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The cultivation of marine invertebrate cells in vitro has garnered significant attention due to the availability of diverse cell types and cellular potentialities in comparison to vertebrates and particularly in response to the demand for a multitude of applications. While cells in the colonial urochordate Botryllus schlosseri have a very high potential for omnipotent differentiation, no proliferating cell line has been established in Botryllus, with results indicating that cell divisions cease 24–72 h post initiation. This research assessed how various Botryllus blood cell types respond to in vitro conditions by utilizing five different refinements of cell culture media (TGM1–TGM5). During the initial week of culture, there was a noticeable medium-dependent increase in the proliferation and viability of distinct blood cell types. Within less than one month from initiation, we developed medium-specific primary cultures, a discovery that supports larger efforts to develop cell type-specific cultures. Specific cell types were easily distinguished and classified based on their natural fluorescence properties using confocal microscopy. These results are in agreement with recent advances in marine invertebrate cell cultures, demonstrating the significance of optimized nutrient media for cell culture development and for cell selection. 

Global warming is one of the most significant and widespread effects of climate change. While early life stages are particularly vulnerable to increasing temperatures, little is known about the molecular processes that underpin their capacity to adapt to temperature change during early development. Using a quantitative proteomics approach, we investigated the effects of thermal stress on octopus embryos. We exposedOctopus berrimaembryos to different temperature treatments (control 19°C, current summer temperature 22°C, or future projected summer temperature 25°C) until hatching. By comparing their protein expression levels, we found that future projected temperatures significantly reduced levels of key eye proteins such as S‐crystallin and retinol dehydrogenase 12, suggesting the embryonic octopuses had impaired vision at elevated temperature. We also found that this was coupled with a cellular stress response that included a significant elevation of proteins involved in molecular chaperoning and redox regulation. Energy resources were also redirected away from non‐essential processes such as growth and digestion. These findings, taken together with the high embryonic mortality observed under the highest temperature, identify critical physiological functions of embryonic octopuses that may be impaired under future warming conditions. Our findings demonstrate the severity of the thermal impacts on the early life stages of octopuses as demonstrated by quantitative proteome changes that affect vision, protein chaperoning, redox regulation and energy metabolism as critical physiological functions that underlie the responses to thermal stress.

 

Salinity tolerance in fish involves a suite of physiological changes, but a cohesive theory leading to a mechanistic understanding at the organismal level is lacking. To examine the potential of adapting energy homeostasis theory in the context of salinity stress in teleost fish,Oreochromis mossambicuswere acclimated to hypersalinity at multiple rates and durations to determine salinity ranges of tolerance and resistance. Over 3000 proteins were quantified simultaneously to analyze molecular phenotypes associated with hypersalinity. A species‐ and tissue‐specific data‐independent acquisition (DIA) assay library of MSMS spectra was created. Protein networks representing complex molecular phenotypes associated with salinity acclimation were generated.O. mossambicushas a wide “zone of resistance” from 75 g/kg salinity to 120 g/kg. Crossing into the zone of resistance resulted in marked phenotypic changes including blood osmolality over 400 mOsm/kg, reduced body condition, and cessation of feeding. Protein networks impacted by hypersalinity consist of electron transport chain (ETC) proteins and specific osmoregulatory proteins. Cytoskeletal, cell adhesion, and extracellular matrix proteins are enriched in networks that are sensitive to the critical salinity threshold. These network analyses identify specific proteome changes that are associated with distinct zones described by energy homeostasis theory and distinguish them from general hypersalinity‐induced proteome changes.

 

ABSTRACT Organisms mount the cellular stress response whenever environmental parameters exceed the range that is conducive to maintaining homeostasis. This response is critical for survival in emergency situations because it protects macromolecular integrity and, therefore, cell/organismal function. From an evolutionary perspective, the cellular stress response counteracts severe stress by accelerating adaptation via a process called stress-induced evolution. In this Review, we summarize five key physiological mechanisms of stress-induced evolution. Namely, these are stress-induced changes in: (1) mutation rates, (2) histone post-translational modifications, (3) DNA methylation, (4) chromoanagenesis and (5) transposable element activity. Through each of these mechanisms, organisms rapidly generate heritable phenotypes that may be adaptive, maladaptive or neutral in specific contexts. Regardless of their consequences to individual fitness, these mechanisms produce phenotypic variation at the population level. Because variation fuels natural selection, the physiological mechanisms of stress-induced evolution increase the likelihood that populations can avoid extirpation and instead adapt under the stress of new environmental conditions. 

