Neutral Red Staining Research Articles

Characterizing the Status of Energetic Metabolism of Dinoflagellate Resting Cysts under Mock Conditions of Marine Sediments via Physiological and Transcriptional Measurements

Similar to the seeds of higher plants, resting cysts, a non-motile, benthic, and dormant stage in the life history of many dinoflagellate species, play vital roles via germination in the seasonal dynamics and particularly the initiation of harmful algal blooms (HABs) of dinoflagellates. It is thus crucial for resting cysts to balance between the energetic catabolism for viability maintenance and the energy preservation for germination during their dormancy. Despite this importance, studies on how resting cysts of dinoflagellates accomplish energetic metabolism in marine sediment have been virtually absent. In this study, using the cosmopolitan HABs-causing species Scrippsiella acuminata as a representative, we measured the transcriptional activity of the most efficient pathway of the energy catabolism tricarboxylic acid (TCA) cycle, cell viability (via neutral red staining), and the cellular ATP content of resting cysts under a set of mock conditions in marine sediments (e.g., 4 °C, darkness, and anoxia) for a maximum period of one year. Based on the correlation analyses among the expression levels of genes, cyst viability, and ATP content, we revealed that the TCA cycle was still a crucial pathway of energetic catabolism for resting cysts under aerobic conditions, and its expression was elevated at higher temperatures, light irradiation, and the early stage of dormancy. Under anaerobic conditions, however, the TCA cycle pathway ceased expression in resting cysts, as also supported by ATP measurements. Our results have laid a cornerstone for the comprehensive revelation of the energetic metabolism and biochemical processes of dormancy of resting cysts in marine sediments.

Viral Titer Quantification of West Nile Virus by Immunostaining Plaque Assay.

Immunostained plaque assay based on the specific antibody binding to viral antigen enables the detection and titration of virus infectivity, especially for viruses that could not form plaques using the classical crystal violet or neutral red staining methods. Here we describe the application of this method to quantify viral titers of wild-type West Nile virus (WNV-WT) and replication-defective WNV-ΔNS1 virus.

Bivalve Oocyte Inviability Revealed Using a Simple Staining Technique

Photo 1. Cerastoderma edule (Linnaeus, 1758). (A) Emerged animals at low tide, (B) submerged animals with foot (F) and inhalant (IS) and exhalent (ES) siphons deployed. Animals in (A) and (B) are in their 3rd year (two annual growth rings are visible, corresponding to periostracal thickening) and thus both sexually mature. (C) Oocytes (OC) double-stained with Neutral Red vital stain and Trypan Blue mortal stain. Left, normal oocyte, right, dead oocyte. TB, undissolved Trypan Blue stain. (D) Oocytes stained with Neutral Red only; normal oocytes (NO) stain red, while dead oocytes do not stain. The TB stain proved effective, but cumbersome and costly to use in routine studies, while the more convenient and cost-efficient Neutral Red stain alone provided the same information. Photo credit: D. Chérel. These photographs illustrate the article “Evolutionary and ecological insights from vital staining of bivalve oocytes: A Red Queen at the sweepstakes?” by Peter G. Beninger, Daphné Chérel, Gaël Le Pennec, and Sandra E. Shumway published in Ecosphere. https://doi.org/10.1002/ecs2.4047

Soybean Meal-Dependent Acute Intestinal Inflammation Delays Osteogenesis in Zebrafish Larvae.

Foods are known to be modulators of inflammation and skeletal development. The intestine plays an essential role in the regulation of bone health mainly through the regulation of the absorption of vitamin D and calcium; in fact, inflammatory bowel diseases are often related to bone health issues such as low bone mineral density, high fracture risk, osteoporosis and osteopenia. Considering the complexity of the pathways involved, the use of a simple animal model can be highly useful to better elucidate the pathogenic mechanisms. Soybean flour with a high saponin content has been used in many studies to induce intestinal inflammation in zebrafish larvae. Using a 50% soybean meal (SBM), we analyzed the effects of this soy-induced inflammatory bowel disease on zebrafish larval osteogenesis. Soybean meal induces intestinal functional alterations and an inflammatory state, highlighted by neutral red staining, without altering the general development of the larvae. Our data show that the chondrogenesis as well as endochondral ossification of the head of zebrafish larvae are not affected by an SBM-diet, whereas intramembranous ossification was delayed both in the head, where the length of the ethmoid plate reduced by 17%, and in the trunk with a delayed vertebral mineralization of 47% of SBM larvae. These data highlight that diet-dependent bowel inflammation can differently modulate the different mechanisms of bone development in different zones of the skeleton of zebrafish larvae.

