Artificial sound impact could put at risk hermit crabs and their symbiont anemones.

The sea anemone Calliactis parasitica, which is found in the East Atlantic (Portugal to Senegal) and the Mediterranean Sea, forms a symbiotic relationship with the red hermit crab, Dardanus calidus, in which the anemone provides protection from predators such as the octopus while it gains mobility, and possibly food scraps, from the hermit crab. Acoustic pollution is recognised by the scientific community as a growing threat to ocean inhabitants. Recent findings on marine invertebrates showed that exposure to artificial sound had direct behavioural, physiological and ultrastructural consequences. In this study we assess the impact of artificial sound (received level 157 ± 5 dB re 1 µPa2 with peak levels up to 175 dB re 1 µPa2) on the red hermit crab and its symbiotic sea anemone. Scanning electron microscopy analyses revealed lesions in the statocyst of the red hermit crab and in the tentacle sensory epithelia of its anemone when exposed to low-intensity, low-frequency sounds. These ultrastructural changes under situations of acoustic stress in symbiotic partners belonging to different phyla is a new issue that may limit their survival capacity, and a new challenge in assessing the effects of acoustic disturbance in the oceanic ecosystem. Despite the lesions found in the red hermit crab, its righting reflex time was not as strongly affected showing only an increase in the range of righting times. Given that low-frequency sound levels in the ocean are increasing and that reliable bioacoustic data on invertebrates is very scarce, in light of the results of the present study, we argue that anthropogenic sound effects on invertebrates species may have direct consequences in the entire ecosystem.

Cross-sensory interference assessment after exposure to noise shows different effects in the blue crab olfactory and sound sensing capabilities.

Underwater noise pollution is an increasing threat to marine ecosystems. Marine animals use sound in communication and orientation processes. The introduction of anthropogenic noise in their habitat can interfere with sound production and reception as well as with the acquisition of vital information through other sensory systems. In the blue crab (Callinectes sapidus), the statocyst is responsible for acoustic perception, and it is housed at the base of its first pair of antennae (antennule). The sensilla of the distal part of these antennule hosts the olfactory system, which is key for foraging. Given the anatomical proximity of the two sensory regions, we evaluated the possible interference of sound exposure with the crab ability to find food, by using an aquatic maze, and looked at the potential impairment of the righting reflex as well as at ultrastructural damages in statocysts. Although a significant effect was observed when looking at the time used by the animal to recover its habitual position ("righting reflex"), which was associated to lesions in the statocyst sensory epithelia, the time required to find food did not increase after the exposure to sound. When the crabs were exposed to natural sounds (marine background noise and sounds of their predators: Micropogonias undulates and Sciaenops ocellatus) they did not show significant differences in foraging behaviour. Although we found no unequivocal evidence of a negative impact of sound on olfactory capabilities, the study showed a clear righting reflex impairment correlated with ultrastructural damages of the statocysts. We argue that crab populations that cannot easily avoid noise sources due to their specific coastal distributions may incur in significant direct fitness costs (e.g. impairment of complex reflexes). This integrated approach to sound effect assessment could be used as a model for other invertebrate species to effectively monitor noise impact in marine environments.

Commercial cuttlefish exposed to noise from offshore windmill construction show short-range acoustic trauma.

The installation of marine renewable energy devices (MREDs, wind turbines and converters of wave, tidal and ocean thermal energy) has increased quickly in the last decade. There is a lack of knowledge concerning the effects of MREDs on benthic invertebrates that live in contact with the seabed. The European common cuttlefish (Sepia officinalis) is the most abundant cephalopod in the Northeast Atlantic and one of the three most valuable resources for English Channel fisheries. A project to build an offshore wind farm in the French bay of Saint-Brieuc, near the English Channel, raised concern about the possible acoustic impact on local cuttlefish communities. In this study, consisting of six exposure experiments, three types of noise were considered: 3 levels of pile-driving and 3 levels of drilling. The objectives were to assess possible associated changes in hatching and larva survival, and behavioural and ultrastructural effects on sensory organs of all life stages of S. officinalis populations. After exposure, damage was observed in the statocyst sensory epithelia (hair cell extrusion) in adults compared to controls, and no anti-predator reaction was observed. The exposed larvae showed a decreased survival rate with an increasing received sound level when they were exposed to maximum pile-driving and drilling sound levels (170 dB re 1 µPa2 and 167 dB re 1 µPa2, respectively). However, sound pressure levels's lower than 163 dB re 1 µPa2 were not found to elicit severe damage. Simulating a scenario of immobile organisms, eggs were exposed to a combination of both pile driving and drilling as they would be exposed to all operations without a chance to escape. In this scenario a decrease of hatching success was observed with increasing received sound levels.

