at a concentration of 100.0 lg/ml for the methanol extract and its fractions obtained from the methanolic extract of Laminaria japonica dissolved in DMSO b The 50% inhibition concentrations (IC50 were calculated from the log dose inhibition curve and expressed as the mean ± SEM of triplicate experiments c Quercetin (1.0 lg/ml) was used as a positive control in the RLAR assay d Aminoguanidine was used as a positive control in the AGE assay EtOAc fraction based on the results of the RLAR and AGE formation assays, it did show a higher yield than the EtOAc fraction Therefore, the CH2Cl2 fraction may contain the majority of the active components against both RLAR and AGE formation Isolation of compounds from the CH2Cl2 fraction The results of the yield and anti-diabetic complications assays together indicated that the CH2Cl2 fraction may be a potent target fraction, with a potential to treat and prevent various diabetic complications Accordingly, the CH2Cl2 fraction was selected for chromatographic resolution of the active compounds using silica gel, RP-18, and Diaion HP20 columns, yielding two active compounds The chemical structures of both compounds were characterized by one-dimensional (1D) NMR as well as by comparison with reported spectral data for and [21–24] The individual structures of compounds and are shown in Fig 2a and b Both were isolated from L japonica for the first time Inhibitory effects of the compounds from the CH2Cl2 fraction on RLAR and AGE formation The inhibitory effects of the compounds and isolated from L japonica against RLAR and AGE formation were evaluated (Table 2) In the RLAR assays, compound exhibited the most potent RLAR inhibitory activity, with an IC50 of 12.3 lM, whereas compound showed no inhibitory activity up to 100 lM Nonetheless, the inhibitory activity of both compounds and against AGE formation was than that of the positive control, aminoguanidine (IC50 = 735.7 lM) Compounds (IC50 = 49.4 lM) and (IC50 = 228.7 lM) demonstrated a 15- and threefold higher activity than aminoguanidine against AGE formation Inhibitory effects of porphyrin derivatives on RLAR and AGE formation To elucidate the structure-inhibitory activity relationship of the porphyrin derivatives, the inhibitory activities of two active porphyrin derivatives from L japonica and four commercially available porphyrin derivatives, including chlorophyll a, protoporphyrin IX, hemin, and hematin (Fig 3), against both RLAR and AGE formation were compared As shown in Table 3, protoporphyrin IX exhibited intermediate RLAR inhibitory activity with an IC50 of 55.5 lM compared to the positive control (quercetin, 1.17 ± 0.17 lM) Hemin showed marginal inhibitory activity with an IC50 of 101.3 lM In contrast, chlorophyll a and hematin exerted no activity toward 123 1074 Fish Sci (2011) 77:1069–1079 a Table In vitro activities of and isolated from Laminaria japonica on RLAR and AGE formation CH2 31 8 H 3C NH N 20 18 H 3C N HN 17 13 Pheophorbide a 12.31 ± 1.19 Pheophytin a [100 Quercetinc Aminoguanidind 1.17 ± 0.17 49.43 ± 1.32 228.71 ± 0.07 735.65 ± 6.71 Values are expressed as the mean ± standard error of the mean (SEM) 13 b CH3 12 13 The 50% inhibition concentrations (lg/ml) were calculated from the log dose inhibition curve and expressed as the mean ± SEM of triplicate experiments c Quercetin was used as a reference compound on lens aldose reductase inhibitory activity d H 3C O O C O 12 15 18 AGEb IC50 (lM) a The concentration that caused IC50 is given as the mean ± SEM of duplicate experiments 17 17 CH3 10 RLARa IC50 (lM) Compounds CH3 Aminoguanidine was used as a reference compound on AGE formation inhibitory activity O O potent inhibitory activity upon AGE formation, with an IC50 of 42.1 lM, followed by chlorophyll a at 69.5 lM Conversely, both hemin and hematin showed marginal inhibitory activity, with respective IC50 values of 280.9 and 299.14 lM H b 3 CH2 CH3 H 3C Quantitative HPLC analysis of two active porphyrin derivatives from L japonica NH N CH3 20 18 10 N H 3C HN 18 17 17 O 12 15 13 17 H 3C O O C O CH3 13 13 12 1 O p2 p16 p1 p17 p18 Fig Structures of and isolated from L japonica a Pheophorbide a, b Pheophytin a RLAR In the AGE formation assay, all the porphyrin derivatives showed higher activities than aminoguanidine (IC50 735.7 lM) Protoporphyrin IX exhibited the most 123 In order to determine the optimized extraction conditions of the two active compounds (1 and 2), we extracted L japonica with different solvents, such as MeOH, CH2Cl2, and acetone; subsequent quantitative analysis of compounds and in these extracts was performed simultaneously in an HPLC-UV detector system The peaks of compounds and occurred at approximately 16.00 and 45.85 in the three extracts (Fig 4) There was a good linearity in the range of 31.25–1.95 lg/ml for compound and 125.0–7.80 lg/ml for compound The regression equations of each calibration curve of compounds and were: y = 8,000,000x ? 