Spawning failure in the sea urchin Strongylocentrotus intermedius in the northwestern Sea of Japan: Potential environmental causes

Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

Contents lists available at ScienceDirect

Journal of Experimental Marine Biology and Ecology journal homepage: www.elsevier.com/locate/jembe

Spawning failure in the sea urchin Strongylocentrotus intermedius in the northwestern Sea of Japan: Potential environmental causes Peter M. Zhadan a,⁎, Marina A. Vaschenko b, Tatyana N. Almyashova a a b

V.I. Ilˆichev Pacific Oceanological Institute, Far East Branch of the Russian Academy of Sciences, Baltiiskaya Street 43, 690041 Vladivostok, Russia A.V. Zhirmunsky Institute of Marine Biology, Far East Branch of the Russian Academy of Sciences, Palchevsky Street 17, 690041 Vladivostok, Russia

a r t i c l e

i n f o

Article history: Received 26 June 2014 Received in revised form 15 December 2014 Accepted 28 December 2014 Available online xxxx Keywords: Broadcast spawning Echinoderms Phytoplankton bloom Reproductive cycle Reproductive failure Temperature

a b s t r a c t The natural populations of the sea urchin Strongylocentrotus intermedius were studied along 350 km of the coast of the Primorye region of Russia (northwestern Sea of Japan). Most of the surveys were carried out in Kievka Bay, where sea urchins were sampled monthly from April 2008 to April 2011. The sea urchin gonads became ripe in late July–August. However, in two out of three reproductive seasons, extremely high percentage of individuals that did not complete their reproductive cycle by spawning (95% and 53% of unspawned females in 2008 and 2009, respectively) was found. A large-scale egg fragmentation was observed to start in middle September. This phenomenon was also found to different extents in other populations of S. intermedius in the studied area. Unique characteristics of the reproductive cycle of S. intermedius displaying spawning failure were revealed: (1) the cleaning process associated with the resorption of numerous undischarged eggs and spermatozoa was unusually long, lasting for approximately 7 months, and (2) the decrease in the gonadal index was significantly lower and occurred 2 months later than in the case of normal spawning. Analysis of environmental factors that potentially modulate the spawning process in S. intermedius populations, such as temperature and phytoplankton concentration (measured as the chlorophyll а (Chl а) concentration), showed that large difference in percentage of spawned sea urchins occurred at the nearly identical temperature profiles. At the same time, the positive correlation (p b 0.05) detected between the Chl а concentration and the proportion of spawned females of S. intermedius indicates that there is a relationship between the spawning of this species and the concentration of phytoplankton. Two major conclusions were drawn from the study results: (1) resorption of undischarged gametes begins regardless of whether spawning takes place, and (2) sea urchin spawning does not occur automatically after gamete maturation but is affected by environmental conditions. We proposed that the phenomenon of spawning failure of S. intermedius seemed to be attributable to low phytoplankton concentration in water column insufficient to trigger the sea urchin spawning. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The sea urchin Strongylocentrotus intermedius (A. Agassiz, 1863) is a commercially important species that inhabits hard substrates in the northern regions of the Asian Pacific coastal waters. It is distributed longitudinally from the Kamchatka Peninsula to the Korean Peninsula and latitudinally from the Russian coast to the Japanese Islands (Agatsuma, 2013; Bazhin, 1998; Kafanov and Pavlyuchkov, 2001). S. intermedius similar to other sea urchin species inhabiting temperate waters exhibits an annual reproductive cycle (Agatsuma, 2013; Fuji, 1960; Khotimchenko et al., 1993). However, there are some differences in spawning schedule of this species among different localities. In the coastal waters around Hokkaido, S. intermedius populations off the Sea of Japan coast to the western Tsugaru Strait spawned in autumn (September–October), while the spawning season of the sea urchins ⁎ Corresponding author. Tel.: +7 423 2312867. E-mail address: [email protected] (P.M. Zhadan).

http://dx.doi.org/10.1016/j.jembe.2014.12.017 0022-0981/© 2015 Elsevier B.V. All rights reserved.

off the eastern Pacific coast and the Okhotsk Sea coast lasted from June to October (see for review Agatsuma, 2013). The sea urchin spawning off the southern coast of the island (Tsugaru Strait and Funka Bay) occurred twice a year, in spring (April–May) and in autumn (from August to November). The annual studies of gonad index and histology conducted in 1970–1980s showed that in Russian waters of the Sea of Japan (Ussuriisky, Vostok and Pos'eta Bays, the secondary bays of Peter the Great Bay), the spawning period of S. intermedius usually took place in late summer–early autumn (see for review Khotimchenko et al., 1993). In 2009–2010, we examined the reproductive cycle of S. intermedius in the same areas and showed that sea urchin spawning schedule in Ussuriisky Bay was different from that of 1970–1980s (Zaslavskaya et al., 2012). In four S. intermedius populations studied, two peaks of spawning were registered, spring (late May–June) and autumn (August–September). Moreover, at the stations located in the adjacent to Vladivostok city zone, sea urchins with the spring spawning schedule dominated. At the same time, the spawning season of the sea

12

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

urchins in Vostok Bay remained unchanged and was characterized by the clearly pronounced autumn spawning (Zaslavskaya et al., 2012). In addition to repeated studies of S. intermedius reproductive cycle in Peter the Great Bay, we examined the seasonal dynamics of gonad condition in the sea urchin populations in Kievka Bay (northwestern Sea of Japan, Primorye region) where such a research has not been formerly conducted. Our interest to this bay is explained by the fact that it is located in sparsely populated area adjoining the State Reserve of Laso, the forest reserve in Russian Far East. This presumes the absence of any important human impact on the environment in the chosen area of study. In the course of our research, we have faced an unexpected phenomenon. In 2008, more than 90% of sea urchin specimens seemed to have not completed their reproductive cycle by spawning. To our knowledge, no previous study has reported that the annual reproductive cycle of the majority of wild sea urchin population might not be completed by spawning. However, in fresh-water fish, the phenomenon of spawning failure has been known for quite some time (Koshelev, 1984). This phenomenon has been observed under conditions that are unfavorable for fish reproduction, when hydro-technical facilities have impeded the access of fish to their spawning habitat or when the water temperature in the spawning habitat has been too low to enable the roe of phytophilous fish species to attach to plants (Koshelev, 1984). Similar findings in invertebrate species are scarce. Nevertheless, in cultivated Pacific oyster Crassostrea gigas from the polluted Manukau Harbor (New Zealand) and Cork Harbor (southern Ireland), an absence of spawning has been reported (Roper et al., 1991; Steele and Mulcahy, 1999), which has mainly been attributed to pollution of the marine environment by tributyltin and infection of the oysters with a parasitic copepod. Spawning failure has been recorded in natural populations of brackish water bivalve Corbicula japonica: in 1995, extremely low abundance of larvae in Lake Abashiri (Hokkaido, Japan) coupled with a mass oocyte atresia in female gonads has been found (Baba et al., 1999). Based on the results of a spawning induction experiment under laboratory conditions, the researchers have suggested that this phenomenon might have been caused by low temperature and low salinity. Despite a great number of studies on echinoderm reproduction, the conditions that are necessary to trigger the spawning of echinoderms, and of sea urchins in particular, remain unclear to a large degree (Mercier and Hamel, 2009). For successful reproduction of marine invertebrates with external fertilization, synchronization of spawning between females and males as well as conditions that are favorable for offspring development appear to be crucial. The necessity of synchronized spawning is determined by the short longevity of gametes and rapid gamete dilution (Levitan and Petersen, 1995; Pennington, 1985). The spawning season should coincide with environmental conditions that promote the successful development of offspring (Himmelman, 1999). Marine biologists have long noted that the timing of phytoplankton blooming is concurrent with the season in which a large number of planktotrophic larvae of bottom invertebrates appear within the plankton (Thorson, 1946). Coincidence between the spawning season of bottom invertebrates and microalgae blooms has been observed in many field studies (Fournier et al., 2012; Gaudette et al., 2006; González-Irusta et al., 2010; Himmelman, 1975; Smith and Strehlow, 1983; Starr et al., 1993). Moreover, it has been shown in laboratory experiments that phytoplankton and their extracts stimulate the spawning of sea urchins and bivalve mollusks (Breese and Robinson, 1981; Starr et al., 1990, 1992), and the incentive effect of phytoplankton can be further enhanced by the addition of sperm to sea water (Reuter and Levitan, 2010; Starr et al., 1990, 1992). Temperature (González-Irusta et al., 2010; Guillou and Lumingas, 1998; Himmelman et al., 2008), lunar phases (Gaudette et al., 2006; Reuter and Levitan, 2010), and water salinity (Wilson, 1969) can also serve as the stimuli that synchronize or trigger the spawning of bottom invertebrates. In addition, many researchers have noted that a

