Fisheries science JSFS , tập 77, số 4, 2011 7

Fish Sci (2011) 77:447–454 DOI 10.1007/s12562-011-0348-5 ORIGINAL ARTICLE Fisheries Cost–profit analysis of Japanese-type set-net through technology transfer in Rayong, Thailand Nopporn Manajit • Takafumi Arimoto • Osamu Baba • Seiichi Takeda • Aussanee Munprasit Kamolrat Phuttharaksa • Received: 11 December 2010 / Accepted: 28 February 2011 / Published online: 21 April 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract The Japanese type of set-net, Otoshi-ami, was introduced to Thailand in 2003 with the aim of assessing its feasibility as a sustainable coastal fisheries management tool for the empowerment of coastal fishers’ communities All preparations for constructing and installing the set-net in the coastal waters off Mae Rumpheung beach, Rayong Province, Thailand, were carried out by local fishers, with technical advice and support from national and international institutions The gear was modified and developed to suit the conditions of the fishing ground and target species Data on the catch and sales trends in Rayong using the set-net were collected for years for statistical analysis; during this period, the gear design and marketing strategies were improved Simulation analysis for evaluating the cost–profit bases was conducted to establish a model for set-net technology transfer in Southeast Asia, based on differing numbers of fishers and operation days The simulation results show the required size of the average daily catch to cover the total cost according to the average unit price, where the economic return point is an average catch of 128 kg, based on a unit price of 25 Baht/kg (0.83 USD/kg), with ten fishermen and a daily operation cost of 3,200 Baht (USD 106.17) Keywords Otoshi-ami Á Set-net Á Community-based management Á Simulation model Á Cost–profit analysis Á Economic return point Á Technology transfer Introduction Set-net is a type of stationary fishing gear with a large-scale trap net It is used in coastal waters to intercept migrating N Manajit Graduate Course of Applied Biosciences, Tokyo University of Marine Science and Technology (TUMSAT), 4-5-7 Minato-ku, Tokyo 108-8477, Japan S Takeda Department of Ocean Sciences, Tokyo University of Marine Science and Technology, Tokyo, Japan Present Address: N Manajit Training Department, Southeast Asian Fisheries Development Center (SEAFDEC/TD), P.O Box 97, Phrasamutchedi, Samut Prakan 10290, Thailand A Munprasit Training Department, Southeast Asian Fisheries Development Center (SEAFDEC/TD), P.O Box 97, Phrasamutchedi, Samut Prakan 10290, Thailand T Arimoto (&) Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Tokyo, Japan e-mail: tarimoto@kaiyodai.ac.jp K Phuttharaksa Eastern Marine Fisheries Research and Development Center (EMDEC), Moo 2, Phe Subdistrict, Maung District, Rayong 21160, Thailand O Baba Department of Marine Policy and Culture, Tokyo University of Marine Science and Technology, Tokyo, Japan 123 448 fish schools in the leader net, subsequently entrapping them inside the chamber trap It is a very common form of fishing in Japan, with landings totaling 550,000 metric tons in 2006, which is about 38% of the total landings of coastal fisheries Otoshi-ami, is the most popular type of set-net system in Japan, consisting of a non-return slope net in the main net system [1–3] Large-scale trap nets are popular in some parts of the world, such as the Newfoundland cod traps [3, 4], Baltic traps for salmon [5, 6], tuna traps in the Mediterranean Sea [7, 8], pound-net [9] in the Chesapeake Bay [10], and deep trap net in the Great Lakes [11] Setnets are also commonly used in the coastal waters off East Asia, Korea [12], China, and Taiwan [13] However, among the Southeast Asian countries, the Philippines is the only country to have set-net fisheries, having imported the technology from Japan around 1950 [13] The International Set-Net Fishing Summit held in Himi City, Japan in 2002 promoted the transfer of set-net technology, emphasizing set-net as a coastal fisheries management tool that could support local fishing communities in developing countries [14] The Training Department of Southeast Asian Fisheries Development Center (SEAFDEC/TD) initiated set-net technology transfer by organizing the fisherman’s group in Rayong Province, Thailand, with collaboration from the Eastern Marine Fisheries Research and Development Center (EMDEC), within the framework of the 2-year project for 2003 and 2004 on Sustainable Coastal Fisheries Management in Southeast Asia: case study in Thailand [15] The Tokyo University of Marine Science and Technology (TUMSAT) and Himi City joined the project to cooperate with partners in Thailand In this paper, statistical data on years of the Rayong set-net project for the period from 2003 to 2010 were analyzed by means of a simulation model The aim was to evaluate the cost–profit balances in order to establish a model of set-net technology transfer and profit-sharing system in Thailand and other countries in Southeast Asia Materials and methods Fishing gear and setting site In 2003, the Otoshi-ami-type fishing gear was designed with a 250-m leader net and a main net system that was 45 m wide and 140 m long and consisted of a playground and chamber trap The initial net design was modified in 2004 to be 20 m wide and 155 m long and was installed at a depth of 13 m in the coastal waters of Mae Rumpheung beach, on the east side of the Gulf of Thailand At the end of 2006, a second set-net was installed at a location in the coastal waters off Mae Rumpheung beach at a depth of 14 m, approximately km distant in a northwestern 123 Fish Sci (2011) 77:447–454 Fig Location map of Rayong set-net project direction from the first set-net unit (see Fig 1) The local fishers were organized to make a group with a committee system, both for the fishing operation and management activities The estimated cost of the one set-net unit gear was 500,000 Baht (16,590 USD, when the exchange rate was USD = 30.14 Thai Baht, according to http://themoneyconverter.com/THB/USD.aspx), with costs mitigated by partially using second-hand local materials for constructing the gear The gear was constructed by 45 fishermen, working alternately as teams of 16 fishermen, and was completed within months through volunteerbased participation of the community members The gear installation work was done in weeks in October 2003 with the assistance of MV PLALUNG (35 GT, 17.5 m LOA, 270 HP) from the SEAFDEC Training Department [13, 15] to facilitate safe and efficient working conditions The sea conditions during the monsoon season in this area along the east side of the Gulf of Thailand are generally quite severe For this reason, the fishing season is from October to April each year [15] The hauling operation is performed every days during the fishing season The catch is preserved in plastic container boxes with iced seawater to keep it fresh and of high quality and sold directly to local customers and dealers from their beach fish shop, which is managed by the fishers’ group themselves Gear design improvement process A poor catch and hard maintenance work were noted for the first year (2003) that the set-net was in operation due to an inappropriate design of the gear and lack of familiarity with the gear by the fishers, resulting in a low number of operation days and a low value of unit price After technical advice and support from the Japan-side counterparts Fish Sci (2011) 77:447–454 449 in 2004, the gear design was improved in three major aspects, namely, net width in the main net system was decreased, the setting of the slope net from the playground to the chamber trap was lowered and narrowed, and 800 sandbags, each 60 kg were used to replace the iron anchors originally employed for gear installation in 2003 The routine work of net maintenance, such as the periodical net cleaning and replacement with spare nets, was also improved: every weeks for the chamber trap, months for the playground, and months for the leader net The operation time for the hauling process originally required 40–60 with three to four Thai traditional boats (6.0 m LOA, 60 HP) The hauling process was improved and made easier in 2006 when a fiber-reinforced plastic (FRP) boat (10.9 m LOA, 30 HP) equipped with an engine-driven line-hauler became available The hauling operation with the larger FRP boat together with two local boats just requires 20 min, so completion of all daily activities, including fish sales from the beach fish shop, takes about 2–3 h This large FRP boat also provided the technical support for the gear installation and maintenance tasks carried out only by the fishers’ group Another improvement effort in terms of gear design was the resetting of the bottom fixing ropes at the chamber corners in 2005, which ensured that chamber shape would be maintained even with the strong current in the fishing ground The portable engine for driving the high-pressure water jet and the capstan winch with the bow net roller was also installed onboard the FRP boat, which contributed to make the daily fishing operation and maintenance activities more convenient and time/labor efficient, although the use of these auxiliary devices resulted in higher fuel consumption, thereby increasing the daily operation costs Catch/sale data analysis and simulation process Seven years of data on the catch and sales trends of the Rayong set-net were statistically analyzed with the aim of improving efforts for gear design and marketing strategy The simulation analysis for evaluating the cost–profit bases was conducted to establish a model of set-net technology transfer in Southeast Asia according to differing the number of fishers, unit price, and operation days The profit is Table Factors associated to each table for the cost–profit simulation process of Rayong set-net Factors Number of operation days Number of fishers Average unit price (Baht/kg) Fisher’s salary (Baht/fisher/day) Variable 50 100 150 10 20 10 25 35 100 200 250 200 evaluated as the sale value against the operational cost, where 100 Thai Baht is equivalent with 3.32 USD, as mentioned above The factors for the simulation process are summarized in Table with a set of variables for each factor The number of operation days in the first year was only 52 days due to initial technical difficulties However, after several improvements in the gear design and operational techniques, this reached around 100 days per year The operation days can be increased by every-day hauling—not every days as currently in the custom in Rayong—and also by technical efforts for a longer fishing period, even during the Monsoon season, for setting the variables as 150 and 200 days The number of fishers participating in the group was originally set as 20 fishers in the first year; since 2006, with the introduction of the large FRP boat and line hauler, this number has dropped to 10–12 For the simulation process, the participation of five and ten fishers was set in comparison to the case of 20 fishers Participation by five fishers is not practical in the case of the Rayong set-net operation, while this size of set-net can be operated by two fishers in Japan, so the participation of five fishers is introduced in consideration for a future condition The average unit price for marketing was set as 10, 25, and 35 Baht/kg, according to the actual trend shown by daily market data at Rayong during the 7-year study period This unit price was calculated from the daily total sale value (Baht) divided by daily total catch weight (kg) Each fisher’s salary is the main component of the operational cost and was set according to the actual payment records for years as 100 Baht/fisher/day in 2002, 200 Baht for 2003–2007, and 250 Baht since 2008 to the present Other components contributing to operational costs include boat charges (1,200 Baht/day), paid to the group members for using their own local boats by compensating for the fuel and oil consumption The cost for ice and food/drink consumption was also fixed and covered in the operational costs The analysis process consists of a simple approach in which ‘‘Profit = Sale – Costs’’, where ‘‘Sale = Catch (kg) Unit price (Baht/kg) Operation days’’ and ‘‘Costs = (number of fishers daily salary ? operational cost) Operation days.’’ The yearly profit is calculated according to the average catch amount (kg/day), for the unit price of 10, 25, and 35 Baht/kg, for the number of fishers of 5, 10, and 20, with a daily salary of 200 Baht, and with the number of operation days as the variable in each simulation According to the result of this simulation process, the catch amount of the economic return point (CERP) was also examined to gain an understanding of the sale value balancing the cost The simulation results were compared with the actual yearly trends of CERP with each different condition of cost payment The actual cost calculation was 123 450 Fish Sci (2011) 77:447–454 Results Catch data analysis dominant and economically important species for 2003–2010 are listed in Table At the initial stage of the project in 2003, the total number of fishing operation days was only 52 days due to gear maintenance problems, which resulted in a low catch of 8.7 tons, as shown in Fig The trend in the catch Total value (×103 Baht) 40 Total catch (×103 kg) changed year by year to adjust for the yearly trend in cost profit balance If the yearly total sale value as the catch weight multiplied by the unit price is equal to the total operation cost, CERP can be calculated as the catch weight for showing no profit, according to the unit price, as CERP = C/Up, where C is the operational cost as the daily fixed cost and salary for 10 fishers, and Up is the average unit price (Baht/kg) Here, the operation cost (C) for the simulation process was set as 2,200 Baht with a daily salary of 100 Baht, 3,200 Baht with a salary of 200 Baht, and 3,700 Baht with a salary of 250 Baht for ten fishers, with a fixed daily cost of 1,200 Baht Total catch (×103 kg) Total value (×103 Baht) 30 20 10 101 108 110 98 91 86 1st year 2nd year 3rd year 4th year 5th year 6th year 7th year (2003-) (2004-) (2005-) (2006-) (2007-) (2008-) (2009-) 52 days 2003 -2004 The catch of the Rayong set-net over the 7-year period comprised mainly pelagic species (91.1%) in terms of total weight, with demersal fishes and cephalopods accounting for only minor shares (7.2 and 1.7%, respectively) The Table Dominant and economic species of Rayong set-net catch in 2003–2010 Scientific name 2004 -2005 2005 -2006 2006 -2007 2007 -2008 2008 -2009 1000 900 800 700 600 500 400 300 200 100 2009 -2010 Year of project implementation Fig Total catch (kg) and total value (Baht) of the Rayong set-net according to the year of implementation with number of operation days Common name Family Scomberomorus commerson Narrow barred Spanish mackerel Scombridae Rastrelliger brachysoma Short mackerel Scombridae Rastrelliger kanagurta Indian mackerel Scombridae Atule mate Yellowtail scad Carangidae Selaroides leptolepis Yellowstripe scad Carangidae Alectis indica Indian threadfish Carangidae Parastomateus niger Black pomfret Carangidae Hemiramphus far Blackbarred halfbeak Hemiramphidae Tylosurus acus melanotus Keel-jawed needlefish Belonidae Ablennes hians Flat needlefish Belonidae Amblygaster clupeoides Bleeker’s smoothbelly sardinella Clupeidae Sardinella gibbosa Goldstripe sardinella Clupeidae Sphyraena jello Sphyraena putnamae Pickhandle barracuda Sawtooth barracuda Sphyraenidae Sphyraenidae Sphyraena obtusata Obtuse barracuda Sphyraenidae Nemipterus hexodon Ornate threadfin bream Nemipteridae Scolopsis taenioptera Lattice monocle bream Nemipteridae Trichiurus lepturus Largehead hairtail Trichiuridae Lutjanus lutjanus Bigeye snapper Lutjanidae Siganus canaliculatus White-spotted spinefoot Siganidae Loligo chinensis Mitre squid Loliginidae Loligo duvauceli Indian squid Loliginidae Sepioteuthis lessoniana Bigfin reef squid Loliginidae Sepia recurvirostra Curvespine cuttlefish Sepiidae Pelagic fishes Demersal fishes Cephalopods 123 451 30 20 10 30 20 10 30 20 10 30 20 10 30 20 10 30 20 10 30 20 10 1st year 40 (2003-) n=52 40 2nd year (2004-) n=101 5th year (2007-) n=98 6th year (2008-) n=91 7th year (2009-) n=86 n=101 40 3rd year (2005-) n=108 n=108 n=110 (2003-) n=52 2nd year (2004-) 20 3rd year (2005-) 4th year (2006-) 1st year 20 20 Frequency (%) Frequency (%) Fish Sci (2011) 77:447–454 40 4th year (2006-) n=110 20 40 5th year (2007-) n=98 20 40 6th year (2008-) n=91 20 40 7th year (2009-) n=86 20 0 100 200 300 400 500 600 700 800 900 1,000 10 15 20 25 30 35 40 45 50 55 60 65 70 Unit price (Baht/kg) Catch (kg) Fig Frequency distribution of the daily total catch (kg) of the Rayong set-net according to the year of implementation Fig Frequency distribution of average unit price (Baht/kg) of the Rayong set-net according to the year of implementation improved after the gear had been modified in 2004, with an increase to around 20–30 tons in that year, with the income increasing year by year, up to 900,000 Baht in 2009–2010 The peak level of daily catch amount category in the first year was 50–100 kg, which shifted upwards year after year, ultimately to reach 200–250 kg in the 7th year of the project, with a number of big catch events ([500 kg catch per day) (Fig 3) year (2003–2004), the peak of the average unit price was as low as 10–15 Baht/kg; in the second and third year (2004–2006), it increased to 20–25 Baht/kg, further increasing to 25–30 Bath/kg after the fourth year (2007) Unit price analysis The management skills of the fishers’ group also improved as they gained experience in selling the landed catch from their beach fish shop, run by the group members themselves The techniques for fish handling onboard were also improved, with short-time sorting of the catch onboard and fish being put into ice boxes containing sea water immediately after being scooped out of the set-net Such improved techniques and skills of the fishers’ group resulted in the improvement of the quality of fish sold and, subsequently, a better income, as shown in Fig 2, and also contributed to the improved trend for unit price The yearly trend of the frequency distribution of the average unit price (Baht/kg) is shown in Fig In the first Cost–profit analysis The simulation results of the cost–profit analysis of the Rayong set-net is shown in Fig as the yearly profit according to the average catch amount (kg/day), based on unit prices of 10, 25, and 35 Baht/kg, respectively (Fig 5, horizontal rows, top to bottom) and 5, 10, and 20 fishers, respectively (Fig 5, vertical columns, left to right) The number of operation days varied in each simulation, with the fixed daily cost as 1,200 Baht/day In the case of 10 fishers and a 25 Baht/kg unit price as the center middle simulation in Fig 5, the CERP is 128 kg/day, showing the increase in yearly profit in accordance to the increase in the number of operation days when the daily average catch is larger than 128 kg CERP In the case of a 10 Baht/kg unit price for ten fishers, as shown in the center top simulation, a profit can not be expected with the higher CERP of 320 kg catch per day, so that increasing the number of operation days can never support a profit increase with a daily average catch smaller than 320 kg/day 123 452 Fish Sci (2011) 77:447–454 200 days 150 days 100 days 50 days 3,000,000 2,000,000 200 days CERP as Fishers 10 Fishers 20 Fishers 10 Baht/kg 220kg CERP as 520kg CERP as 320kg 1,000,000 0 100 200 300 400 500 100 200 300 400 500 100 200 300 400 500 Yearly profit (Baht) -1,000,000 3,000,000 25 Baht/kg 2,000,000 CERP as 88kg CERP as 208kg CERP as 128kg 1,000,000 0 100 200 300 400 500 100 200 300 400 500 100 200 300 400 500 -1,000,000 3,000,000 35 Baht/kg 2,000,000 CERP as 91kg CERP as 63kg CERP as 149kg 1,000,000 0 100 200 300 400 500 100 200 300 400 500 100 200 300 400 500 -1,000,000 Average Daily Catch (kg) Fig Simulation results of cost–profit analysis of the Rayong set-net according to the average catch amount (kg/day) for a unit price of 10, 25, and 35 Baht/kg, respectively (horizontal rows) and for 5, 10, and 20 fishers respectively (vertical columns) Salary CERP curve 250 Baht Daily average catch (kg) 400 350 1st year (2003-) 2nd year (2004-) 3rd year (2005-) 4th year (2006-) 5th year (2007-) 6th year (2008-) 7th year (2009-) 200 Baht 300 250 100 Baht 200 150 100 -1 Cy ERP = 3700/Up = 3704.8x -1 = 3199.9x Cy ERP = 3200/Up -1 = 2201.7x = 2200/Up Cy ERP 50 0 10 15 20 25 30 35 40 45 Unit price (Baht/kg) Fig Estimation of economic return point (kilograms) of the Rayong set-net according to the unit price (Baht/kg) and the fishers’ salary (Baht/day) in the case for ten fishers and 100 fishing operation days Comparison of the CERP estimation based on ten fishers for 100 days of operation and the unit price with the fishers’ daily salary as variable (100, 200, and 250 Baht) is shown in Fig as a plot of the actual yearly results for years The lowest CERP is 88 kg in the first year (2003); this was derived from the low number of operation days, 123 namely, 52 days, which was plotted below the simulated curve for the daily salary of 100 Baht, and the cost payment was minimized by the support from the project budget The CERP in this case is much lower than the simulation, but it can be the minimum case without the profit sharing as the bonus at the end of the season, which was decided by the fishers’ committee to be adjusted to the poor catch trend in the first year After the improvement in gear design and operational techniques in the second year (2004), the profit level increased, so as to be able set the higher daily salary as 200 Baht, where the actual data plotting can show the good fit with the simulated curve for the two subsequent years (second and third year) In 2008 (sixth year), the daily salary for fishers was set as 250 Baht; the simulated curve for CERP was then elevated due to the higher cost The actual data plotting for the sixth to seventh year showed that CERP was higher than the simulated curve, which was affected by the higher cost setting for the large FRP boat together with the use of auxiliary devices for fuel consumption; therefore, the daily operation cost was revised to 1,600 Baht from the original setting of 1,200 Baht/day Fish Sci (2011) 77:447–454 Discussion During the 7-years period covered by this analysis, starting with the transfer of technology of the Japanese-type set-net in Rayong, the catch and sale value trends have increased through technical improvements in gear design and improved operation and management skills, including fish