Comparative assessment of microplastics and microalgae as vectors of mercury and chlorpyrifos in the copepod Acartia tonsa.

Microplastics (MPs) raise concerns not only as pollutants themselves, but also due to their ability to act as vectors of pollutants adsorbed from seawater, transferring them to marine organisms. However, the relevance of MPs as carriers of pollutants compared to microalgae needs further exploration. This study compared the role of MPs (2-10 µm non-oxidized and 10-15 µm oxidized high-density polyethylene) and natural organic particles (Rhodomonas lens microalgae, MA) as carriers of mercury (Hg, 2.3 µg Hg/L) and chlorpyrifos (CPF, 1.0 µg CPF/L) to adult Acartia tonsa copepods, after 24-48 h exposure. Dose-response experiments were first performed with adult female copepods exposed to oxidized MPs (0.25-4.0 mg/L), waterborne Hg (0.01-10.0 µg/L) and Ox MPs + Hg (0.25-4.0 mg oxidized MPs/L + 0.50-8.0 µg Hg/L) for 48 h, to complement previous studies that focused on the pesticide CPF. Effects were evaluated with four replicates for physiological and reproductive responses (6 females/replicate), biochemical techniques (40 individuals/replicate) and Hg/CPF bioaccumulation measurements (1000 individuals/replicate). Copepods accumulated Hg/CPF similarly from dissolved pollutants (6204 ± 2265 ng Hg/g and 1251 ± 646 ng CPF/g) and loaded MPs (3125 ± 1389 ng Hg/g and 1156 ± 266 ng CPF/g), but significantly less from loaded MA (21 ± 8 ng Hg/g and 173 ± 80 ng CPF/g). After 24-48 h, copepods exposed to MPs + Hg/CPF showed generally greater biological effects than those exposed to dissolved Hg/CPF or to MA + Hg/CPF, although differences were not statistically significant. MA + CPF had significantly lower AChE inhibition (1073.4 nmol min-1 mg-1) and MA + Hg lower GRx induction (48.8 nmol min-1 mg-1) compared to MPs + Hg/CPF and dissolved Hg/CPF (182.8-236.4 nmol min-1 mg-1 of AChE and 74.1-101.7 nmol min-1 mg-1 of GRx). Principal component analysis suggested different modes of action for Hg and CPF.

Rational synthesis of total damage during cryoprotectant equilibration: modelling and experimental validation of osmomechanical, temperature, and cytotoxic damage in sea urchin (Paracentrotus lividus) oocytes.

Sea urchins (e.g., Paracentrotus lividus) are important for both aquaculture and as model species. Despite their importance, biobanking of urchin oocytes by cryopreservation is currently not possible. Optimized cryoprotectant loading may enable novel vitrification methods and thus successful cryopreservation of oocytes. One method for determining an optimized loading protocol uses membrane characteristics and models of damage, namely osmomechanical damage, temperature damage (e.g., chill injury) and cytotoxicity. Here we present and experimentally evaluate existing and novel models of these damage modalities as a function of time and temperature. In osmomechanical damage experiments, oocytes were exposed for 2 to 30 minutes in hypertonic NaCl or sucrose supplemented seawater or in hypotonic diluted seawater. In temperature damage experiments, oocytes were exposed to 1.7 °C, 10 °C, or 20 °C for 2 to 90 minutes. Cytotoxicity was investigated by exposing oocytes to solutions of Me2SO for 2 to 30 minutes. We identified a time-dependent osmotic damage model, a temperature-dependent damage model, and a temperature and time-dependent cytotoxicity model. We combined these models to estimate total damage during a cryoprotectant loading protocol and determined the optimal loading protocol for any given goal intracellular cryoprotectant concentration. Given our fitted models, we find sea urchin oocytes can only be loaded to 13% Me2SO v/v with about 50% survival. This synthesis of multiple damage modalities is the first of its kind and enables a novel approach to modelling cryoprotectant equilibration survival for cells in general.

The Importance of Developing and Standardizing Gamete, Embryo and Larvae Handling in Aquatic Animals.

