Biomedical Model Organism Research Articles

Variation and Variability in Skeletal Ossification of the Gray Short-tailed Opossum, Monodelphis domestica.

By reconstructing and comparing the sequence of ontogenetic (embryonic development and post-natal growth) events across species, developmental biologists have gained unique insights into the key processes underlying the evolution of modern lineages and their extinct relatives. However, despite the importance of intraspecific variation to evolutionary transformation and lineage divergence, variation in the sequence of developmental events is seldom acknowledged. Thus, how much variation or variability should be expected during ontogeny remains poorly understood and it is an open question to what extent it impacts interspecific comparisons of developmental patterns. To address this crucial question, we studied the skeletal development of the important biomedical and developmental model organism, Monodelphis domestica. We investigated cranial, forelimb, and hindlimb elements using ontogenetic sequence analysis (OSA) to quantify and assess the full range of variation and variability in the sequence of ossification. Our study documented that previously unrecognized variation exists during M. domestica ontogeny-with over 5000 sequences for the full 92 event analysis. Further, OSA revealed unexpectedly high variability (i.e., the propensity to express variation) in the sequence of ossification for the skull and across the entire skeleton. Reconstructed modal sequences were generally in agreement with previously recognized patterns, including earlier ossification of the facial skeleton and a slight offset between forelimb and hindlimb development. However, the full range of variation shows that the majority of specimens in our analysis followed developmental trajectories distinct from those recovered by prior studies. This level of variation is quite remarkable and demonstrates the importance of assessing intraspecific ontogenetic variation. By quantifying sequence polymorphism and studying how developmental variation and variability differ among species, we can clarify more precisely how developmental patterns differ among species and gain insights into how ontogeny itself evolves.

Modeling of cryopreservation pathway operation at an aquatic biomedical stock center for zebrafish

Aquatic biomedical model organisms play a substantial role in advancing our understanding of human health, however, comparably little work has been directed towards developing dependable, high-throughput storage programs for valuable genetic resources. The Zebrafish International Resource Center (ZIRC) has developed a standardized cryopreservation pathway and stored thousands of genetic lines in their repository for use by the biomedical research community. This has yet to be replicated in other facilities, and an overall repository-level pathway has never been analyzed for aquatic species. To encourage repository development for other biomedical models and to improve the ZIRC storage process and system, this study used discrete-event simulation modeling to systematically analyze the cryopreservation pathway for efficiency, and to identify improvements. The models reflected “real-world” working conditions and were used to simulate key outputs, such as production capacity over time (throughput) and steps in the process that limit production (bottlenecks). With these models, recommendations were identified to eliminate waiting times and increase efficiency. These included following proper husbandry protocols because male quality significantly affected production time, and the use of part-time operators to assist with steps that had longer Waiting Times (i.e., time samples spent in a queue) to increase production capacity. Simulation process modeling is a powerful tool that can improve the operations of existing repositories. It can also support repository development at other biomedical stock centers, and at other facilities devoted to aquatic species such as research, conservation, and aquaculture production hatcheries.

Automated staging of zebrafish embryos with deep learning.

The zebrafish (Danio rerio) is an important biomedical model organism used in many disciplines. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype. However, the detection and quantification of these delays is often achieved through manual observation, which is both time-consuming and subjective. We present KimmelNet, a deep learning model trained to predict embryo age (hours post fertilisation) from 2D brightfield images. KimmelNet's predictions agree closely with established staging methods and can detect developmental delays between populations with high confidence using as few as 100 images. Moreover, KimmelNet generalises to previously unseen data, with transfer learning enhancing its performance. With the ability to analyse tens of thousands of standard brightfield microscopy images on a timescale of minutes, we envisage that KimmelNet will be a valuable resource for the developmental biology community. Furthermore, the approach we have used could easily be adapted to generate models for other organisms.

Antifungal activity of Allyl isothiocyanate by targeting signal transduction pathway, ergosterol biosynthesis, and cell cycle in Candida albicans.