62,039 and y = 6,000,000x ? 198,908, respectively, where y and x correspond to the peak area and concentration, respectively The respective correlation coefficients of compounds and were 0.9970 and 0.9999 The relative quantities of the two compounds in the CH2Cl2, MeOH, and acetone extracts were expressed as contents (mg/g) calculated from the calibration curves The contents of compounds and in the MeOH, CH2Cl2, and acetone extracts were 0.3 and 2.24, 1.13 and 14.3, and 3.15 and 42.93 mg/g extract, respectively (Fig 5) These results indicated that acetone was the optimal extraction solvent for these active constituents Fish Sci (2011) 77:1069–1079 1075 Fig Structures of and isolated from L japonica and commercially available porphyrin derivatives used in this study CH CH CH CH H 3C H3C N N X N N H3C CH O O H H Table In vitro inhibitory activities of commercially available porphyrin derivatives on RLAR and AGE formation Compounds Chlorophyll a Protoporphyrin IX Hemin Hematin Quercetinc Aminoguanidind RLARa IC50 (lM) – AGEb IC50 (lM) 69.45 ± 1.94 55.45 ± 1.54 42.06 ± 1.03 101.33 ± 2.61 – 280.92 ± 1.83 299.14 ± 2.04 1.17 ± 0.17 735.65 ± 6.71 Values are expressed as the mean ± standard error of the mean (SEM) a The concentration that caused IC50 is given as the mean ± SEM of duplicate experiments b The 50% inhibition concentration (lg/ml) were calculated from the log dose inhibition curve and expressed as the mean ± SEM of triplicate experiments c Quercetin was used as a reference compound on lens aldose reductase inhibitory activity d Aminoguanidine was used as a reference compound on AGE formation inhibitory activity Discussion Increased AR-related polyol pathway activity plus the formation and accumulation of AGEs have been implicated in the onset of diabetic complications, including O N H 3C CH H COOC O CH M M N N N CH O O O R1 atherosclerosis, cardiac dysfunction, retinopathy, neuropathy, and nephropathy Extensive experimental evidence suggests that synthetic ARIs and AGEIs could be effective in preventing and treating diabetic complications However, these experimental treatments were withdrawn from clinical trials due to low efficacy, poor pharmacokinetics, and insufficient safety [27, 28], leading pharmaceutical companies and many researchers to focus on discovering potent and safe ARIs and AGEIs in nature [12, 13] Our group has directed its efforts to the search for safe and nutritionally packed marine natural products A number of seaweed species have been used in both traditional medicine and diverse foodstuffs by coastal peoples of many areas around the world, particularly Asia and the Caribbean [29] In particular, Asian populations have used seaweed to treat such medical conditions as fever, stomach ailments, swelling, cancer, diabetes, goiter, and other glandular problems, as well as to add flavor and texture to food [29–31] With the increasing number of reports being published on the bioactivity of seaweeds, such as antioxidative and antiinflammatory effects [32], immunostimulating activity [33], antiherpetic activity [34], antifungal activity [35], antiviral and immunoregulatory activity [36], and anticoagulant activity [37], interest in the nutritional value and health benefits of seaweed have been increasing among Western countries In this context, we have determined that 123 1076 Fish Sci (2011) 77:1069–1079 Fig Contents of and in the MeOH extract, CH2Cl2 extract, and acetone extract of L japonica Fig High-performance liquid chromatography chromatograms of the MeOH, CH2Cl2, and acetone extract from L japonica a MeOH extract, b CH2Cl2 extract, c Acetone extract Peak [retention time (RT) 16.0 min, 1], Peak [RT 45.8 min, 2] L japonica, an edible brown alga, has an inhibitory effect on both RLAR