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Background The spatial distribution of spawners and temporal parameters of spawning in motile invertebrates with external fertilization might influence reproductive success. However, to date, data on the prespawning and spawning behaviors of broadcast spawners in the field have been scarce and mostly qualitative. The present study was intended to clarify the behavioral adaptations of two sea urchin species, Strongylocentrotus intermedius and Mesocentrotus nudus, using quantitative analysis of their behavior during mass spawning events under natural conditions. Methods We analyzed in situ video recordings of sea urchin behavior obtained during six spawning seasons (2014–2019). The total number of specimens of each sea urchin species and the numbers of spawning males and females were counted. Quantitative parameters of sea urchin spawning (numbers of gamete batches, release duration of one gamete batch, time intervals between gamete batches and total duration of spawning) and movement (step length of spawners and nonspawners before and during spawning and changes in distances between males/nonspawners and females) were determined. Results For each species, 12 mass spawning events were recorded in which 10 or more individuals participated. The temporal dynamics of the numbers of males and females participating in mass spawning were well synchronized in both species; however, males began to spawn earlier and ended their spawning later than females. In both species, the most significant intersex difference was the longer spawning duration in males due to the longer pause between gamete batches. The total duration of gamete release did not differ significantly between sexes. The average duration of sperm release during mass spawning events was longer than solitary male spawning. Males and females showed significant increases in the locomotion rate 35 min before the start of spawning and continued to actively move during spawning. An increase in movement rate before spawning in males and females was induced by environmental factor(s). Nonspawners of both species showed increased locomotion activity but in the presence of spawning neighbors and less prominently than spawners. On a vertical surface, both echinoids moved strictly upward. On flat surfaces, males, females and nonspawners of both echinoids became closer during spawning. Discussion We showed that two sea urchin species with planktotrophic larvae display similar behavioral adaptations aimed at enhancing reproductive success. The high sensitivity of sea urchins, primarily males, to some environmental factors, most likely phytoplankton, may be considered a large-scale adaptation promoting the development of mass spawning events. The longer spawning duration in males and increased movement activity before and during spawning in both sexes may be considered small-scale adaptations promoting approach of males and females and enhancing the chances of egg fertilization.

Simultaneous video recording of the sea urchin spawning behavior and recording of environmental factors enabled us to found synergism of the effects of circadian rhythm and phytoplankton on the spawning activity of the sea urchin Strongylocentrotus intermedius (Agassiz, 1864).

Background In the predator–sea urchin–macrophyte trophic cascade, the ecological effect of sea urchins as grazers depends both on their density and the changes in foraging activity, which are influenced by various disturbing factors. However, the complete duration of the alarm reactions of echinoids has not been studied until now. Here, we tested a hypothesis that two cohabiting sea urchins, Mesocentrotus nudus and Strongylocentrotus intermedius, which differ morphologically, might display different behavioral responses to high hydrodynamic activity and predation. Methods We used continuous time-lapse video recording to clarify behavioral patterns of M. nudus and S. intermedius in presence of a large quantity of food (the kelp Saccharina japonica) but under different weather conditions and different types of predation threat: (1) calm weather conditions, (2) stormy weather conditions, (3) predation risk associated with the presence of several sea star species and (4) predation risk associated with an alarm stimulus (crushed conspecifics or heterospecifics). Three separate video recording experiments (134 days in total) were conducted under field conditions. Video recording analysis was performed to determine the number of specimens of each sea urchin species in the cameras’ field of view, size of sea urchins’ groups, movement patterns and the duration of the alarm responses of both sea urchin species. Results We showed that in the presence of kelp, M. nudus and S. intermedius exhibited both similar and different behavioral responses to hydrodynamics and predation threat. Under calm weather, movement patterns of both echinoids were similar but M. nudus exhibited the higher locomotion speed and distance traveled. Furthermore, S. intermedius but not M. nudus tended to group near the food substrate. The stormy weather caused a sharp decrease in movement activity followed by escape response in both echinoids. Six starfish species failed to predate on healthy sea urchins of either species and only a few attacks on ailing S. intermedius specimens were successful. The alarm response of S. intermedius lasted approximately 90 h and 20 h for starfish attacks on ailing conspecifics and for simulated attacks (crushed conspecifics or heterospecifics), respectively and involved several phases: (1) flight response, (2) grouping close to the food, (3) leaving the food and (4) return to the food. Phase three was the more pronounced in a case of starfish attack. M. nudus only responded to crushed conspecifics and exhibited no grouping behavior but displayed fast escape (during 4 h) and prolonged (up to 19 days) avoidance of the food source. This outcome is the longest alarm response reported for sea urchins. Discussion The most interesting finding is that two cohabiting sea urchin species, M. nudus and S. intermedius, display different alarm responses to predation threat. Both alarm responses are interpreted as defensive adaptations against visual predators.

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 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.