Fertility preservation: improved neovascularization and follicle viability in cryopreserved bovine ovarian cortex transplants by remaining medulla tissue

To investigate the advantages of cryopreserved medulla-containing ovarian cortex grafts with those of commonly used sole cortex grafts for fertility preservation by analyzing tissue quality, neovascularization processes, and the number of vital follicles. Experimental setting of cryopreserved bovine ovarian cortex tissue grafts with or without medulla tissue. Laboratory animal research at Ulm University, Ulm, Germany. Bovine ovaries and fertilized chicken eggs. Experimental setting of bovine ovarian tissue samples grafted on the chicken chorioallantoic membrane (CAM) after cryopreservation and thawing to examine histologic tissue integrity, apoptosis and proliferation immunohistochemically, blood vessel counts and determine the presence of neutral red-stained vital follicles. We used hematoxylin and eosin staining to visualize tissue structures, immunohistochemistry with anti-caspase 3 to detect apoptosis, anti-Ki67 to examine proliferation, blood vessel count on the chicken CAM to investigate neovascularization processes, and neutral red staining to evaluate vital follicles. We demonstrated that in all analyzed tissue samples, after cryopreservation, thawing, and grafting on the chicken CAM, there was excellent tissue integrity and quality, as shown by extremely rare apoptosis processes analyzed using immunohistochemical caspase 3 staining (sole cortex, 0.54%; thin medulla-containing cortex, 0.43%; thick medulla-containing cortex, 0.13%; and sole medulla, 2.82%). Moreover, we detected increased neovascularization in the vicinity of medulla and medulla-containing grafts (small blood vessels: cortex 8.7, thin medulla-containing cortex 9.9, thick medulla-containing cortex 9.7, and medulla 9.8; very small blood vessels: cortex 7.0, thin medulla-containing cortex 13.0, thick medulla-containing cortex 12.0, and medulla 15.0), with higher Ki67-detected proliferation (cortex, 17.58%; thin medulla-containing cortex, 20.28%; thick medulla-containing cortex, 20.56%; and medulla, 29.9%). Additionally, we identified an increased number of vital follicles in medulla-containing cortex grafts compared with the number of vital follicles in sole cortex tissue (cortex, 256.1; thin medulla-containing cortex, 338.2; thick medulla-containing cortex, 346.6; and medulla, 8.1). In this experimental setting, bovine medulla-containing cortex tissue had excellent tissue structure and quality after cryopreservation and thawing and increased neovascularization and an augmented vital follicle count after grafting than the commonly used sole cortex tissue. Therefore, we suggest reconsidering the current cryopreservation and grafting processes in humans for fertility preservation by favoring retain medulla tissue at the ovarian cortex.

Cryopreservation Competence of Chicken Oocytes as a Model of Endangered Wild Birds: Effects of Storage Time and Temperature on the Ovarian Follicle Survival.

