Variation in Manila clam (Ruditapes philippinarum) fecundity in eastern Hokkaido, Japan.
KEY WORDS: Manila clam, Ruditapes philippinarum, Manila clam, fecundity, gametogenic development, monoclonal antibodies, population dynamics
The Manila or Asari (Japanese) clam Ruditapes philippinarum has been one of the most popular marine foods among Japanese people since ancient times. It has been found in shell middens dating from the Jornon Period, before the 10th century bc (cf. Morse 1879). In Japan, the annual landings of several fishery cooperatives (defined as fisheries production) of the clam reached 14-16 x [10.sup.4] t/y during the 1970s and 1980s, but has decreased by 75% to 4 X [10.sup.4] t/y during the 2000s (Ishii & Sekiguchi 2002). Reclamation of tidal flats, where clams are harvested, is one of the main causes of the reduction in fisheries production. However, production in this fishery continued to decline even after the cessation of reclamation (Masu et al. 2008).
The Manila clam has a pelagic life stage, and research on the early life stage, including the pelagic larval period, has focused on the decrease in the fisheries output and population numbers (Ishii & Sekiguchi 2002). Ishii and Sekiguchi (2002) suggested that the reduction in the clam population in Ariake Bay was caused by 3 factors: (1) a shortage of larval supply, (2) a decrease in size of the spawning stock, and (3) an increase in larval mortality resulting from environmental deterioration. To maintain or recover the clam fishery, the ecological network linked by plankton and the larval dynamics of the clam, including larval transport to discrete habitats, have been investigated to reconstruct and examine measures to maintain clam fisheries production (Matsumura et al. 2001, Kasuya et al. 2004). Although some information on the early life stage of the clam is available for the management and recovery of its stocks, how fecundity has changed remains unclear. The current study investigated the variations in condition and fecundity of the Manila clam and its associated environmental conditions in its fishing grounds in a midlatitude estuary in eastern Hokkaido, Japan, to evaluate the clam fecundity response to environmental factors--in particular, food availability. In southern parts of Japan, such as Honshu and Kyusyu islands, Manila clams usually spawn from spring to autumn and there are several stages in the reproductive cycle (Matsumoto et al. 2014). However, the Manila clam in eastern Hokkaido exhibits a single synchronized spawning event during the summer (Yamamoto & Iwata 1956). Therefore, we hypothesized that the relationship between clam fecundity and environmental factors would be clear in this area. Moreover, a simple technique--enzyme-linked immunosorbent assay--was used to quantify the number of eggs in the clams; this method has been applied effectively to other bivalve species (Long et al. 2008).
MATERIALS AND METHODS
The study was conducted in Manila clam intertidal fishing grounds in the Akkeshi-ko estuary, Hokkaido, northern Japan (Fig. 1). This estuary is semiclosed and is connected to Akkeshi Bay (open to the Pacific Ocean-controlled cold Oyashio Current) via a narrow channel (width, 500 m). Two rivers flow into the estuary: the Beakanbeushi River from the north and the Tokitai River from the east. The water depth is usually less than 2 m, and this shallow area is covered by seagrass (Zostera spp.). The Manila clam fishing grounds are located between the center and near the mouth of the estuary, whereas the Pacific oyster Crassostrea gigas is cultured throughout the estuary. The clam fishing grounds were constructed artificially on the dyed oyster bed by sand capping.
Two study sites (A and B) were established in the estuary. Site A (43.043[degrees] N, 144.884[degrees] E) and site B (43.042[degrees] N, 144.861[degrees] E) were located in the center area and near the mouth of the estuary, respectively. The salinity at site A fluctuated more widely than that at site B. but remained more than 20 at both sites (Iizumi et al. 1995).