combination of several natural factors can contribute to spawning synchronization. Moreover, recent analysis of the reproductive cycle of Paracentrotus lividus (González-Irusta et al., 2010) suggests that in sea urchins possessing fully mature gonads, spawning can begin even in the absence of external stimuli. In the present study, we investigated the unique characteristics of the reproductive cycle of S. intermedius sea urchins showing incomplete spawning and potential environmental factors leading to sea urchin spawning failure.

2. Materials and methods 2.1. Sea urchin sampling Most surveys were carried out in Kievka Bay (42°50′ N, 133°41′ E) (Fig. 1) where sea urchins were sampled monthly from April 2008 to April 2011, with the exception of the period from July 27 to October 1, 2010, when sampling interval was 8–14 days. In two cases (January and November 2009), sampling was not performed because of abundant snowfalls. In November 2008, just after we found out that a vast majority of sea urchins sampled in Kievka Bay in October did not spawn, we carried out a one-time survey to examine the gonads of sea urchins sampled from Vostok Bay (42°53′ N, 132°43′ E). There were two reasons to do it: 1) In this bay, the reproductive cycle of S. intermedius has been examined earlier for 5 years, in 1971–1975 (Yakovlev, 1976), and spawning failure has not been observed; and 2) According to satellite data (The Center for Regional Satellite Monitoring of Environment: http://www.satellite.dvo.ru/en.html), the chlorophyll а (Chl a) concentration in Vostok Bay significantly exceeded that in Kievka Bay. In 2009, we extended the study area from Vostok Bay to Rudnaya Bay (44°20′ N, 145°49′ E), the extreme north point from which sea urchins may be delivered to the laboratory for one day. Monthly analysis of sea urchin gonads was performed during the period from May to November (in the pre-spawning, spawning and postspawning periods: Yakovlev, 1976) in Vostok and Rudnaya Bays. In addition, a one-time analysis of the sea urchin gonads sampled in November in the Bays of Vrangel (42°45′ N, 133°3′ E), Shepalova (42°41′ N, 133°4′ E), Sokolovskaya (42°52′ N, 133°53′ E) and Kit (43°4′ N, 134°11′ E) was conducted. This work was done to reveal the spatial (geographic) distribution of cases of spawning failure in S. intermedius populations. Adult sea urchins were collected from 2 to 6 m depths on bedrock and bolder habitats by haphazardly selecting from the area of approximately 100–150 m in length and 8–15 m in width. Study sites differed in their macroalgae composition. The Kievka, Rudnaya, Kit, and Sokolovskaya Bays were dominated by Saccharina japonica, Phyllospadix iwatensis and Zostera marina; those of Vostok Bay, by S. japonica and Ulva fenestrata; and Vrangel Bay, by Z. marina and U. fenestrata. According to the data for 1996–1998, the median S. intermedius density in the area from Shepalova Bay to Kievka Bay was 2 no. m−2, while that in the area from Kievka Bay to Olga Bay, located 85 km to the south, was 3 no. m−2 (Borisovetz et al., 2000). The sample size was 50 or slightly more specimens for every collection and all sites. Sea urchin transportation was performed in accordance with recommendations of Buznikov et al. (2003) and Dale et al. (2005). Just after sampling, sea urchins were placed into thermocontainers with plastic ice boxes and transported by a car to the stationary laboratory located in Vladivostok. Time of transportation was from 3 h (Vostok Bay located 160 km away from Vladivostok) to 9 h (Rudnay Bay located 530 km away from Vladivostok). Then thermo-containers with sea urchins were stored in the refrigerator. Total time from sampling to animal analysis did not exceed 24 h. The advantages of this transportation method are both effective maintenance of sea urchin viability and prevention of spontaneous spawning. A total of 1899 males and 1438 females were examined. The diameter of the sea urchin tests

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

13

Fig. 1. Schematized map of the study area in the northwestern Sea of Japan. In the insets, the surveyed sites are presented in larger scale.

was 55.5 ± 7.8 mm and 55.7 ± 7.8 mm (mean ± SD) for females and males, respectively. 2.2. Examination of the gonadal state The gonads of 30 animals with test diameter of 50–75 mm were weighed to determine the gonad index (GI), which was calculated using the following formula: GI (%) = [(gonad wet weight / total wet weight) × 100]. After weighing, the pieces taken from the medial part of the gonad were fixed in Bouin's fluid for histological examination. For all animals in the sample, small pieces of the gonads were used to prepare smears for further determination of sex and the condition of the gametes under a light microscope. The percentage of individuals that released gametes during sampling or after dissection was calculated and referred to a group of sea urchins artificially induced to release gametes (AIRG). Routine histological procedure was used. Fixed pieces of the gonads were rinsed in 70% alcohol, dehydrated in increasing concentrations of alcohol, then subsequently transferred to a mixture of alcohol and

chloroform, chloroform, and a mixture of chloroform and paraffin, and embedded into paraffin. Histological sections (6 μm thickness) were stained with hematoxylin and eosin and examined under a light microscope (Olympus B-46, Japan) equipped with a C5060-ADU digital camera (Olympus, Japan) using Videotest 5.0 image analysis software (Saint Petersburg, Russia). From August to October, the majority of females and almost all males released gametes during sampling or just after dissection. Some individuals released up to 4 ml of gametes that did not give an opportunity for correctly distinguishing between “mature” and “partially spawned” stages of the sea urchin reproductive cycle, in accordance with the most frequently used classification (Byrne, 1990). Therefore, the stage of gonad maturity was determined for each individual in accordance with the classification proposed for the sea urchin S. intermedius (Fuji, 1960) and used by Matsui et al. (2008) with a slight modification. We distinguished five stages of gonad maturity for both males and females: (1) recovering, (2) growing, (3) premature, (4) mature and (5) spent. In addition, we distinguished one more stage for unspawned females, in which the gonadal cleaning process was highly prolonged. Therefore, we referred to this period of the reproductive

14

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

cycle as the stage of prolonged oocyte resorption (6). In unspawned males, the gonad acini contained a large number of spermatozoa across two reproductive cycles. We did not differentiate the gonads with undischarged and new generation of spermatozoa, therefore, we continued to refer to this period as the mature stage (4). The monthly average size of the oocytes was determined by measuring the theoretical diameter of 50 oocytes and eggs per individual (from 500 to 900 cells per sample). The area of each oocyte (μm2) was measured by drawing its perimeter on the computer screen, followed by transformation into a theoretical diameter, D, based on the relationship D = √4S/π (Lango-Reynoso et al., 2000). Only the oocytes that displayed a well-defined germinal vesicle and, in the case of small previtellogenic oocytes, a nucleolus, as well as fully cross-sectioned eggs were measured. Oogonia and relict eggs exhibiting obvious signs of degeneration were not measured.