Simple SummaryMany avian species are classified as endangered, and the development of the gamete preservation technique is essential to preserve their genetic diversity. Since cryopreservation and transplantation of ovarian tissues are currently the only methods of female fertility preservation in birds after birth, transporting the ovary to the laboratory in good condition is key for successful female fertility preservation. Here, we report using chickens as a model of wild birds for which storage at low temperature (4 °C), but not room temperature, can maintain the ovary, ensuring oocyte survival, morphology and protein expression of germ cell markers for at least 3 days. Furthermore, a vitrification study confirmed that oocytes after ovary storage at a low temperature can be preserved by ovarian tissue cryopreservation. On the other hand, ovaries stored at room temperature decreased oocyte survival, while increasing gene expression of apoptosis and oxidation stress markers. Our study not only verifies the fact that the storage of hen ovaries at low temperatures protects oocytes, which can be preserved by vitrification, but it also contributes to understanding the effect of ovary storage on the integrity and cryotolerance of immature follicles within the hen ovary.For the conservation of endangered avian species, developing gamete preservation technologies is essential. However, studies in oocytes have not been widely conducted. In this study, assuming that the ovaries are transported to a research facility after death, we investigated the effect of ovary storage on oocytes for the purpose of cryopreserving avian female gametes by using a chicken as a model of endangered avian species. After excision, the ovaries were stored at either a low temperature (4 °C) or room temperature for 1–3 days. Ovarian follicles stored under different conditions for each period were examined by neutral red staining, histology, and gene and protein expression analysis. In addition, the pH of the storage medium after preserving the ovaries was measured. Then, ovarian tissues were vitrified to determine the cryopreservation competence. Storing the ovarian tissues at 4 °C kept the follicles viable and morphologically normal for 3 days with slow decline. In contrast, although different storage temperature did not influence follicle viability and morphology after only 1 day of storage, ovarian tissues stored at room temperature rapidly declined in structurally normal follicles, and viable follicles were rarely seen after 3 days of storage. Gene and protein expression analysis showed that apoptosis had already started on the first day, as shown by the higher expression of CASP9 under room temperature conditions. Furthermore, high expression of SOD1 and a rapid decline of pH in the storage medium under room temperature storage suggested the influence of oxidative stress associated with low pH in this condition on the follicle survivability in hen ovarian tissues. Our cryopreservation study also showed that ovarian tissues stored at 4 °C could recover after cryopreservation even after 3 days of storage. The described storage conditions and cryopreservation methods, which preserve chicken follicle survival, will lay the foundation of ovarian tissue preservation to preserve the fertility of wild female birds.

Evolutionary and ecological insights from vital staining of bivalve oocytes: A Red Queen at the sweepstakes?

AbstractTo determine the proportion of inviable oocytes that are spawned along with normal oocytes in marine bivalves, we turned to a time‐honored technique not previously used on invertebrate oocytes: the neutral red vital stain. Despite the difficulty in obtaining induced spawns from field‐collected Cerastoderma edule (L), the results and insights from this simple procedure to answer a simple question were unexpected and far‐reaching. Mean proportions of inviable oocytes in the spawns of cockle C. edule ranged from 35% to 85%, with the vast majority represented by atresic (autolyzing) oocytes. These levels are in agreement with previous quantitative histological observations of prespawning atresia in both C. edule and the Manila clam Tapes philippinarum. Such elevated prefertilization inviability mirrors the known high level of postfertilization inviability in bivalves. Together, these high levels of inviability may account for much of the early mortality in the type III survivorship curves typical of marine bivalves. The combination of high fecundity and high oocyte inviability suggests a Red Queen‐type reproductive dilemma in these species. In addition, the great degree of interindividual variation observed in oocyte inviability suggests that cockles, and perhaps other intertidal bivalves, present a Sweepstakes Reproductive Strategy, in which highly variable individual fecundity confronts a highly variable environment. The neutral red technique promises to be a useful tool for investigating prespawning inviability in other marine taxa, as well as for aquaculture, conservation, restocking, ecophysiology, and environmental monitoring.

Measuring viability of dinoflagellate cysts and diatoms with stains to test the efficiency of facsimile treatments possibly applicable to ships’ ballast water and sediment