In recent years, the inclusion of Candida albicans on the list of infections that pose a threat due to drug resistance has urged researchers to look into cutting-edge and effective antifungal medications. In this regard, the current study investigated the probable mode of action of allyl isothiocyanate (AITC) against Candida albicans. In this study, planktonic assay, germ tube inhibition assay, adhesion, and biofilm formation assay were performed to check the growth and virulence factors. Furthermore, ergosterol assay, reactive oxygen production analysis, cell cycle analysis, and quantitative real-time polymerase chain reaction analysis were performed with the aim of finding the mode of action. A biomedical model organism, like a silkworm, was used in an in vivo study to demonstrate AITC anti-infective ability against C. albicans infection. Allyl isothiocyanate completely inhibited ergosterol biosynthesis in C. albicans at 0.125 mg/ml. Allyl isothiocyanate produces reactive oxygen species in both planktonic and biofilm cells of C. albicans. At 0.125 mg/ml concentration, AITC arrested cells at the G2/M phase of the cell cycle, which may induce apoptosis in C. albicans. In quantitative real-time polymerase chain reaction analysis, it was found that AITC inhibited virulence factors, like germ tube formation, at 0.125 mg/ml concentration by downregulation of PDE2, CEK1, TEC1 by 2.54-, 1.91-, and 1.04-fold change, respectively, and upregulation of MIG1, NRG1, and TUP1 by 9.22-, 3.35-, and 7.80-fold change, respectively. The in vivo study showed that AITC treatment successfully protected silkworms against C. albicans infections and increased their survival rate by preventing internal colonization by C. albicans. In vitro and in vivo studies revealed that AITC can be an alternative therapeutic option for the treatment of C. albicans infection.

Automated staging of zebrafish embryos using machine learning.

The zebrafish ( Danio rerio), is an important biomedical model organism used in many disciplines, including development, disease modeling and toxicology, to better understand vertebrate biology. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype, and accurate characterization of such delays is imperative. Despite this, the only way at present to identify and quantify these delays is through manual observation, which is both time-consuming and subjective. Machine learning approaches in biology are rapidly becoming part of the toolkit used by researchers to address complex questions. In this work, we introduce a machine learning-based classifier that has been trained to detect temporal developmental differences across groups of zebrafish embryos. Our classifier is capable of rapidly analyzing thousands of images, allowing comparisons of developmental temporal rates to be assessed across and between experimental groups of embryos. Finally, as our classifier uses images obtained from a standard live-imaging widefield microscope and camera set-up, we envisage it will be readily accessible to the zebrafish community, and prove to be a valuable resource.

Automated staging of zebrafish embryos using machine learning

The zebrafish (Danio rerio), is an important biomedical model organism used in many disciplines, including development, disease modeling and toxicology, to better understand vertebrate biology. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype, and accurate characterization of such delays is imperative. Despite this, the only way at present to identify and quantify these delays is through manual observation, which is both time-consuming and subjective. Machine learning approaches in biology are rapidly becoming part of the toolkit used by researchers to address complex questions. In this work, we introduce a machine learning-based classifier that has been trained to detect temporal developmental differences across groups of zebrafish embryos. Our classifier is capable of rapidly analyzing thousands of images, allowing comparisons of developmental temporal rates to be assessed across and between experimental groups of embryos. Finally, as our classifier uses images obtained from a standard live-imaging widefield microscope and camera set-up, we envisage it will be readily accessible to the zebrafish community, and prove to be a valuable resource.

The conserved cellular roles of CLN proteins: Novel insights from Dictyostelium discoideum

The neuronal ceroid lipofuscinoses (NCLs), collectively referred to as Batten disease, are a group of fatal neurodegenerative disorders that primarily affect children. The etiology of Batten disease is linked to mutations in 13 genes that encode distinct CLN proteins, whose functions have yet to be fully elucidated. The social amoeba Dictyostelium discoideum has been adopted as an efficient and powerful model system for studying the diverse cellular roles of CLN proteins. The genome of D. discoideum encodes several homologs of human CLN proteins, and a growing body of literature supports the conserved roles and networking of CLN proteins in D. discoideum and humans. In humans, CLN proteins have diverse cellular roles related to autophagy, signal transduction, lipid homeostasis, lysosomal ion homeostasis, and intracellular trafficking. Recent work also indicates that CLN proteins play an important role in protein secretion. Remarkably, many of these findings have found parallels in studies with D. discoideum. Accordingly, this review will highlight the translatable value of novel work with D. discoideum in the field of NCL research and propose further avenues of research using this biomedical model organism for studying the NCLs.