Seagrass Posidonia is impaired by human-generated noise.

The last hundred years have seen the introduction of many sources of artificial noise in the sea environment which have shown to negatively affect marine organisms. Little attention has been devoted to how much this noise could affect sessile organisms. Here, we report morphological and ultrastructural changes in seagrass, after exposure to sounds in a controlled environment. These results are new to aquatic plants pathology. Low-frequency sounds produced alterations in Posidonia oceanica root and rhizome statocysts, which sense gravity and process sound vibration. Nutritional processes of the plant were affected as well: we observed a decrease in the number of rhizome starch grains, which have a vital role in energy storage, as well as a degradation in the specific fungal symbionts of P. oceanica roots. This sensitivity to artificial sounds revealed how sound can potentially affect the health status of P. oceanica. Moreover, these findings address the question of how much the increase of ocean noise pollution may contribute in the future to the depletion of seagrass populations and to biodiversity loss.

A critical period of susceptibility to sound in the sensory cells of cephalopod hatchlings.

The cephalopod statocyst and lateral line systems are sensory organs involved in orientation and balance. Lateral lines allow cephalopods to detect particle motion and are used for locating prey or predators in low light conditions. Here, we show the first analysis of damaged sensory epithelia in three species of cephalopod hatchlings (Sepia officinalis, Loligo vulgaris and Illex coindetii) after sound exposure. Our results indicate lesions in the statocyst sensory epithelia, similar to what was found in adult specimens. The novelty is that the severity of the lesions advanced more rapidly in hatchlings than in adult animals; i.e. the degree of lesions seen in hatchlings immediately after noise exposure would develop within 48 h in adults. This feature suggests a critical period of increased sensitivity to acoustic trauma in those species as has been described in developing mammalian cochlea and avian basilar papilla. The hair cells in the lateral lines of S. officinalis followed the same pattern of damage occurrence, while those of L. vulgaris and I. coindetii displayed a decreasing severity of damage after 24 h. These differences could be due to dissimilarities in size and life stages between the three species.

Evidence of Cnidarians sensitivity to sound after exposure to low frequency underwater sources.

Jellyfishes represent a group of species that play an important role in oceans, particularly as a food source for different taxa and as a predator of fish larvae and planktonic prey. The massive introduction of artificial sound sources in the oceans has become a concern to science and society. While we are only beginning to understand that non-hearing specialists like cephalopods can be affected by anthropogenic noises and regulation is underway to measure European water noise levels, we still don't know yet if the impact of sound may be extended to other lower level taxa of the food web. Here we exposed two species of Mediterranean Scyphozoan medusa, Cotylorhiza tuberculata and Rhizostoma pulmo to a sweep of low frequency sounds. Scanning electron microscopy (SEM) revealed injuries in the statocyst sensory epithelium of both species after exposure to sound, that are consistent with the manifestation of a massive acoustic trauma observed in other species. The presence of acoustic trauma in marine species that are not hearing specialists, like medusa, shows the magnitude of the problem of noise pollution and the complexity of the task to determine threshold values that would help building up regulation to prevent permanent damage of the ecosystems.

Taxonomic and Environmental Variability in the Elemental Composition and Stoichiometry of Individual Dinoflagellate and Diatom Cells from the NW Mediterranean Sea.