Expansion of harmful algal bloom (HAB) species through ships' ballast water and sediment has been an increasing concern. Determining whether a microalgal cell, particularly for the toxic and HAB-forming species, is "viable" or "dead" is fundamental to understanding the effectiveness of the many ballast-water treatments that have been considered. To this end, we screened a variety of stains to assess the viability of dinoflagellate (Gymnodinium catenatum, GC) cysts and diatom (Corethron hystrix) vegetative cells to test the efficiency of ballast water treatments. Results showed that the stains fluorescing red or green are not sound candidates for viability measurements due to the interference of chlorophyll-induced red fluorescence or cytosolic green autofluorescence, while the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide is limited by its toxicity, pseudo-positive judgment and the consequent confusion between cysts and vegetative cells. We further demonstrated that the stain Neutral Red (NR) is a sound candidate as the "vital stain" and can be easily applied for functionally defining the viability of both dinoflagellate cysts and diatoms. Another stain, the Evans Blue (EB), could be used as a "mortal stain" for the vegetative diatom cells but not a sensitive indicator of viability for GC cysts. The NR staining for GC cysts generally needs a higher dosage (0.005%) and longer staining time (24 h) than that were used for staining zooplankton, diatoms, and vegetative cells of dinoflagellates. In all cases, EB staining defined a "percentage of viable cells" significantly higher than that defined by NR. We conclude that the viability of a population is highly dependent on the species of stains used thus must be referred as a method-defined indicator.

Copepod carcasses in the western Bay of Bengal and associated ecology.

Although a portion of all copepods in the natural environment are physiologically inactive or dead, there is little data on this aspect in Indian seas. Using the classical neutral red live staining technique, the dead percentage of copepods in the western Bay of Bengal (BoB) is determined for the first time in this study, which takes into account hydrography and zooplankton samples collected on April 2015 (Spring Intermonsoon [SIM]) from 6 inshore and 8 offshore locations in the western BoB. The offshore parts of the research area were infested with a persistent and extensive warm-core eddy/gyre during the sampling time, as demonstrated by satellite data of sea surface temperature, mean sea level anomaly, and surface currents. As a result, the mixed layer depth in the offshore zone was higher, whereas nutrients, chlorophyll a, and turbidity were significantly lower than inshore. Copepods dominated the zooplankton community (> 80%), with abundances ranging from 112 to 2580 No m-3 in the study area. Calanoids (particularly Paracalanidae) dominated nearshore waters (35-65%), whereas cyclopoids (notably Corycaeidae and Oncaeidae) dominated offshore (49-71%). There were 48 species of copepods identified in total, and all of them had dead individuals (carcasses). Offshore waters had a larger percentage of copepod carcasses (27-39%) than inshore seas (18-28%). The larger percentage of carcasses is possibly related to a higher top-down feeding pressure on cyclopoids in offshore waters due to extreme oligotrophy and the dominance of the microbial food web.

Study on Multi-Dimensional Optimization of Detailed Laboratory Analysis Method for Ship Ballast Water

Ballast water can bring aquatic organisms into foreign ecosystems and cause the risk of biological invasion. Therefore, it needs to be treated before discharge, and the treated water needs to be analyzed to determine whether it meets the specified standard. At present, there is no unified method for the detection of plankton worldwide, and there are some problems in the existing methods, such as unreasonable and inaccurate. In this study, neutral red (NR), 5-chloromethyfluorescein diacetate (CMFDA) and fluorescein diacetate (FDA) were used to dye the 10-50 µm plankton of three common phyla in ballast water, and were determined by fluorescence microscope. The results showed that for 10-50 µm plankton, the staining effect of double fluorescence staining was obvious, while the fluorescent dyes could only stain part of marine diatoms, and the sample could not be fixed. The optimal dyeing concentration of neutral red was 1/5000, the dyeing time was 20 min, and the overall dyeing efficiency was above 99%. Fixing agent was used after neutral red staining, when fixing with Lugol’s solution, the lower the temperature, the better the preservation effect. At -4 °C, the storage time of the sample could be increased to 12 h, and the proportion of plankton was more than 96%. In conclusion, neutral red staining can be used as a supplement to fluorescence staining, and the the storage time of samples can be prolong by pretreatment and fixation the samples.