Software, San Diego, CA). Statistical analysis was not performed on data presented as percentage of AIRG sea urchins due to lack of replication. 3. Results 3.1. Sex ratio The overall mean male/female ratio from all the samples taken was 1.397 which differed significantly from 1 (one sample t-test) (Table 1). In Kievka Bay, the overall mean sex ratio was 1.488 whereas in Rudnaya and Vostok Bays, it did not deviate significantly from 1. At the same time, one way ANOVA revealed no significant differences in the overall mean sex ratios between the stations (2 df, MS = 0.594, F = 2.221, p = 0.120). Similarly, there were no significant differences between the years in Kievka Bay (3 df, MS = 0.029, F = 0.071, p = 0.964). No hermaphrodites were observed.

2.3. Temperature and chlorophyll a determination 3.2. Dynamics of gonadal development in S. intermedius from Kievka Bay Satellite monitoring data (Aqua satellite equipped with MODIS sensor) pertaining to the temperature of the water surface and Chl a concentration in the surveyed area over the period of 2008–2009 were obtained from the Center for the Collective Usage of Regional Satellite Monitoring of the Far East Branch of the Russian Academy of Sciences. The data were processed using SeaDAS and Glance 1.80 softwares. The area over which the satellite monitoring data were processed was 8–40 pixels (one pixel = 1 km2), corresponding to the nearest pixels to the site of sea urchin sampling. To determine the Chl a concentration, the ОС3 algorithm was used. Sea surface temperature estimated from infrared satellite data is close to in situ temperature data (Emery et al., 2001). At the same time, due to increased turbidity, bottom reflectance and atmospheric effects, remotely sensed Chl a in coastal waters is a less accurate indicator of actual chlorophyll concentrations than in the open sea (D'Sa et al., 2002; Liu et al., 2003). Therefore, in our work we used Chl a concentrations estimated from satellite data for the purpose of revealing only regional differences in chlorophyll levels in the surveyed area. In summer 2010, there were too few clear days suitable for satellite monitoring; however, from July to October 2010, we had the opportunity to measure the Chl a concentration, water temperature and salinity directly at the site of sea urchin sampling near the bottom with a multi-parameter sonde YSI 6920V2 (YSI-Incorporated, USA). Measurements were performed daily except for storm days. To calibrate the sonde, various volumes (1–2 l) of sea water were filtered through Whatman glass-fiber filters GF/F (nominal pore size 0.7 μm) immediately after sampling. The filters were preserved frozen (−20 °C) until subsequent analysis, which was performed within a month. Pigments were extracted with 90% acetone and measured by spectrophotometry, Chl a concentrations were calculated by using the SCORE-UNESCO equations (UNESCO, 1966). 2.4. Statistical analysis For normally distributed data, unpaired t-test, Pearson r analysis and one sample t-test were performed. Parametric methods were not employed when the variance homogeneity (Barlett's test and Fisher ttest: p b 0.05) and normal distribution (D'Agostino and Pearson normality test: p b 0.05) of the data were not met. In the case of non-normally distributed data, a non-parametric Kruskal–Wallis ANOVA on ranks followed by Dunn's test as a post-hoc test, non-parametric Mann– Whitney test and Spearman r analysis were performed. An exception was made for the analysis of seasonal dynamic of the GI values. GI data had normal distribution in monthly samples of 2008 but in 2009 and 2010, a few samples did not correspond to Gaussian distribution. We failed to normalize the GI data of 2009 and 2010; therefore, we analyzed non-transformed data by parametric one-way and two-way ANOVA followed by Tukey's multiple comparison test. All statistical analyses were performed using the GraphPad Prism 6.0 for Windows (GraphPad

3.2.1. Gonad index Not one sea urchin sample exhibited significant difference in the GI between sexes (unpaired t test and Mann–Whitney U test, all p N 0.05), therefore, we pooled the data from both sexes for further statistical analysis. To determine if there were significant differences between the months for each year, one-way ANOVA was applied. All the years showed significant differences (Table 2). In all the years, the GI of S. intermedius reached its maximum in the period from April to July which corresponds to growing and premature stages of gonadal development (Figs. 2 and 3). Inter-year comparison of the GI values for this period by two-way ANOVA showed no significant effect of the main factor, year, but revealed significant effect of the factor month as well as of the interaction between month and year (Table 3). In contrast, significant effects of the year and month as well as of the interaction between them were revealed in the period from August to October (Table 3). Tukey's tests showed that in 2008, a first significant decrease in the GI occurred in October (p b 0.001), and then the GI remained stable (p N 0.05) until April of 2009 (Fig. 2). In 2009, a significant decrease in the GI was registered in August (p b 0.05), and again it remained stable until May of the next year (all p N 0.05). In 2010, the GI decreased stepwise from August to October (differed from July by Tukey's tests, p b 0.0001). The 3-year minimum for the GI was observed during the period from October 2010 to February 2011 (Fig. 2), when the GIs were 5.8 and 4.5 times lower than the corresponding GI values recorded in 2008 and 2009, respectively. 3.2.2. Gonad maturity At the beginning of this study (April 2008), the gonadal state of the females corresponded to the growing stage of the reproductive cycle (2), whereas in males, the growing (2), premature (3) and mature (4) stages were observed (Fig. 3). In August, the gonads of both sexes had reached the premature (3) and mature (4) stages. Nevertheless, in a major portion of the sea urchin population, the reproductive cycle did not appear to be completed by spawning. The percentages of spent females in the September and October samples were 12% and 5%, respectively. In September, the destruction of undischarged eggs Table 1 Sex ratios and significance of deviations from a 1:1 sex ratio in the sea urchin Strongylocentrotus intermedius (one sample t-test). Site

Males

Females

Mean sex ratio ± SD

df

t

p value

Kievka Rudnaya Vostok Overalla

1300 250 200 1899

945 236 186 1438

1.488 ± 0.525 1.142 ± 0.362 1.175 ± 0.441 1.397 ± 0.525

38 6 7 57

5.487 1.036 1.118 5.749

b0.0001 0.340 0.304 b0.0001

a The animals from all the samples including one-time samples from the Bays of Vrangel, Shepalova, Sokolovskaya and Kit.

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23 Table 2 Comparison of the gonad index (GI) of the sea urchin Strongylocentrotus intermedius between the months for each year of study (2008_2010) by one-way ANOVA. GI/month

df

MS

F

p value

2008 2009 2010

8 9 11

455.5 579.2 1668

18.22 28.09 126.5

b 0.0001 b 0.0001 b 0.0001

(oocyte resorption stage) had begun in 12% of females (Fig. 3). The gonads of these females contained both morphologically normal and fragmented eggs (Fig. 4A). Examination of the morphological features of undischarged eggs that had degenerated in the gonads of unspawned females of S. intermedius clarified some details of this process. First, the eggs disintegrate into numerous spherical fragments, still surrounded by the membrane (Fig. 4A), and these spherical fragments are subsequently released (Fig. 4B) and presumably absorbed by ovarian nutritive phagocytes (NPs). In morphological terms (Fig. 4), these fragments are similar to “mosaic” globules, which showed abundant accumulation in the cytoplasm of NPs of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus as a result of cleaning processes in the post-spawning gonad (Masuda and Dan, 1977). In October, the percentage of females in the oocyte resorption stage (6) increased to 45%, reaching 94% and 86% in November and December, respectively (Fig. 3). The dynamics of an increase in the portion of females in the mature stage (4) containing normal eggs during breeding season positively, with a 3 month lag, correlated with the dynamics of the portion of females in the oocyte resorption stage (6) of the reproductive cycle containing fragmented eggs (89.8 ± 0.8 days lag, r = 0.950, p = 0.0132, α = 0.05). At the end of December, virtually all of the eggs present in the females had degraded into small fragments (Fig. 4B). Egg fragments were visible in smears and histological slides up to June 2009 when the females in the oocyte resorption stage (6) were not found (Fig. 3). Females in the growing stage (2) of the reproductive cycle first appeared in March 2009 (Figs. 3 and 4D) and were dominant in the April–June samples. In July and August, the gonadal state of the majority of females corresponded to the premature (3) and mature (4) stages of gonad maturity. In 2009, the reproductive cycle of a significant portion of the sea urchin population from Kievka Bay was not again completed by spawning. In the sea urchin samples collected in September, 58% of spawned females were found (Fig. 3). In October, the gonads of 54% contained eggs that were being destroyed. Females with gonads containing eggs at different stages of destruction were found in the sea urchin samples during the period from October 2009 through June 2010 (Fig. 3). It was notable that after breeding season of 2008, in November 2008February 2009, only a small percentage (from 6% to 18%) of females could be considered to be in the recovering stage (1) of the reproductive cycle due to the long duration of the cleaning process in their gonads. In December 2009-February 2010, the portion of such females was approximately 50% (Fig. 3). In 2008–2009, spawning failure was also observed in males. In 2008, spawned males were found in the sea urchin samples of November and