Water temperature was measured with a data logger (TidbiT V2; Onset Computer Corporation, MA) every hour on the seabed of both Ashing grounds. After omitting the low-tide data, average daily temperatures were calculated. The chlorophyll a (Chi a) fluorescence level was measured with water-quality measurement instruments (YSI6600, YSI Incorporated; ACL2180-TPM, Alec Electronics Co. Ltd.). Measurements were taken every 10 min; the minimum value recorded within 1 h was used as the representative value for that hour because abnormally high values were recorded frequently. The fluorescence levels obtained were converted to Chi a concentration values, which were determined with an extraction and measurement fluorometer (model 10-AU-005-CE; Turner Designs Inc., Sunnyvale, CA). Current velocity was measured with magnetic current meters (Compact-EM; Alec Electronics Co. Ltd., Kobe, Japan). Measurements were taken in burst mode with 1 measurement every 0.5 s for 5 min at 2-h intervals and then averaged. The water-quality measurement instrument and current meter sensors were mounted 15 cm above the seabed.
Manila clams were collected 30 times from 2 sites between April 2010 and November 2012, except during ice-cover season (December to March) for monitoring of nutritional condition, fecundity, and gametogenic development. Approximately 15-30 clams with a shell length (SL) of 30-50 mm were collected randomly. In March 2011, the clams and sediment from half of site B were washed away from the Ashing grounds by a tsunami. Therefore, clams were collected only from half the initial area at site B in subsequent years, where new sand and clams had accumulated. After measuring the SL, shell height (SH), shell width (SW), and total wet weight (WW), the clams were dissected and the soft body tissue wet weight was recorded. The soft body tissue was then Axed in Davidson's fixative or stored at -80[degrees]C for reproductive staging and fecundity assessment, respectively. The condition index (CI) of the clams was calculated as follows:
CI = Soft tissue weight / SL X SH X SW x [10.sup.5]
where soft tissue WW was measure in grams WW and SL. SH, and SW were measured in millimeters.
Gametogenic development of the clams was determined by histological analysis. Sections were cut from around the middle of the body from the fixed samples. The sections were dehydrated in tissue dehydration solution and embedded in paraffin. The paraffin blocks were sliced at a thickness of 5-8 pm and stained with hematoxylin-eosin stain. The reproductive stage was then determined according to Park and Choi (2004), in which the gametes are assigned to 1 of 6 stages--indifferent, early developing, late developing, ripe, spawning, and spent--by microscopic observation.
Quadrate sampling (0.20 X 0.20 m; depth, 0.15 m) was conducted for monitoring the clam biomass and density at both sites in July 2010, April 2011, and August 2012. Basically, 6 samples were collected randomly from the fishing grounds. After the tsunami, samples were collected every 30 m on a 150-m line transect over site B, which included spots where sediments and clams had either been lost or newly accumulated despite the disturbance. After counting the number of Manila clams and other animals that remained on the 2-mm sieve, clam biomass (total WW) and density were determined. Clam SL was measured and the pooled size-frequency distribution was determined for each site.
Egg Yolk Protein Quantification
Fecundity was determined in female clams (stored at -80[degrees]C until analysis) at peak maturity. The sampling day, when the spawning stage was observed during histological preparations and the Cl had increased, was regarded as the peak day of maturity. The number of eggs in whole soft-body tissue was determined using indirect ELISA with monoclonal antibodies specific to Manila clam yolk protein according to Park and Choi (2004). Frozen whole tissue was freeze-dried and homogenized using a sonicator with 10-15 mL buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing a protease inhibitor cocktail (#04080-11 for general use; Nacalai Tesque Inc., Kyoto, Japan). The homogenate supernatant was separated by centrifugation (15,000g for 20 min at 4[degrees]C) and stored at -80[degrees]C until analysis. The supernatants were diluted to 2-100 times with 0.5% bovine serum albumin-phosphate-buffered saline buffer before ELISA. A 100-[micro]L sample was added to a 96-well polystyrene microplate and incubated at 4[degrees]C overnight. After incubation, the plate was washed twice with phosphate-buffered saline containing 0.05% detergent (Tween20; Promega Co. Ltd., Madison, WI); 250 [micro]L 1% bovine serum albumin and 5% skim milk in SBB was added as a blocking agent. After incubation at room temperature for 1 h and washing 5 times, the monoclonal antibody (Hamaguchi & Usuki 2006) was added in 100-[micro]L aliquots to each well and incubated at room temperature for 2 h. After washing 5 times, a 100-[micro]L aliquot of polyclonal goat antimouse immunoglobulin peroxidase conjugate (diluted 1:4,000) was added to each well. After incubation at room temperature for 2 h. 100 [micro]L substrate-chromogen containing 3,3',5,5'-tetramethylbenzidine (TMB+; Dako Co. Ltd., Glostrup, Denmark) was added as a color agent. Last, 50 [micro]L 2 N sulfuric acid was added as the stop solution. The optical density of the end product was measured at 450 nrn using a microplate reader.