and AGE formation and, therefore, on diabetic complications In order to evaluate the effects of L japonica on such diabetic complications, we measured the inhibitory activities of the MeOH extract and its fractions on both RLAR and AGE formation As shown Table 1, the EtOAc fraction showed the most significant 123 inhibitory activity on both RLAR and AGE formation, followed by the CH2Cl2 fractions Previous findings on L japonica suggested that L japonica must contain fucoidan and alginic acid, which possess antioxidant activity [38, 39], indicating that both compounds may be, at least partly, associated with the inhibitory activity of L japonica against diabetic complications in which the antioxidant system has been implicated Although the data were not shown, fucoidan and alginic acid exhibited no inhibitory activity against AGE formation at a higher concentration (200 lg/ml), indicating that both compounds not contribute to the effects against diabetic complications Although the CH2Cl2 fraction was less active than the EtOAc fraction in both the RLAR and AGE formation assays, it showed a higher yield than the EtOAc fraction Therefore, repeated column chromatography of the CH2Cl2 fraction on silica gel, RP-18, and Diaion HP20 columns yielded two porphyrin derivatives: and This is the first time that compounds and have been isolated from L japonica Earlier studies have shown these porphyrin derivatives to possess a variety of biological activities Compound has been implicated in antioxidant [40] and inhibitory effects on the growth of a number of human cancer cells, such as pigmented melanoma [41], colon cancer [42], and pancreatic carcinoma [43], and animal studies have shown compound to have potent antitumor and anti-inflammatory activities towards ICR mouse Fish Sci (2011) 77:1069–1079 skin [44] Compound has been indicated as having potent anti-hepatitis C virus [45], antioxidant [46], and antiinflammatory activities [47], and as promoting neurite outgrowth in PC12 cells [48] However, to the best of our knowledge, the inhibitory activities of and on RLAR and AGE formation have not been studied Therefore, we examined the inhibitory effects of compounds and from L japonica against diabetic complications, based on the results of RLAR and AGE formation assays Compound exhibited potent inhibitory activities against both RLAR and AGE formation, while compound was found to be active only against AGE formation In the AGE formation assay, compound (IC50 = 49.4 lM) demonstrated a fivefold higher activity than compound (IC50 = 228.7 lM), indicating that the presence of carboxyl group and the absence of the phytyl group at the C-172 position of the porphyrin derivatives influence inhibitory activity For a more detailed elucidation of the structure–inhibitory activity relationship of the porphyrin derivatives, we assayed and compared the inhibitory activities of four commercially available porphyrin derivatives and two active porphyrin derivatives isolated from L japonica towards both AGE formation and RLAR The RLAR inhibitory activities of compound and protoporphyrin IX were stronger than those of compound and chlorophyll a, indicating that a carboxyl group rather than a phytyl group at the C-172 position contributes to their inhibitory effects In the AGE formation assay, all of the porphyrin derivatives showed higher activities than the positive control, aminoguanidine, suggesting that the presence of a porphyrin ring at least partly contributed to the AGE inhibitory activities of porphyrin derivatives Wolff et al [49] reported that free radicals stimulate AGE production through the autoxidation of sugars Free radicals also act on albumin, leading to AGE formation from high levels of glucose and albumin [50] a-Tocopherol, a potent antioxidant, has been shown to be an antiglycating substance in vitro [51] Matsushita and Iwami [52] reported that porphyrin derivatives contain more than ten conjugative double bonds in the porphyrin ring skeleton; this skeleton appears to be an important chemical structure for antioxidant activity Endo et al [53] also demonstrated that porphyrin derivatives provide the hydrogen donor to reduce free radicals, such as DPPH Therefore, these porphyrin derivatives may exert the necessary inhibitory effects on AGE formation via free radical scavenging The inhibitory activities