An investigation into the transgenerational management of the stress response in semelparous and iteroparous salmonids
By: Samuel Mayer
In recent years fish stocking has become an increasingly important practice. Factors such as climate change, habitat degradation and overfishing have driven the decline of many fish populations. Primarily, fish stocking provides stocks for recreational fishing, and serves as the backbone of the fish farming industry, but it also plays a crucial role in stabilizing wild fish populations, helping to avoid local and regional extinction. The increasing economic, recreational, and ecological role of fish stocking has led to an increase in research on the physiological effects of stocking. At this point much of the discourse in this area focuses on how chronic stress affects growth, information relevant in maximizing recreational and fish farming yields. Yet this research limits itself to studies in current generations and neglects to assess the long term transgenerational effects of stressors on existing fish populations, a more valuable focus for conservation purposes.
There is however a small subset of research looking into the transgenerational effects of maternal and paternal chronic stress. These studies focus on the effect of stocking density on key physiological indicators of chronic stress such as, Cortisol Levels, IgM Levels, and serum lysozyme, with Cortisol being the primary focus. (Lund et al, S. Hinch, 2014; D. Montero,
1999). The focus of these studies is limited to the expression levels of specific hormones and proteins. Consequently, there is a lack of information regarding genetic processes behind these stress induced changes in expression and how they may be transmitted inter-generationally.
Research into different fish species has shown that the regulation of cortisol in response to chronic stress varies greatly between species, indicating an array of potential regulatory mechanisms at play that may be unique to different clades and even to individual species. Often used as the standard to measure the effects of chronic stress, cortisol is seen to increase with varying levels to a plateau in some species. In many others it increases only temporarily after which point it is then downregulated, likely to prevent or reduce the potential detrimental effects of a sustained overexpression (Barry et al, 2001; Hinch et al, 2014). Unfortunately, due to the inherent variation in transgenerational stress management strategies, model organisms may not be the most suitable for studying the mechanisms at play here. Furthermore, it is important to investigate multiple species to try to determine whether a pattern can be found in transgenerational transmission of modified stress response genes. Therefore we aim to answer the following question: What are some of the possible mechanisms controlling the epigenetic
inheritance of modified stress related genes and how does it vary between different species?
We believe a good place to start is by building a model around the basic reproductive strategies observed in teleost fishes, semelparity and iteroparity. Semelparity describes any fish species which only has one reproductive cycle in its lifetime; fish are observed to spawn once and then die. Iteroparity on the other hand, describes any fish which have multiple cycles of reproduction through their lifetime. There are some fish species in which some populations express iteroparity and others semelparity, based on the conditions in which they live, even

though they are identified as the same species (Crespi and Teo, 2002). It is possible that the ways in which fish reproduce, affects the ways in which they manage epigenetic inheritance (Hinch et al, 2014).
We propose a model in which iteroparous fishes with many chances to produce viable offspring, have fewer mechanisms in place to remove methylated histone proteins and cytosine base groups in sex cells. On the other hand semelparous fishes, with only a single chance to produce viable offspring, should have more mechanisms in place to regulate how methylated products are passed onto offspring. In order to test this model we will focus on salmonids, comparing the effects of parental stocking density induced chronic stress on the resulting histone and cytosine methylation patterns in offspring of O. merka (Sockeye Salmon), O. mykiss
(Steelhead Trout) and S. salar (Atlantic Salmon). The reason for choosing these species is that they are all closely related, being salmonids, they have similar migration patterns for spawning, but they differ in the number of times in which they reproduce. O. Merka are completely semelparous, O. mykiss have some semelparous and some iteroparous populations, and lastly S. salar are an entirely iteroparous species (Crespi and Teo, 2002).
The question this study aims to answer is whether the reproductive strategies of salmonid species are correlated with different degrees of regulation of the epigenetic inheritance in cortisol related genes.. We predict that S. salar, being entirely iteroparous, will have the least regulation of epigenetic transmission and as such offspring methylation patterns will very closely resemble that observed in their parent’s cortisol genes. O. Mykiss, a species which demonstrates both iteroparity and semelparity, will have slightly more regulation. There will be some differences between the methylation patterns observed in O. Mykiss cortisol genes of parents and their offspring. Lastly, O. merka, being an entirely semelparous species will show the greatest difference between methylation patterns observed between parent and their offspring.
Developing an understanding of this concept can help with conservation efforts for many reasons. It can help to provide evidence to develop a preliminary idea of which fish species is more likely to pass on epigenetic adaptations, thus helping the species as a whole adapt to changing environmental stressors. Araki et al (2008) showed how hatchery reared wild Chinook salmon have a decreased survival rate in the wild. As such it is evident that research into fish restocking practices is vital to help understand what can be improved to help breed populations better suited for the wild. Consequently, the most important implication of identifying that the stress response in some species of salmonids are more heavily downregulated by high stocking densities is that it indicates the need for determining ideal breeding densities for each species.
Methods:
The first step in the design of the experiment is to set up two spawning grounds for each of the species being assessed. Each species will be subject to a treatment in which a breeding pool is at a low stocking density while another other pool is being maintained at a higher density. The density of the spawning grounds is being used as a tool to induce chronic stress; it has been shown to be a sufficient treatment to do so (Sadhu et al, 2014) The low stocking density pools on