Artificial reproduction in aquatic animals usually involves the collection and handling of gametes both from males and females in a way that secures their quality and optimizes the fertilization event [...].

Influence of microplastics on the toxicity of chlorpyrifos and mercury on the marine microalgae Rhodomonas lens.

The growing use of plastics, including microplastics (MPs), has enhanced their potential release into aquatic environments, where microalgae represent the basis of food webs. Due to their physicochemical properties, MPs may act as carriers of organic and inorganic pollutants. The present study aimed to determine the toxicity of polyethylene MPs (plain and oxidized) and the model pollutants chlorpyrifos (CPF) and mercury (Hg) on the red microalgae Rhodomonas lens, to contribute to the understanding of the effects of MPs and associated pollutants on marine ecosystems, including the role of MPs as vectors of potentially harmful pollutants to marine food webs. R. lens cultures were exposed to MPs (1-1000 µg/L; 25-24,750 particles/mL), CPF (1-4900 µg/L), Hg (1-500 µg/L), and to CPF- and Hg-loaded MPs, for 96 h. Average specific growth rate (ASGR, day-1), cellular viability and pigment concentration (chlorophyll a, c2 and carotenoids) were measured at 48 and 96 h. No significant effects were observed on the growth pattern of the microalgae after 96-h exposure to plain and oxidized MPs. However, a significant increase in cell concentration was detected after 48-h exposure to plain MPs. A decrease of the ASGR was noticed after exposure to CPF, Hg and to CPF/Hg-loaded MPs, whereas viability was affected by exposure to MPs, CPF and Hg, alone and in combination. Chlorophyll a and c2 significantly decreased when microalgae were exposed to plain MPs and CPF, while both pigments significantly increased when exposed to CPF-loaded MPs. Similarly, chlorophyll and carotenoids content significantly decreased after exposure to Hg, whereas a significant increase in chlorophyll a was observed after 48-h exposure to Hg-loaded MPs, at the higher tested concentration. Overall, the presence of MPs modulates the toxicity of Hg and CPF to these microalgae, decreasing the toxic effects on R. lens, probably due to a lower bioavailability of the contaminants.

Handling, Reproducing and Cryopreserving Five European Sea Urchins (Echinodermata, Klein, 1778) for Biodiversity Conservation Purposes.

In this work, five local sea urchin species found in European waters were studied. Four were regular species: Sphaerechinus granularis, Psammechinus miliaris, Echinus esculentus (Linnaeus, 1758) and the edible sea urchin Paracentrotus lividus; and one was an irregular species, Echinocardium cordatum. These five species of sea urchins have been studied regarding their fertility, toxicity of cryoprotecting agents, cryopreservation of different cell types and chilling injury. The baseline fertility is similar in P. lividus, P. miliaris and S. granularis. Nonetheless, the sperm:egg ratio, contact time and development of the fertilization envelope would need to be studied further on a case-by-case basis. Sperm can be maintained inactively in the gonad (4 °C), and oocytes also maintain quality in sea water (4 °C), even after 72 h. Sperm was cryopreserved for four species with some post-thaw intra specific variability, and embryo cryopreservation was only possible for S. granularis. Overall, this study provided a wider vision of the biology and reproduction of these species that will help us develop tools for their biodiversity conservation through cryopreservation.

Cryopreservation of Marine Invertebrates: From Sperm to Complex Larval Stages.

Marine invertebrates represent the vast majority of marine biodiversity; they are extremely diverse playing a key role in marine ecosystems, thus playing an important role at the socioeconomic level. Some invertebrates such as sea urchins, ascidians, and horse-shoe crabs are very well-known model organisms for research and biocompound discovery. In this chapter we revisit the importance of cryopreservation for the conservation and rational use in research, fisheries management, or aquaculture and provide comprehensive protocols for the cryopreservation of sperm, embryos, and larvae.

Cryopreservation of Algae.

Cryopreservation has been successfully used in the banking and maintenance of cultures of microorganisms, from bacteria to yeasts, since the onset of cryobiology. Biobanking of marine biological resources is crucial for development of scientific knowledge as researchers rely on guaranteed access to reliable, stable resources. Culture collections play a key role in the provision of marine biological resources as they ensure long-term ex situ storage of biological resources that are made available for public and private sector research and education. In this chapter, we provide protocols for cryopreservation of different types of algae cultures.