of the tested porphyrin derivatives on AGE formation increased in the following order: protoporphyrin IX [ [ chlorophyll a [[ These results indicate that the presence of the carboxyl group and the absence of the phytyl group at the C-172 position of the porphyrin derivatives seem to play key roles in the inhibitory effects of these derivatives on AGE formation Endo and others 1077 proposed that while the porphyrin structure has an effect on the reduction of the free radical, magnesium chelation apparently strengthens the antioxidant activity of chlorophyll a [53] Metal chelation would lead to the concentration of electron density toward the centrally bound metal and away from the porphyrin backbone of the chlorophyll molecule, resulting in an increased ability of the conjugated porphyrin system to donate electrons [54] The HPLC profiles demonstrated that the acetone extract of L japonica contains substantial levels of and 2, more than other solvents such as MeOH and CH2Cl2 Based on these results, we conclude that acetone is well-suited for the extraction of these compounds Taken together, our results suggest that L japonica, and particularly and from L japonica, may be potentially useful as a functional food resource for the prevention of diabetic complications Acknowledgments This 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Published online: 25 September 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract Various physiological functions of dietary glucosylceramides (GlcCer) have been reported, such as preventing colon cancer and improving skin barrier function One potential GlcCer source used as a foodstuff is sea cucumber In this study, our objective was to determine the effect of dietary GlcCer prepared from sea cucumber on plasma and liver lipids in cholesterol-fed mice ICR mice were fed four different diets (control diet, sea cucumber GlcCer supplemented diet, high cholesterol supplemented diet, and high cholesterol ? sea cucumber GlcCer supplemented diet) Dietary GlcCer decreased total cholesterol significantly in ICR mice The mRNA expression of LDL receptor was increased significantly, while the expression of the gene CYP7A1, which is involved in bile acid formation, was decreased significantly compared with the control (diet without cholesterol) These results suggest that the expression of the cholesterol homeostasis gene in liver is modulated due to the cholesterol lowering effect of dietary GlcCer Z Hossain Á T Sugawara (&) Á T Hirata Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan e-mail: sugawara@kais.kyoto-u.ac.jp Z Hossain e-mail: zakirh1000@yahoo.com T Hirata e-mail: hiratan@kais.kyoto-u.ac.jp Z Hossain Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh K Aida Central Laboratory, Nippon Flour Mill Co Ltd, Atsugi, Kanagawa 234-0041, Japan e-mail: kaida@nippn.co.jp Keywords Glycosylceramide Á Sphingolipids Á Sea cucumber Á Cholesterol Á Lipid metabolism Á Mice Introduction The roles of functional foods in preventing various chronic diseases (e.g., cardiovascular disease, allergies, cancer) have attracted much interest recently Sphingolipids are highly bioactive compounds that participate in the regulation of cell growth, differentiation, diverse cell functions, and apoptosis [1, 2] The nutritional significance and the food functional importance of sphingolipids were disregarded for decades It has, however, been reported that dietary supplementation with sphingolipids has diverse physiological effects, such as improving skin barrier function [3, 4], protecting the colon against cancer [5, 6], and inhibiting inflammation [7, 8] Sphingolipids are found in egg, milk, meat, fish, soybean, and so on [9] Dietary sphingolipids can be hydrolyzed by digestive enzymes in the small intestine, although they are relatively hard to hydrolyze and absorb compared with glycerolipids [10–12] On the other hand, it has been reported that sphingomyelin (SM), which is a major phosphosphingolipid in animals, inhibits luminal absorption of cholesterol [13, 14] One potential mechanism for this suppression may be associated with SM, which may decrease micellar solubilization and the transfer of cholesterol from the micellar matrix to intestinal cells In addition, it seems that free sphingoid bases liberated in the intestinal tract may be important in the inhibitory effect