Botryllus schlosseri, is a model marine invertebrate for studying immunity, regeneration, and stress‐induced evolution. Conditions for validating its predicted proteome were optimized using nanoElute® 2 deep‐coverage LCMS, revealing up to 4930 protein groups and 20,984 unique peptides per sample. Spectral libraries were generated and filtered to remove interferences, low‐quality transitions, and only retain proteins with >3 unique peptides. The resulting DIA assay library enabled label‐free quantitation of 3426 protein groups represented by 22,593 unique peptides. Quantitative comparisons of single systems from a laboratory‐raised with two field‐collected populations revealed (1) a more unique proteome in the laboratory‐raised population, and (2) proteins with high/low individual variabilities in each population. DNA repair/replication, ion transport, and intracellular signaling processes were distinct in laboratory‐cultured colonies. Spliceosome and Wnt signaling proteins were the least variable (highly functionally constrained) in all populations. In conclusion, we present the first colonial tunicate's deep quantitative proteome analysis, identifying functional protein clusters associated with laboratory conditions, different habitats, and strong versus relaxed abundance constraints. These results empower research onB. schlosseriwith proteomics resources and enable quantitative molecular phenotyping of changes associated with transfer from in situ to ex situ and from in vivo to in vitro culture conditions.

 

CRISPR/Cas9 gene editing is effective in manipulating genetic loci in mammalian cell cultures and whole fish but efficient platforms applicable to fish cell lines are currently limited. Our initial attempts to employ this technology in fish cell lines using heterologous promoters or a ribonucleoprotein approach failed to indicate genomic alteration at targeted sites in a tilapia brain cell line (OmB). For potential use in a DNA vector approach, endogenous tilapia beta Actin (OmBAct), EF1 alpha (OmEF1a), and U6 (TU6) promoters were isolated. The strongest candidate promoter determined by EGFP reporter assay, OmEF1a, was used to drive constitutive Cas9 expression in a modified OmB cell line (Cas9-OmB1). Cas9-OmB1 cell transfection with vectors expressing gRNAs driven by the TU6 promoter achieved mutational efficiencies as high as 81% following hygromycin selection. Mutations were not detected using human and zebrafish U6 promoters demonstrating the phylogenetic proximity of U6 promoters as critical when used for gRNA expression. Sequence alteration to TU6 improved mutation rate and cloning efficiency. In conclusion, we report new tools for ectopic expression and a highly efficient, economical system for manipulation of genomic loci and evaluation of their causal relationship with adaptive cellular phenotypes by CRISPR/Cas9 gene editing in fish cells.

 

Using abundance measurements of 1,490 proteins from four separate populations of three-spined sticklebacks, we implemented a system-level approach to correlate proteome dynamics with environmental salinity and temperature and the fish's population and morphotype. We identified robust and accurate fingerprints that classify environmental salinity, temperature, morphotype, and the population sample origin, observing that proteins with specific functions are enriched in these fingerprints. Highly apparent functions represented in all fingerprints include ion transport, proteostasis, growth, and immunity, suggesting that these functions are most diversified in populations inhabiting different environments. Applying a differential network approach, we analyzed the network of protein interactions that differs between populations. Looking at specific population combinations of differential interaction, we identify sets of connected proteins. We find that these sets and their corresponding enriched functions reflect key processes that have diverged between the four populations. Moreover, the extent of divergence, i.e., the number of enriched functions that differ between populations, is highest when all three environmental parameters are different between two populations. Key nodes in the differential interaction network signify functions that are also inherent in the fingerprints, most prominently proteostasis-related functions. However, the differential interaction network also reveals additional functions that have diverged between populations, notably cytoskeletal organization and morphogenesis. The strength of these analyses is that the results are purely data driven. With such an unbiased approach applied on a large proteomic data set, we find the strongest signals given by the data, making it possible to develop more discriminatory and complex biomarkers for specific contexts of interest.