Anticancer and antioxidant activity of ethanolic extract of Clove (Syzygium aromaticum) on oral squamous cell carcinoma cell lines (KB cell lines)- An In-vitro study

Cloves (Syzygium aromaticum) as been used as traditional medicine for many years and they possess antibacterial, antifungal and antiviral properties. Clove is known for its anticancer property on various cancer cell lines and is well established, but its anticancer effect on OSCC cell lines is less known. Aim of the study was to determine the anticancer and antioxidant effect of Syzygium aromaticum extract on OSCC cell lines (KB cell lines) and compare the same with normal mouse fibroblasts cell lines (L292 cell lines). KB cell lines and L292 cell lines were commercially obtained. Clove was obtained from local market and ethanolic extract (EC) of clove was prepared. Anticancer activity was assessed by MTT, neutral red, DAPI and Double staining assay and antioxidant assay was carried out by FRAP, PM and DPPH assay. The antioxidant property of EC of clove increased with increase in the concentration in a dose dependent manner. Both MTT and Neutral Red assay showed increase in cell death with increase in concentration of EC of clove. Double staining and DAPI showed increase in cell death when treated with EC of clove. The anticancer and antioxidant activity of EC of clove was comparable with standard drug used in the assay. This in vitro study demonstrates effective anticancer and antioxidant activity on KB cell lines when compared to standard control. However, further studies are to be conducted in order to characterize other potential antitumor components of the clove, so that it can be used as therapeutic agent in treating oral carcinoma.

Macroscopic detection of demyelinated lesions in mouse PNS with neutral red dye

Lysophosphatidylcholine (LPC)-induced demyelination is a versatile animal model that is frequently used to identify and examine molecular pathways of demyelination and remyelination in the central (CNS) and peripheral nervous system (PNS). However, identification of focally demyelinated lesion had been difficult and usually required tissue fixation, sectioning and histological analysis. Recently, a method for labeling and identification of demyelinated lesions in the CNS by intraperitoneal injection of neutral red (NR) dye was developed. However, it remained unknown whether NR can be used to label demyelinated lesions in PNS. In this study, we generated LPC-induced demyelination in sciatic nerve of mice, and demonstrated that the demyelinated lesions at the site of LPC injection were readily detectable at 7 days postlesion (dpl) by macroscopic observation of NR labeling. Moreover, NR staining gradually decreased from 7 to 21 dpl over the course of remyelination. Electron microscopy analysis of NR-labeled sciatic nerves at 7 dpl confirmed demyelination and myelin debris in lesions. Furthermore, fluorescence microscopy showed NR co-labeling with activated macrophages and Schwann cells in the PNS lesions. Together, NR labeling is a straightforward method that allows the macroscopic detection of demyelinated lesions in sciatic nerves after LPC injection.

Probing the Energetic Metabolism of Resting Cysts under Different Conditions from Molecular and Physiological Perspectives in the Harmful Algal Blooms-Forming Dinoflagellate Scrippsiella trochoidea.

Energetic metabolism is essential in maintaining the viability of all organisms. Resting cysts play important roles in the ecology of dinoflagellates, particularly for harmful algal blooms (HABs)-causative species. However, the energetic metabolism underlying the germination potency maintenance of resting cysts of dinoflagellate have been extremely scarce in studies from physiological and, particularly, molecular perspectives. Therefore, we used the cosmopolitan Scrippsiella trochoidea as a representative of HABs-forming and cyst-producing dinoflagellates in this work to obtain novel insights into the molecular mechanisms, regulating the energetic metabolism in dinoflagellate resting cysts, under different physical condition. As the starting step, we established a cDNA subtractive library via suppression subtractive hybridization (SSH) technology, from which we screened an incomplete sequence for the β subunit of ATP synthase gene (β-F1-ATPase), a key indicator for the status of cell’s energetic metabolism. The full-length cDNA of β-F1-ATPase gene from S.trochoidea (Stβ-F1-ATPase) was then obtained via rapid amplification of cDNA ends (RACE) (Accession: MZ343333). Our real-time qPCR detections, in vegetative cells and resting cysts treated with different physical conditions, revealed that (1) the expression of Stβ-F1-ATPase in resting cysts was generally much lower than that in vegetative cells, and (2) the Stβ-F1-ATPase expressions in the resting cysts under darkness, lowered temperature, and anoxia, and during an extended duration of dormancy, were significantly lower than that in cysts under the condition normally used for culture-maintaining (a 12 h light:12 h dark cycle, 21 °C, aerobic, and newly harvested). Our detections of the viability (via Neutral Red staining) and cellular ATP content of resting cysts, at the conditions corresponding to the abovementioned treatments, showed that both the viability and ATP content decreased rapidly within 12 h and then maintained at low levels within the 4-day experimentation under all the three conditions applied (4 °C, darkness, and anoxia), which are well in accordance with the measurements of the transcription of Stβ-F1-ATPase. These results demonstrated that the energy consumption of resting cysts reaches a low, but somehow stable, level within a short time period and is lower at low temperature, darkness, and anoxia than that at ambient temperature. Our work provides an important basis for explaining that resting cysts survive long-term darkness and low temperature in marine sediments from molecular and physiological levels.