The tissue homogenate supernatant from 10 clams collected from site B in late August 2012 was used as the standard sample.
A dilution series of this standard sample was analyzed in each plate, and a standard regression curve was constructed. The amount of egg protein in the clam homogenates relative to the standard sample was estimated from this regression curve. Moreover, mature eggs were collected from the females in late August 2012 by making an incision in the soft body tissue. The number of eggs was counted using a microscope, and the relative amount of egg protein in these eggs was also quantified. After determining the protein content per egg in each clam, the maximum value was used for determining the number of eggs in both the standard samples and the clams. Clam fecundity is represented as the number of eggs per unit of soft body weight (1 g WW) or per individual.
Progression of sexual maturation in the Manila clam was assessed based on the rate of increase of the gonadal index (G) according to Toba and Miyama (1995) as follows.
G = 0.0377T - 0.0168.
where T is the average daily temperature. When G reached 3, the clams were considered ripe.
Differences in Chi a concentration and current velocity between the 2 sites were tested each month using Wilcoxon's signed-rank test, and a P value less than 0.05 was considered statistically significant.
The effects of independent variables--site (A and B), year (2010, 2011, and 2012), and clam size (SL)--on Cl, number of eggs per unit of soft body weight, and individual were estimated using a generalized linear regression model (GLM) for which site, year, and their interaction were considered fixed factors, and SL was considered a numerical factor. All dependent variables were assumed to follow the gamma distribution (log link). During the analysis of number of eggs per unit of soft body weight, the numbers of eggs per individual and soft body weight were used as independent and offset variables, respectively. Model selection based on Akaike's information criterion (AIC) was then performed; those with the minimum AICs were regarded as the best model. The effects of the independent variables in these models were also tested with a Wald test, and a P value less than 0.05 was considered statistically significant.
Water temperature increased gradually from less than 5[degrees]C in early spring to around 20[degrees]C in late summer at both sites (Fig. 2). Daily variation in temperature exceeded 10[degrees]C in summer at both sites. The gonadal index of the clams, which was calculated from accumulated temperature, reached 3. The clams were considered sexually mature on the August 9 and 11, 2010; August 7 and 11, 2011; and August 12 and 18, 2012; at sites A and B. respectively. Monthly average Chi a concentrations ranged between 1.3 [micro]g Chl a/L and 2.8 [micro]g Chi a/L at site A, and between 2.8 [micro]g Chi a/L and 5.5 [micro]g Chi a/L at site B (Fig. 3). Chlorophyll a concentration at site B was significantly greater than that at site A in all seasons (Wilcoxon's signed-rank test, V = 1,341-18,117, n = 182-473, P< 0.001). Monthly average current velocity at sites A and B ranged between 6.8 cm/sec and 15.6 cm/sec, and between 9.5 cm/sec and 17.0 cm/sec, respectively (Fig. 4). Current velocity at site B was significantly greater than that at A in most months (Wilcoxon's signed-rank test, V = 396-15,413, n = 65-320, P < 0.001), except August 2012 (Wilcoxon's signed-rank test, V = 1,023, n = 65, P = 0.75).