Automated staging of zebrafish embryos using machine learning

The zebrafish (Danio rerio), is an important biomedical model organism used in many disciplines, including development, disease modeling and toxicology, to better understand vertebrate biology. The phenomenon of developmental delay in zebrafish embryos has been widely reported as part of a mutant or treatment-induced phenotype, and accurate characterization of such delays is imperative. Despite this, the only way at present to identify and quantify these delays is through manual observation, which is both time-consuming and subjective. Machine learning approaches in biology are rapidly becoming part of the toolkit used by researchers to address complex questions. In this work, we introduce a machine learning-based classifier that has been trained to detect temporal developmental differences across groups of zebrafish embryos. Our classifier is capable of rapidly analyzing thousands of images, allowing comparisons of developmental temporal rates to be assessed across and between experimental groups of embryos. Finally, as our classifier uses images obtained from a standard live-imaging widefield microscope and camera set-up, we envisage it will be readily accessible to the zebrafish community, and prove to be a valuable resource.

Altered protein secretion in Batten disease.

ABSTRACTThe neuronal ceroid lipofuscinoses (NCLs), collectively known as Batten disease, are a group of neurological diseases that affect all ages and ethnicities worldwide. There are 13 different subtypes of NCL, each caused by a mutation in a distinct gene. The NCLs are characterized by the accumulation of undigestible lipids and proteins in various cell types. This leads to progressive neurodegeneration and clinical symptoms including vision loss, progressive motor and cognitive decline, seizures, and premature death. These diseases have commonly been characterized by lysosomal defects leading to the accumulation of undigestible material but further research on the NCLs suggests that altered protein secretion may also play an important role. This has been strengthened by recent work in biomedical model organisms, including Dictyostelium discoideum, mice, and sheep. Research in D. discoideum has reported the extracellular localization of some NCL-related proteins and the effects of NCL-related gene loss on protein secretion during unicellular growth and multicellular development. Aberrant protein secretion has also been observed in mammalian models of NCL, which has allowed examination of patient-derived cerebrospinal fluid and urine for potential diagnostic and prognostic biomarkers. Accumulated evidence links seven of the 13 known NCL-related genes to protein secretion, suggesting that altered secretion is a common hallmark of multiple NCL subtypes. This Review highlights the impact of altered protein secretion in the NCLs, identifies potential biomarkers of interest and suggests that future work in this area can provide new therapeutic insight.

CRISPR Toolbox for Genome Editing in Dictyostelium.

The development of new techniques to create gene knockouts and knock-ins is essential for successful investigation of gene functions and elucidation of the causes of diseases and their associated fundamental cellular processes. In the biomedical model organism Dictyostelium discoideum, the methodology for gene targeting with homologous recombination to generate mutants is well-established. Recently, we have applied CRISPR/Cas9-mediated approaches in Dictyostelium, allowing the rapid generation of mutants by transiently expressing sgRNA and Cas9 using an all-in-one vector. CRISPR/Cas9 techniques not only provide an alternative to homologous recombination-based gene knockouts but also enable the creation of mutants that were technically unfeasible previously. Herein, we provide a detailed protocol for the CRISPR/Cas9-based method in Dictyostelium. We also describe new tools, including double knockouts using a single CRISPR vector, drug-inducible knockouts, and gene knockdown using CRISPR interference (CRISPRi). We demonstrate the use of these tools for some candidate genes. Our data indicate that more suitable mutants can be rapidly generated using CRISPR/Cas9-based techniques to study gene function in Dictyostelium.

UHPLC-IM-Q-ToFMS analysis of maradolipids, found exclusively in Caenorhabditis elegans dauer larvae

Lipid identification is one of the current bottlenecks in lipidomics and lipid profiling, especially for novel lipid classes, and requires multidimensional data for correct annotation. We used the combination of chromatographic and ion mobility separation together with data-independent acquisition (DIA) of tandem mass spectrometric data for the analysis of lipids in the biomedical model organism Caenorhabditis elegans. C. elegans reacts to harsh environmental conditions by interrupting its normal life cycle and entering an alternative developmental stage called dauer stage. Dauer larvae show distinct changes in metabolism and morphology to survive unfavorable environmental conditions and are able to survive for a long time without feeding. Only at this developmental stage, dauer larvae produce a specific class of glycolipids called maradolipids. We performed an analysis of maradolipids using ultrahigh performance liquid chromatography-ion mobility spectrometry-quadrupole-time of flight-mass spectrometry (UHPLC-IM-Q-ToFMS) using drift tube ion mobility to showcase how the integration of retention times, collisional cross sections, and DIA fragmentation data can be used for lipid identification. The obtained results show that combination of UHPLC and IM separation together with DIA represents a valuable tool for initial lipid identification. Using this analytical tool, a total of 45 marado- and lysomaradolipids have been putatively identified and 10 confirmed by authentic standards directly from C. elegans dauer larvae lipid extracts without the further need for further purification of glycolipids. Furthermore, we putatively identified two isomers of a lysomaradolipid not known so far.Graphical abstract