Sodium Sulfite-Induced Mast Cell Pyroptosis and Degranulation.

Sodium sulfite, a common food additive, has been proved to cause allergic reaction. Pyroptosis is an inflammatory form of programmed cell death with plasma membrane lysis. In this study, we found that sodium sulfite triggered pyroptosis, which depended on reactive oxygen species (ROS)/NOD-like receptor protein 3 (NLRP3) in RBL-2H3 mast cells. Sodium sulfite increased the generation of ROS and the expression of NLRP3, caspase-1, gasdermin D N-terminal (GSDMD-N), interleukin-1β (IL-1β), and interleukin-18 (IL-18). The ROS scavenger N-acetyl-L-carnosine (NAC) and the NLRP3 inhibitor MCC950 reversed these effects. Furthermore, using a lactate dehydrogenase kit, propidium iodide staining, scanning electron microscopy, colocalization of GSDMD-N with histamine, and neutral red staining, we found that sodium sulfite notably induced cell membrane rupture. Because β-Hexosaminidase and histamine play a key role in allergic reactions, we detected the release of β-Hexosaminidase and histamine. The data showed that the release of β-Hexosaminidase and histamine induced by sodium sulfite was increased with dose independence, which were inhibited after treatment with NAC or MCC950. Overall, evidence suggested that pyroptosis induced by sodium sulfite may rupture the cell membrane and result in degranulation of mast cells. Our study may provide new insights for the mechanism by which sodium sulfite induces mast cell death and sensitization.

Long-Term Suppression of c-Jun and nNOS Preserves Ultrastructural Features of Lower Motor Neurons and Forelimb Function after Brachial Plexus Roots Avulsion.

Brachial plexus root avulsions cause debilitating upper limb paralysis. Short-term neuroprotective treatments have reported preservation of motor neurons and function in model animals while reports of long-term benefits of such treatments are scarce, especially the morphological sequelae. This morphological study investigated the long-term suppression of c-Jun- and neuronal nitric oxide synthase (nNOS) (neuroprotective treatments for one month) on the motor neuron survival, ultrastructural features of lower motor neurons, and forelimb function at six months after brachial plexus roots avulsion. Neuroprotective treatments reduced oxidative stress and preserved ventral horn motor neurons at the end of the 28-day treatment period relative to vehicle treated ones. Motor neuron sparing was associated with suppression of c-Jun, nNOS, and pro-apoptotic proteins Bim and caspases at this time point. Following 6 months of survival, neutral red staining revealed a significant loss of most of the motor neurons and ventral horn atrophy in the avulsed C6, 7, and 8 cervical segments among the vehicle-treated rats (n = 4). However, rats that received neuroprotective treatments c-Jun JNK inhibitor, SP600125 (n = 4) and a selective inhibitor of nNOS, 7-nitroindazole (n = 4), retained over half of their motor neurons in the ipsilateral avulsed side compared. Myelinated axons in the avulsed ventral horns of vehicle-treated rats were smaller but numerous compared to the intact contralateral ventral horns or neuroprotective-treated groups. In the neuroprotective treatment groups, there was the preservation of myelin thickness around large-caliber axons. Ultrastructural evaluation also confirmed the preservation of organelles including mitochondria and synapses in the two groups that received neuroprotective treatments compared with vehicle controls. Also, forelimb functional evaluation demonstrated that neuroprotective treatments improved functional abilities in the rats. In conclusion, neuroprotective treatments aimed at suppressing degenerative c-Jun and nNOS attenuated apoptosis, provided long-term preservation of motor neurons, their organelles, ventral horn size, and forelimb function.