handling, marketing, and selling It is important that the design of the set-net fits with the specific oceanographic conditions of the fishing ground and with the migration route and behavior of the target species Fish behavior in relation to trap design is the key factor for optimizing design [3] Nomura [16] described the importance of fish behavior in relation to the design of set-nets, especially the behavioral response to the leader net and the main net system Akiyama and Arimoto [17] also indicated a possible approach to have better catch performance of set-net according to the gear design and subsequently performed a data analysis of catch trend for different gear designs of set-net [18] The modification of the gear design in 2004 in Rayong was based on the popular design of a small scale set-net unit used in the coastal area of Toyama Bay, Japan, with the aim of increasing the entrapping efficiency in the chamber net through the use of a narrower main net system and decreasing the chance of escape with narrower and lower sloped net settings The learning experience in the Rayong case requires continuous effort in terms of rethinking set-net design based on monitoring of the catch trend over a long-term period The setting site decision is another factor, especially in relation to the sea bed topography [3, 16] for estimating the migration route of fish schools towards the coastal waters Following the installation of the second set-net unit in 2006, the annual total catch increased, while the catch did not achieve double the amount of the single unit (Fig 2) This result suggests that these two neighboring units share the same stock migrating to the Mae Rumpheung water due to the close proximity of the two units The appropriate setting distance of neighboring units should be considered in any future expansion phase of the set-net technology transfer program The simulation results on cost–profit balances of the Rayong set-net show that a higher unit price with few fishers can enable an income and profit increase under community-based management In this management strategy under the committee system for a fishers’ group, the yearly profit is partially shared among fishers as a bonus at the end of the fishing season, partially spent as the expenditure for daily/yearly gear maintenance (e.g., repair and replace parts of the gear), and partially saved for preparing new units of set-net gear (as in 2006) or for introducing new devices (such as the line-hauler or high pressure water jet net cleaner in 2008) 453 For the simulation process of CERP in Fig 6, a simple approach was carried out to calculate the CERP value for each year, without considering the bonus-sharing among the fishers, maintenance costs, and saving for future additional costs, as mentioned above The initial cost to construct and install the gear was provided from the project budget in the case of the Rayong set-net The depreciation process was also not considered in this project, while the maintenance costs and savings for new unit introduction are important for future sustainability Even using this simple approach for considering the cost–profit balance, the optimum operational cost can be set through the decision-making for daily operation costs on the fisher’s salary, fuel/ice and boat charge, according to the number of fishers and operation days The catch and sale trends of newly introduced set-net gear are difficult to predict even with the results of a prior survey, as was carried out in Rayong by SEAFDEC [13, 15] The fishers’ group in Rayong was forced to set the cost payment factors to adjust with the sales and profit trends year by year through their decision-making process under the committee system This is also another learning process in the Rayong case and will be applicable to other cases of set-net technology transfer The pilot project on technology transfer of the community-based set-net in Rayong, Thailand, is a possible solution for empowering small-scale fishers to work together as a group, with the potential of successfully increasing their income Another positive impact of set-net is the environmentally friendly aspects [19] of the technology This technology transfer program should be studied by long-term monitoring, and future extension phases should be promoted in other regions of Thailand and Southeast Asian countries Acknowledgments The authors would like to thank the participants of the Rayong set-net fishers group for their full engagement and cooperation in the set-net operation and management We also express our sincere gratitude to the participating institutes for their support in both the practical and theoretical aspects of set-net technology transfer: Eastern Marine Fisheries Research and Development Center (EMDEC); Department of Fisheries, Thailand, and Training Department of Southeast Asian Fisheries Development Center (SEAFDEC/TD); Tokyo University of Marine Science and Technology (TUMSAT) funded by Japan Society for the Promotion of Science (JSPS); Himi City, Toyama Prefecture, Japan, funded by Japan International Cooperation Agency (JICA) References Gabriel O, Lange K, Dahm E, Wendt T (2007) Fish catching methods of the world, 4th edn Blackwell, London Akiyama S (2010) Teichi-ami In: Takeuchi T et al (eds) A handbook on fisheries science (in Japanese) Seibutsu Kenkyusha, Tokyo, pp 256–258 He P, Inoue Y (2010) Large-scale fish traps: gear design, fish behavior, and conservation challenges In: He P (ed) Behavior of 123 454 10 11 12 Fish Sci (2011) 77:447–454 marine fishes: capture processes and conservation challenges Blackwell, Singapore, pp 159–181 Story GM, Kirwin WJ, Widdowson JDA (1990) Dictionary of Newfoundland English, 2nd edn University of Toronto Press, Toronto Siira A, Suuronen P, Kauppinen Ikonen E, Erkinaro J (2006) Survival of Atlantic salmon captured in and released from a commercial trap-net: potential for selective harvesting of stocked salmon Fish Res 80:280–294 Suuronen P, Siira A, Kauppinen T, Riikonen R, Lehtonen E, Harjunpaa H (2006) Reduction of seal-induced catch and gear damage by modification of trap-net design: design principles for a seal-safe trap-net Fish Res 79:129–138 Ravier C, Fromentin J-M (2001) Long-term fluctuations in the Eastern Atlantic and Mediterranean bluefin tuna population ICES J Mar Sci 58:1299–1317 Wardle CS, Videler JJ, Arimoto T, Franco JM, He P (1989) The muscle twitch and the maximum swimming speed of giant bluefin tuna, Thunnus thynnus L J Fish Biol 35:129–137 Bridger JP, Foster JJ, Margetts AR, Strange ES (1981) Glossary of United Kingdom fishing gear terms Fishing News Books, Farnham Reid GK Jr (1955) The pound-net fishery in Virginia Part History, gear description and catch J Commer Fish Rev 17:1–15 Hubert WA (1983) Passive capture techniques In: Nielsen LA, Johnson DL (eds) Fisheries techniques Southern Printing, Blacksburg, pp 95–122 Yun IB, Lee JH, Kwon BG, Cho YB, Yoo JB, Kim SH, Kim BY (2004) A study on improvement for fishing gear and method of the Pound-net-I-Net shapes of the commercial net in the flow Bull Korean Soc Fish Tech 40(4):268–281 123 13 Training Department (2008) Set-net fishing technology transfer for sustainable coastal fisheries management in Southeast Asia Report TD/RES/107 Southeast Asian Fisheries Development Center, Phrasamutchedi, pp 1–214 14 Fisheries and Fishing Port Division (2003) Report on the set net training program in Himi Fisheries and Fishing Port Division, Himi, pp 1–117 15 SEAFDEC/TD and DOF Thailand (2005) Final report of set-net project/Japanese trust fund I: introduction of set-net fishing to develop the sustainable fisheries management in Southeast Asia: case study in Thailand 2003–2005 Report TD/RP/74 SEAFDEC/TD and DOF Thailand, Phrasamutchedi, pp 1–402 16 Nomura M (1980) Influence of fish behavior on use and design of setnets In: Bardach JE et al (eds) Fish behaviour and its use in the capture and culture of fishes: ICLARM Conference Proceedings 5, International Center for Living Aquatic Resources Management, Manila, pp 446–471 17 Akiyama S, Arimoto T (2001) Set-net—capture process research and prospects for the development of selective fishing gear and techniques (in Japanese) Nippon Suisan Gakkaishi 67:134–135 18 Akiyama S, Arimoto T (2000) Analysis of accumulation performance of differing set-net designs Fish Sci 66:78–83 19 Munprasit A, Amornpiyakrit T, Yasook N, Yingyuad W, Manajit N, Arimoto T (2005) Fishing methods and catch composition of stationary fishing gear in Thailand (in Japanese with English abstract) In: Proc Steering Committee for the Colloquium on Fishing Technology Jap Soc Fish Sci 50:34–35 Fish Sci (2011) 77:455–466 DOI 10.1007/s12562-011-0343-x ORIGINAL ARTICLE Fisheries Simulation of copepod biomass by a prey–predator model in Hiuchi-nada, central part of the Seto Inland Sea: does copepod biomass affect the recruitment to the shirasu (Japanese larval anchovy Engraulis japonicus) fishery? Hiromu Zenitani • Naoaki Kono • Youichi Tsukamoto Received: 10 October 2010 / Accepted: March 2011 / Published online: 27 April 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract We have modeled the prey–predator dynamics between nutrients, phytoplankton, and copepods in Hiuchinada, central part of the Seto Inland Sea The model parameters were estimated by stepwise regression using data sampled from 2001 to 2005 We re-created the fluctuations in copepod biomass in the spring–summer of 2001–2004 by model simulation and investigated the relationship between the re-created copepod biomass and anchovy Engraulis japonicus reproductive success rate in Hiuchi-nada The anchovy reproductive success rate was proportional to the copepod biomass during the last 10 days of May, a period that immediately preceded anchovy recruitment This relationship indicates that a possible key factor in the regulation of anchovy population levels is the fluctuation in abundance of the copepod assemblage and that the crucial period for anchovy recruitment in Hiuchinada would be the period just before anchovy recruitment to the shirasu (body length: approx 20–35 mm) fishery These results provide a potential framework for forecasting the anchovy recruitment level that is based on both larval abundance and survival rate as estimated from the biomass of copepods in the pre-recruitment period of anchovy H Zenitani (&) Fisheries Research Institute, Toyama Prefectural Agricultural, Forestry, and Fisheries Research Center, Namerikawa, Toyama 936-8536, Japan e-mail: hiromu-zenitani@pref.toyama.lg.jp N Kono National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Hastukaichi, Hiroshima 739-0452, Japan Y Tsukamoto Seikai National Fisheries Research Institute, Fisheries Research Agency, Nagasaki, Nagasaki 851-2213, Japan Keywords Japanese anchovy Á Prey–predator model Á Recruitment Á Seto Inland Sea Introduction The Seto Inland Sea of Japan is well known for its high fishery production and is one of the most productive areas of enclosed or semi-enclosed waters in the world (Seto Inland Sea, 21 t km-2 year-1; North Sea, t km-2 year-1; Chesapeake Bay, t km-2 year-1; Baltic Sea, t km-2 year-1; Mediterranean Sea, t km-2 year-1) [1] The Japanese anchovy Engraulis japonicus is an important commercial species in the Seto Inland Sea (2006 annual catch: 60 103 t, accounting for 36% of the