Clam Condition Factor
The average clam CI increased from 14.5 in spring to 19.6 at site B in summer 2010 (Fig. 5A). In summer 2011, clam CI also increased to 18.6 but remained below 16 in 2012 (Fig. 5B, C). Clam CI at site A did not exhibit an obvious increase in summer in any year. During the GLM analysis of clam CI at peak maturity, the AIC of the full model was 595, and that of the model without SL was the lowest with 594. A Wald test revealed CI at site B was significantly greater than at site A, and CI in 2010 was significantly greater than that in 2011 but lower than that in 2012. In addition, the interactions between site (B) and years (2012 and 2013) were also significant (Table 1).
Histological analysis revealed simple gametogenic development from spring to summer, and spawning during summer at both sites (Fig. 6). Spawning individuals were first observed in July in the clams from site A, but in August at site B in 2010 and 2011. However, spawning individuals were first observed in August at both sites in 2012. At site A, spent gonads (degeneration) appeared before mature gonads were observed every year. The clams at site B did not exhibit such abnormal tissue in their gonads before spawning in 2010, but reached the spent stage before a sufficient number of mature individuals were recorded in 2011 and 2012. The clams at site A reached peak maturity in July 2010 and 2011, and the beginning of August 2012. The clams at site B, however, reached peak maturity at the beginning of August every year.
At site A, the number of eggs per unit of soft body weight of clams was 4.88 [+ or -] 7.16 X [10.sup.4] (mean [+ or -] SD) eggs/g WW, 2.20 [+ or -] 2.49 X [10.sub.4] eggs/g WW, and 1.48 [+ or -] 1.27 X [10.sup.4] egg/g WW on August 8, 2010; July 13, 2011; and August 7, 2012; when the clams reached peak maturity, respectively (Fig. 7A). At site B, the number of eggs per unit soft body weight of clams was 3.06 [+ or -] 7.16 X [10.sup.5] eggs/g WW, 5.39 [+ or -] 2.49 X [10.sup.4] eggs/g WW, and 9.34 [+ or -] 8.42 X [10.sup.3] eggs/g WW on August 12, 2010; August 5, 2011; and August 7, 2012; respectively. The number of eggs per individual was 1.48 [+ or -] 2.34 X [10.sup.5] eggs/individual in the clams at site A and 1.70 [+ or -] 1.81 X [10.sup.6] eggs/individual in the clams at site B during peak maturity in 2010 (Fig. 7B). In 2011. the number of eggs was 4.82 [+ or -] 5.31 X [10.sup.4] eggs/individual and 1.69 [+ or -] 1.00 X [10.sup.5] eggs/individual at sites A and B, respectively. In 2012, the number of eggs was 3.73 [+ or -] 3.40 X [10.sup.4] eggs/individual and 3.42 [+ or -] 3.61 X [10.sup.4] eggs/individual at sites A and B, respectively.
Generalized linear model analysis without offset revealed that the AICs of the null models for the number of eggs and full models including all factors (e.g., site, year, SL, and interactions between site and year) were 1,703 and 1,644, respectively. Generalized linear model analysis with offset also revealed that the AICs of the null models and full models were 1,680 and 1,639, respectively. These full models had a minimum AIC. Wald tests revealed the number of eggs per unit of soft body weight at site B was significantly greater than at site A and that SL was not a significant factor (Table 2). The number of eggs per individual at site B was significantly greater than at site A (Table 3), and the SL was a significant function of this independence, with a positive coefficient (estimate [+ or -] SE, 0.16 [+ or -] 0.05; Fig. 8). Wald tests revealed that the interactions between site (B) and year (2012) was significant, and the number of eggs per unit of soft body weight and individual in 2012 was less than that in 2010 and 2011 at site B.