15

December only (7% and 12%, respectively) (Fig. 3). In 2009, the percentages of spawned males were 17% in September and 24% in October, respectively (Fig. 3). Undischarged and new generations of spermatozoa were present in the sea urchin testes from April 2008 up to September 2010 (Fig. 3). In 2010, the reproductive cycle of S. intermedius in the population from Kievka Bay ended in virtually complete spawning that was synchronous in females and males (Fig. 3): only one female (in September) and one male (in December) showed signs typical of an absence of complete spawning. Following complete spawning, a lengthy period of gonad restoration was observed: females and males showing a gonadal state corresponding to the recovering stage (1) of the reproductive cycle were found in the sea urchin samples from November 2010 up to March 2011 (Fig. 3). The seasonal dynamics of the sea urchins that were artificially induced to release gametes (AIRG) by sampling procedure or dissection generally corresponded to the data obtained from the histological analysis (Fig. 5). On September 15, 2008, the percentage of AIRG females reached 91%, and 13% of females released degenerating eggs in addition to normal eggs. Among released eggs, morphologically normal ones were observed from June to November. In December, the percentage of AIRG females reached 100%; however, only degenerating eggs and egg fragments were released. AIRG females were observed through March 2009 (Fig. 5). In 2009, the dynamics of AIRG females were qualitatively similar to that in 2008, with the following exception: the percentage of females releasing degenerating eggs during the autumn and winter months was two times lower. As it is evident from Fig. 5, the line reflecting the portion of females which released fragmented eggs is shifted to the right-hand side relatively the line reflecting the portion of females which released normal eggs. This shift corresponds to the time period of 3 months. As for histological data, the dynamics of an increase in the portion of females which released normal eggs during breeding season positively, with a 3 month lag, correlated with the dynamics of an increase in the portion of females releasing fragmented eggs (89.8 ± 0.8 days lag; r = 0.966, p = 0.0073, α = 0.05). It may be proposed that this 3 month lag corresponds to a life-time of mature eggs in the sea urchin gonad. AIRG males were found in all samples of 2008 and 2009 (Fig. 5), the maximum (approximately 100%) was observed during the period from August 2008 to March 2009. A high proportion of AIRG males (87% to 57%) were also recorded from July to December of 2009. In 2010, the percentage of AIRG males increased to 100% in July–August; then, after spawning in September, it dropped to zero. AIRG females exhibited similar dynamics (Fig. 5). 3.2.3. Oocyte diameter The data on the size dynamics of female reproductive cells (oocytes and eggs) corresponded well with the data on the dynamics of the stages of the reproductive cycle and GI values. Generally, the changes in the median cell diameter were similar for three years (Fig. 6). However, some peculiarities were observed in the period from August to October. In September and October of 2008 and 2009, the median cell diameter was close to that in August, reflecting a high share of

Fig. 2. Temporal dynamics of the gonad index (mean ± SD) in the sea urchin Strongylocentrotus intermedius from Kievka Bay (northwestern Sea of Japan) in 2008–2011.

16

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

Fig. 3. Temporal dynamics of the percentages of the reproductive cycle stages and gonad index in Strongylocentrotus intermedius females and males from Kievka Bay (northwestern Sea of Japan) in 2008–2011. Numbers above the columns show the number of sea urchins from which the frequency was calculated.

undischarged eggs. In these years, the median oocyte diameter showed no significant differences between September and October while in November and December, when no morphologically normal eggs were present in the gonads (fragmented eggs were not taken into account), it decreased sharply (Fig. 6). In 2010, the median oocyte diameter went down quickly in September when the ovaries became spent (Fig. 6). 3.3. Occurrence of spawning failure in S. intermedius populations along the coastline of the Primorye region and dynamics of environmental variables 3.3.1. Sea urchin spawning In 2008, a higher proportion of females spawned in Vostok Bay compared to Kievka Bay (Table 4). In Vostok Bay, only 1 of 14 females did not spawn. This female displayed a large gonad with degenerating eggs. In 2009, the maturation of the sea urchins in Vostok and Rudnaya Bays (extreme south and north stations) occurred almost simultaneously with that in centrally positioned Kievka Bay. During the first half of August, the gonads were mature in 94% and 100% of the sea urchins in Table 3 Comparison of the gonad index of the sea urchin Strongylocentrotus intermedius across the years of study (2008_2010) by two-way ANOVA. Factor

df

April–July Year Month Year/month

2 3 6

August–October Year Month Year/month

2 3 6

MS 63.70 346.3 97.62

3335 875 126.5

F

p value 2.597 14.12 3.979

0.0760 b 0.0001 0.0007

176.7 44.78 6.705

b 0.0001 b 0.0001 b 0.0001

Vostok and Rudnaya Bays, respectively (data not shown). It presumes the maturation synchrony in S. intermedius populations at other stations. However, the percentage of spawned individuals was quite different (Table 4). It varied from 100% in Vostok and Sokolovskaya Bays to 32% in Kit Bay (Table 4). 3.3.2. Temperature In contrast to large difference in percentage of spawned females in Kievka and Vostok Bays, the temperature profiles of the two sites during the spawning season in 2008 were nearly identical. They were strongly correlated and appeared to be well synchronized (Pearson r = 0.81, p b 0.0001, α = 0.05, Fig. 7A). An abrupt temperature decrease caused by upwelling, which is typically observed in this season (Zuenko, 2008), occurred on September 28 at both stations simultaneously (Fig. 7A). The dynamics of the surface water temperature in August–September 2009 were similar at all 7 stations (Fig. 8). A sharp decrease in temperature due to upwelling was observed at all stations on August 23 and 31. After that, the second order oscillation of temperature was observed at all stations. Comparison of the data on temperature dynamics (Fig. 8) and sea urchin spawning success at different localities (Table 4) showed that despite the coincidence of temperature changes at the majority of stations, the proportions of females that failed to spawn were different between the stations. 3.3.3. Chlorophyll a concentration The concentrations of Chl a observed in August–September differed among the stations (Figs. 7B and 9, Table 4). In 2008, the maximum and mean concentrations of Chl a in Vostok Bay were 5.5 and 3.5 times higher, respectively, than those in Kievka Bay (Fig. 7B). In 2009, the highest peak concentrations of Chl a were observed at the stations located close to settlements (Table 4, Fig. 9). Positive correlations were found between Chl a concentrations and both the population size (Population

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

17

Fig. 4. Morphological features of the gonads of Strongylocentrotus intermedius females from Kievka Bay (northwestern Sea of Japan) which have not spawned in summer–autumn season of 2008. (A) Appearance of normal and degenerating (arrows) eggs in the gonad smear. (B) Numerous fragments of degenerating eggs in the gonad smear. Note the fragments still enveloped by the membrane. (C) Histological section of the ovary in December 2008 showing numerous fragments of relict eggs in the acinus lumen, the arrow indicates small fragments still enveloped by the membrane. (D) Histological section of the ovary in March 2009 showing the fragments of relict eggs (arrows) in the acinus lumen along with the developing oocytes at the acinus periphery. Designations: NPs — nutritive phagocytes, Oc — oocyte. Scale bar: A, B = 100 μm, C = 20 μm, D = 50 μm.