total fish production) [2] Hiuchi-nada is located in the central part of the Seto Inland Sea and is a major Japanese anchovy spawning (Fig 1) [3] and fishing ground (2005 annual catch in Hiuchinada was 15 103 t, accounting for 27% of that in the Seto Inland Sea) [4] Hiuchi-nada, located between Kurushima strait and Bisan strait, has a size of about 50 30 km and an average depth of about 20 m The water exchange between the bay and straits in the summer has been found to be important to the density structure and nutrient supply in the bay [5, 6] A part of the water in Hiuchi-nada enters the Kurushima strait through the surface layer and moves into the Bisan strait through the bottom layer, which can be considered as a compensatory effect to the intrusion of water from the two straits [7] There is a clockwise circular residual current in the western part of Hiuchi-nada, whereas a counterclockwise circular residual current occurs in the eastern part of Hiuchi-nada [8] Hiuchi-nada can be regarded as a natural test area for larval anchovy growth and survival studies due to its largely enclosed condition, as anchovy eggs 123 a 14.1 ± 0.4a 47.6 ± 1.3 17.5 ± 0.4b Aspartic acid Threonine 234 ± 6.2c 236 ± 7.0c Alanine N.D 11.0 ± 0.4 N.D N.D 6.65 ± 0.3 a-Amino-n-butyric acid Valine Cysteine Cystathionine Isoleucine a a 14.6 ± 0.8b a 11.2 ± 0.1 20.5 ± 0.6c a Tyrosine b-Alanine c 101 ± 3.0c a 8.13 ± 0.2c a 13.5 ± 0.4 88.6 ± 2.0b b b-Amino-iso-butyric acid Ornithine Histidine Tryptophan Arginine 4060b Total 4350c 34.6 ± 4.0 18.3 ± 0.7 15.8 ± 0.5 1.80 ± 0.1 b b 3220a 29.2 ± 3.5 a 99.8 ± 0.6c 16.1 ± 0.7 b 23.8 ± 0.1 c 3.97 ± 0.0a N.D 5.58 ± 0.2c N.D N.D 8.20 ± 0.3 b 8.08 ± 0.6a 13.9 ± 0.2 c 12.3 ± 0.9 a 8.40 ± 1.5 ab 2.10 ± 0.0 N.D 12.8 ± 0.1 N.D 1.85 ± 0.0 b 180 ± 1.4a 5.05 ± 0.0b 155 ± 1.1a N.D N.D 232 ± 1.0 a 17.0 ± 0.1 c 19.9 ± 0.1c 55.2 ± 0.4 c 2210 ± 20 a 98 ± 4.9e Jan-2007 d b g e e b d bc 4060b 196 ± 10 d 108 ± 2.0d 17.1 ± 0.4 49.1 ± 1.6 g 6.95 ± 0.2b N.D 5.53 ± 0.2bc N.D N.D 17.3 ± 0.5 f 21.9 ± 0.6d 27.9 ± 0.6 30.6 ± 1.0 21.9 ± 1.8 2.15 ± 0.0 N.D 32.7 ± 0.7 N.D 1.60 ± 0.0 a 234 ± 5.2c 6.35 ± 0.0c 269 ± 6.3c N.D N.D 359 ± 8.0 d 40.6 ± 0.8 e 41.9 ± 1.1f 61.2 ± 1.3 2370 ± 40 139 ± 4g Feb-2007 Different superscript letters within the same row indicate significant differences at P \ 0.05 82.9 ± 6.0 Proline 13.7 ± 0.4 6.72 ± 0.1b 5.17 ± 0.1a 4.77 ± 0.2a a N.D Ethanolamine Lysine N.D 5.20 ± 0.1b b N.D N.D c-Aminobutyric acid a 7.57 ± 0.2 Phenylalanine 6.83 ± 0.9 11.8 ± 0.1 10.6 ± 0.8 b 9.38 ± 0.3 a Leucine a a 7.63 ± 1.4 ab 1.80 ± 0.0 N.D 10.9 ± 0.1 N.D 1.85 ± 0.3 a a 2.83 ± 0.0 Citrulline ab 7.68 ± 0.8cd 262 ± 6.1c 6.50 ± 0.2c 267 ± 7.0c a-Aminoadipic acid Glycine c N.D N.D Sarcosine N.D N.D b Glutamine 250 ± 6.0 7.6 ± 0.2 298 ± 8.0 c 11.7 ± 0.4 a Glutamic acid Serine b 40.1 ± 1.0 3230 ± 60 b o-Phosphoserine 2840 ± 60 48.8 ± 1.8a d Nov-2006 Harvest (month-year) Taurine 56.8 ± 2.8b e Dec-2006 Miyazaki Growing area e a a c b e c a 4500cd 135 ± 3.0 c 91.9 ± 1.7b 12.0 ± 0.9 26.8 ± 0.5 d 13.5 ± 0.4d N.D 7.07 ± 0.1d N.D N.D 11.1 ± 0.4 d 19.2 ± 0.6c 18.0 ± 0.2 20.9 ± 0.3 15.2 ± 0.2 c 2.68 ± 0.1c 3.05 ± 0.2a 19.8 ± 0.4 1.55 ± 0.1 N.D 216 ± 3.0b 5.00 ± 0.0b 219 ± 3.0b 25.7 ± 1.7 85.5 ± 2.7 297 ± 7.0 c 30.9 ± 0.6 d 37.6 ± 0.7e 71.5 ± 1.3 3070 ± 60 d 47.9 ± 1.4a Mar-2007 Table Free amino acid concentrations of Japanese oyster (mean ± SD, mg/100 g dry matter, n = 3) c c c b f d c 5030e 410 ± 11 e 104 ± 3.0cd N.D 42.9 ± 2.2 f 20.2 ± 1.0f N.D 13.0 ± 0.5e N.D 7.65 ± 0.2 13.0 ± 0.4 e 80.4 ± 2.6f 25.7 ± 0.9 27.9 ± 0.5 16.8 ± 0.1 d 2.08 ± 0.3ab N.D 19.7 ± 1.3 4.07 ± 0.3 2.47 ± 0.3 427 ± 12f 8.43 ± 0.3d 368 ± 11e N.D N.D 439 ± 14 ef 83.0 ± 2.2 f 60.7 ± 2.0g 123 ± f 2660 ± 100 67.4 ± 2.4c Nov-2006 Miyagi b 5020e 378 ± 4.0 d 80.0 ± 1.4a N.D 32.7 ± 0.7 e 7.25 ± 0.2b N.D 14.9 ± 0.3f N.D 4.40 ± 0.1 b 8.72 ± 0.1 c 61.9 ± 0.9e 14.9 ± 0.2 a 17.1 ± 0.6 9.6 ± 1.5 b 2.15 ± 0.0b N.D 11.3 ± 0.2 a 1.78 ± 0.0 a 3.23 ± 0.0 d 321 ± 6.1d 4.20 ± 0.0a 345 ± 7.0d N.D N.D 436 ± 7.1 ef 81.4 ± 1.5 f 30.1 ± 0.4d 149 ± g 2960 ± 30 d 48.0 ± 1.7a Dec-2006 4660d 337 ± 57 de 83.9 ± 17ab N.D 31.2 ± 9.5 cdef 9.67 ± 4.6abcdef N.D 12.7 ± 0.3e N.D 5.02 ± 2.2 bc 9.52 ± 2.0 abcde 71.6 ± 6.5ef 16.7 ± 8.1 abcdefg 19.0 ± 7.8 bcde 11.2 ± 5.2 bc 2.13 ± 0.1b N.D 12.6 ± 5.3 ab 2.23 ± 1.6 ab 2.30 ± 0.1 b 296 ± 110cdef 5.45 ± 2.6abcd 313 ± 49cd N.D N.D 413 ± 19 e 73.5 ± 8.3 f 35.8 ± 21e 146 ± 20 fg 2690 ± 20 c 64.2 ± 2.2c Jan-2007 b c g f d i g c 5830f 461 ± 5.0 f 153 ± 1.0f N.D 98.0 ± 0.2 h 31.0 ± 0.2g N.D 23.9 ± 0.0f 9.30 ± 0.0 7.98 ± 0.1 39.0 ± 0.3 h 109 ± 1.0h 51.3 ± 0.2 72.9 ± 0.3 42.4 ± 0.1 g 3.73 ± 0.1e N.D 57.3 ± 0.4 4.83 ± 0.1 2.52 ± 0.1 b 332 ± 1.1e 6.65 ± 0.1c 547 ± 2.2g N.D 266 ± 7.0 490 ± 2.2 168 ± 1.0 h 102 ± 1.0i 279 ± i 2350 ± 10 123 ± 1f Feb-2007 5090e 436 ± 12e 131 ± 2.0e N.D 43.0 ± 0.6f 15.6 ± 0.5e N.D 19.2 ± 0.3g 8.73 ± 0.4 13.20 ± 0.3d 26.6 ± 0.4g 105 ± 2.0g 38.4 ± 0.4h 50.1 ± 0.7f 30.2 ± 0.6f 3.32 ± 0.1d N.D 37.7 ± 0.6e 4.13 ± 0.1c 2.37 ± 0.9b 329 ± 5.0d N.D 489 ± 8.3f N.D 175 ± 5.2b 451 ± 9f 143 ± 2.0g 82.9 ± 1.5h 186 ± 3h 2180 ± 30a 87.3 ± 2.2d Mar-2007 Fish Sci (2011) 77:687–696 691 123 123 c ab ab 95 ± 34a 1580 ± 180ab 540 ± 140a ab ab 1740 ± 180 55 ± 38a 1580 ± 140ab 421 ± 130a Alanine Citrulline Valine Methionine 598 ± 280ab ab 620 ± 460ab ab Tyrosine 36300 37600 b 1370 ± 230a 2350 ± 290 ab 34800 ab 1390 ± 250a 2160 ± 230 ab 2190 ± 210 694 ± 41 c 1300 ± 140 ab 351 ± 110ab 2360 ± 260 ab 1500 ± 200 ab 370 ± 130a 105 ± 23a 1560 ± 150ab 1620 ± 150 ab 2520 ± 220 a 5170 ± 500 bc 1570 ± 150 ab 1550 ± 130 b 3650 ± 360ab 4730 ± 230d a 31600 a 1350 ± 130a 1860 ± 240 ab 1990 ± 290 ab 683 ± 110 a 1120 ± 150 a 398 ± 230a 2030 ± 250 ab 1250 ± 150 427 ± 95a 51 ± 46a 1360 ± 160a 1440 ± 130 a 2250 ± 140 a 4760 ± 530 ab 1370 ± 140 a 1370 ± 150 a 3250 ± 380a 4610 ± 3000cd Feb-2007 34500 ab 1520 ± 340ab 2130 ± 400 ab 2100 ± 390 ab 721 ± 130 c 1220 ± 240 ab 757 ± 510ab 2200 ± 400 ab 1320 ± 260 ab 659 ± 180a 61 ± 53a 1450 ± 260ab 1550 ± 240 ab 2400 ± 410 ab 5200 ± 830 c 1480 ± 230 ab 1470 ± 250 ab 3550 ± 590ab 4670 ± 410cd Mar-2007 Different superscript letters within the same row indicate significant differences at P \ 0.05 Total 1600 ± 300ab Proline ab 2300 ± 270 Arginine b 2530 ± 300 b 2430 ± 230 Lysine c 820 ± 98 c 1340 ± 160 c 760 ± 57 c 1300 ± 100 Histidine Phenylalanine 2540 ± 290 2490 ± 270 1560 ± 200 Leucine 1540 ± 160 ab Isoleucine ab 1760 ± 180 2680 ± 300 5660 ± 620 ab 2610 ± 240 5530 ± 580 ab Glycine Glutamic acid 1600 ± 140 1630 ± 140 c Serine 1580 ± 160 ab 1640 ± 230 ab Threonine b 3910 ± 400b 3820 ± 350ab Aspartic acid b 5020 ± 330d 4240 ± 280bc Taurine Jan-2007 a 30400 a 1420 ± 340a 1850 ± 170 ab 1900 ± 220 547 ± 60 a 1140 ± 100 a 677 ± 110ab 2010 ± 200 a 1280 ± 140 a 425 ± 120a 77 ± 21a 1360 ± 120a 1680 ± 180 ab 2140 ± 220 a 4530 ± 450 ab 1370 ± 110 a 1360 ± 150 a 3120 ± 280a 3490 ± 300a Nov-2006 Dec-2006 Harvest (month-year) Nov-2006 Miyagi Growing area Miyazaki Table Total amino acid concentrations of Japanese oyster (mean ± SD, mg/100 g dry matter, n = 3) ab 31000 a 1450 ± 210a 1760 ± 160 a ab 1960 ± 250 573 ± 71 ab 1160 ± 110 a 585 ± 120ab 2040 ± 210 a 1300 ± 150 456 ± 58a 85 ± 27a 1380 ± 140a 1570 ± 150 ab 2220 ± 230 a 4590 ± 450 a 1420 ± 150 a 1380 ± 150 a 3230 ± 310a 3800 ± 290ab Dec-2006 35400 ab 1740 ± 280ab 2110 ± 190 ab ab 2270 ± 250 662 ± 74 ab 1360 ± 140 ab 676 ± 200ab 2370 ± 250 ab 1550 ± 200 ab 539 ± 190a 97 ± 32a 1620 ± 160ab 1680 ± 160 ab 2490 ± 230 ab 5190 ± 500 bc 1610 ± 150 ab 1580 ± 140 b 3790 ± 360ab 4080 ± 380ab Jan-2007 b 39400 b 2310 ± 100c 2330 ± 210 b bc bc 2350 ± 150 696 ± 40 1460 ± 90 b 974 ± 360b 2490 ± 160 b 1550 ± 100 b 669 ± 210a 68 ± 53a 1720 ± 90b 1870 ± 90 2920 ± 90 b 6000 ± 340 c 1780 ± 100 b 1770 ± 130 b 4160 ± 250b 4260 ± 100bc Feb-2007 33500a 1910 ± 150b 1920 ± 130ab 2000 ± 230ab 617 ± 64ab 1260 ± 120ab 683 ± 180ab 2130 ± 210ab 1350 ± 140ab 589 ± 51a 63 ± 28a 1500 ± 150ab 1620 ± 140ab 2430 ± 190a 5040 ± 430c 1490 ± 120a 1470 ± 130a 3470 ± 250ab 3920 ± 320ab Mar-2007 692 Fish Sci (2011) 77:687–696 36.7 ± 1.1a a 9.80 presented in Table The minimum and maximum contents were obtained from oysters harvested at Miyagi in November (30400 mg/100 g on a dry weight) and February (39400 mg/100 g dry weight), respectively 36.3 ± 1.1 Fatty acid composition 34.5 ± 2.1 a The fatty acids found in oysters from the two growing areas are presented in Table In the case of saturated fatty acids, palmitic acid (16:0) made an important contribution to this fraction, i.e., from 21.1% to 24.9% of total fatty acid In general, oysters from Miyagi showed a higher percentage of monoenoic acid than oysters from Miyazaki In November and December, oysters from Miyazaki showed significantly lower monoenoic acid content (14.8–15.5%) than oysters from Miyagi (20.6–22.4%) Polyenoic fatty acid predominantly contained eicosapentaenoic acid (EPA 20:5n-3) and docosapentaenoic acid (DHA 22:6n-3) EPA content did not show significant differences between harvesting months EPA percentages in oysters at Miyazaki and Miyagi were in the range of 11.8–12.8% and 13.8–14.8% of total lipid, respectively In general, the DHA content at Miyazaki was higher than at Miyagi The highest DHA content/total fatty acid fraction was found in oysters from Miyazaki harvested in November (17.5%) Different superscript letters within the same row indicate significant differences at P \ 0.05 42.2 ± 3.5 c RPolyenoic 40.8 ± 1.1 c 38.4 ± 1.8 ab 39.5 ± 1.5 bc 38.9 ± 2.4 ab 35.2 ± 1.9 a 35.2 ± 1.6 a 12.7 ± 1.2b 12.0 ± 0.6ab 10.5 8.70 11.6 ± 0.4ab 10.9 10.2 ± 1.4ab 9.55 ± 1.4a 11.9 12.8 13.5 13.1 ± 0.9b 14.2 ± 0.3b 17.5 ± 1.8b 12.3 Others 12.4 ± 0.4a 20:5n-3 22:6n-3 12.8 ± 1.1a 14.8 ± 1.0a RMonoenoic 13.8 14.4 ± 0.4b 14.5 ± 0.5b 10.8 14.2 ± 0.0b 13.8 ± 0.8ab 14.8 ± 1.0b 7.64 15.5 ± 0.4a 7.46 Others 5.52 ± 0.4 5.99 ± 0.2 11.8 ± 1.2a 6.07 12.3 ± 0.4a 12.5 ± 0.8a 14.2 ± 0.3b 14.2 ± 2.8ab 12.3 22.4 ± 0.6c 9.54 21.3 ± 0.8b 21.3 ± 0.7bc 9.45 9.89 21.0 ± 1.2bc 9.64 20.6 ± 0.9b 8.81 19.9 ± 1.5b 8.00 5.76 ± 0.5 7.06 ± 0.7 19.5 ± 1.1b 10.0 ± 0.6 9.06 ± 0.8 5.32 ± 0.4 9.54 ± 0.6 c 18:1n-7 19.2 ± 1.0b 11.0 ± 0.8c c 10.4 ± 0.6 1.28 ± 0.1a 1.36 ± 0.3a c c 1.42 ± 0.1a a ab b 6.07 ± 0.2b 1.35 ± 1.0a 18:1n-9 ab 2.34 ± 0.0a 43.0 ± 0.5b RSaturated ab 5.74 ± 0.6b 5.77 ± 0.5b 2.05 ± 0.5a 1.85 ± 0.0a 7.35 40.9 ± 0.8a 7.19 42.3 ± 1.0ab 44.3 ± 1.3b 7.85 6.39 43.8 ± 0.8b 5.98 44.2 ± 0.6b 8.44 41.2 ± 0.3a 8.15 6.50 43.6 ± 0.6b 6.40 Others 10.7 41.0 ± 0.3a 5.50 ± 0.3 5.21 ± 0.1 4.63 ± 0.2 5.37 ± 0.6 5.18 ± 0.4 4.29 ± 0.3 5.05 ± 0.1 4.93 ± 0.1 18 42.4 ± 0.8ab 4.40 ± 0.3a 4.99 ± 0.1 2.37 ± 0.1a b b 2.43 ± 0.4 3.12 ± 0.2 ab 3.29 ± 0.3 b 2.50 ± 0.3 2.44 ± 0.4 b a 4.02 ± 0.7 b 5.28 ± 0.2 b 5.26 ± 0.3 18 Iso 693 b 2.28 ± 0.1 21.1 ± 0.4ab a 22.5 ± 0.5 22.6 ± 0.9 a ab 23.3 ± 0.5 ab 24.9 ± 1.2 a 20.6 ± 0.8 20.7 ± 0.7 a b 20.4 ± 0.4 c 22.3 ± 0.9 c 22.5 ± 0.4 16 5.68 ± 0.2c 5.34 ± 0.5bc b bc 5.92 ± 0.2c 5.78 ± 0.1c bc c 5.40 ± 0.2bc 4.29 ± 0.2ab ab ab 4.53 ± 0.5b 3.03 ± 0.5a a ab 4.47 ± 0.1b 3.91 ± 0.7ab 14 b Mar-2007 Nov-2006 Mar-2007 Feb-2007 Nov-2006 Harvest (month-year) Jan-2007 Dec-2006 Miyazaki Growing area Table Fatty acid composition of Japanese oyster (%, w/w, mean ± SD, n = 3) Miyagi Dec-2006 Jan-2007 Feb-2007 Fish Sci (2011) 77:687–696 Discussion Biometric parameters and condition indices The Miyazaki area has warm sea water because of the Black Current, whereas sea water