Biomass and Density
Average Manila clam biomass ranged between 5.44 kg WW/[m.sup.2] and 7.27 kg WW/[m.sup.2] at site A, and between 4.36 kg WW/[m.sup.2] and 12.64 kg WW [m.sup.2] at site B (Fig. 9A). At site B, the biomass SD (n = 6) increased after the tsunami because the clams were swept away along with the sediment. Clam density was consistently between 1,864 [+ or -] 1,044 individuals/[m.sup.2] and 2,769 [+ or -] 533 individuals/[m.sup.2] at site A (Fig. 9B). In contrast, at site B, clam density decreased from 5,731 [+ or -] 1,094 individuals/ [m.sup.2] in 2010 to 1,421 [+ or -] 1,747 individuals/[m.sup.2] in 2011, because of the tsunami, then increased to 15,315 [+ or -] 8,964 individual/[m.sup.2] in 2012 because small juveniles less than 10 mm in SL were recruited abundantly. However, clam density varied a lot within site B. Clams were distributed more densely in areas of sand accumulated by the tsunami (>8,200 individuals/[m.sup.2]) than in areas where sediment and clams had been washed out by the tsunami or where sand capping was conducted in winter 2011 (<2,200 individual/m). In addition. Mya japonica and Ceratostoma inornatus were also collected, but their biomass and density were less than 1% of that of the Manila clam.
Postsettlement dispersal is a key process affecting the population dynamics of many soft-sediment benthic invertebrates (Lundquist et al. 2004) and its control is a target in Manila clam in stock enhancement in Japan (Kakino 2006). However, food availability is also a determining factor in the performance of filter feeders, including bivalves, growth nutritional condition, and reproductive output, which affects population dynamics in supply-side ecology (Lewin 1986). In scallops, variation of reproductive output depending on food supply has been reported in Argopecten irradians (Barber & Blake 1983, Bricelj et al. 1987), Mizuhopecten yezoensis (Baba et al. 2009), and Placopecten magellanicus (MacDonald & Thompson 1985, Barber et al. 1988). In addition to food concentration, the flow of water also affects the availability of food for filter feeders. In motionless seawater and very low velocities, seston, including food, becomes locally depleted around filter feeders; however, increases in velocity serve to remove localized seston depletion (Wildish & Kristmanson 1997). Kakino (1996) reported that high current velocity modifies Manila clam shell morphology by inducing growth through greater food supply. In this study, both Chl a concentration and current velocity were greater at site B, located near a channel between the estuary and the bay with greater water volume than at site A, located at the center of the estuary, respectively. Moreover, there might be intraspecific competition for food at these fishing grounds where clam biomass and density reached more than 10 kg/[m.sup.2] and [10.sup.4] individual/[m.sup.2], respectively. However, the high velocity would make competition less intense by overcoming local food depletion above the sediment surface at site B but not at site A. which is located near the center of a shallow estuary and a developed seagrass bed where current velocity is low in summer (Hasegawa et al. 2008). Kasim and Mukai (2009) reported that benthic algae, including epiphytes, released from seagrass were found in the gut contents of clams along with phytoplankton. However, higher Chl a concentration at site B near the bay suggests the importance of food supply from the bay system to the clams. Sobral and Widdows (2000) reported that clearance rates in the carpet shell clam, Ruditapes decussatus, declined with velocities greater than 8 cm/sec; the maximum current velocity reached more than 50 cm/sec at site B. However, measurement of current velocity was conducted 15 cm above the seabed; at this depth, velocity would have a minor influence.