Size of the Russian Federation, 2013) (Spearman r = 0.96, p = 0.003, α = 0.005) and the percentage of unspawned S. intermedius females (Spearman r = 0.90, p = 0.012, α = 0.01). 3.4. Dynamics of gonadal development in S. intermedius and environmental variables in July–October 2010 in Kievka Bay Analysis of the gonadal state of the sea urchins during the period between July and October 2010, carried out at intervals of 8–14 days, revealed at least three spawning events. Tukey's tests (one way ANOVA) showed two subsequent steps of decrease in the GI from August 6 to August 31 (both p b 0.0001) (Fig. 10A). Third spawning event between

August 31 and September 8 was followed by a dramatic increase in completely spawned males and females (Fig. 10B and C). It indicates that spawning started between August 6 and 18 and was completed between August 31 and September 8. On August 6, 31% of females were in the premature stage of the reproductive cycle (Fig. 10B). It indicates that in the S. intermedius females, the process of gonad maturation was not completely synchronous, and in parallel with spawning, ripening of the gonads occurred. Generally, sea urchin spawning in 2010 was successful, but one unspawned female of the 22 examined was found in the sample collected on September 22 (Fig. 10B). The water salinity during the first spawning event (August 6–18) was relatively stable, whereas during the second (August 18–31) and

Fig. 5. Temporal dynamics of the percentages of Strongylocentrotus intermedius females and males from Kievka Bay (northwestern Sea of Japan) that were artificially induced to release gametes by sampling procedure or dissection in 2008–2011. Every point on the graph represents a minimum of 50 individuals.

18

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

Fig. 6. Temporal dynamics of the median oocyte diameter of the sea urchin Strongylocentrotus intermedius from Kievka Bay (northwestern Sea of Japan) in 2008–2010. The boxes represent the 25th to 75th percentiles, the solid lines within the boxes show the median values, the whiskers are the minimum and maximum values. Different letters represent significant median difference compared to the previous month (a: p b 0.05, b: p b 0.0001; one-way ANOVA, Kruskal–Wallis statistics, Dunn's multiple comparison test).

third (August 31–September 8) spawning events, a short-term decrease in salinity by 2–5 psu occurred (Fig. 10D). The temperature varied from 17 to 23 °С during the first and third spawning events and was fairly stable during the second spawning event (Fig. 10D). An abrupt decrease in temperature associated with upwelling occurred on September 11, when the spawning of sea urchins was nearly completed. During the entire spawning period, the Chl a concentration was relatively high. It ranged from 0.83 to 2.7 mg m−3 during the first and second spawning events and rose up to 10.1 mg m−3 during the third spawning event (Fig. 10D). 4. Discussion Despite the seasonal ripening of gonads by all individuals in S. intermedius population, examination of the gonadal development (GI dynamics, AIRG dynamics and histology) throughout the year suggests that spawning may not occur for most individuals in a population in some years. We noted this pattern both for some years at a single site and for some locations within a single year. Thus, spawning in the sea urchin S. intermedius does not occur automatically following gonad maturation but requires special environmental conditions. To our knowledge, the present work is the first to show that the annual reproductive cycle of broadcast-spawning marine invertebrates from natural populations may not be completed by spawning. The other reported case referred to brackish water bivalve mollusk C. japonica from Lake Abashiri (Hokkaido, Japan) (Baba et al., 1999). The observations of extremely low abundance of planktonic larvae of C. japonica in the lake in 1995 and 1996, compared to other years

Fig. 7. Temporal variation of the temperature (A) and chlorophyll a concentration (B) in Vostok and Kievka Bays (northwestern Sea of Japan) in summer–autumn season of 2008 (the satellite data). Data are presented as mean ± SD. Horizontal dashed line in (B) denotes the concentration of chlorophyll a corresponding to 1 mg m−3.

(1989, 1990, 1994 and 1997), and extremely high amount of atretic oocytes in the ovaries after spawning season of 1995 led to the conclusion that most mollusks failed to spawn in 1995. The most pronounced spawning failure in S. intermedius population was recorded in 2008 in Kievka Bay. However, there was a decline in the GI in October in the absence of full-fledged spawning. There may be three reasons for this. First, partial spawning of some individuals might take place but because the majority of ripe individuals started to release their gametes right away after catching it made it difficult to differentiate between naturally occurring and artificially induced gamete release. Second, as we had noted, the sea urchins with overmatured gonads after September exhibited more intensive release of gametes resulting from sampling procedure or dissection than in the previous months. Third, according to our observations, sea urchins with mature gonads did not feed on, as their intestines were virtually empty. Therefore, starvation might result in a decrease in the mass of the gonad, whose nutrient reserves are consumed to satisfy the energy needs of the organism (Lares and Pomory, 1998).

Table 4 Occurrence of spawning failure in Strongylocentrotus intermedius populations along the coastline of the Primorye region and some characteristics of the sites surveyed. The data on the chlorophyll a (Chl a) concentration are presented as mean ± SD. “–” denotes the absence of data. Characteristics

Portion of unspawned S. intermedius females (%) 2008 2009 Peak Chl a concentration (mg m−3) 2008 2009 Settlement population size (thousand people)

Site Vostok

Vrangel

Sokolovskaya

Rudnaya

Kievka

Shepalova

Kit

7 0

– 8

– 0

– 23

95 50

– 36

_– 68

4.9 ± 1.6 15.5 ± 1.3 25.5

– 7.2 ± 1.3 18

– 6.6 ± 2.6 8.5

– 4.9 ± 1.5 2.4

0.9 ± 0.4 2.3 ± 1.1 0

– 1.6 ± 0.2 0

_– 1.2 ± 0.3 0

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

Fig. 8. Temporal variation of the surface water temperature at different surveyed sites in the northwestern Sea of Japan in summer–autumn season of 2009 (the satellite data).

19

After spawning, the gonadal acini still contain small quantity of undischarged gametes. These gametes subsequently undergo resorption, which involves the NPs (Fuji, 1960; Holland and Giese, 1965; Reunov et al., 2004a, b; Walker et al., 2005, 2007). The data obtained in the present work indicate that this mechanism is triggered regardless of whether spawning occurs and therefore significantly limits the lifetime of mature eggs. According to our data, the lifetime of eggs in S. intermedius ovaries is approximately 3 months whereas sperm cells in the testes keep their viability for longer time. In the absence of spawning, sea urchin gonads contain an enormous amount of undischarged mature gametes which have to be phagocytosed. The processes of phagocytosis of the eggs and spermatozoa by NPs have certain features stipulated by the differences in size of female and male gametes. Residual spermatozoa are phagocytosed by testicular NPs, become a part of their heterophagosomes, and are then subsequently digested (Reunov et al., 2004a, b; Walker et al., 2005, 2007). Undischarged eggs are too large to undergo phagocytosis without being disintegrated. Therefore, they are first subjected to destruction (atresia), the mechanisms of which have not been well studied in the sea urchins. Masuda and Dan (1977) revealed a high activity of acidic phosphatase in sea urchin residual eggs and assumed that they are destructed with the participation of lysosomes (i.e., through autophagy). They observed the

Fig. 9. Temporal variation of the chlorophyll a concentration (mean ± SD) at different surveyed sites in the northwestern Sea of Japan in summer–autumn season of 2009 (the satellite data). Horizontal dashed lines indicate the concentration of chlorophyll a corresponding to 1 mg m−3.

