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 3h 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.
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.
BackgroundIn 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.MethodsWe 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.ResultsWe 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.DiscussionThe 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.
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