in the Miyagi area has low temperature owing to the Chishima Current and cultivation depth (20 m) The warmer conditions explain the more favorable oyster growth at Miyazaki Therefore, commercial-scale 2-year cultivation of oysters is common at Miyagi, whereas 1-year cultivation is common at Miyazaki CICG reflects the fullness of an oyster cup, with higher CICG indicating larger oyster meat Despite this difference in age of oysters at harvest time, CIGC did not show a significant difference between oysters from Miyagi and Miyazaki when they were harvested in November, January, February, and March It is reported that Alaskan oysters cultivated for year showed CIGC values of 84–155 [19], whereas oysters from Miyazaki and Miyagi showed CIGC values ranging from 132 to 160 and from 147 to 169, respectively A higher CIGC value indicates that the oyster cup is fuller; therefore, the cups of Japanese oysters harvested from November to March were well filled with oyster meat CIE is an indicator of economic quality of oysters, with higher CIE indicating economically higher 123 694 quality In this study, the CIE values of oysters from Miyazaki and Miyagi ranged from 0.41 to 0.50 and from 0.32 to 0.41, respectively During each month of cultivation, Miyazaki oysters tended to show higher CIE values than Miyagi oysters Oysters from Alaska [19] and Ireland [20] were reported to have CIE values of 0.35–0.41 and 0.43–0.75, respectively The CIE values of oysters of our study were within the ranges of these reports, which means an economically reasonable size to harvest Proximate composition, and mineral analysis In general, oysters from Miyagi showed higher glycogen contents in all of the months of collection than oysters from Miyazaki The highest glycogen content (4.99%) was found in oysters at Miyagi harvested in February Therefore, longer cultivation and low cultivation temperature may be associated with accumulation of glycogen, which may be reflected in the quality and taste of oysters [19] Despite the absence of significant differences in the two condition indices, the age of oysters reflects the accumulation of glycogen The glycogen contents of oysters in Mediterranean Sea were reported to be 2.09–11.52% [21] while Japanese oyster from Standard Table of Food Composition in Japan [22] showed 4.7% The calcium content of oysters harvested in November at Miyazaki was the highest at 7.73 mg/g dry sample, which was much lower than that reported for Italian oysters C gigas [2] Italian oysters were reported to have calcium contents ranging from 12 to 27 mg/g dry sample while Japanese oyster from Standard Table of Food Composition in Japan [22] showed 5.87 mg/g (after conversion to dry matter basis) The location and characteristics of the growing area such as stream and temperature are supposed to affect the mineral composition of oysters The magnesium contents ranged from 5.49 mg/g for oysters harvested in November at Miyagi to 12.2 mg/g for oysters harvested in December at Miyazaki, while Japanese oyster from Standard Table of Food Composition in Japan [22] showed 4.93 mg/g The magnesium content of oyster harvested in December at Miyazaki was significantly the highest (12.15 mg/g) among all of the samples Different ocean environment including stream and temperature is known to affect the chemical composition of oysters We have also tried to determine seawater minerals The sodium content of oyster correlated to the sodium content of sea water with correlation coefficient r = 0.9755 for Miyazaki and r = 0.9865 for Miyagi (data not shown) There were no significant changes in the potassium content with the month of harvest, in agreement with the report on the seasonal potassium content of oysters in Italy [2] The potassium content of oyster correlated to the potassium content of sea water The copper contents of oysters ranged from 201 to 306 lg/ 123 Fish Sci (2011) 77:687–696 g, while Japanese oyster from Standard Table of Food Composition in Japan [22] showed 50 lg/g In December, February, and March, oysters harvested at Miyagi showed significantly higher copper contents than oysters harvested from November to March at Miyazaki In general, the iron content was high in the oysters from Miyazaki, and oysters from Miyazaki harvested in January showed a significantly higher Fe content than those harvested in other months The iron contents of oysters in this study ranged from 220 to 373 lg/g, which were much higher than those of oysters from Italy (116–191 lg/g) [2] and Japanese oyster from Standard Table of Food Composition in Japan (130 lg/g) [22] The zinc contents of oysters from Miyagi and Miyazaki ranged from 511 to 598 lg/g, while Japanese oyster from Standard Table of Food Composition in Japan [22] showed 880 lg/g Oysters harvested in January at Miyazaki showed the highest zinc content The zinc contents of the oysters in this study were much higher than the zinc contents of Italian oysters C gigas [2] but smaller than given in Standard Table of Food Composition in Japan [22] The phosphate contents of oysters from Miyagi and Miyazaki ranged from 11.3 to 15.6 lg/g, while Japanese oyster from Standard Table of Food Composition in Japan [22] showed 6.67 mg/g The phosphate content in the oysters harvested at Miyazaki was higher than at Miyagi, but vice versa in January Free amino acid and total amino acid compositions Taurine is reported to be a predominant FAA in oysters [3, 19], and taurine has been recognized to play an important role in human physiological functions [8, 9] In this study, taurine was also the predominant FAA in all the samples, accounting for 30–60% of total FAA Oysters were reported to show no seasonal difference in total FAA content, but showed a significant difference in total FAA content between cultivation areas [3, 19] The same observations were also reported for different edible bivalves such as Pacific lions-paw scallops [23] and mussels [24] The total FAA contents of oysters in this study showed slight difference by harvesting month, but the results related to the effect of cultivation areas were in agreement with those in previous studies Japanese oyster was reported to contain 30670 mg/ 100 g of total amino acids [25] The oysters harvested in February at Miyagi tended to show higher glycine and proline contents at 2920 and 2310 mg/100 g, respectively (shown in Table 4, nonsignificant effect observed) while Standard Tables of Food Composition in Japan, amino acid composition of foods mentions glycine and proline contents of 2470 and 1930 mg/100 g [25], respectively There were no significant changes in total amino acid Fish Sci (2011) 77:687–696 composition except for taurine, serine, glycine, histidine, and proline From proximate analysis, the protein contents determined by nitrogen analysis were approximately 45000 mg/100 g dry matter The highest total amino acid content of 39400 mg/100 g was much lower than 45000 mg/100 g, indicating that nitrogen-containing compounds such as ammonia and urea may be present at certain percentages, as shown by the proximate protein analysis by the Kjeldahl method (nitrogen converting factor 6.25) Fatty acid composition The saturated fatty acids accounted for 41.0–44.2% of the total fatty acids, while Japanese oyster from Standard Table of Food Composition in Japan [22] showed 29.5% The monounsaturated and polyunsaturated fatty acids accounted for 14.8–22.4% and 35.2–42.2%, respectively, while Japanese oyster from Standard Table of Food Composition in Japan [22] showed 23.1% and 41.0% Surh et al [4] and Oliveira et al [19] reported the fatty acid composition of oysters From their reports, the calculated percentages of saturated, monounsaturated, and polyunsaturated fatty acids were 35.2–40.2%, 16.2–21.0%, and 37.1–50.4% except for the spawning season in Korea, and 23.1–31.5%, 17.6–23.7%, and 44.5–55.2% in Alaska, respectively Compared with these reports [4, 19, 22], oysters in this study contained higher percentage of saturated fatty acids and lower percentages of polyunsaturated fatty acids of total lipid The percentages of palmitic acid in the extracted lipids of oysters from Miyazaki and Miyagi were higher than those of Alaskan oysters (17.5%, [19]) but similar to those of Irish oysters C gigas [20] Alaskan oysters were reported to have 17.7–24.2% EPA and 13.3–20.0% DHA [19], and Irish oysters were reported to have 10.8–15.2% EPA and 11.0–15.5% DHA [20] Curz-Romeo et al [26] reported the EPA and DHA contents of oysters from Ireland to be 23.1% and 9.4%, respectively From comparison of these reports, we consider that the harvesting month has smaller effects on fatty acid compositions than the cultivation area We have determined many parameters of oysters cultivated in two areas with different durations until harvest From this study, we conclude that oysters from Miyazaki showed good quality in March with high CICG, glycogen, and lipid contents Oysters from Miyagi showed excellent quality in February with high CICG and high FAA contents These analysis data including not only condition indices but also biochemical composition might be useful parameters allowing establishment of evaluation criteria Acknowledgments The authors thank Dr Makoto Terayama (Miyazaki Prefectural Fisheries Experimental Station) and Mr Nasumi Tomikawa (Kesennuma Miyagi Prefectural Fisheries Experimental Station) for the generous supply of oyster samples 695 References Chew KK (1990) Global bivalve shellfish introductions World Aquac 21:9–22 Orban E, Di Lena G, Masci M, Nevigato T, Casini I, Caproni R, Gambelli L, Pellizzato M (2004) Growth, nutritional quality and safety of oysters (Crassostrea gigas) cultured in the lagoon of Venice (Italy) J Sci Food Agric 84:1929–1938 Hosoi M, Kubota S, Toyohara M, Toyohara H, Hayashi I (2003) Effect of salinity change on free amino acid content in Pacific oyster Fish Sci 69:395–400 Surh J, Ryu J-S, Kwon H (2003) Seasonal variations of fatty acid compositions in various Korean shellfish J Agric Food Chem 51:1617–1622 Crosby MP, Gale LD (1990) A review and evaluation of bivalve condition index methodologies with a suggested standard method J Shellfish Res 9:233–237 Hand RE, Nell JA (1999) Studies on triploid oysters in Australia XII Gonad discolouration and meat condition of diploid and triploid Sydney rock oysters (Saccostrea commercialis) in five estuaries in New South Wales, Australia Aquaculture 171:181–194 Imai T, Sasaki S (1961) Study on the breeding of the Japanese oyster, Crassostrea gigas Tohoku J Agric Res 12:125–171 Fuke S (1994) Taste-active components of seafoods with special reference to umami substances In: Shahidi F, Botta JR (eds) Seafoods: chemistry, processing technology and quality Blackie Academic & Professional, Glasgow, pp 115–139 Konosu S, Yamaguchi K (1982) The flavor components in fish and shellfish In: Martin RE, Flick G, Hebard CE, Ward DR (eds) Chemistry and biochemistry of marine food products AVI publishing Company, Westport, CN, pp 367–404 10 Leaf A, Weber PC (1988) Cardiovascular effects of n-3 fatty acids New Engl J Med 318:549–557 11 AOAC (1995) Official methods of analysis Association of Official Analytical Chemists, Washington, DC ¨ zden O ¨ , Erkan N, Deval MC (2009) Trace mineral profiles of 12 O the bivalve species Chamelea gallina and Donax trunculus Food Chem 113:222–226 