20

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

Fig. 10. Temporal dynamics of gonadal development in Strongylocentrotus intermedius and environmental variables in summer–autumn season of 2010 in Kievka Bay (northwestern Sea of Japan). (A) Gonad index (GI: mean for both sexes ± SD). (B) The percentages of the reproductive cycle stages (columns) and the GI values in females. (C) The percentages of the reproductive cycle stages and the GI values in males. (D) Temporal variation of the temperature, chlorophyll a concentration and salinity (data of direct measurements in the bottom boundary layer). Numbers above the columns in (B) and (C) show the number of sea urchins from which the frequency was calculated. Horizontal dashed line in (D) indicates the concentration of chlorophyll a corresponding to 1 mg m−3. Solid vertical lines denote standard deviation.

appearance of numerous “mosaic” globules containing cortical granules in NPs of female post-spawning gonad and suggested that this indicates the mode of disposal of relict ova. The large-scale fragmentation of undischarged eggs which we revealed both in gonadal smears (Fig. 4B) and in histological sections (Fig. 4C) of the ovaries of unspawned S. intermedius represents a good illustration of the mechanism of sea urchin egg disintegration suggested by Masuda and Dan (1977). A similar pattern of degeneration of growing oocytes, i.e. their disintegration into spherical fragments, was described for the crinoid Antedon mediterranea (Barbaglio et al., 2009) and for the mussel Mytilus galloprovincialis (Ortiz-Zarragoitia et al., 2011). The resulting fragments appeared to be phagocytosed by gonadal accessory cells and/or hemocytes.

The resorption of relict gametes in the sea urchin gonad normally lasts for 2–3 months (Walker et al., 2007). The much greater duration (over 7 months) of the cleaning process observed in the present work is in all likelihood due to the much larger volume of the sex cells that were subject to resorption in seasons of 2008–2009. Development of a new generation of oocytes in S. intermedius took place from December to August. Thus, in the absence of spawning, the growth and differentiation of the new generation of oocytes occur simultaneously with the cleaning process. We have no data to believe that there is any natural disaster responsible for the prohibition of spawning in S. intermedius populations in some years and some locations. Therefore we proposed that the

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

phenomenon of spawning failure can be attested to the fact that a particular stimulus is needed to trigger the spawning in the natural population of S. intermedius. The factors ensuring offspring survival may include this stimulus, i.e., an optimal ambient temperature for larval development, and a sufficiently high concentration of phytoplankton (food for the larvae). The available data on the role of temperature in the stimulation of sea urchin spawning are rather contradictory. In field studies, some researchers have noted the coincidence of spawning with a rise in temperature (Byrne et al., 1998; González-Irusta et al., 2010; Himmelman et al., 2008), whereas others have observed that spawning coincides with a temperature decrease (Byrne et al., 1998; King et al., 1994; Lamare and Stewart, 1998; Tsuji et al., 1989). Attempts to stimulate the spawning of sea urchins in laboratory experiments under temperature conditions typically encountered during their spawning season have failed (Reuter and Levitan, 2010). Thus, it seems that temperature does not have a consistent effect on sea urchin spawning activity, taking into consideration that temperature changes might be accompanied by changes in other environmental variables (see for review Himmelman, 1999; Himmelman et al., 2008). The results of the present work are in agreement with the notion that temperature and its variations may not serve as proximal cues triggering the spawning of sea urchins. The significant difference in the percentages of spawned S. intermedius females between Kievka and Vostok Bays observed in 2008 as well as at 7 stations along the coast of the Primorye region in 2009 that were under similar temperature conditions, including substantial temperature changes associated with upwelling, support this point of view. The coincidence of sea urchin spawning with increases in phytoplankton concentrations observed in field studies is considered by a number of researchers as evidence that phytoplankton may serve as the primary stimulus to trigger spawning (Gaudette et al., 2006; González-Irusta et al., 2010; Himmelman, 1975; Levitan, 2002; Starr et al., 1993). This hypothesis has been confirmed by data from laboratory experiments demonstrating that the addition of various species of microalgae (both combined with sperm and in the absence of it) to water stimulates the spawning of sea urchins (Reuter and Levitan, 2010; Starr et al., 1990, 1992). These results provide good support for the notion that phytoplankton stimulates the spawning of the most sensitive males, whose sperm promote synchronous mass spawning (Gaudette et al., 2006; Reuter and Levitan, 2010; Starr et al., 1990,

21

1992). At the same time, researchers have noted that other environmental factors, such as the lunar phase, might influence the intensity of spawning (Reuter and Levitan, 2010). Moreover, some contradictory results have been obtained in studies addressing the roles of phytoplankton and sperm in spawning stimulations. For example, Reuter and Levitan (2010) did not detect a consistent reaction to phytoplankton in experiments with the sea urchin Lytechinus variegatus, and McCarthy and Young (2004) did not observe a reaction to sperm in this species in the field. In a field study conducted at a high temporal resolution, mass spawning of the representative of echinoderm Cucumaria frondosa with lecithotrophic larvae was recorded under conditions of a rise in temperature and a decrease in the phytoplankton concentration (Hamel and Mercier, 1995). The positive correlation between Chl a concentrations and the percentage of spawned S. intermedius females suggests that there is a relationship between the spawning of this species and the concentration of phytoplankton (Table 4). Primary production in nearshore waters substantially depends on the influx of biogenic elements from the terrigenous runoff, upwelling and anthropogenic sources (see for review: Cloern et al., 2013; Hecky and Kilham, 1988; Howarth, 2008). It seems that in 2008 the supply of Kievka Bay with nutrients was poor due to minimum-level runoff (Fig. 11) and late (September 24, see Fig. 7A) development of upwelling which have started after the beginning of the resorption of undischarged eggs in the sea urchin ovaries. Therefore, biogenic elements delivered by upwelling and the associated development of phytoplankton cannot have influenced the spawning success of S. intermedius at this time. The intermediate spawning success observed in S. intermedius population of Kievka Bay in 2009 might be due to weak upwelling activity and relatively low river runoff. In 2010, which was characterized by high-level runoff (Fig. 11), the spawning of S. intermedius in Kievka Bay was almost totally completed by September 6. It seems reasonable to assume that under conditions of low river flow the phytoplankton concentration may depend on the level of anthropogenic pressure in the coastal area. The positive correlations between settlement population size, the peak Chl a concentration and the percentage of unspawned S. intermedius females observed in different areas of the coastal zone of the Primorye region in 2009 support the relationships between these variables (Table 4). In conclusion, it should be noted that the study area is characterized by spatially different levels of anthropogenic pressure and significant

Fig. 11. Temporal dynamics of the runoff of the Lazovka River, the main tributary of the Kievka River that flows into Kievka Bay (northwestern Sea of Japan), in August–September of 1980–2010.