Seasonal variability in non-consumptive mortality of Arctic zooplankton.

Recent observations from high-latitude marine ecosystems indicate that non-consumptive mortality may be particularly high in Arctic zooplankton during the polar night. Here we have estimated the contribution of dead organisms to the mesozooplankton community in the high Arctic (Svalbard 78–81oN) during the polar night (January), in spring (May) and in late summer (end of August). To identify in situ dead organisms, we used Neutral Red Stain. The dead zooplankton fraction consisted mainly of copepods, while the contribution of dead non-copepods was low in all seasons. The absolute abundance of dead copepods varied little between seasons; however, the relative contribution of dead copepods was highest in January with 11–35% of the copepods classified as dead, in contrast to 2–12% in spring and summer. Furthermore, there were species-specific differences: copepods of the genus Calanus contributed more to the dead fraction of the copepod community during the polar night compared to spring and summer, leading to a higher “dead” biomass in winter. We conclude that non-consumptive winter mortality is considerable in calanoid copepods in the Arctic and an important but so far neglected component of the passive carbon flux, providing carbon in larger portions for higher trophic level consumers during the low-productive winter.

A Vital Staining Practice That Discerns Ancestry within Groups of Settling Larvae of a Brooding Coral

Xenogeneic and allogeneic encounters following aggregated and clustered settlements of coral larvae (planulae) may carry important ecological consequences in shaping coral reefs’ communities. However, larval settlement behaviors and settlement location choices in the presence of conspecifics or heterospecifics have not been examined in detail, due to a lack of experimental tools. One potential approach is the employment of vital staining of planulae with dyes that do not impair larval metamorphosis processes, are stable for prolonged periods, and do not diffuse to un-labeled counterpart planulae. For these purposes, we examined the use of neutral red (NR) dye, as an identification marker, on the planulae of Stylophora pistillata, a Red Sea branching coral species. To examine possible NR impacts on larval settlement in the presence of conspecific planulae, we followed the settlement ratios of kin, non-kin, and mixed assemblages, as a proxy for metamorphosis success. We found no differences in settlement rates of stained vs. unstained larvae, lack of stain diffusion to other larvae and that NR stain is maintained for more than a week under a still water regimen. Thus, staining with NR may serve as a useful experimental tool, opening new opportunities in studying larval settlement patterns in sessile marine organisms.

Methylenetetrahydrofolate Reductase Deficiency Reduces Brain Microglia in Zebrafish During Embryonic Development and Is Not Corrected by Folic Acid

ObjectivesNeuronal development and function is dependent on the interaction between the central nervous system and immune system. Microglia are resident macrophages of the brain critical for regulating neuronal activity during embryonic development. 5-methyltetrahydrofolate (5MTHF), the bioactive folate form, is essential for fetal brain development and immune function. Common variants in methylenetetrahydrofolate reductase (MTHFR), required for conversion of folic acid (FA) to 5-MTHF, limits its production. High dose FA supplementation is recommended but high FA may have the converse effect of reducing MTHFR activity. The objective of this study was to determine the effects of mthfr deficiency and its interaction with FA during embryonic development on microglia in a zebrafish model. MethodsThe mthfr gene in zebrafish was disrupted using two CRISPR mutagenesis methods. A set of 4 guide RNAs (gRNAs) + cas9 protein or cas9 alone (control) were injected to assay F0 zebrafish, or 2 gRNAs + cas9 mRNA were used to induce a germline mutation. To visualize macrophages at 4 days post fertilization (dpf) in live zebrafish, the transgenic mpeg1: mcherry line was used. In a subset of embryos, FA was added at 0, 50, 75, or 100- μM from 0–4dpf. At 4dpf, live neutral red staining for microglia was performed and the number in the optic tectum was quantified. 5MTHF, s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) were assayed in whole zebrafish at 5dpf. ResultsIn vivo imaging revealed a reduction in macrophage number (∼30%, P < 0.001) in the head region of mthfr disrupted zebrafish, but not in the periphery. mthfr zebrafish also had less microglia compared to controls (15%, P < 0.001). These changes were associated with lower 5MTHF (90%, P < 0.0001) and SAM: SAH (∼50%, P < 0.001) at 5dpf indicative of lower methylation potential. Exposure with FA did not correct the phenotype and at 100μM FA, control zebrafish also showed a decrease in microglia similar to mthfr zebrafish, confirming inhibitory effects of the high FA dose. Conclusionsmthfr deficiency reduces microglia in zebrafish but supplementation with FA does not prevent and may exacerbate the negative effects. The 5MTHF form of folate may be a better alternative to FA for brain health in patients with underlying genetic conditions. Funding SourcesSupported by CIHR-INMD.