of dietary sphingolipids on cholesterol absorption [15] The plasma cholesterol level is dependent on several parameters, including the endogenous synthesis, secretion, 123 1082 and catabolism of the various plasma lipoproteins Other major contributors to the amount of cholesterol entering the body each day include the amount of cholesterol in the diet and the rate at which the dietary cholesterol is absorbed [16, 17] For example, a 90% reduction in cholesterol absorption in moderately hypercholesterolemic subjects has been shown to reduce plasma cholesterol and LDL levels by 35% [18] Physiologically active substances, including glucosylceramide (GlcCer) and some related compounds, have been extracted from a variety of sea cucumber species [19, 20] Dry sea cucumber contains *200 mg GlcCer per 100 g dry weight [21] GlcCer has been isolated from some plant sources and used as a food ingredient, but the GlcCer contents of such plants are very low (1–40 mg/100 g dry weight) [22] Thus, sea cucumber may be a suitable source of dietary GlcCer However, the sphingoid base structures in sea cucumber are more complicated than those in mammals [23], and there is little information on the dietary functions of these sphingoid bases that are not found in mammals The aim of the present study was to evaluate the effects of dietary GlcCer from sea cucumber on plasma and liver lipids in cholesterol-fed mice Materials and methods Preparation of GlcCer GlcCer was prepared from sea cucumber using a silica gel column after lipid extraction and saponification, as described previously [6, 21] Its purity was above 96%, as determined by an HPLC equipped with an evaporative light-scattering detector [22] Fish Sci (2011) 77:1081–1085 Table Compositions of the diets used in the experiment Ingredient C g/kg diet S HC HCS Cornstarch 397.5 397.5 397.5 397.5 Casein 200.0 200.0 200.0 200.0 Dextrinized cornstarch 132.0 132.0 132.0 132.0 Sucrose 100.0 95.0 92.5 87.5 Soybean oil 70.0 70.0 70.0 70.0 Cellulose 50.0 50.0 50.0 50.0 Mineral mix Vitamin mix 35.0 10.0 35.0 10.0 35.0 10.0 35.0 10.0 L-Cystine 3.0 3.0 3.0 3.0 Choline bitartrate 2.5 2.5 2.5 2.5 5.0 5.0 2.5 2.5 Cholesterol Sodium cholate Sea cucumber SL 5.0 5.0 C control diet, S sea cucumber sphingolipid supplemented diet, HC high cholesterol supplemented diet, HCS high cholesterol ? sea cucumber sphingolipid supplemented diet Sampling procedures At the end of the feeding experiment, the mice were sacrificed after blood collection under light ether anesthesia Blood samples were centrifuged at 1,0009g for 15 at 4°C to separate the plasma Plasma samples were stored at -80°C until lipid analysis The liver, spleen, and small intestine were taken, weighed, frozen in liquid nitrogen, and kept at -80°C A portion of the liver was soaked in RNA later and kept at -80°C for the mRNA expression experiment Determination of lipids Animals and diets All animals were treated in accordance with the guidelines on the treatment of animals drafted by the experimentation committee of Kyoto University, Japan Four-week old male ICR mice (Japan SLC, Inc., Hamamatsu, Japan) were housed at 25°C with a 12 h light-dark cycle and acclimatized with a commercial diet (MF, Oriental Yeast, Kyoto, Japan) for week Four groups of mice each were fed for weeks with semisynthetic diets (Table 1) These four groups were fed a control diet (C), a sea cucumber GlcCer supplemented diet (S), a high cholesterol supplemented diet (HC), and a high cholesterol plus sea cucumber GlcCer supplemented diet (HCS), respectively During the feeding period, each group of mice was housed with free access to their assigned diet and water The body weights and the food intakes of the mice were measured every day All of the prepared diets were stored at 0°C and replaced daily 123 Triacylglycerols and total cholesterol of plasma and liver were colorimetrically determined by commercially available enzyme kits (Wako Pure Chemical, Osaka, Japan) according to the manufacturer’s protocol For the liver lipid analysis, total lipids were extracted with ml of a mixture of chloroform and methanol (2:1, v/v) from 0.5 ml of 25% liver homogenate The total lipids were dissolved in ml of Triton X-100 before performing colorimetric assays of the triacylglycerols and cholesterol [24] Determination of mRNA expression of enzymes related to lipid