Here we present, for the first time, the elemental concentration, including C, N and O, of single phytoplankton cells collected from the sea. Plankton elemental concentration and stoichiometry are key variables in phytoplankton ecophysiology and ocean biogeochemistry, and are used to link cells and ecosystems. However, most field studies rely on bulk techniques that overestimate carbon and nitrogen because the samples include organic matter other than plankton organisms. Here we used X-ray microanalysis (XRMA), a technique that, unlike bulk analyses, gives simultaneous quotas of C, N, O, Mg, Si, P, and S, in single-cell organisms that can be collected directly from the sea. We analysed the elemental composition of dinoflagellates and diatoms (largely Chaetoceros spp.) collected from different sites of the Catalan coast (NW Mediterranean Sea). As expected, a lower C content is found in our cells compared to historical values of cultured cells. Our results indicate that, except for Si and O in diatoms, the mass of all elements is not a constant fraction of cell volume but rather decreases with increasing cell volume. Also, diatoms are significantly less dense in all the measured elements, except Si, compared to dinoflagellates. The N:P ratio of both groups is higher than the Redfield ratio, as it is the N:P nutrient ratio in deep NW Mediterranean Sea waters (N:P = 20-23). The results suggest that the P requirement is highest for bacterioplankton, followed by dinoflagellates, and lowest for diatoms, giving them a clear ecological advantage in P-limited environments like the Mediterranean Sea. Finally, the P concentration of cells of the same genera but growing under different nutrient conditions was the same, suggesting that the P quota of these cells is at a critical level. Our results indicate that XRMA is an accurate technique to determine single cell elemental quotas and derived conversion factors used to understand and model ocean biogeochemical cycles.

Ultrastructure of the Odontocete organ of Corti: scanning and transmission electron microscopy.

The morphological study of the Odontocete organ of Corti, together with possible alterations associated with damage from sound exposure, represents a key conservation approach to assess the effects of acoustic pollution on marine ecosystems. By collaborating with stranding networks from several European countries, 150 ears from 13 species of Odontocetes were collected and analyzed by scanning (SEM) and transmission (TEM) electron microscopy. Based on our analyses, we first describe and compare Odontocete cochlear structures and then propose a diagnostic method to identify inner ear alterations in stranded individuals. The two species analyzed by TEM (Phocoena phocoena and Stenella coeruleoalba) showed morphological characteristics in the lower basal turn of high-frequency hearing species. Among other striking features, outer hair cell bodies were extremely small and were strongly attached to Deiters cells. Such morphological characteristics, shared with horseshoe bats, suggest that there has been convergent evolution of sound reception mechanisms among echolocating species. Despite possible autolytic artifacts due to technical and experimental constraints, the SEM analysis allowed us to detect the presence of scarring processes resulting from the disappearance of outer hair cells from the epithelium. In addition, in contrast to the rapid decomposition process of the sensory epithelium after death (especially of the inner hair cells), the tectorial membrane appeared to be more resistant to postmortem autolysis effects. Analysis of the stereocilia imprint pattern at the undersurface of the tectorial membrane may provide a way to detect possible ultrastructural alterations of the hair cell stereocilia by mirroring them on the tectorial membrane.

HOMOTHALLIC AUXOSPORULATION IN PSEUDO-NITZSCHIA BRASILIANA (BACILLARIOPHYTA)(1).

Most pennate diatoms are allogamous, and various types of mating systems have been described. In Pseudo-nitzschia, reproductive stages have been identified in some species, and it is generally accepted that the genus is mainly heterothallic. Here we report homothallic auxosporulation of Pseudo-nitzschia brasiliana Lundholm, Hasle et G. A. Fryxell. To our knowledge, this is the first verified description of homothallic sexual reproduction in the genus. Auxospore formation was observed in all 16 subclones derived from three initial clonal cultures of P. brasiliana. Pairing was followed by production of two gametes per gametangium, which fused to give two zygotes. Each zygote (early auxospore) was initially spherical and adhered to one girdle band of the parental frustule. The two auxospores tended to expand parallel to each other and perpendicular to the parental frustule. Elongation was synchronous, slightly asynchronous, or totally asynchronous. The entire process of sexual reproduction, from gamete formation to the appearance of the initial vegetative cells, took 2-4 d. The occurrence of sex in a homothallic species seems an advantageous life strategy for this species in that any encounter between cells of the right size class is potentially sexual.

New application of air-drying techniques for studying Ephemeroptera and Plecoptera eggs by scanning electron microscopy.

Hexamethyldisilizane (HMDS) and tetramethylsilane are organic compounds that are volatile at ambient temperature and which can therefore be used for air-drying biological samples for SEM studies. The techniques using these compounds provide results that are comparable with those obtained by critical point drying, but which involve a very simple process that saves time and money. Both techniques were applied to SEM studies of Ephemeroptera and Plecoptera eggs in order to assess their suitability as alternative methods to critical point drying for these kinds of biological material. The results show no morphological differences between eggs HMDS air-dried and critical point-dried.