Sexual maturation in the Manila clam was assessed based on accumulated temperature data, which suggested a similar rate of maturation between sites, with peak maturity in August. Histological analysis also revealed the synchronous progress in sexual maturation in the gonads from spring to summer, and the clams had only 1 peak maturity at both sites, However the ripe stage, which was identified by histological analysis, appeared earlier in the clams at site A than site B, and the clams at site A reached maturity in July during 2010 and 2011, which was 1 mo earlier than the clams at site B. Assessment of sexual maturation based on temperature might not be suitable for the eastern Hokkaido clam population (43[degrees]N) because the accumulated temperature (Toba & Miyama 1995) was determined in the clam population in Chiba, Japan, which is south of Hokkaido and has a warmer climate without the cold current (35[degrees]N). Moreover, the difference in genotype frequency in both allozymes (Oniwa et al. 1988) and the mitochondrial COI gene (Sekine et al. 2006, Mao et al. 2011), and the frequency in shell marking type (Chow et al. 2013) were detected between the eastern Hokkaido clams and those from other areas of Japan, including Chiba. However, estimated maturation of the clams was in accordance with that observed in the histological preparations at site B. Park et al. (2006) reported a delay in spawning period with reduced reproductive output in clams that were heavily infected and experienced an energy drain and chemical secretion by the protozoan Perkinsus olseni, causing mass mortalities; however, infection by Perkinsus sp. was not detected in the clams in the current study area (Hamaguchi et al. 1998, Nishihara 2010). Toba and Miyama (1995) suggested that abundant food supply promoted gonadal development. In contrast, the result from the current study revealed that gonadal development in the clams was promoted at site A with lower food supply.
Generalized linear model analysis revealed the nutritional condition of clams was better primarily at site B than site A during 2010 and 2011, but not in 2012. Food availability at site B was more favorable than at site A during 2012, the same as other years. Therefore, other factors would have affected the worse nutritional condition of clams at site B during 2012. At site B, the variation in clam biomass and density increased at site B after the tsunami and the repairing of the grounds in 2011. The clams collected were those that remained and had accumulated after the tsunami; clam density increased via high recruitment in autumn 2011. Although juvenile biomass was not high, weight-specific clearance rates increased with decreases in clam size (Nakamura 2004). Food competition intensified, leading to a worsening in nutritional condition of clams at site B in 2012.
In addition to the nutritional condition, fecundity of the clams (number of eggs per individual) varied between sites, and clam fecundity was several times greater at food-rich site B than site A. Histological analysis also revealed abnormal degenerated tissue in the gonads (spent stages appearing before the clam had matured), and clam fecundity and CI decreased at site B after the tsunami disturbance in 2011 and the abundant recruitment of juveniles in 2012. In particular, clam fecundity at site B decreased greatly in 2012. Olafsson et al. (1994) reviewed the influence of food levels and animal density, including intraspecific competition, on the growth rate, survival, and fecundity of marine invertebrates. They reported that density-dependent inhibition of adult invertebrates to recruitments is common. The current study on Manila clams also suggests that fecundity varies with food availability, which was determined by both environmental factors and density of its juveniles.
Generalized linear model analysis revealed that the number of eggs per individual was a function of clam size, but clam size was not a significant function of weight based number of eggs. Park and Choi (2004) also reported a positive relationship between clam size and fecundity, but the maximum recorded was more than [10.sup.6] eggs (SL, 42 mm), which was greater than the maximum in the current study (6 X [10.sup.5] eggs; SL, 47 mm). Fecundity of marine invertebrates increases monotonically with size in most species. The current study revealed the Manila clam increased its fecundity through weight gain at the favorable site (site B), in terms of food availability in the estuary. Many invertebrates, including clams (Green & Hobson 1970), can also increase fecundity by spawning repeatedly during the year (Spight & Emlen 1976). Manila clams usually spawn from spring to autumn in more southern regions of Japan (Matsumoto et al. 2014). However, the clams in the study area spawn only during the summer because of the slow progress of sexual maturation at low temperatures, which are controlled by the cold Oyashio current. Moreover, abnormal degenerated tissue was observed in the gonads of clams at site A during every year, and at site B after dense recruitment. This result suggests that egg number was decreased after the beginning of maturation if food availability was not sufficient.
The current study quantified size-dependent fecundity, but age-related fecundity was not investigated. Gamete production in Mercenaria mercenaria continues into old age as a simple function of size (Peterson 1986), which was also evident in the current study, although age was not considered directly. Future investigations should be expanded to elucidate the relationship between age and fecundity, which will facilitate proper management of deteriorated clam populations in Japanese coastal areas to maximize production for recovery.