Development of a method to cryopreserve Greenshell mussel™ (Perna canaliculus) veliger larvae.

Cryopreservation of larvae of Greenshell™ mussel Perna canaliculus, the most cultivated species in New Zealand, can provide flexibility for selective breeding programmes and enhance its global production. In this study, we set out to develop a reliable protocol for freezing D-stage larvae of Greenshell™ mussels that ensured long-term survival for successful rearing of thawed larvae in the hatchery. The effects of different combinations of cryoprotecting agents (CPA), varying CPA equilibration times, larval concentrations per straw as well as different larval development stages (48 h vs 72 h old) were evaluated by assessing the behavioural response (swimming activity, algal consumption), shell size and survival of larvae, up to 4 days post-thawing. The protocol yielding the best larval performances was a combination of the following CPA (final concentrations): 14% ethylene-glycol (EG) + 0.6 M trehalose (TRE) + 1% polyvinyl-pyrrolidone (PVP), prepared with Milli-Q water. Stocking densities ranging from 50,000 to 150,000 larvae per straw (0.25 mL) and a 20 min equilibration time gave the best results, while no significant differences in fitness were found between larvae cryopreserved at 48 h nor 72 h-old. Using the improved cryopreservation protocol, over 50% of previously cryopreserved D-larvae were able to survive after 4 days of rearing, compared with 65% in the unfrozen control. More importantly, about one third of thawed larvae were able to swim and feed, and to potentially develop further. These findings contribute to enhance the selective breeding programmes for this species.

First steps towards Echinometra lucunter embryo cryopreservation.

We have studied the sensitivity to cryoprotecting agents of different embryos of the local sea urchin, Echinometra lucunter which is the species used for embryo-larval bioassays in Brazil. We have located significant differences between both species sensitivity to cryoprotecting agents; while for P. lividus propylene glycol was the less toxic compound for most development stages, whereas for E. lucunter is was the most toxic and the least toxic was Dimethyl sulfoxide. There is a significant difference between development stages as well; in the case of P. lividus, the blastula embryo was the most resistant to the cryoprotecting agents, meanwhile for E. lucunter, it was the fertilized oocyte. This is a very promising result, a really early embryo that is not extremely sensitive to Me2SO. Our next aim is to develop a cryopreservation protocol for E. lucunter early embryos and test them in an embryo-larval bioassay.

A preliminary study of osmotic dehydration in zebrafish embryos: Implications for vitrification and ultra-fast laser warming.

To date, traditional cryopreservation techniques have not been amenable to zebrafish (Danio rerio) embryos, due in part to their large yolky eggs, which have a low surface to volume ratio and limited permeability to water and cryoprotectants. However, recent vitrification and ultra-rapid warming studies in mice have demonstrated successful preservation by dehydrating 85% or more of their total water content. We hypothesized that this approach may help overcome the barriers to embryo cryopreservation among D. rerio. The purpose of this study was to determine the osmotic tolerance limit of D. rerio embryos under conditions relevant to cryopreservation. We found that embryos undergoing gastrulation (30%-70% epiboly) were particularly sensitive to osmotic dehydration/rehydration. By contrast, a subset of embryos dehydrated during or after segmentation (20-22 somite, prim 5) survived 3 h in a 2 M sucrose solution but exhibit developmental delay, edema and trunk necrosis 2-4 days post-treatment.

Intracellular ice formation in mouse zygotes and early morulae vs. cooling rate and temperature-experimental vs. theory.