Reproductive health of the common cupped oyster Crassostrea angulata cultivated in Taiwan was estimated through histological examination and semiquantitative analysis of their gonads. Oysters were collected in 4 aquaculture sites along the western coast of Taiwan (Hsiangshan, Chiku, Beimen, and Putai), where the major sources of industrial pollution are located, and off Penghu Island in the southern part of the Taiwan Strait, a comparatively clean resort area. The oysters from Hsiangshan had the lowest gonad maturity index (GMI) and the highest index of pathological alterations (IPA; P < 0.001); greater numbers of oysters with undifferentiated gonads (30%) and a green mantle (66.7%) were also present here. Most prominent gonad pathologies were atresia of oocytes and hemocytic infiltration of gonadal tissue. Oysters from Penghu had the highest GMI and the lowest IPA, whereas oysters from Chiku, Beimen, and Putai had intermediate values. Soft tissues of oysters from Hsiangshan contained 3,761.0 ± 1,201.0 µg/g dry weight Cu, 3,761.0 ± 1,201.0 µg/g Zn, and 1.5 ± 0.06 µg/g Cd, which is about 17 times, 3 times, and 1.5 times greater than the corresponding metal concentrations in the tissues of oysters from Penghu. Gametogenesis impairment in oysters cultivated in Taiwan are likely caused by environmental pollution.

Accumulation of lipofuscin-like pigments (LLPs) has been shown to be an appropriate index of both age and stress in some aquatic invertebrates. In the present study, LLP was quantified by measuring its autofluorescence intensity (ex 450 nm/em 512 nm) in nutritive phagocytes (NPs) of sea urchins Strongylocentrotus intermedius inhabiting polluted and relatively clean areas of Japan Sea. To avoid variations in LLP content related to sea urchin reproductive condition, only developing gonads with acini occupied mostly by NPs were used for LLP quantification as well as semiquantitative histopathological analysis. LLP concentrations ranged from 0.0 to 4.57 ± 0.53% area fraction in female gonads and from 0.0 to 4.61 ± 0.35% in male gonads. The presence of specimens with extremely high LLP concentrations (>1.5%) in all examined samples, including specimens from the reference station, as well as the absence of strong correlations between LLP concentrations and several parameters related to pollution (heavy-metal concentrations in sea urchin gonads and concentrations of heavy metals, DDT, hexachlorocyclohexane, and total petroleum hydrocarbons in sediments), allow us to conclude that LLP content in sea urchin NPs can not be used as a biomarker in marine pollution monitoring.

A comparative light- and electron microscopic study of the male gonads of the bivalve mollusk Modiolus kurilensis from the reference and polluted sites in Amursky Bay (Sea of Japan) was conducted. Testicular acini in the mussels from the reference site had well-ordered structure (vertical spermatogenic columns located among the accessory cells bodies) whereas in the testes of the mollusks from the polluted site, the accessory and spermatogenic cell populations were disarranged. Mussels from the polluted station had about 26% of spermatogenic cells with marginal localization of nuclear chromatin, swollen outer nuclear membrane and heavily vacuolated cytoplasm and about 8% of spermatozoa with transformed or destructed acrosome; in mussels from the reference station, these values were close to zero. The accessory cells in the mussels from the polluted site were underdeveloped, and their phagocytic activity was inhibited. Our ultrastructural observations provide evidence that both spermatogenic and accessory cells are targets of environmental pollution in marine mussels.

In August-September 2001, 15 samples of bottom sediments were collected in the inner, middle and open parts of Amursky Bay near Vladivostok, Russia, and barfin plaice Pleuronectes pinnifasciatus was sampled from the inner and the middle locations of the bay. In the sediments from all three sites, ele- vated concentrations of several heavy metals, i.e. Zn (102-115 μg/g dry weight), Ni (70-73 μg/g), and Cu (27-35 μg/g) were discovered. The contents of oil hydrocarbons were very close to or slightly higher than the maximal normal environmental background level, 100 μg/g dry weight. The sediments contained neg- ligible amounts of hexachlorocyclohexane, while DDT concentrations were quite high (1.7-16.3 ng/g dry weight). Generally, there were no substantial differences in the pollution levels of the locations studied, and our results resembled those reported for Amursky Bay in the 1990s. Surprisingly, in 2001 "fresh" DDT comprised 70-85% of the total DDT content in sediment from all the locations studied. In fish liver, total DDTs concentrations were 212.8 and 122.54 ng/g wet weight for the inner and the middle locations, respectively, and "fresh" DDT comprised 35 and 64% of DDTs, respectively. These results provide evi- dence of recent input of DDT from an unknown source into the ecosystem of Amursky Bay. Histopatho- logical changes revealed in the plaice liver (vacuolization of hepatocytes, coagulative necrosis of hepatocytes, inflammatory reaction, and necrosis of epithelial cells of bile ducts) are probably connected with an intensive metabolism of DDT in the fish organism. No histological and histomorphometric dif- ferences were found in the state of the interrenal tissue. Similar condition of the liver and the interrenal tis- sue in barfin plaice sampled from the inner and the middle locations of Amursky Bay may be explained by the absence of great differences in the pollution levels of these sites.