22

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23

year-to-year variations in the magnitude of river runoff and the upwelling schedule. We hypothesized that the phenomenon of spawning failure in natural populations of S. intermedius seemed to be attributable to combination of environmental factors responsible for low primary productivity in water column during the sea urchin spawning season. Acknowledgments This work was supported by the Russian Foundation for Basic Research [09-04-98562-r_vostok_a and 11-04-98523-r_vostok_a] and the Far Eastern Branch of the Russian Academy of Sciences [09-III-A06-194, 09-I-P16-04, 12-III-А-06-087, 12-I-P4-02 and 15-I-6-007 o]. [ST] References Agatsuma, Y., 2013. Strongylocentrotus intermedius. In: Lawrence, J.M. (Ed.), Sea Urchins: Biology and Ecology. Elsevier B.V., pp. 438–447. Baba, K., Tada, M., Kawajiri, T., Kuwahara, Y., 1999. Effects of temperature and salinity on spawning in brackish water bivalve Corbicula japonica in Lake Abashiri, Hokkaido, Japan. Mar. Ecol. Prog. Ser. 180, 213–221 (http://www.int-res.com/articles/meps/ 180/m180p213.pdf). Barbaglio, A., Biressi, A., Melone, G., Bonasoro, F., Lavado, R., Porte, C., Candia Carnevali, M.D., 2009. Reproductive cycle of Antedon mediterranea (Crinoidea, Echinodermata): correlation between morphology and physiology. Zoomorphology 128, 119–134. http://dx.doi.org/10.1007/s00435-008-0079-z. Bazhin, A.G., 1998. The sea urchin genus Strongylocentrotus in the seas of Russia: taxonomy and ranges. In: Mooi, R., Telford, M. (Eds.), Proceedings of the 9th International Echinoderm Conference, San Francisco, August 1996. Echinoderms. Balkema, Rotterdam, pp. 563–566. Borisovetz, E.E., Bregman, Y.E., Viktorovskaya, G.I., Kalinina, M.V., 2000. Biology of the sea urchin Strongylocentrotus intermedius (A. Agassiz) in the northwestern coastal waters of the Sea of Japan. Distribution and size composition of the populations. Proceedings of the Pacific Research Institute of Fisheries and Oceanography (TINRO-Center). 127, pp. 416–439 (in Russian with English summary). Breese, W.P., Robinson, A., 1981. Razor clams, Siliqua patula (Dixon): gonadal development, induced spawning and larval rearing. Aquaculture 22, 27–33. http://dx.doi. org/10.1016/0044-8486(81)90130-7. Buznikov, G.A., Slotkin, T.A., Lauder, J.M., 2003. Sea urchin embryos and larvae as biosensors for neurotoxicants. Curr. Protoc. Toxicol. http://dx.doi.org/10.1002/0471140856. tx0106s16 (Chapter 1: Unit1.6). Byrne, M., 1990. Annual reproductive cycles of the commercial sea urchin Paracentrotus lividus from an exposed intertidal and a sheltered subtidal habitat on the west coast of Ireland. Mar. Biol. 104, 275–289 (http://link.springer.com/article/10.1007% 2FBF01313269#page-1). Byrne, M., Andrew, N.L., Worthington, D.G., Brett, P.A., 1998. Reproduction in the diadematoid sea urchin Centrostephanus rodgersii in contrasting habitats along the coast of New South Wales, Australia. Mar. Biol. 132, 305–318 (http://link.springer. com/article/10.1007%2Fs002270050396#page-1). Cloern, J.E., Foster, S.Q., Kleckner, A.E., 2013. Review: phytoplankton primary production in the world's estuarine-coastal ecosystems. Biogeosci. Discuss. 10, 17725–17783. http://dx.doi.org/10.5194/bgd-10-17725-2013. D'Sa, E.J., Hu, C., Muller-Karger, F.E., Carder, K.L., 2002. Estimation of colored dissolved organic matter and salinity fields in case 2 waters using SeaWiFS: examples from Florida Bay and Florida Shelf. P. Indian As-Earth 111, 197–207. http://dx.doi.org/10. 1007/BF02701966#page-1. Dale, T., Siikavuopio, S.I., Aas, K., 2005. Roe enhancement in sea urchin: effects of handling during harvest and transport on mortality and gonad growth in Strongylocentrotus droebachiensis. J. Shellfish Res. 24, 1235–1239. Emery, W.J., Castro, S., Wick, G.A., Schluessel, P., Donlon, C., 2001. Estimating sea surface temperature from infrared satellite and in situ temperature data. Bull. Am. Meteorol. Soc. 82, 2773–2785. http://dx.doi.org/10.1175/1520-0477(2001)082b2773:ESSTFIN2. 3.CO;2. Fournier, J., Levesque, E., Pouvreau, S., Le Pennec, M., Le Moullac, G., 2012. Influence of plankton concentration on gametogenesis and spawning of the black lip pearl oyster Pinctada margaritifera in Ahe atoll lagoon (Tuamotu archipelago, French Polynesia). Mar. Pollut. Bull. 65, 463–470. http://dx.doi.org/10.1016/j.marpolbul.2012.03.027. Fuji, A., 1960. Studies on the biology of the sea urchin. III. Reproductive cycle of two sea urchins, Strongylocentrotus nudus and S. intermedius, in southern Hokkaido. Bull. Fac. Fish. Hokkaido Univ. 11, 43–48 (http://eprints.lib.hokudai.ac.jp/dspace/ bitstream/2115/23098/1/11%282%29_P49-57.pdf). Gaudette, J., Wahle, R.A., Himmelman, J.H., 2006. Spawning events in small and large populations of the green sea urchin Strongylocentrotus droebachiensis as recorded using fertilization assays. Limnol. Oceanogr. 51, 1485–1496. http://dx.doi.org/10.4319/lo. 2006.51.3.1485. González-Irusta, J.M., De Cerio, F.G., Canteras, J.C., 2010. Reproductive cycle of the sea urchin Paracentrotus lividus in the Cantabrian Sea (northern Spain): environmental effects. J. Mar. Biol. Assoc. U.K. 90, 699–709. http://dx.doi.org/10.1017/S002531540999110X. Guillou, M., Lumingas, L.J.L., 1998. The reproductive cycle of the ‘blunt’ sea urchin. Aquacult. Int. 6, 147–160. http://dx.doi.org/10.1023/A:1009290307840.