Examining bivalve fecundity: oocyte viability revealed by Neutral Red vital staining

Estimation of realized fecundity (Freal, number of viable oocytes produced) is an essential, yet seldom-achieved element in the understanding of marine animal production and population dynamics. We used the Neutral Red (NR) vital stain to determine oocyte viability in spawns and gonad strippings of four species of commercially-important bivalves: Cerastoderma edule (L), Crassostrea gigas Thunberg, Mytilus edulis L, and Tapes philippinarum (Adams and Reeve). The utility of Trypan Blue as a complementary mortal stain was also assessed, and found to be unnecessary. Normal, live oocytes stained with NR, and were either spherical (mature oocytes) or pedunculated (immature oocytes); atresic/abnormal oocytes also stained with NR, allowing their ready recognition due to their abnormal shapes and cytoplasmic retraction. Dead oocytes did not stain with NR. Across the species studied, a considerable and highly variable proportion of spawned or stripped oocytes was either dead or non-viable; quantitative counts were performed for Cerastoderma edule, the only species for which > 5 spawns occurred. The high level and variability (34–85%) of dead or non-viable oocytes is consistent with a reproductive Red Queen dilemma, in which greater oocyte numbers do not translate to commensurately greater real fecundities, and also with a Sweepstakes Reproductive Success strategy, in which a large range of Freal confronts the considerable variability of intertidal environmental conditions. Neutral Red vital staining is a promising tool for the elucidation and optimization of crucial yet previously intractable aspects of bivalve hatchery production, genetic improvement, restocking, stock management, and conservation.

In vitro study on the effect of double freezing on ovarian tissue preservation in cancer women

Ovarian tissue cryopreservation (OTC) is very debated in women with breast cancer, chemotherapy and mutations in breast cancer 1 and breast cancer 2 (BRCA) genes, due to the high risk of developing ovarian cancer after ovarian tissue reimplantation. Objective: The present study aims to evaluate the effects of a double slow freezing/rapid thawing procedure on the quality of human ovarian tissue, in terms of preservation of tissue components and follicle viability by light microscopy. Methods: To this end the ovarian tissue of three women, donated for research purposes, was subjected to a single (T1) and double (T2) OTC procedure. For each woman fresh (CTRL-T0), T1 and T2 samples were fixed in formaldehyde for light microscopy analysis. In addition, for T1 and T2 samples of each woman an additional sample was thawed, in vitro cultured for 4 hours (h) and subsequently subjected to neutral red staining combined with histological evaluation. Results: After re-freezing, a good preservation of follicles and stroma was observed by histological and immunohistochemical (Ki67 and Bcl2) analysis. Moreover, a high follicle survival rate resulted after in vitro culture and Neutral Red staining followed by histological evaluation. Discussion: Although the analyzed tissue was not reimplanted in human or/and animal models, these preliminary data, suggest the possibility of twice freezing ovarian tissue as a potential new way to treat women with breast cancer at risk of recurrence or ovarian cancer. In these women to safeguard the women’s health, the transplanted ovarian tissue could be removed after childbearing and re-frozen for later motherhood request.