metabolism Total RNA was extracted from the liver of mouse using an RNeasy mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions To quantify the mRNA expression level, real-time quantitative RT-PCR was Fish Sci (2011) 77:1081–1085 performed in a thermal cycler (Bio-Rad, Hercules, CA, USA) using SYBR green PCR reagents The following primers were used: Fas, 50 -ACCATGCCAACCTGGTAAAA-30 (sense), 50 -CAGTGTTCACAGCCAGGAGA-30 (anti-sense); Srebp1c 50 -GGCTGGCCAATGGACTACTA-30 (sense), 50 -GGC TGAGGTTCCAAAGCAGA-30 (anti-sense); Cyp7al, 50 -AG ACCGCACATAAAGCCCGG-30 (sense), 50 -CTTTCATT-G CTTCAGGGCTC-30 (anti-sense); HmgcoAred, 50 -TACAA CGCCCACGCAGCA-30 (sense), 50 -ACCAACCTTCCTAC CTCAGCAA-30 (anti-sense), and Ldlr, 50 -AGCCATTTT CAGTGCCAATC-30 (sense), 50 -GAGGAGGGCTGTTGTC TCAC-30 (anti-sense) The primer pair of Gapdh was 50 -TGGGATCGAGTGAAGGACCT-30 (sense), 50 -CTCCT CCTGCCACTTCTTCTG-30 (anti-sense) The reaction solution (final volume of 20 ll) contained ll of sample, 10 ll of SYBR green dye (Bio-Rad), and ll of each primer The thermal cycling conditions were as follows: 48°C for 30 to prevent DNA carryover, an initial denaturation of 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 s, and an annealing temperature of 55°C for Statistical analyses Data are presented as the mean ± SD and were analyzed by Student’s t test or one-way ANOVA with Fisher’s PLSD test to identify significant differences between the dietary groups P \ 0.05 was considered significant 1083 Fig Body weights of the mice during the experimental period The hepatic expressions of five genes were studied by applying real-time RT-PCR to liver samples from a mouse fed the experimental diet without cholesterol (Fig 2) The mRNA expressions of genes such as Fas and Srebp-1c involved in fatty acid and TG synthesis tended to be higher in mice that were fed dietary sea cucumber GlcCer, but not significantly The mRNA expression of Ldlr was significantly increased, while HmgcoAred showed a tendency to increase compared with the control (diet without cholesterol) The gene Cyp7a1, which is involved in bile acid formation, was significantly decreased compared with the control Results Dietary sea cucumber GlcCer did not affect body weight (Fig 1) Daily food consumption was similar among the four groups: 36.5 ± 4.8, 33.9 ± 5.5, 37.3 ± 5.1, and 35.5 ± 5.2 g/day/eight mice for the C, S, HC, and HCS groups, respectively Based on these data, the calculated daily intakes of cholesterol in the HC and HCS groups were approximately 2.3 and 2.2 mg/day/mouse Liver and spleen weights were significantly increased with the high cholesterol diet (Table 2) In contrast, these increases in liver and spleen weights were significantly suppressed in mice fed GlcCer Sea cucumber GlcCer was used to evaluate the effect of GlcCer on plasma and liver triacylglycerol (TG) and cholesterol concentrations in mice Dietary sea cucumber GlcCer without cholesterol supplement increased plasma TG and decreased plasma total cholesterol (TC) significantly compared with the control group, but liver TG and TC did not alter significantly (Table 3) Although HCS did not change plasma TG and TC compared with the HC group, HCS decreased liver TC significantly compared with the HC group (Table 3) Discussion In our results, dietary sea cucumber GlcCer decreased plasma cholesterol concentrations in mice This cholesterol-lowering effect is possibly, at least in part, mediated by inhibiting the intestinal absorption of cholesterol, and would eventually lead to the protection of the liver from cholesterol-induced steatosis In agreement with this prediction, dietary GluCer significantly suppressed the increase in liver weight caused by a high-cholesterol diet Intestinal absorption of cholesterol depends on bile acids, and is favored by the presence of TG-derived fatty acids in the intestine, which forms a mixture of micelles with bile acids in which cholesterol is solubilized [13] It has been reported that dietary SM inhibits the luminal absorption of cholesterol [14] The formation of stable cholesterol and SM (or sphingosine) complexes could be the cause of the reduced intestinal absorption of cholesterol Because of the diverse chemical structures of the various sphingolipid species, a wide range of physical and chemical properties are expected, so the present results may be due to