This paper describes the histology of the digestive diverticula of Mizuhopecten yessoensis (Jay) sampled from one reference and three polluted stations in Peter the Great Bay, Sea of Japan. A wide spectrum of morphological changes has been identified in scallops from polluted sites. Some changes were typical of only one of the polluted stations, such as the occurrence of new cell types, transitive cells with attributes of both digestive and basophilic cells, altered basophilic cells at Station B, the occurrence of atrophic tubules at Station C and mass shedding of basophilic cells in tubule lumina at Station D. Other changes were common in scallops from all polluted sites, such as vacuolization and shedding of digestive cells and necrosis of digestive tubules. Two different responses of the basophilic cells may represent an adaptive-compensatory reaction of the digestive diverticula to damage, i.e. loss of digestive cells and disruption of intracellular digestion. The regeneration of the digestive diverticula through the formation of new tubules was observed in scallops from all stations but at Station D there was incomplete regeneration as evidenced by the smaller numbers of digestive tubules per unit area of digestive diverticula. Histological changes in the scallop digestive diverticula seem to be related to pollution of Peter the Great Bay.

We studied here neuron ultrastructure, synaptic plasticity and subcellular localization of NADPH-diaphorase (NADPH-d), a cytochemical marker for nitric oxide syntase, in the pedal ganglia of the Gray mussel Crenomytilus grayanus sampled from the polluted and reference sites in Amursky Bay (Sea of Japan) at lower and higher water temperature (in the beginning and the end of August, respectively). At lower temperature, neuroplastic changes in mussel ganglia prevailed: a sharp increase in the number of cytosomes in NADPH-d-positive neurons and a sharp decrease in the number of mitochondria in both NADPH-d-positive and NADPH-d-negative neurons. At higher temperature, neurodegenerative changes prevailed: disruption of a part of NADPH-d-negative axons and interneuronal contacts, formation of concentric lamellar structures in the neuropils, and accumulation of autophagosomes in NADPH-d negative neurons. The results suggest that the stress-induced production of nitric oxide in cytosomes of mussel neurons and plasticity of gap junctions have a neuroprotective effect.

NADPH-diaphorase (NADPH-d) is a histochemical marker for nitric oxide synthase (NOS) and is widely used to identify nitric oxide (NO) producing cells in the central nervous system (CNS) of both vertebrates and invertebrates. NADPH-d histochemistry was used to quantitatively characterize putative NO-producing neurons in the CNS of the Gray mussel Crenomytilus grayanus subjected to two kinds of stress, environmental pollution and hypoxia, the latter caused by the mollusk transportation in a small volume of water. Mussels were sampled from one relatively clean (reference) and four polluted sites in Amursky and Ussuriysky Bays (Peter the Great Bay, Sea of Japan) in August, 2003. The number of NADPH-d-positive neurons was estimated and enzyme activity was determined from the optical density of the formazan precipitate in the CNS ganglia at 0, 3, and 72 h after sampling. Just after sampling, NADPH-d-positive neurons were found in the cerebropleural, visceral, and pedal ganglia. The number and staining intensity of NADPH-d-positive neurons were significantly higher in the pedal ganglia than the other two ganglia. There were significant differences in the number of NADPH-d-positive neurons and enzyme activity between the mussels from the reference and heavily polluted stations. The proportion and staining intensity of NADPH-d-positive neurons were maximum in the pedal ganglia of the mussels from the heavily polluted station in Amursky Bay. Transportation of mussels in a limited volume of water for 3 h resulted in a significant increase in the proportion and staining intensity of NADPH-d-positive neurons in all ganglia. In mollusks from all stations kept in aerated aquaria for 72 h, both the proportion and staining intensity of NADPH-d-positive neurons did not differ significantly from the initial level. However, the differences in the proportion and staining intensity of NADPH-d-positive neurons between the reference and heavily polluted stations were significant. The present results suggest that NO is involved in mollusk nerve cell adaptation to environmental changes.

The morphology of gonads and development of offspring of the scallop Mizuhopecten yessoensis sampled from six stations in Peter the Great Bay (Sea of Japan) were studied. The retardation of gametogenesis, oocytes resorption, autolysis of spermatozoa and their phagocytosis were observed in the gonads of the scallops from polluted sites. The number of hermaphrodites was about 6% against 0.3–0.4% in the scallop populations from clean areas. In the offspring development, a decrease in fertilisation success, diminution in percent of normal trochophores, D-veligers, veligers, and retardation of larval growth were recorded. The scallop populations inhabiting polluted areas of Peter the Great Bay seem to be incapable of normal reproduction. Development of offspring was a more sensitive index of disturbance of the reproductive function than morphology of scallop gonads. Analysis of the offspring development of common species of marine invertebrates is suggested to be used as a sensitive indicator of adverse environmental conditions.

This study reports on the effect of PAF on sperm motility, fertilization and early embryo development in the sea urchin Strongylocentrotus intermedius . PAF proved to be the amplifier of sperm motility and fertilizing capability at 10 −8 -10 −9 M and was toxic at higher concentrations. BN 52201 did not counteract the PAF action. The advantages of using sea urchins for PAF research in embryology are discussed.