Assessing Pressure-Volume Relationship in Developing Heart of Zebrafish In-Vivo.

During embryogenesis, the developing heart transforms from a linear peristaltic tube into a multi-chambered pulsatile pump with blood flow-regulating valves. In this work, we report how hemodynamic parameters evolve during the heart's development, leading to its rhythmic pumping and blood flow regulation as a functioning organ. We measured the time course of intra-ventricular pressure from zebrafish embryos at 3, 4, and 5 days post fertilization (dpf) using the servo null method. We also measured the ventricular volume and monitored the opening/closing activity of the AV and VB valves using 4D selective plane illumination microscopy (SPIM). Our results revealed significant increases in peak systolic pressure, stroke volume and work, cardiac output, and power generation, and a total peripheral resistance decrease from zebrafish at 4, 5 dpf versus 3 dpf. These data illustrate that the early-stage zebrafish heart's increasing efficiency is synchronous with the expected changes in valve development, chamber morphology and increasing vascular network complexity. Such physiological measurements in tractable laboratory model organisms are critical for understanding how gene variants may affect phenotype. As the zebrafish emerges as a leading biomedical model organism, the ability to effectively measure its physiology is critical to its translational relevance.

Origins and Long-Term Patterns of Copy-Number Variation in Rhesus Macaques.

Mutations play a key role in the development of disease in an individual and the evolution of traits within species. Recent work in humans and other primates has clarified the origins and patterns of single-nucleotide variants, showing that most arise in the father’s germline during spermatogenesis. It remains unknown whether larger mutations, such as deletions and duplications of hundreds or thousands of nucleotides, follow similar patterns. Such mutations lead to copy-number variation (CNV) within and between species, and can have profound effects by deleting or duplicating genes. Here, we analyze patterns of CNV mutations in 32 rhesus macaque individuals from 14 parent–offspring trios. We find the rate of CNV mutations per generation is low (less than one per genome) and we observe no correlation between parental age and the number of CNVs that are passed on to offspring. We also examine segregating CNVs within the rhesus macaque sample and compare them to a similar data set from humans, finding that both species have far more segregating deletions than duplications. We contrast this with long-term patterns of gene copy-number evolution between 17 mammals, where the proportion of deletions that become fixed along the macaque lineage is much smaller than the proportion of segregating deletions. These results suggest purifying selection acting on deletions, such that the majority of them are removed from the population over time. Rhesus macaques are an important biomedical model organism, so these results will aid in our understanding of this species and the disease models it supports.

Surprisingly long survival of premature conclusions about naked mole-rat biology.

Naked mole-rats express many unusual traits for such a small rodent. Their morphology, social behaviour, physiology, and ageing have been well studied over the past half-century. Many early findings and speculations about this subterranean species persist in the literature, although some have been repeatedly questioned or refuted. While the popularity of this species as a natural-history curiosity, and oversimplified story-telling in science journalism, might have fuelled the perpetuation of such misconceptions, an accurate understanding of their biology is especially important for this new biomedical model organism. We review 28 of these persistent myths about naked mole-rat sensory abilities, ecophysiology, social behaviour, development and ageing, and where possible we explain how these misunderstandings came about.

Inhibitory Effect of Morin Against Candida albicans Pathogenicity and Virulence Factor Production: An in vitro and in vivo Approaches.