the other hand act as a control to create a baseline to compare changes in methylation patterns occurring between parents of high density treatments and their offspring. Wild females of each species that are in the process of migrating to their spawning grounds will be captured and brought to the spawning pools, densities will be established and males will be added to allow for fertilization.
Immediately after the eggs are laid they will be isolated in order to be able link them directly to their mothers. Once the eggs hatch the offspring will be collected and allowed to incubate in tanks where all of the stocking densities of all juvenile populations are the same. By subjecting all juveniles to the same treatment, the effects of the specific adult treatment groups may be isolated for in order to identify if any methylation patterns are carried through to offspring.
Tissue sample will then be taken from parental fish and from their respective offspring to run genetic analyses to compare the two.
The genetic analyses will look for both methylated cytosine groups through bisulphite-PCR sequencing as well as analyze the H3K9me3 states of the same regions using Chromosome
Immunoprecipitation (ChIP) with an H3K9me3 specific antibody. This study will investigate the
StAR gene as well as the P450c17 gene. StAR was selected as it codes for the protein responsible for shuttling cholesterol across the cell membrane into the cell for hormone biosynthesis (Mommsen et al, 1999; Sewer and Waterman, 2001; Nemtollahi et al, 2009). This is a good gene to analyze as the downregulation of StAR expression would reduce/regulate the amount of cholesterol coming into the cell effectively capping the amount/rate of cortisol production. P450c17, unlike StAR codes a protein directly involved in cortisol biosynthesis by catalyzing a series of isomerizations and hydroxylations of pregnenolone, a precursor of cortisol
(Mommsen et al, 1999; Sewer and Waterman, 2001; Nemtollahi et al, 2009). This is also a good gene to assess as it can be used to determine if there is methylated regulation occurring directly in the process of synthesizing cortisol.
A few controls are necessary in this experiment, the first of which is listed above, a control in which parental fish are at a low density to create a baseline of the methylation patterning when chronic stress is not induced. For the bisulphite sequencing: A non-template control will be used which is relatively standard procedure, this shows that the changing methylation pattern are unique to the template strand. Additionally, unmethylated and fully methylated samples of DNA will be used as useful controls by which to compare the amount of methylation present on the target StAR and P450c17 genes (Darst et al, 2011). For the ChIP analysis: Similar to the bisulphite sequencing, the ChIP analysis will make use of positive and negative controls. This shows the response to ChIP analysis of a highly H3K9me3 saturated regions as well as one with extremely low to none of this methylation so that there is an upper and lower reference point to compare the patterns observed in the analysis of StAR and P450c17. The positive control will be the telomeric regions of the same chromosomes in which StAR and P450c17 are found as this is a highly methylated region. The beta-actin gene, a housekeeping gene which is constitutively produced in the cell will be used as a negative control as its high level of activity lends it a very low methylation state (Pena, 2012).