13 PS Chen Jr, Tribara YT, Warner H (1956) Microdetermination of phosphorus Anal Chem 28:1756–1758 14 Carroll NV, Longley RW, Roe JJ (1956) The determination of glycogen in liver and muscle by use of anthrone reagent J Biol Chem 220:583–593 15 Ogushi M, Harada R (1999) Proximate composition and extractable components of sun-dried gonads of sea cucumber (Konoko) and corresponding marine products J Jpn Soc Nutr Food Sci 52(2):79–84 16 Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification Can J Biochem Physiol 37:911–917 17 AOCS (1989) Official method Ce 1b-89 Official method for marine oil fatty acid composition by GLC In: Firestone D (ed) Official methods and recommended practices of the American Oil Chemists’ Society, 4th edn AOCS Press, Champaign 18 Hochberg Y (1988) A sharper Bonferonni procedure for multiple tests of significance Biometrika 75:800–803 19 Oliveira ACM, Himelbloom B, Crapo CA, Verholt C, Fong Q, RaLonde R (2006) Quality of Alaskan maricultured oysters (Crassostrea gigas): a one-year survey J Food Sci 71:532–543 20 Linehan LG, O’Conner TP, Burnell G (1999) Seasonal variation in the chemical composition and fatty acid profile of Pacific oysters (Crassostrea gigas) Food Chem 64:211–214 21 Dridi S, Romdhane MS, Elcafsi M (2007) Seasonal variation in weight and biochemical composition of the pacific oyster, Crassostrea gigas in relation to the gametogenic cycle and 123 696 environmental conditions of the Bizert lagoon, Tunisia Aquaculture 26:238–248 22 The Ministry of Education, Culture, Sports, Science, Technology, Japan (2010) Standard table of food composition in Japan Government Publications Service Center, Tokyo 23 Beltran-Lugo AI, Maeda-Martines AN, Pacheco-Aguilar R, Nolasco-Soria HG (2006) Seasonal variations in chemical, physical, textural, and microstructural properties of adductor muscles of pacific lions-paw scallop (Nodipecten Subnodosus) Aquaculture 258:619–632 24 Fuentes A, Fernandez-Segovia I, Escriche I, Serra JA (2009) Comparison of physico-chemical parameters and composition of 123 Fish Sci (2011) 77:687–696 mussels (Mytilus galloprovincialis Lmk.) from different Spanish origins Food Chem 112:295–302 25 The Ministry of Education, Culture, Sports, Science, Technology, Japan (2010) Standard tables of food composition in Japan, amino acid composition of foods Government Publications Service Center, Tokyo 26 Curz-Romeo MC, Kerry JP, Kelly AL (2008) Fatty acids, volatile compounds and colour changes in high-pressure-treated oysters (Crassostrea gigas) Innov Food Sci Emerg Tech 9:54–61 Fish Sci (2011) 77:697–705 DOI 10.1007/s12562-011-0372-5 ORIGINAL ARTICLE Food Science and Technology Partial characterization of alkaline proteases from viscera of vermiculated sailfin catfish Pterygoplichthys disjunctivus Weber, 1991 Ana Gloria Villalba-Villalba • Ramo´n Pacheco-Aguilar • Juan Carlos Ramirez-Suarez • Elisa Miriam Valenzuela-Soto Francisco Javier Castillo-Ya´n˜ez • Enrique Ma´rquez-Rı´os • Received: August 2010 / Accepted: 25 April 2011 / Published online: 11 June 2011 Ó The Japanese Society of Fisheries Science 2011 Abstract Vermiculated sailfin catfish (Pterygoplichthys disjunctivus, Weber, 1991), a member of the Loricariidae family and an invasive species of several inland waters around the world, possess an enormous digestive tract representing about 10% of fish weight Thus, the aim of this study was to partially characterize proteases from their digestive tracts Azocasein digestion of the crude extract of intestine at different pH values and temperatures revealed the presence of alkaline proteases with optimum activities at pH 9.0 and 50°C Incubation assays of the crude extract with inhibitors such as phenyl methyl sulfonyl fluoride, N-a-p-tosyl-L-lysine chloromethyl ketone, N-tosyl-phenyalanine chloromethyl ketone, benzamidine, pepstatin A and ethylenediamine tetra-acetic acid showed that trypsin and chymotrypsin are the main alkaline proteinases present Zymography showed that the crude extract of A G Villalba-Villalba Á R Pacheco-Aguilar (&) Á J C Ramirez-Suarez Á E M Valenzuela-Soto Centro de Investigacio´n en Alimentacio´n y Desarrollo (CIAD), Carretera a La Victoria Km 0.6 Apdo Postal 1735, 83000 Hermosillo, Sonora, Mexico e-mail: rpacheco@ciad.mx A G Villalba-Villalba e-mail: anagloria@estudiantes.ciad.mx J C Ramirez-Suarez e-mail: jcramirez@ciad.mx E M Valenzuela-Soto e-mail: elisa@ciad.mx F J Castillo-Ya´n˜ez Á E Ma´rquez-Rı´os Universidad de Sonora, 83000 Hermosillo, Sonora, Mexico e-mail: jcastillo@guayacan.uson.mx E Ma´rquez-Rı´os e-mail: emarquez@guayacan.uson.mx Pterygoplichthys disjunctivus viscera contained proteases with molecular masses ranging from 21.5 to 116 kDa Trypsin and chymotrypsin were inhibited by the following ions in decreasing order: Hg2?, Fe2?, Cu2?, Li?, Mg2?, K?, while Mn2?, and Ca2? had no effect Activities decreased continuously as the NaCl concentration increased from to 30% These results constitute important background information for future studies and for the potential biotechnological use of the crude digestive extract from this invasive species Keywords Pterygoplichthys disjunctivus Á Serin proteases Á Trypsin Á Chymotrypsin Á Visceras Introduction Most analytical studies in enzymology have focused on mammal or microorganism enzymes, mainly because samples from these two types of organisms are easy to obtain However, there is increasing interest in learning more about digestive enzymes from other different species, such as fish from different aquatic environments, either marine or freshwater [1] This is due to the different performance characteristics that these enzymes can offer to the food industry Proteases constitute one of the most important groups of industrial enzymes, and account for at least 60% of all global enzyme sales [2, 3] These proteases may have some unique properties for industrial applications, such as in the detergent, food, pharmaceutical, leather and silk industries [4] Acid proteases in the stomach and alkaline proteases in the intestines are the most important digestive proteolytic enzymes in fish viscera As a general rule, alkaline proteases from tropical fish have higher thermal stabilities, higher activities over a wide 123 698 range of pH values, and longer shelf lives [5, 6] than their mammalian counterparts The vermiculated sailfin catfish (Pterygoplichthys disjunctivus, Weber, 1991) is a loricariid catfish that is native to the Madeira River drainage of the Amazon Basin in Brazil and Bolivia [7], but has successfully invaded several inland waters around the world [8] It was introduced to Me´xico as an ornamental fish or a cleaner of fish tanks, but has since proceeded to invade most of the inland waters south of Me´xico However, this fish has a special characteristic that can be considered an advantage: its abundant viscera For most fish, the viscera accounts for about 5% of the total body mass [9], but for vermiculated sailfin catfish, it corresponds to 10–25% of the total body weight (Ramirez-Suarez JC, personal communication, 2008) The whole fish is considered to be of little practical use in the region Nevertheless, the aforementioned characteristics make the proteases of this fish a target for the food industry Nowadays, biotechnology can provide the means to transform this raw material into a valuable product, such as an enzyme extract [10] Little is known about the physiological biochemistry of the vermiculated sailfin catfish, as most of the literature refers to its biology However, since this species can become an important fishing resource due to its heightened ability to invade water bodies around the world, studies into its possible uses must be conducted In addition, as no information is available regarding the use of active proteases from vermiculated sailfin catfish, the aim of this study was to characterize the alkaline proteases from its viscera and to generate basic information about this by-product Materials and methods Fish samples Vermiculated sailfin catfish specimens were obtained from the Adolfo Lo´pez Mateos Dam (also commonly known as ‘‘El Infiernillo’’), located at the boundary of the Mexican states of Michoaca´n and Guerrero (18°520 –18°150 N and 101°540 –102°550 W) Samples were cryogenically frozen in situ with liquid N2, placed between layers of CO2, and transported by airplane to the CIAD Seafood Products Quality and Biochemistry Laboratory located in Hermosillo, Sonora, Me´xico Preparation of the crude enzyme extract At the laboratory, thawed vermiculated sailfin catfish specimens were dissected, their intestines (containing juice and food) were removed and immediately frozen, and they were maintained at -80°C until further analysis Intestines 123 Fish Sci (2011) 77:697–705 (100 g) were homogenized at 20,0009g with 200 ml of 50 mM Tris–HCl buffer pH 8.0, 10 mM CaCl2 and 0.5 M NaCl for and centrifuged at 18,0009g for 30 at 2–4°C The supernatant (enzyme extract) was frozen and kept at -80°C until further analysis [11, 12] Optimum pH and temperature The effect of pH on the total protease activity was evaluated using universal buffer from pH 5–11 at 25°C for 15 [13]; 100 mM glycine–HCl buffer was used for pH at 25°C and 100 mM glycine–NaOH for pH 12 In order to study the effect of temperature on enzyme activity, the extract was incubated at 25, 30, 40, 50, 60, 70 and 80°C for 15 in 50 mM Tris–HCl buffer under optimal pH (the pH was directly measured in the reaction mixture at each indicated temperature) Optimum pH and temperature assays were performed using 1% azocasein pH and thermal stabilities The effects of pH and temperature on enzyme stability (total proteases, trypsin and chymotrypsin) were evaluated by measuring the residual activity after incubation at various pH values (from to 12) for 60 at 25°C using the buffers described in the ‘‘Optimum pH and temperature’’ section The enzyme solution temperature stability was evaluated by incubation at various temperatures (30, 40, 50, 60, 70 and 80°C) for 60 Total protease activity Total protease activity was assayed using 1% azocasein in 50 mM Tris–HCl at pH [14] Triplicates of 10 ll of enzyme extract were mixed with 0.5 ml of buffer and 0.5 ml of substrate solution Reaction mixtures were incubated for 15 at 25°C Proteolysis was stopped by adding 0.5 ml 20% trichloroacetic acid (TCA), and the mixture was centrifuged in microcentrifuge tubes for 15 at 10,0009g One unit of the enzyme was defined as the activity that increased the absorbance by 1.0 at 366 nm for Trypsin and chymotrypsin activities of extract Amidase activity assay In order to elucidate the possible trypsin amidase activity, the extract was treated with mM N-benzoyl-DL-arginine p-nitroanilide (BAPNA) as substrate, while chymotrypsin amidase activity was evaluated using 0.1 mM Suc-AlaAla-Pro-Phe-p-nitroanilide (SAPNA) as substrate [15] Thus, appropriately diluted enzyme solution (10 ll) was Fish Sci (2011) 77:697–705 added to 990 ll of substrate solution at 25°C and the production of p-nitroaniline was measured by monitoring its increase in absorbance at 410 nm every 30 s for 10 One unit of the enzyme was defined as the activity that produced lM p-nitroaniline released/min, using 8800 M-1 cm-1 as the extinction coefficient of p-nitroaniline at 410 nm Esterase activity assay The esterase of trypsin was