Candida albicans is considered an exclusive etiologic agent of candidiasis, a very common fungal infection in human. The expression of virulence factors contributes highly to the pathogenicity of C. albicans. These factors include biofilm formation, yeast-to-hyphal transition, adhesins, aspartyl proteases, and phospholipases secretion. Moreover, resistance development is a critical issue for the therapeutic failure of antifungal agents against systemic candidiasis. To circumvent resistance development, the present study investigated the virulence targeted therapeutic activity of the phyto-bioactive compound morin against C. albicans. Morin is a natural compound commonly found in medicinal plants and widely used in the pharmaceutical and cosmetic products/industries. The present study explicated the significant inhibitory potential of morin against biofilm formation and other virulence factors’ production, such as yeast-hyphal formation, phospholipase, and exopolymeric substances, in C. albicans. Further, qPCR analysis confirmed the downregulation of biofilm and virlence-related genes in C. albicans upon morin treatment, which is in correspondence with the in vitro bioassays. Further, the docking analysis revealed that morin shows strong affinity with Hwp-1 protein, which regulates the expression of biofilm and hyphal formation in C. albicans and, thereby, abolishes fungal pathogenicity. Moreover, the anti-infective potential of morin against C. albicans-associated systemic candidiasis is confirmed through an in vivo approach using biomedical model organism zebrafish (Danio rerio). The outcomes of the in vivo study demonstrate that the morin treatment effectively rescues animals from C. albicans infections and extends their survival rate by inhibiting the internal colonization of C. albicans. Histopathology analysis revealed extensive candidiasis-related pathognomonic changes in the gills, intestine, and kidney of animals infected with C. albicans, while no extensive abnormalities were observed in morin-treated animals. The results evidenced that morin has the ability to protect against the pathognomonic effect and histopathological lesions caused by C. albicans infection in zebrafish. Thus, the present study suggests that the utilization of morin could act as a potent therapeutic medication for C. albicans instigated candidiasis.

A new mechanism for cannabidiol in regulating the one-carbon cycle and methionine levels in Dictyostelium and in mammalian epilepsy models.

Background and PurposeEpidiolex™, a form of highly purified cannabidiol (CBD) derived from Cannabis plants, has demonstrated seizure control activity in patients with Dravet syndrome, without a fully elucidated mechanism of action. We have employed an unbiased approach to investigate this mechanism at a cellular level.Experimental ApproachWe use a tractable biomedical model organism, Dictyostelium, to identify a protein controlling the effect of CBD and characterize this mechanism. We then translate these results to a Dravet syndrome mouse model and an acute in vitro seizure model.Key ResultsCBD activity is partially dependent upon the mitochondrial glycine cleavage system component, GcvH1 in Dictyostelium, orthologous to the human glycine cleavage system component H protein, which is functionally linked to folate one‐carbon metabolism (FOCM). Analysis of FOCM components identified a mechanism for CBD in directly inhibiting methionine synthesis. Analysis of brain tissue from a Dravet syndrome mouse model also showed drastically altered levels of one‐carbon components including methionine, and an in vitro rat seizure model showed an elevated level of methionine that is attenuated following CBD treatment.Conclusions and ImplicationsOur results suggest a novel mechanism for CBD in the regulating methionine levels and identify altered one‐carbon metabolism in Dravet syndrome and seizure activity.

The Alliance of Genome Resources: Building a Modern Data Ecosystem for Model Organism Databases.

Model organisms are essential experimental platforms for discovering gene functions, defining protein and genetic networks, uncovering functional consequences of human genome variation, and for modeling human disease. For decades, researchers who use model organisms have relied on Model Organism Databases (MODs) and the Gene Ontology Consortium (GOC) for expertly curated annotations, and for access to integrated genomic and biological information obtained from the scientific literature and public data archives. Through the development and enforcement of data and semantic standards, these genome resources provide rapid access to the collected knowledge of model organisms in human readable and computation-ready formats that would otherwise require countless hours for individual researchers to assemble on their own. Since their inception, the MODs for the predominant biomedical model organisms [Mus sp. (laboratory mouse), Saccharomyces cerevisiae, Drosophila melanogaster, Caenorhabditis elegans, Danio rerio, and Rattus norvegicus] along with the GOC have operated as a network of independent, highly collaborative genome resources. In 2016, these six MODs and the GOC joined forces as the Alliance of Genome Resources (the Alliance). By implementing shared programmatic access methods and data-specific web pages with a unified “look and feel,” the Alliance is tackling barriers that have limited the ability of researchers to easily compare common data types and annotations across model organisms. To adapt to the rapidly changing landscape for evaluating and funding core data resources, the Alliance is building a modern, extensible, and operationally efficient “knowledge commons” for model organisms using shared, modular infrastructure.