specific 123 1084 Fish Sci (2011) 77:1081–1085 Table Effects of dietary sphingolipids on body, liver, and spleen weights (in g) for the weeks of the experimental period Organs C S HC HCS Body 37.69 ± 2.83 34.72 ± 1.96 37.64 ± 2.62 36.31 ± 3.33 Liver Spleen 1.44 ± 0.22a 0.12 ± 0.01 1.40 ± 0.11a a a,b 0.13 ± 0.02 2.35 ± 0.36c 1.91 ± 0.16b c 0.16 ± 0.03b 0.20 ± 0.05 C control diet, S sea cucumber sphingolipid supplemented diet, HC high cholesterol supplemented diet, HCS high cholesterol ? sea cucumber sphingolipid supplemented diet Values in rows with different letters are significantly different according to Fisher’s PLSD test (P \ 0.05) Table Plasma and liver lipids of the animals fed different diets for the weeks of the experimental period Plasma (mg/dL) Liver (mg/g) Lipids C S HC HCS TG 114 ± 40b 161 ± 25c 54 ± 13a TC b 153 ± 31 a 114 ± 24 216 ± 44 c TG 39.8 ± 17.0 47.7 ± 27.9 22.0 ± 15.5 38.0 ± 22.3 TC 3.8 ± 0.6a 2.9 ± 0.7a 36.6 ± 4.7c 32.2 ± 6.9b 74 ± 17a 179 ± 43b,c C control diet, S sea cucumber sphingolipid supplemented diet, HC high cholesterol supplemented diet, HCS high cholesterol ? sea cucumber sphingolipid supplemented diet, TG triacylglycerol, TC total cholesterol Values in rows with different letters are significantly different by Fisher’s PLSD test (P \ 0.05) Fig Effects of sea cucumber sphingolipid on the expression levels of Cyp7al, HmgcoAred, Ldlr, Fas, and Srebp-1c mRNA in mouse liver The mouse was fed a sea cucumber sphingolipid supplemented diet for weeks Expressions of Cyp7al, HmgcoAred, Ldlr, Fas, and Srebp-1c were determined by real-time quantitative RT-PCR analysis Data were normalized to GAPDH mRNA levels and are shown as the mean ± SD *P \ 0.01 and **P \ 0.05 versus control according to Student’s t test complex formation with bile acids and the disturbance of bile acid micelles in the intestinal lumen, as well as other factors It was reported that short-term dietary supplement with GlcCer significantly increased serum SM levels without influencing cholesterol levels in rats [25] It is known that two types of cholesterol-raising fatty acids in the diet, saturated fatty acids and trans fatty acids, increase the serum low density lipoprotein cholesterol concentration [26, 27] The increase in cholesterol associated with the sphingolipid-rich diet is likely due to the fatty acids resulting from sphingolipid digestion However, dietary 123 sphingolipids are relatively hard to hydrolyze and absorb compared with glycerolipids [10–12] Indeed, it was also reported that long-term (two generations in duration) dietary supplements of sphingolipids significantly decreased cholesterol (30%) but not SM levels in rats [28] Reducing the cholesterol pool in the liver leads to a reduction in bile acid synthesis, as reflected by reduced expression in the liver of the bile acid synthesis gene Cyp7a1, concomitant with increased expression of genes involved in hepatic cholesterol synthesis (HmgcoAred) and hepatic cholesterol uptake from plasma (Ldlr) To maintain its lipid homeostasis, the liver may compensate for the decreased sphingolipid-mediated dietary and biliary cholesterol and fatty acid supply from the intestine by increasing its endogenous cholesterol and fatty acid synthesis, as reflected in the trend for increased hepatic mRNA concentrations of HmgcoAred, Ldlr, Fas, and Srebp-1c One major regulator of fatty acid synthesis is Srebp-1c, and it was reported that cholesterol feeding resulted in a large increase in the expression of Srebp-1c mRNA in the livers of mice [29] In summary, the sea cucumber GlcCer supplemented diet significantly decreased plasma cholesterol in ICR mice It also decreased liver cholesterol Further study is needed to identify the mechanisms of action by sea cucumber sphingoid bases on intestinal or liver physiology in order to elucidate the scientific basis for their use in the prevention of chronic diseases Acknowledgments This work was supported by the Program for Promotion and Applied Researches for Innovations in Bio-oriented Industry (BRAIN) Fish Sci (2011) 77:1081–1085 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the optimal fat intake? Am J Med 113:25S–29S 28 Kobayashi T, Shimizugawa T, Osakabe T, Watanabe S, Okuyama H (1997) A long-term feeding of sphingolipids affected the levels of plasma cholesterol and hepatic triaceylglycerol but not tissue phospholipids and sphingolipids Nutr Res 17:111–114 29 Knight BL, Hebbachi A, Hauton D, Brown AM, Wiggins D, Patel DD, Gibbons GF (2005) A role for PPARa in the control of SREBP activity and lipid synthesis in the liver Biochem J 389:413–421 123 [...]