determined using mM N-a-ptosyl-L-arginine methyl ester hydrochloride (TAME) as a substrate according to Hummel [16] Thus, an appropriately diluted enzyme solution (10 ll) was added to 990 ll of substrate solution at 25°C The release of tosyl arginine was measured at 247 nm One unit of the enzyme was defined as the hydrolysis of lM of TAME for min, using 540 M-1 cm-1 as the extinction coefficient of tosyl arginine at 247 nm Inhibition assays of extract proteases In order to elucidate the type of enzymes presented on the extract, a series of inhibitors were evaluated Thus, phenylmethylsulfonyl fluoride (PMSF) (100 mM in 2-propanol) as a serine class protease inhibitor, N-a-p-tosyl-L-lysine chloromethyl ketone (TLCK) (10 mM in dimethyl sulfoxide, DMSO) and benzamidine (10 mM in DMSO) as specific trypsin inhibitors, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) (5 mM in methanol) as a specific chymotrypsin inhibitor, and ethylendiamine tetra-acetic acid (EDTA) (10 mM in distilled water) and pepstatin A (1 mM in DMSO) as specific inhibitors of metalloproteases and aspartic proteases, respectively, were used All of these inhibitors were preincubated at a ratio of 1:1 for h at 25°C Control assays were performed without any addition of protease inhibitor Residual activity was reported as the percentage of the control activity (activity measured in the absence of inhibitor was considered 100%) Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and zymography Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out according to Laemmli [17] using gels of 14% polyacrylamide with 0.1% SDS For protein band analysis, a volume of enzyme extract containing 25 lg of protein was mixed with two volumes of sample buffer Zymography was performed according to the procedure of Laemmli [17], except that samples were not heated and no reducing agents were added After electrophoresis, the gel was soaked for 30 in 1.25% casein in 50 mM Tris–HCl at pH 8.0 and 4°C Then the gel was 699 immersed in the same solution at 37°C for 60 and soaked in trichloroacetic acid for 30 to stop the reaction, before being washed in distilled water, fixed and stained with 0.05% Coomassie Blue solution In order to characterize the types of proteases present in the extracts by SDSPAGE, enzyme extracts were incubated with the same protease inhibitors Solutions of PMSF (100 mM), benzamidine (10 mM), TLCK (10 mM), TPCK (5 mM), EDTA (10 mM) and pepstatin A (1 mM) were added separately to enzyme extracts containing mU in the ratio 1:2 (inhibitor:extract) and incubated at 25°C for 60 [18] Distilled water (replacing inhibitors) was used as the control Samples were mixed in sample buffer (no heat and no reducing agents were added), loaded into gels, and incubated as described above Effects of metal ions The effects of KCl, LiCl, CaCl2, MnCl2, MgCl2, CuSO4, FeSO4 and HgSO4 (5 mM) on trypsin and chymotrypsin activity were carried out using BAPNA and SAPNA as the substrate, respectively Enzyme solution (30 ll) was mixed with 70 ll of ion solution, incubated at 25°C for 30 min, and then 900 ll of substrate were added to the mixture The production of p-nitroaniline was measured by monitoring its increase in absorbance at 410 nm every 30 s for 10 The activities of the enzymes in the absence of metal ions were taken as controls Effect of NaCl Trypsin and chymotrypsin activities were assayed in the presence of NaCl at varying final concentrations (0, 5, 10, 15, 20, 25 and 30%, w/v), using TAME and SAPNA as the substrate, respectively The activities of the enzymes in the absence of NaCl were taken as controls Protein concentration determination The protein concentration in each sample was determined according to Bradford [19], using bovine serum albumin as the standard Results Optimum pH and temperature The intestinal extract obtained from the vermiculated sailfin catfish viscera showed its maximum activity at pH (Fig 1a), which can thus be considered its optimum pH at 25°C As expected, this result indicates the existence of a group of alkaline proteases functioning in the intestinal tract of the species The optimum temperature (at which the 123 700 Fish Sci (2011) 77:697–705 Fig Optimal pH (a) and optimal temperature (b) of total alkaline proteases from Pterygoplichthys disjunctivus viscera extract The activity was assayed at pH and 25°C, using 1% azocasein in 50 mM Tris–HCl as a substrate Values are expressed as the mean of triplicate determinations extracted proteases showed their highest activities) was 50°C; the activity decreased abruptly at 60°C, indicating the denaturation of the enzymes in the extract and the subsequent loss of activity (Fig 1b) The trypsin and chymotrypsin activity assays gave 0.60 ± 0.08 U/mg of protein and 3.06 ± 0.12 U/mg of protein, respectively This assay showed that the chymotrypsin activity was fivefold that of the trypsin activity pH and thermal stabilities The effect of pH on total alkaline protease activity is shown in Fig 2a Enzyme activity was stable ([95%) for 60 at pH and (activity was highest at this latter pH); however, the total alkaline protease activity remained high (93% of activity) at pH Extreme pH values, such as 4, 5, and 12, affected the enzyme activity of the extract the most, reducing it by up to 95, 70 and 60% after just of incubation, respectively Figure 2b and c show the effect of pH on the trypsin and chymotrypsin activities present in the enzyme extract Trypsin activity was stable (C90%) at 123 Fig Effect of pH on the stability of all alkaline proteases (a), trypsin (b) and chymotrypsin (c) from Pterygoplichthys disjunctivus viscera extract The stability was tested by incubating the enzyme extract for 60 at 25°C using azocasein, BAPNA and SAPNA, respectively pH values between and 10 for 60 At pH and 7, the trypsin activities were 63 and 84%, respectively; however, trypsin activity decreased to 22% at pH 4, while all of the activity was completely lost at pH 12 On the other hand, the chymotrypsin activity was more stable than that of trypsin at all pH values, showing stabilities of 90% or higher between pH and 10 during the 60 of incubation time Besides, the activity declined Fish Sci (2011) 77:697–705 701 for 30 min, but dropped by about 20% after incubation at 50°C for 60 However, enzyme activities declined by about 80% when incubated at 60°C for 60 When higher temperatures were used, such as 70 and 80°C, the activity decreased abruptly during the first of incubation, indicating enzyme denaturation and a subsequent loss of activity Sodium dodecyl sulfate–polyacrylamide gel electrophoresis and zymography Fig Thermal stabilities of total alkaline proteases (a), trypsin (b) and chymotrypsin (c) from Pterygoplichthys disjunctivus viscera extract The thermal stabilities were tested by incubating the enzyme extract for 60 at pH 9, using azocasein, BAPNA and SAPNA, respectively by only 40% at extreme alkaline pH (pH 11 and 12) The lowest activity was observed at pH and 5: 25 and 50% of the remaining chymotrypsin activity, respectively (Fig 2c) The thermostability assay of total alkaline proteases (Fig 3a), trypsin (Fig 3b) and chymotrypsin (Fig 3c) showed similar stability patterns for these enzymes: enzyme activities remained high (B90%) at 30 and 40°C The protein composition of the enzyme extract was evaluated by SDS-PAGE Several protein bands were found in the enzyme extract obtained from this species (Fig 4a) A wide range of proteins with molecular weights between 6.5 and 116 kDa were found On the other hand, a zymogram of the protease activity zones of the enzyme extract from vermiculated sailfin catfish viscera treated with several inhibitors is shown in Fig 4b Different classes of proteases were detected in the presence of their specific inhibitors through the disappearance or reduced intensity of the corresponding bands compared to the control (without inhibitors) The zymogram indicated that most of the enzymes present in the extract were serine proteases, since the presence of PMSF (a serine protease inhibitor) led to the disappearance or a reduction in the intensity of all bands between 10 and 116 kDa TPCK, a chymotrypsin inhibitor, caused a reduction in band intensity around 30 kDa (which apparently corresponds to chymotrypsin; see upper arrow in Fig 4a), while TLCK and benzamidine—trypsin inhibitors—inhibited a band around 25 kDa (which apparently corresponds to trypsin; see the lower arrow in Fig 4a) On the other hand, EDTA and pepstatin A (which are not serine protease inhibitors) did not produce any reductions in band intensity A low molecular weight protease exhibiting high activity was found at around 10–12 kDa As observed, several bands that showed activity on the control were inhibited according to the specific inhibitor used, as also shown by the protease inhibition analysis, which used the same type of inhibitors Inhibition assays of extract proteases The effects of different protease inhibitors on the total extract activity were evaluated (Fig 5) PMSF reduced the alkaline protease activity by up to 68.9 ± 3.3% (from 1.48 to 0.47 U/mg, control vs inhibitor), confirming the presence of this type of protease (to which trypsin and chymotrypsin belong) Thus, to verify the presence of these enzymes in the extract, several protease-specific inhibitors were used TLCK (which presented the most effective alkaline protease inhibition for the extract) and benzamidine inhibited activity by 97.0 ± 0.5 (from 1.48 to 0.074 U/mg, control vs 123 702 Fish Sci (2011) 77:697–705 Fig a SDS-PAGE electrophoresis gel image showing different bands of proteins and proteases from Pterygoplichthys disjunctivus viscera extracts Lanes show (MW) molecular weight markers; enzyme extract Upper arrow is most probably chymotrypsin Lower arrow is most probably trypsin b Zymogram showing the effects of protease inhibitors Lanes: control, extract enzyme without inhibitors; PMSF; TPCK; TLCK; benzamidine; EDTA; pepstatin A; control, extract enzyme without inhibitors Table Effect of metal ions on enzyme activity Metal ions Activity (%) Trypsin Fig Effects of specific inhibitors on the proteases from Pterygoplichthys disjunctivus viscera extract Values are expressed as the mean ± SD from triplicate determinations inhibitor) and 45.7 ± 0.4% (from 1.48 to 0.82 U/mg, control vs inhibitor), respectively; these results confirm the presence of trypsin in the extract On the other hand, TPCK, a chymotrypsin-activity inhibitor, resulted in inhibition of 61.4 ± 0.5% (from 1.48 to 0.30 U/mg, control vs inhibitor), confirming the presence of this enzyme in the extract EDTA showed inhibition of 15.8 ± 1.5% (from 1.48 to 1.30 U/mg, control vs inhibitor), indicative of the presence of metalloproteases in the extract Pepstatin A reduced enzyme activity by 4.2 ± 0.7% (from 1.48 to 1.43 U/mg, control vs inhibitor); however, inhibitions of \10% of the activity are considered to be negligible Effects of metal ions The effects of ions on the activities of trypsin and chymotrypsin are shown in Table The enzymes showed 123 Chymotrypsin Control (no ions) 100 100 CaCl2 100 100 MnCl2 100 100 KCl 95 96 92 MgCl2 95 LiCl 90 93 CuSO4 FeSO4 87 73 85 77 HgSO4 63 61 Enzyme extract was preincubated with metal ions (5 mM) at 25°C for 30 The trypsin and chymotrypsin activities were determined using BAPNA and SAPNA as