The authors are grateful to Y. Fuchigami, Y. Toya, R. Matsuda, and S. Tanaka for assistance with analysis. Also, they thank E. Negishi and M. Suzuki, Fisheries Cooperative Association of Akkeshi and Akkeshi Marine Station, Hokkaido University, for assistance with field research. The authors thank Dr. S. Chow and the members of the Manila clam fecundity research project for his valuable comments. They thank the anonymous reviewer and Dr. S. E. Shumway for improving the manuscript. This study was supported by the Ministry of Agriculture, Forest, and Fisheries and the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
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* Corresponding author. E-mail: firstname.lastname@example.org DOI: 10.2983/035.033.0308
NATSUKI HASEGAWA, (1) * SAYUMI SAWAGUCHI, (2) TATSUYA UNUMA, (3) TOSHIHIRO ONITSUKA (3) AND MASAMI HAMAGUCHI (4)
(1) National Research Institute of Aquaculture, Fisheries Research Agency, 422-1 Nakatsuhamaura, Minami-ise, Mie 516-0193, Japan; (2) Seikai National Fisheries Research Institute, Fisheries Research Agency, 1551-8 Taira-machi, Nagasaki, Nagasaki 851-2213, Japan; (3) Fisheries Research Agency, Hokkaido National Fisheries Research Institute, 116 Katsurakoi, Kushiro, Hokkaido 085-0802, Japan; (4) National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima 739-0452, Japan
TABLE 1. Result of generalized linear model analysis of condition index of the Manila clam Ruditapesphilippinarum in the Akkeshi-ko estuary, Japan. Independent variables Coefficient SE t value P value estimate Intercept 2.61 0.12 22.22 <0.001 Site (B) 0.28 0.03 10.00 <0.001 Year (2011) -0.12 0.03 -4.33 <0.001 Year (2012) 0.08 0.03 2.34 0.02 Shell length 0.002 0.003 0.55 0.55 Site (B)X year (2011) 0.08 0.04 2.23 0.03 Site (B)X year (2012) -0.28 0.04 -6.88 <0.001 TABLE 2. Result of generalized linear model analysis of number of eggs/1 g wet-weight of the soft-body Manila clam Ruditapes philippinarum in the Akkeshi-ko estuary, Japan. Independent variables Coefficient SE t value P value estimate Intercept 7.29 1.95 3.74 <0.001 Site (B) 1.44 0.44 3.28 0.002 Year(2011) -0.43 0.49 -0.86 0.329 Year (2012) -0.85 0.546 -1.85 0.07 Shell length 0.09 0.05 1.81 0.08 Site (B) X year (2011) -0.72 0.69 -1.05 0.30 Site (B) X year (2012) -2.31 0.62 -3.73 <0.001 To evaluate the effect of the independent variables on the egg numbers per soft-body weight, soft-body weight was used as offset in the models. TABLE 3. Result of generalized linear model analysis of number of eggs per individual of the Manila clam Ruditapes philippinamm in the Akkeshi-ko estuary, Japan. Independent variables Coefficient SE t value P value estimate Intercept 5.37 2.02 2.66 0.01 Site (B) 1.71 0.45 3.77 <0.001 Year(2011) -0.43 0.51 -0.84 0.40 Year (2012) -0.74 0.48 -1.56 0.13 Shell length 0.146 0.05 3.26 0.002 Site (B)X year (2011) -0.81 0.71 -1.14 0.26 Site (B)X year (2012) -2.63 0.64 -4.10 <0.001
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|Author:||Hasegawa, Natsuki; Sawaguchi, Sayumi; Unuma, Tatsuya; Onitsuka, Toshihiro; Hamaguchi, Masami|
|Publication:||Journal of Shellfish Research|
|Date:||Dec 1, 2014|
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