In this study, mature female mice of the ICR strain were induced to superovultate, mated, and collected at either zygote or early morula stages. Embryos suspended in 1 M ethylene glycol in PBS containing 10 mg/L Snomax for 15 min, then transferred in sample holder to Linkam cryostage, cooled to and seeded at 7 °C, and then observed and photographed while being cooled to -70 °C at 0.5-20 °C/min. Intracellular ice formation (IIF) was observed as abrupt ''flashing''. Two types of flashing or IIF were observed in this study. Extracellular freezing occurred at a mean of -7.7 °C. In morulae, about 25% turned dark within ±1 °C of extracellular ice formation (EIF). These we refer to as "high temperature'' flashers. In zygotes, there were no high temperature flashers. All the zygotes flashed at temperatures well below the temperature for EIF. Presumably high temperature flashers were a consequence of membrane damage prior to EIF or damage from EIF. We shall not discuss them further. In the majority of cases, IIF occurred well below -7.7 °C; these we call ''low temperature'' flashers. None flashed with cooling rate (CR) of 0.5 °C/min in either zygotes or morulae. Nearly all flashed with CR of 4 °C/min or higher, but the distribution of temperatures is much broader with morulae than with zygotes. Also, the mean flashing temperature is much higher with morulae (-20.9 °C) than with zygotes (-40.3 °C). We computed the kinetics of water loss with respect to CR and temperature in both mouse zygotes and in morulae based on published estimates of Lp and it is Ea. The resulting dehydration curves combined with knowledge of the embryo nucleation temperature permits an estimate of the likelihood of IIF as a function of CR and subzero temperature. The agreement between these computed probabilities and the observed values are good.

Roles of intracellular ice formation, vitrification of cell water, and recrystallisation of intracellular ice on the survival of mouse embryos and oocytes.

Mazur and collaborators began examining the validity of initial views regarding mouse oocyte and embryo vitrification and found that most are partially or fully wrong. First, the relative effects of warming and cooling rates on the survival of mouse oocytes subjected to a vitrification procedure were determined. The high sensitivity to warming rate strongly suggests that the lethality of slow warming is a consequence of either the crystallisation of intracellular glassy water during warming or the recrystallisation during slow warming of small intracellular crystals that had formed during cooling. Warming rates of 107°C min-1 were achieved in 0.1-µL drops of ethylene glycol-acetamide-Ficoll-sucrose (EAFS) solution plus a small amount of India ink on Cryotops warmed using an infrared laser pulse. Under these conditions, survival rates of 90% were obtained even when mouse oocytes were suspended in 0.3× EAFS, a concentration that falls in the range that many cells can tolerate. A second important finding was that the survival of oocytes is more dependent on the osmotic withdrawal of much of the intracellular water before vitrification than it is on the penetration of cryoprotective solutes into the cells. Herein we review the roles of internal ice formation, vitrification and recrystallisation. It remains to be seen how widely these findings will be applicable to other types of cells and tissues from other species.

The survival of mouse oocytes shows little or no correlation with the vitrification or freezing of the external medium, but the ability of the medium to vitrify is affected by its solute concentration and by the cooling rate.

As survival of mouse oocytes subjected to vitrification depends far more on the warming rate than on the cooling rate, we wished to determine whether the lack of correlation between survival and cooling rate was mirrored by a lack of correlation between cooling rate and vitrification of the medium (EAFS), and between survival and the vitrification of the medium. The morphological and functional survival of the oocytes showed little or no relation to whether or not the EAFS medium vitrified or froze. We studied if the droplet size and the elapsed time (between placing the droplet on the Cryotop and the start of cooling) affects the result through modification of the cooling rate and solute concentration. Dehydration was rapid; consequently, the time between the placing the droplets into a Cryotop and cooling must be held to a minimum. The size of the EAFS droplet that is being cooled does not seem to affect vitrification. Finally, the degree to which samples of EAFS vitrify is firmly dependent on both its solute concentration and the cooling rate.

Advances in the cryopreservation of sea-urchin embryos: Potential application in marine water quality assessment.