the substrate, respectively Control experiments were performed under identical conditions without metal ions 100% activity (i.e., the same activity as seen in the control experiments) with Ca2?, Mn2?, and more than 90% activity with K?, Mg2? and Li? Ions such as Cu2? and Fe2? led to activities of around 85 and 70%, respectively, while both enzymes showed around 65% activity in the presence of Hg2? Effect of NaCl The effects of NaCl on the activities of trypsin and chymotrypsin are shown in Fig A continuous decrease in both trypsin and chymotrypsin activity was observed with increasing NaCl concentration, possibly due to denaturation caused by the salting-out phenomenon However, the activities of trypsin and chymotrypsin remained high (67 and 65%, respectively), even at 30% NaCl Fish Sci (2011) 77:697–705 Fig Effects of NaCl concentration on the activities of trypsin and chymotrypsin at pH and 25°C using TAME and SAPNA as the substrate, respectively Discussion This study presents a partial characterization of the alkaline proteases in the viscera of vermiculated sailfin catfish (Pterygoplichthys disjunctivus, Weber, 1991) The crude intestinal extract from this fish was found to possess high proteolytic activity in the alkaline pH range 8–10, indicating the presence of a group of alkaline proteases with an optimum pH for activity of Similar results were found by Natalia et al [20] for alkaline proteinases from extracts from the pancreas and intestine of Scleropages formosus (using azocasein as substrate), which showed maximum activities between pH and 10 On the other hand, Das and Tripathi [21] reported that the optimal pH range of alkaline proteases from the digestive tract of grass carp Ctenopharyngodon idella digestive tract was in the range 7.6–8.4, although bovine albumin was used as the substrate Similarly, Chiu and Pan [22] reported that the optimal pH values for trypsin and chymotrypsin activity were and 7, respectively Chong et al [23] reported that intestine alkaline proteases from Symphysodon aequifasciata showed two high-activity peaks, one in the pH range 7.5–9 and the other at 11.5–12.5, using casein as the substrate The literature indicates that the optimal pH of alkaline proteases from digestive extracts, and the substrates used for their analysis, varies slightly among fish species However, in most cases, the optimum pH range for trypsins and chymotrypsins from the digestive organs of fish species ranged from to 10 [14] Species can offer new catalytic options, such as wide ranges of catalytic activity and stability Hence, the effects of pH and temperature on both the activity and stability of the extract were analyzed Results showed that maximum 703 alkaline protease stability when incubated for 60 occurred in the pH and temperature ranges of 7–10 and 30–50°C, respectively These stability ranges are probably due to the structures of the alkaline proteases, which are better suited to activity under these conditions, as they promote a better enzyme–substrate interaction [12] Some enzymes can show greater activities at higher temperatures and pH values (such as the enzymes in the present study) compared to normal physiological conditions, indicating that these enzymes can work normally under unusual conditions Temperature is an important factor in an enzymatic reaction, because enzyme activity can be altered by changes at its catalytic site In the present study, trypsin and chymotrypsin most effectively catalyzed reactions at 50°C, and showed the same thermostability pattern Similar results were obtained by Dı´az-Tenorio et al [24], who reported that 55°C was the optimum temperature for digestive proteases from Callinectes bellicouss The SDS-PAGE and zymogram analysis of an extract obtained from the intestine of Pterygoplichthys disjunctivus showed that a variety of proteases are synthesized by its digestive gland Most of them hydrolyzed natural and synthetic substrates at alkaline pH The specific inhibition (PMSF, TLCK, benzamidine and TPCK) and substrate (BAPNA and SAPNA) analyses, as well as the zymogram, indicated that the alkaline proteases present were mainly trypsin and chymotrypsin However, further analysis must be done to identify the nature of these enzymes A low molecular weight protease (around 10–12 kDa) found in the extract appeared to be a serine protease, since it was inhibited by PMSF and TLCK inhibitors; however, its identity remains unknown Similar results were found by Celis-Guerrero et al [18] for the spiny lobster Panulirus interruptus, which suggests that this type of protease is commonly found in such specimens The methods used in the present study suggest that trypsin and chymotrypsin are the main proteases present in the viscera of Pterygoplichthys disjunctivus, since they showed molecular weights of between 20 and 29 kDa, similar to the trypsins from Monterey sardine Sardinops sagax caerulea viscera (MW of 25 kDa) [10], Theragra chalcograma and Katsuwonus pelamis (both 24 kDa) [25, 26], and to chymotrypsin from Monterey sardine Sardinops sagax caerulea (MW of 26 kDa) [27] and the isoforms from the hepatopancreas of crucian carp Carassius auratus (27 and 28 kDa) [28] In contrast, Moyano et al [29] detected trypsins ranging from 23.5 to 95 kDa in size in seabream Sparus aurata It has been reported that trypsin activity is generally higher in carnivorous and omnivorous fish [30], while chymotrypsin activity is generally higher for herbivorous species [31] Vermiculated sailfin catfish, a bottom detritus and benthic algae feeder [32] (herbivorous species), 123 704 followed this pattern, with chymotrypsin being the most important protease involved in the hydrolysis of food proteins However, both types of proteases are believed to play a collaborative role in protein digestion in the intestine of this species [33] It has been shown that some tropical fish trypsins (i.e., those from nile tilapia intestines Oreochromis niloticus [5] and spotted goatfish Pseudupeneus maculatus [6]) are sensitive to metal ions [34], especially to Hg2? and Cu2? Interestingly, the trypsin and chymotrypsin from Pterygoplichthys disjunctivus were found to be highly active in the presence of all of the metal ions tested, even Hg2? and Cu2? 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Comp Biochem Physiol 95:625–634 31 Jonas E, Ragyanssszki M, Olah J, Boross L (1983) Proteolytic digestive enzymes of carnivorous (Silurus glanis L.), herbivorous 705 (Hypophtlamichthys molitrix Val.) and omnivorous (Cyprinus carpio) fishes Aquaculture 30:145–154 ¨ zdilek SY (2007) Possible threat for Middle East inland water: 32 O an exotic and invasive species, Pterygoplichthys disjunctivus (Weber, 1991) in Asi River, Turkey (Pisces: Loricariidae) EU J Fish Aquat Sci 24:303–306 33 Glass H, MacDonald N, Moran R, Stark J (1989) Digestion of protein in different marine species Comp Biochem Physiol 94:607–611 34 Cohen T, Gertler A, Birk Y (1981) Pancreatic proteolytic enzymes from carp Cyprinus carpio II Kinetic properties and inhibition studies of trypsin, chymotrypsin and elastase Comp Biochem Physiol B 69:647–653 123 [...]... 0.130 0. 17 1. 47 0.2493 a2 1, a2 2, c2 3 0.138 0.12 0. 97 0.4235 a2 1, b 2, c2 3 0.088 0.44 5.68 0.0049 a2 2, b 2, c2 3 0.088 0.43 5.62 0.0051 d(CO)/dt = (a3 ? b3AL ? c3WT)CO ? d3 a3 1, a3 2, b 3, c 3, d3 5 2.5 17 0.65 7. 30 0.0005 a3 = a31 ? a32CH a3 1, a3 2, c 3, d3 4 2.350 0.65 9. 57 0.0001 a3 1, a3 2, c3 3 2.623 0. 57 9.53 0.0003 a3 1, a3 2, d3 3 3.883 0.36 4.06 0.0195 a3 1, c 3, d3 3 3.222 0. 47 6.39 0.0028 a3 2, c 3, d3 3 2.481... 0.04 07 a1 = a11CO WT ? a12CH WT a1 2, b 1, c 1, d1 4 0.0010 0.41 3.64 0.0211 a1 2, b 1, c1 3 0.0013 0.18 1.58 0.2230 a1 2, b 1, d1 3 0.0012 0.18 1.65 0.2063 a1 2, c 1, d1 3 0.0012 0.19 1 .77 0.1833 b 1, c 1, d1 3 0.0012 0.20 1.88 0.1624 d(CH)/dt = (a2 ? b2CO ? c2WT)CH ? d2 a2 1, a2 2, b 2, c 2, d2 5 0.058 0.68 8. 57 0.0002 a2 = TN SD (a21 ? a22CH)/CH a2 1, a2 2, b 2, c2 4 0.058 0.65 9.85 0.0001 a2 1, a2 2, b2 3 0.130 0. 17. .. 10 .7 8.9 6.1 4.3 7. 0 2.5 2.2 7. 0 4 .7 1.3 5.6 4.2 13 .7 3 .7 1.6 3.2 5.0 PR(i) (mm day-1) 4.3 6.6 7. 0 7. 2 7. 0 3 .7 5.8 4.4 5.2 2.6 1.2 5.5 3.2 5.8 6.4 2 .7 3.3 7. 2 4.4 3.2 4.6 4.4 4.1 5.8 4.5 SD(i) (h day-1) 20.4 18.3 16 .7 15 .7 12.6 24.3 22.5 21.3 17. 5 19.8 15.9 22.8 20.8 19.0 16.3 14.9 12.4 27. 0 23.5 21.6 20.8 18.1 16.1 13 .7 19.8 21 .7 18.3 16.8 15.1 13.5 WT(i) (°C) 0.26 0.11 0. 17 0.14 0.24 0.19 0. 17 0.19... Estuarine Research, East China Normal University, Shanghai 20006 2, China e-mail: liuyong 770 7@yahoo.com.cn Y Liu Á J Cheng Key and Open Laboratory of Marine and Estuarine Fisheries Certificated by the Ministry of Agriculture, East China Sea Fisheries Institute, Chinese Academy of Fishery Sciences, Shanghai 20009 0, China Y Chen School of Marine Sciences, University of Maine, Orono, ME 0446 9, USA adaptive... feasibility, this type can be another option for use in tori-lines if used with a main tori-line of sufficient length K Yokota (&) Marine Fisheries Research and Development Center, Fisheries Research Agency, 2-3-3 Minatomirai, Nishi-ku, Yokohama, Kanagawa 220-611 5, Japan e-mail: yokotaks@affrc.go.jp H Minami National Research Institute of Far Seas Fisheries, Fisheries Research Agency, 5 -7- 1 Orido, Shimizu-ku,... Research Committee, Oceanographical Society of Japan (ed) Coastal oceanography of Japanese Islands Tokai University Press, Tokyo, pp 694–6 97 9 Zenitani H, Kono N, Tsukamoto Y, Masuda R (2009) Effect of temperature, food availability, and body size on daily growth rate of Japanese anchovy Engraulis japonicus larvae in Hiuchi-nada Fish Sci 75 :1 177 –1188 10 Zenitani H, Kono N, Tsukamoto Y (20 07) Relationship... Sea, Japan Fish Oceanogr 16: 473 – 478 11 Heath MR (1992) Field investigations of the early life stages of marine fish In: Blaxter JHS, Southward AJ (eds) Advances in marine biology, vol 28 Academic Press, London, pp 1– 174 ˜ iquen CM (2003) From 12 Chavez FP, Ryan J, Lluch-Cota SE, N anchovies to sardines and back: multidecadal change in the Pacific Ocean Science 299:2 17 221 13 Takasuka A, Oozeki Y, Aoki... 0.14 0.24 0.19 0. 17 0.19 0.21 0. 17 0.15 0.51 0. 27 0.13 0. 07 0.06 0.13 0. 07 0.21 0.34 0.19 0.12 0.12 0.06 0.38 0.21 0.15 0. 17 0.06 0.16 TN(i) (lM) 1. 67 1.22 1.29 1.51 1. 07 6.29 6.60 2 .72 1. 37 1.48 1.24 1.81 1.42 1.10 1.22 1.33 1.02 3 .75 2.50 3.12 1.29 2.55 2.18 1.64 1.32 1.96 0.90 1.35 1 .73 1.88 CH(i) (lg L-1) 8 .70 18.23 14.55 16. 17 15.89 9.12 4.14 1.30 9 .72 1.84 10. 87 5.98 11.85 11.48 26.00 16.45 14.55... [1 4, 2 3, 24] In this study, the RSE was used to summarize and compare both the accuracy and precision of parameter estimation for the covariance function [2 5, 26 ], the RAE was used to compare the accuracy of stock size estimation [ 27 ], and the RE was used to evaluate the direction of the bias in the estimation of parameters or abundance However, it was interesting to 123 474 Fish Sci (2011) 77 :4 67 478 ... annealing J Environ Qual 27: 1 078 –1086 16 Cochran WG (1 977 ) Sampling techniques, 3rd edn Wiley, New York 17 Miller TJ, Skalski JR, Ianelli JN (20 07) Optimizing a stratified sampling design when faced with multiple objectives ICES J Mar Sci 64: 97 109 18 Liu Y, Cheng J, Li S (2004) A study on the distribution of Setipinna taty in the East China Sea Mar Fish 26:255–260 19 Liu Y, Cheng J, Chen X (2006) Studies