The sphingolipidome of the model organism Caenorhabditis elegans.

Sphingolipids are important lipids and integral members of membranes, where they form small microdomains called lipid rafts. These rafts are enriched in cholesterol and sphingolipids, which influences biophysical properties. Interestingly, the membranes of the biomedical model organism Caenorhabditis elegans contain only low amounts of cholesterol. Sphingolipids in C. elegans are based on an unusual C17iso branched sphingoid base. In order to analyze and the sphingolipidome of C. elegans in more detail, we performed fractionation of lipid extracts and depletion of glycero- and glycerophospholipids together with in-depth analysis using UPLC-UHR-ToF-MS. In total we were able to detect 82 different sphingolipids from different classes, including several isomeric species.

Visualising the Cardiovascular System of Embryos of Biomedical Model Organisms with High Resolution Episcopic Microscopy (HREM).

The article will briefly introduce the high-resolution episcopic microscopy (HREM) technique and will focus on its potential for researching cardiovascular development and remodelling in embryos of biomedical model organisms. It will demonstrate the capacity of HREM for analysing the cardiovascular system of normally developed and genetically or experimentally malformed zebrafish, frog, chick and mouse embryos in the context of the whole specimen and will exemplarily show the possibilities HREM offers for comprehensive visualisation of the vasculature of adult human skin. Finally, it will provide examples of the successful application of HREM for identifying cardiovascular malformations in genetically altered mouse embryos produced in the deciphering the mechanisms of developmental disorders (DMDD) program.

Workshop report: Cryopreservation of aquatic biomedical models

The genetic resources of aquatic biomedical model organisms are the products of millions of years of evolution, decades of scientific development, and hundreds of millions of dollars of research funding investment. Genetic resources (e.g., specific alleles, transgenes, or combinations) of each model organism can be considered a form of scientific wealth that can be accumulated and exchanged, typically in the form of live animals or germplasm. Large-scale maintenance of live aquatic organisms that carry these genetic resources is inefficient, costly, and risky. In situ maintenance may be substantially enhanced and backed up by combining cryopreserved germplasm repositories and genetic information systems with live animal culture. Unfortunately, cryopreservation has not advanced much beyond the status of an exploratory research for most aquatic species, lacks widespread application, and methods for successful cryopreservation remain poorly defined. For most aquatic species biological materials other than sperm or somatic cells are not comprehensively banked to represent and preserve a broad range of genetic diversity for each species. Therefore, new approaches and standardization are needed for repository-level application to ensure reproducible recovery of cryopreserved materials. Additionally, development of new technologies is needed to address preservation of novel biological materials, such as eggs and embryos of aquatic species. To address these goals, the Office of Research Infrastructure Programs (ORIP) of the National Institutes of Health (NIH) hosted the Cryopreservation of Aquatic Biomedical Models Workshop on January 7 to 8, 2017, in conjunction with the 8th Aquatic Animal Models of Human Disease Conference in Birmingham, Alabama. The goals of the workshop were to assess the status of germplasm cryopreservation in various biomedical aquatic models and allow representatives of the scientific community to develop and prioritize a consensus of specific actionable recommendations that will move the field of cryopreservation of aquatic resources forward. This workshop included sessions devoted to new approaches for cryopreservation of aquatic species, discussion of current efforts and approaches in preservation of aquatic model germplasm, consideration of needs for standardization of methods to support reproducibility, and enhancement of repository development by establishment of scalable high-throughput technologies. The following three broad recommendations were forwarded from workshop attendees: 1Establish a comprehensive, centralized unit (“hub”) to programmatically develop training for and documentation of cryopreservation methods for aquatic model systems. This would include development of species-specific protocols and approaches, outreach programs, community development and standardization, freezing services and training of the next generation of experts in aquatic cryopreservation. 2Provide mechanisms to support innovative technical advancements that will increase the reliability, reproducibility, simplicity, throughput, and efficiency of the cryopreservation process, including vitrification and pipelines for sperm, oocytes, eggs, embryos, larvae, stem cells, and somatic cells of all aquatic species. This recommendation encompasses basic cryopreservation knowledge and engineering technology, such as microfluidics and automated processing technologies. 3Implement mechanisms that allow the various aquatic model stock centers to increase their planning, personnel, ability to secure genetic resources and to promote interaction within an integrated, comprehensive repository network for aquatic model species repositories.