Hamel, J.F., Mercier, A., 1995. Spawning of the sea cucumber Cucumaria frondosa in the St Lawrence Estuary, eastern Canada. SPC Beche-de-mer Inform. Bull. 7, 12–18. Hecky, R.E., Kilham, P., 1988. Nutrient limitation of phytoplankton in freshwater and marine environments: a review of recent evidence on the effects of enrichment. Limnol. Oceanogr. 33, 796–822 (http://aslo.org/lo/toc/vol_33/issue_4pt2/0796.pdf). Himmelman, J.H., 1975. Phytoplankton as a stimulus for spawning in three marine invertebrates. J. Exp. Mar. Biol. Ecol. 20, 199–214. http://dx.doi.org/10.1016/00220981(75)90024-6. Himmelman, J.H., 1999. Spawning, marine invertebrates. In: Knobil, E., Neill, J.D. (Eds.), Encyclopedia of Reproduction vol. 4. Academic Press, New York, pp. 524–533. Himmelman, J.H., Dumont, C.P., Gaymer, C.F., Vallie'res, C., Drolet, D., 2008. Spawning synchrony and aggregative behaviour of cold-water echinoderms during multi-species mass spawnings. Mar. Ecol. Prog. Ser. 361, 161–168. http://dx.doi.org/10.3354/ meps07415. Holland, N.D., Giese, A.C., 1965. An autoradiographic investigation of the gonads of the purple sea urchin (Strongylocentrotus purpuratus). Biol. Bull. 128, 241–258 (http:// www.biolbull.org/content/128/2/241.full.pdf+html). Howarth, R.W., 2008. Coastal nitrogen pollution: a review of sources and trends globally and regionally. Harmful Algae 8, 14–20. http://dx.doi.org/10.1016/j.hal.2008.08.015. Kafanov, A.I., Pavlyuchkov, V.A., 2001. Ecology of the commercial sea urchins (genus Strongylocentrotus) of continental Japan Sea. Proceedings of the Pacific Research Institute of Fisheries and Oceanography (TINRO-Center). 128, pp. 349–373 (in Russian with English summary). Khotimchenko, Y.S., Deridovich, I.I., Motavkin, P.A., 1993. Biology of Reproduction and Regulation of Gametogenesis in Echinoderms. Nauka Press, Moscow (In Russian). King, C.K., Hoegh-Guldberg, O., Byrne, M., 1994. Reproductive cycle of Centrostephanus rodgersii (Echinoidea), with recommendations for the establishment of a sea urchin fishery in New South Wales. Mar. Biol. 120, 95–106. Koshelev, B.V., 1984. Ecology of Reproduction in Fishes. Nauka Press, Moscow (in Russian). Lamare, M.D., Stewart, B.G., 1998. Mass spawning by the sea urchin Evechinus chloroticus (Echinodermata: Echinoidea) in New Zealand fiord. Mar. Biol. 132, 135–140 (http:// link.springer.com/article/10.1007%2Fs002270050379#page-1). Lango-Reynoso, F., Chaves-Villalba, J., Cochard, J., Le Pennec, M., 2000. Oocyte size, a means to evaluate the gametogenic development of the Pacific oyster Crassostrea gigas (Thunberg). Aquaculture 190, 183–199. http://dx.doi.org/10.1016/S00448486(00)00392-6. Lares, M.T., Pomory, C.M., 1998. Use of body components during starvation in Lytechinus variegatus (Lamarck) (Echinodermata: Echinoidea). J. Exp. Mar. Biol. Ecol. 225, 99–106. http://dx.doi.org/10.1016/S0022-0981(97)00216-5. Levitan, D.R., 2002. Density-dependent selection on gamete traits in three congeneric sea urchins. Ecology 83, 464–479. http://dx.doi.org/10.1890/0012-9658(2002)083[0464: DDSOGT]2.0.CO;2. Levitan, D.R., Petersen, C., 1995. Sperm limitation in the sea. Trends Ecol. Evol. 10, 228–231. http://dx.doi.org/10.1016/S0169-5347(00)89071-0. Liu, Y., Islam, M.A., Gao, J., 2003. Quantification of shallow water quality parameters by means of remote sensing. Prog. Phys. Geogr. 27, 24–43. http://dx.doi.org/10.1191/ 0309133303pp357ra. Masuda, R.I., Dan, J.C., 1977. Studies on the annual reproductive cycle of the sea urchin and the acid phosphatase activity of relict ova. Biol. Bull. 153, 577–590. http://dx. doi.org/10.2307/1540607. Matsui, T., Agatsuma, Y., Ogasawara, M., Taniguchi, K., 2008. Coincidence in reproduction of the sea urchin Strongylocentrotus intermedius in Hirota Bay, on the Pacific Ocean off Northern Honshu, and in the Sea of Japan off Hokkaido, Japan. J. Shellfish Res. 27, 1283–1289. http://dx.doi.org/10.2983/0730-8000-27.5.1283. McCarthy, D.A., Young, C.M., 2004. Effects of water-borne gametes on the aggregation behavior of Lytechinus variegatus. Mar. Ecol. Prog. Ser. 283, 191–198. http://dx.doi.org/ 10.3354/meps283191. Mercier, A., Hamel, J.-F., 2009. Endogenous and exogenous control of gametogenesis and spawning in Echinoderms. Adv. Mar. Biol. 55, 1–302. http://dx.doi.org/10.1016/ S0065-2881(09)55008-0. Ortiz-Zarragoitia, M., Garmendia, L., Barbero, M.C., Serrano, T., Marigómez, I., Cajaraville, M.P., 2011. Effects of the fuel oil spilled by the Prestige tanker on reproduction parameters of wild mussel populations. J. Environ. Monit. 13, 84–94. http://dx.doi.org/ 10.1039/c0em00102c. Pennington, J.T., 1985. The ecology of fertilization of echinoid eggs: the consequences of sperm dilution, adult aggregation, and synchronous spawning. Biol. Bull. 169, 417–430 (http://www.biolbull.org/content/169/2/417.full.pdf). Population Size of the Russian Federation by the Municipalities to January 01, 2013. Federal State Statistics Service, Moscow (In Russian). Reunov, A.A., Yurchenko, A.V., Kalachev, A.V., Au, D.W.T., 2004a. An ultrastructural study of phagocytosis and shrinkage in nutritive phagocytes of the sea urchin Anthocidaris crassispina. Cell Tissue Res. 318, 419–428. http://dx.doi.org/10.1007/s00441-0040901-y. Reunov, A.A., Kalachev, A.V., Yurchenko, O.V., Au, D.W.T., 2004b. Selective resorption in nutritive phagocytes of the sea urchin Anthocidaris crassispina. Zygote 12, 71–73. http://dx.doi.org/10.1017/S0967199403002429. Reuter, K.E., Levitan, D.R., 2010. Influence of sperm and phytoplankton on spawning in the echinoid Lytechinus variegatus. Biol. Bull. 219, 198–206 (http://www.biolbull. org/content/219/3/198.full.pdf+html). Roper, D.S., Pridmore, R.D., Cummings, V.J., Hewitt, J.E., 1991. Pollution related differences in the condition cycles of Pacific oysters Crassostrea gigas from Manukau Harbour. Mar. Environ. Res. 31, 197–214. http://dx.doi.org/10.1016/0141-1136(91)90011-V. Smith, J.R., Strehlow, D.R., 1983. Algal-induced spawning in the marine mussel Mytilus californicanus. Invert. Repr. Dev. 6, 129–133. http://dx.doi.org/10.1080/01651269. 1983.10510032.

P.M. Zhadan et al. / Journal of Experimental Marine Biology and Ecology 465 (2015) 11–23 Starr, M., Himmelman, J.H., Therriault, J.C., 1990. Direct coupling of marine invertebrate spawning with phytoplankton blooms. Science 247, 1070–1074. http://dx.doi.org/ 10.1126/science.247.4946.1071. Starr, M., Himmelman, J.H., Therriault, J.C., 1992. Isolation and properties of a substance from the diatom Phaeodactylum tricornutum which induces spawning in the sea urchin Strongylocentrotus droebachiensis. Mar. Ecol. Prog. Ser. 79, 275–287 (http:// www.int-res.com/articles/meps/79/m079p275.pdf). Starr, M., Himmelman, J.H., Thermiault, J.C., 1993. Environmental control of green sea urchin, Strongylocentrotus droebachiensis, spawning in St Lawrence estuary. Can. J. Fish. Aquat. Sci. 50, 894–901. http://dx.doi.org/10.1139/f93-103. Steele, S., Mulcahy, M.F., 1999. Gametogenesis of the oyster Crassostrea gigas in southern Ireland. J. Mar. Biol. Assoc. U.K. 70, 673–686. http://dx.doi.org/10. 1017/S0025315498000836. Thorson, G., 1946. Reproduction and larval development of Danish marine bottom invertebrates, with special reference to the planktonic larvae of the Sound (Øresund). Meddelelser fra Kommissionen for Danmarks Fiskeri- Og Havunder SoegelserSerie: Plankton 4 pp. 1–523. Tsuji, S., Yoshiya, M., Tanaka, M., Kuwahara, A., Uchino, K., 1989. Seasonal changes in distribution and ripeness of gonad of a sea urchin Strongylocentrotus nudus in the western part of Wakasa Bay. Bull. Kyoto Inst. Ocean. Fish. Sci. 12, 15–21. UNESCO, 1966. Determinations of photosynthetic pigments in seawater. Monogr. Oceanogr. Methodol. 1, 11–18 (http://unesdoc.unesco.org/images/0007/000716/ 071612eo.pdf).

23

Walker, C.W., Harrington, L.M., Lesser, M.P., Fagerberg, W.R., 2005. Nutritive phagocyte incubation chambers provide a structural and nutritive microenvironment for germ cells of Strongylocentrotus droebachiensis, the green sea urchin. Biol. Bull. 209, 31–48 (http://www.biolbull.org/content/209/1/31.long). Walker, C.W., Unuma, T., Lesser, M.P., 2007. Gametogenesis and reproduction of sea urchins. In: Lawrence, J.M. (Ed.), Edible Sea Urchins: Biology and Ecology. Elsevier Science, Amsterdam, pp. 11–33. Wilson, B.R., 1969. Survival and reproduction of the mussel Xenostrobus securis (Lamark) (Mollusca; Bivalvia; Mytilidae) in a western Australia Estuary. Pt. II: reproduction, growth and longevity. J. Nat. Hist. 3, 93–120. http://dx.doi.org/10.1080/ 00222936900770111. Yakovlev, S.N., 1976. The seasons of reproduction of the sea urchins Strongylocentrotus nudus and S. intermedius in Vostok Bay, the Sea of Japan. In: Kasyanov, V.L. (Ed.), Biological Studies of Vostok Bay. Far East Science Center. USSR Academy of Sciences, Vladivostok, pp. 136–142 (in Russian). Zaslavskaya, N.I., Vashchenko, M.A., Zhadan, P.M., 2012. The genetic structure of populations of the sea urchin Strongylocentrotus intermedius from the northwestern Sea of Japan in connection with a shift in spawning time. Russ. J. Mar. Biol. 38, 325–338. http://dx.doi.org/10.1134/S1063074012040104. Zuenko, Y., 2008. Fisheries Oceanography of the Japan Sea. Publishing House of the Pacific Research Institute of Fisheries and Oceanography (TINRO-Center), Vladivostok (in Russian).