... Pei-Ning Rd, Keelung 20 2, Taiwan, ROC H.-J Lu Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, 2 Pei-Ning Rd, Keelung 20 2, Taiwan, ROC M Kang (&) Myriax Software Pty Ltd, 110 Murray Street, Hobart, TAS 700 0, Australia e-mail: size100@hotmail.com C.-C Lai Á L.-J Wu Coastal and Offshore Resource Research Center, Taiwan Fishery Research Institute, No.1- 1, Yugang... Fish Sci (2 011) 77:903–913 DOI 10.1007/s12562- 011- 0401-4 ORIGINAL ARTICLE Fisheries Ex situ and in situ measurements of juvenile yellowfin tuna Thunnus albacares target strength Hsueh-Jung Lu • Myounghee Kang Hsing-Han Huang • Chi-Chang Lai Long-Jin Wu • • Received: 10 March 2 011 / Accepted: 18 August 2 011 / Published online: 17 September 2 011 Ó The Japanese Society of Fisheries Science 2 011 Abstract... overhead cover Hydrobiologia 609:205–217 16 Scheuerell MD, Schindler DE (2004) Changes in the spatial distribution of fishes in lakes along a residential development gradient Ecosystems 7:98–106 17 Prchalova M, Kubecka J, Cech M, Frouzova J, Drastık V, Hohausova E, Juza T, Kratochvıl M, Matena J, Peterka J, Rıha M, Tuser M, Vasek M (2009) The effect of depth, distance from dam and habitat on spatial distribution... Koolkalya S, Thapanand T, Tunkijjanujij S, Havanont V, Jutagate T (2006) Aspects in spawning biology and migration of the mud crab Scylla olivacea in the Andaman Sea, Thailand Fish Manag Ecol 13:391–397 8 Walton ME, Le Vay L, Truong LM, Ut VN (2006) Significance of mangrove-mudflat boundaries as nursery grounds for the mud crab, Scylla paramamosain Mar Biol 149 :111 9–1207 9 Walton ME, Le Vay L, Lebata JH, Binas... Fisheries Fishery biology of mud crabs Scylla spp at Iriomote Island, Japan: species composition, catch, growth and size at sexual maturity Cynthia Yuri Ogawa • Katsuyuki Hamasaki Shigeki Dan • Shuichi Kitada • Received: 10 June 2 011 / Accepted: 30 August 2 011 / Published online: 25 September 2 011 Ó The Japanese Society of Fisheries Science 2 011 Abstract The fishery biology of mud crabs Scylla spp was examined... Conserv 138:180–188 11 Lebata MJHL, Le Vay L, Primavera JH, Walton ME, Bin˜as JB (2007) Baseline assessment of fisheries for three species of mud crabs (Scylla spp.) in the mangroves of Ibajay, Aklan, Philippines Bull Mar Sci 80:891–904 12 Ewel KC (2008) Mangrove crab (Scylla serrata) population may sometimes be best managed locally J Sea Res 59 :114 –120 13 Bonine KM, Bjorkstedt EP, Ewel CK, Palik M (2008)... and Billfish, South Pacific Commission, Majuro, Marshall Islands, 8–18 Aug 2004 5 IATTC (2008) Annual report of the Inter-American Tropical Tuna Commission for 2006 IATTC, La Jolla 6 Simmonds J, MacLennan D (2005) Fisheries acoustics: theory and practice, 2nd edn Blackwell, Oxford 7 Hazen EL, Horne JK (2004) Comparing the modeled and measured target-strength variability of walleye pollock, Theragra... Research, Canberra, pp 48–58 28 Wood SN (2006) Generalized additive models: an introduction with R Chapman and Hall/CRC, Boca Raton 29 R Development Core Team (2 011) R: a language and environment for statistical computing R Foundation for Statistical Computing, Vienna 30 Zuur AF, Ieno EN, Walker NJ, Saveliev AA, Smith GM (2009) Mixed effects models and extensions in ecology with R Springer, New York... crabs, should be indispensable for sustainable utilisation of mud crab resources [ 5, 8, 14] Traditionally, mud crabs were grouped into one species, Scylla serrata [17] However, the species identification of the genus Scylla has been controversial [1 8, 19 ], and the researchers have reported that mud crabs include several species/morphs in many places including the Philippines [20 ], Vietnam [21 ], Malaysia... Vietnam [21 ], Malaysia [22 ], India [23] and Japan [ 3, 1 8, 24] Recently, taxonomy of the genus Scylla has been resolved as four distinct species, i.e ., S serrata (Forska˚l ), S tranquebarica (Fabricius ), S olivacea (Herbst) and S paramamosain Estampador by Keenan et al [2] based on both morphometric and genetic characteristics Since then, biological and ecological studies in relation to fisheries have focused