Among the most widely used biological techniques in marine pollution assessment, the sea-urchin embryo-larval bioassay is in an advanced developmental stage. Cryopreservation might help to overcome the problem of the spawning seasonality and therefore strengthen the use of those embryo-larval bioassays. This work investigates different factors influencing cryopreservation of sea-urchin embryos, including the cooling rates and holding temperatures, the seeding, or the impact of plunging into liquid nitrogen. The blastula stage yielded better results than the fertilised egg, and the most effective cryoprotectants combination was dimethyl sulfoxide 1.5M plus trehalose 0.04M. The optimised protocol developed begins with an initial hold at 4°C for 2min, followed by cooling at -1°Cmin(-1) to -12°C. At this point a seeding step was incorporated with a 2min hold, followed by a second cooling at -1°Cmin(-1) to -80°C. After a final hold of 2min the cryovials are transferred into liquid nitrogen for storage. Although the cryopreservation processes might cause a delay in the development of sea-urchin embryos, high larval growth (71-98% of controls) was obtained when cryopreserved blastulae were further incubated for 72-96h in artificial seawater. We conclude that embryo-larval bioassays with cryopreserved sea-urchin blastulae are suitable for use in marine pollution monitoring programs and may be considered as an acceptable solution to the reproductive seasonality of sea-urchin species.

De estar "toda Teletubbie" a ser Verónica: utilidad de las sesiones de observación (sesiones diagnósticas) para trazar el proceso evolutivo de un sufrimiento mental / From Being "Completely Teletubbie" to Being Veronica: Usefulness of Observation Sessions (Diagnostic Sessions) in Tracing the Evolutionary Process of a Mental Suffering

Introducción: En este estudio se usa un modelo evolutivo de los trastornos y del desarrollo del psiquismo que permite tener una visión global del paciente, en el cual concurre no sólo lo que podría aportar la genética, sino todas las distorsiones producto de las experiencias con el ambiente. Las teorías sobre el desarrollo psíquico han mostrado que el nacimiento, la construcción y la organización del psiquismo de un individuo deben comprenderse como una progresión evolutiva. Objetivo: Mostrar a través de una sesión de observación cómo se comprende la organización psíquica de una paciente y el origen y posterior evolución de sus trastornos. Método: Observación de un caso particular de interacción de una paciente con su madre, su abuela y un equipo de trabajo y, luego, este último reflexionó sobre diversas hipótesis, que surgieron de la comprensión de las relaciones entre la psicopatología de la paciente y las teorías del desarrollo psíquico. Resultados: En el caso ilustrado se descartó el diagnóstico de autismo con el cual venía remitida la paciente, pues los síntomas de esta paciente podrían verse desde la perspectiva de las primeras relaciones interpersonales. Las teorías actuales sobre el desarrollo psíquico permiten comprender que no se trata de algo "que le dio", sino que se fue gestando desde muy temprano en la vida emocional de este ser. Conclusión: En esta sesión de observación se ve cómo estas citas, que inicialmente fueron establecidas para diagnosticar, conducen a una profunda comprensión del paciente, que al ser compartida con sus padres, tiene también un efecto terapéutico y se constituyen así en "consultas terapéuticas", como las propuestas por D. W. Winnicott.

Introduction: In this study an evolutionary model of the disorders and the development of psychism is used, that allows achiving an overall vision of the patient and which combines not only what could contribute to genetics, but all the distortions resulting from experiences with the environment. The theories about psychic development have shown that the birth, building and organization of the psychism of an individual must be understood as an evolutionary progression. Objective: To show, by means of an observation session, how the psychic organization of a female patient and the subsequent evolution of her disorders may be understood. Method: Observing an individual case of interaction between a female patient and her mother, grandmother and a work team. Subsequently this team discussed different hypothesis that emerged from the understanding of the relationships between the patient’s psicopathology and the psychic development theories. Results: In this case the diagnosis of autism, with which the patient was remitted, was ruled out because the symptoms of this patient could be seen from the perspective of first interpersonal relationships. Current theories on psychic development allow understanding that it is not "just something that she got", but that it started developing very early in the emotional life of this being. Conclusion: In this observation session it is apparent how these visits, which initially were established to reach a diagnosis, lead to a deep understanding of the patient that, when shared with her parents, have a therapeutic effect and become "therapeutic consultations" as those proposed by D.W. Winnicott.

Cryopreservation of sea urchin embryos (Paracentrotus lividus) applied to marine ecotoxicological studies.

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me(2)SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to -12 degrees C at 1 degrees C/min, holding for 2 min for seeding, cooling to -20 degrees C at 0.5 degrees C/min, and then cooling to -35 degrees C at 1 degrees C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.