Discussion of Possible Results:
The first possible outcome if in line with our prediction would be that methylation patterns of P450c17 and StAR are stratified by reproductive stragies of species. If this is the case, we would expect to see in the high density treatments that both P450c17 and StAR are in their highest methylated states in S. salar offpsirng, followed by O. mykiss offspring. Lastly O. merka offspring will have the least methylation of theses cortisol genes and therefore the least similar methylation pattern to parents. In this model some degree of methylation is observed on both of the genes in all of the offspring. This is consistent with our prediction that iteroparous fish will have less mechanism in place to manage epigenetic inhertiance than semelparous fish. Another possible outcome within the bound of our prediction is that in O. merka fish, neither of these genes are observed in a methylated state, in O. Mykiss one of the two genes is present in a methylated state, and in S. salar both are present in a methylated state. Both of these findings would indicate that regulation of the cortisol response and its epigenetic inheritance is occurring within genes involved directly in cortisol synthesis and not in genes that are involved in binding to the promotors and enhancer regions of these genes.
Alternatively we could find that, as these genes are all part of the essential process for cortisol synthesis, none of them are observed in a methylated state and as such there is no trend correlated with reproductive strategy. This finding would not be in vain however, as the fact that there is an epigenetic process at play here has already been shown. As such the observation of no trend indicates merely that the genes analyzed here are not responsible for this process.
Therefore further research into other regulatory elements as well as other genes involved in cortisol biosynthesis is necessary to uncover the mechanisms at play.

References
Araki, H., Berejikian, B.A., Ford, M.J., Blouin, M.S. (2008). Fitness of hatchery-reared salmonids in the wild.
Evolutionary Applications, 1 (2)
Barry, T. P., Unwin, M. J., Malison, J. A., & Quinn, T. P. (2001). Free and total cortisol levels in semelparous and iteroparous chinook salmon.
Journal of Fish Biology, 59 (6)
Cresp, B. J., & Teo, R. (2002).
Comparative phylogenetic analysis of the evolution of semelparity and life history in salmonid fishes Evolution, 56 (5)
Darst, R. P., Pardo, C. E., Ai, L., Brown, K. D., & Kladde, M. P. (2011). Bisulfite sequencing of
DNA.
Medline, 7
Hinch, S. G., Sopinka, N. M., Middleton, C. T., Hills, J. A., & Patterson, J. A. (2014). Mother knows best, even when stressed? Effects of maternal exposure to a stressor on offspring performance at different life staes in a wild semelparous fish.
Behavioural Ecology, 175
Lund, T., Gjerdrem, T., Bentsen, H. B., Eide, D. M., Larsen, H. J. S., & Roed, K. H. (1995).
Genetic variation in immune parameters and associations to survival in atlantic salmon.
Journal of Fish Biology, 46
Mommsen, T. P., Vijayan, M. M., & Moon, T. W. (1999). Cortisol in teleosts: Dynamics, mechanisms of action, and metabolic regulation. Reviews in Fish Biology and Fidheries, 9

Montero, D., Izquierdo, M. S., Tort, L., Robaina, L., & Vergara, J. M. (1999). High stocking density produces crowding stress altering some physiological and biochemical parameters in gilthead seabream, sparus aurata , juveniles.
Fish Physiology and Biochemisty, 20
Nematollah, M. A., van Pelt-Heerschap, H., & Komen, J. (2009).
Transcript levels of five enzymes involved in cortisol synthesis and regulation during the stress response in common carp: Relationship with cortisol General and Comparative
Endocrinology, 164
Peña, A. A., Bols, N. C., & Marshall, S. H. (2010). An evaluation of potential reference genes for stability of expression in two salmonid cell lines after infection with either piscirickettsia salmonis or IPNV.
Biomedical Central Ltd., 3 (1)
Sadhu, N., Krupesha Sharma, S. R., Joseph, S., Dube, P., & Philipose, K. K. (2014). Chronic stress due to high stocking density in open sea cage farming induces variation in biochemical and immunological functions in asian seabass.
Fish Physiology and
Biochemistry, 40
Sewer, K. L., & Waterman, M. R. (2001). Insights into transcriptional regulation of steroidogenic enzymes and StAR.
Review in Endorcrine and Metabolic Disorders, 2 (3)