Newton, James Raymond (2013) Investigating the genetics of thermal tolerance and adaptation to temperature amongst populations of Australian barramundi (Lates calcarifer). PhD thesis, James Cook University.

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Abstract

Australian barramundi (Lates calcarifer), are distributed over much of the northern and northeastern coast where they inhabit rivers, estuaries and near coastal waters spanning some 16 degrees of latitude (10ºS - 26ºS). Over their distribution, populations of barramundi experience differences in thermal environmental conditions that vary from warmer and more consistent tropical conditions at northern latitudes (mean yearly range of 23.2 – 32 ºC), to cooler and more variable conditions at southern latitudes (mean yearly range of 18.5 – 27.7 ºC). Australian barramundi populations show strong genetic structuring and this, coupled with exposure to varying thermal environments, may have led to temperature tolerance differences among these populations indicative of local adaptation. As barramundi are currently cultured to some degree within all mainland states of Australia, the replication of optimal culture conditions, at significant cost to farmers, is necessary to ensure the success of primary breeding objectives. Identifying the underlying genetic mechanisms contributing towards peak growth and survival under a range of culture temperatures in barramundi would therefore be of significant benefit to the aquaculture industry. Research described in this thesis aimed to identify whether or not barramundi populations at the extreme of their Australian distribution exhibited evidence for local phenotypic thermal adaptation and, if so, whether underlying genetic differences could be established.

To demonstrate evidence of local adaptation, initial experiments aimed to differentiate between two genetically distinct populations of barramundi based upon their tolerance to increased water temperatures. To determine this, loss of swimming equilibrium (LOSE) was used as a predictor of upper thermal tolerance in fish from a warm-water adapted (Darwin, Northern Territory) and cool-water adapted (Gladstone, Queensland) population. Barramundi from both populations were simultaneously exposed to an increase in water temperature from 28 ºC to 40 ºC at the rate of 2 ºC/h. LOSE was recorded as the time taken for individual fish to demonstrate loss of swimming equilibrium after water heating began. Significant differences in upper thermal tolerance, suggestive of local adaptation, were evident between the two populations with warm adapted, northern barramundi demonstrating a significantly longer time until LOSE at 40 ºC than cool adapted, southern barramundi (518.5 ± 8.0 min and 452.8 ± 8.0 min respectively, ANOVA; F₁, ₂₂ = 7.86, P≤0.01). However, as LOSE challenge tests are not practical to the identification of commercial broodstock; the response to temperature was also evaluated within dissociated caudal fin cells as a means of providing a sensitive and non-invasive method with which to determine upper thermal tolerance in whole animals. Prior to measurements of LOSE, small fin clips were taken from each fish and enzymatically digested to produce 'free' caudal fin cells. Cells were incubated at 40 º C for 1 h as a thermal stress, before cell staining with Propidium Iodide (stains dead cells) and Calcein AM (stains live cells) was used, allowing for the determination of a dead/live cell ratio. Thermal tolerance results generated from dissociated caudal fin cells strongly correlated cell viability with LOSE measurements (average r = 0.69), confirming that this method can be used to discriminate between populations with different thermal tolerances without having to directly thermally challenge valuable broodfish. In doing so, these results provide strong evidence that thermal tolerance differences amongst barramundi populations arise due to significant contribution from differences at the genetic level.

Having demonstrated that divergent populations of barramundi show strong evidence for genetic adaptation to temperature, the expression of a group of genes likely to be involved in this species' response to an acute heat stress was examined. The acute heat shock response, as indicated by the expression of genes within the cellular stress (Hsp90α, Hsp90β, Hsc70, Hsp70), metabolic (CiSy, CcoII, Ldh) and growth (Igf1, Mstn1) related pathways, was examined following an increase in water temperature from 28 ºC to 36 ºC over 30 min. Barramundi were maintained at the acute stress temperature of 36 ºC for 1 hr before being returned to 28 ºC and allowed to recover at this temperature for a further 2 weeks. Muscle tissue sampling over the experimental period allowed for the expression quantification of stress, metabolic and growth related genes via real time quantitative PCR (RT-qPCR), where a robust and reliable normalization approach identified both α-tub and Rpl8 as appropriate genes for the analysis of gene expression in response to an acute heat stress. Hsp90α and Hsp70 of the inducible heatshock response pathway showed a massive up-regulation of gene expression in response to heat stress, whilst the constitutive heat shock genes Hsp90β and Hsc70 showed no change over the course of the experiment and a small increase after 2 weeks of recovery respectively. Of the three genes representing the metabolic pathway (CiSy, CcoІІ and Ldh) only CcoІІ changed significantly showing a decrease in gene expression which may suggest a small suppression of aerobic metabolism. Igf1 of the growth pathway showed no significant differences in response to an acute heat stress, whilst Mstn1 increased at the beginning of the heat stress, but returned to basal levels soon after. Overall, the results demonstrate that an acute heat stress in L. calcarifer caused a significant increase in the expression of genes from the cellular stress response pathway along with a potential decrease in aerobic metabolism and only relatively minor changes to the growth pathway. These results highlight the hardy nature of L. calcarifer and demonstrate the importance of an adaptive gene expression response in coping with the sudden temperature changes routinely encountered on a daily basis within its natural environment.

Having identified key genes from temperature responsive pathways, differences in the phenotypic performance of barramundi populations to temperature (as highlighted in Chapter 2), were interpreted using gene expression data. Following on from the analysis of gene expression in response to an acute heat stress, key genes from the cellular stress, metabolic and growth pathways were analysed via RT-qPCR in both a warm (Darwin, Northern Territory) and cool (Gladstone, central Queensland) water adapted barramundi population reared at either hot (36 ºC), control (28 ºC) or cool (22 ºC) temperatures for 106 days. Growth indicators were periodically measured during the growth trial and white muscle tissue was also sampled at day 0 (T=0), day 3 (T=3), day 9 (T=9) and day 106 (T=106) for gene expression analysis. At a rearing temperature of 22 ºC, a higher final weight in cool adapted barramundi over warm adapted barramundi (145.9 ± 11.1 g and 89.9 ± 3.5 g, respectively) was underpinned by a significantly faster induction of the cellular stress response and greater expression of Hsp90α. Conversely, no changes in heat shock protein (Hsp) gene expression were observed in barramundi reared at either 36 ºC or 28 ºC. Genetically regulated adaptation to cool temperatures in barramundi therefore seems to be correlated with changes to the cellular stress response pathway. Regulation of metabolic and growth associated genes to temperature were consistent between populations and were not affected by a control (28 ºC) or a cool (22 ºC) rearing temperature. At 36 ºC, both warm and cool adapted barramundi exhibited a significant decrease in CcoII expression consistent with expectations associated with alterations in aerobic capacity, however, CiSy expression remained unchanged. The impaired growth of both populations reared at 36 ºC was accompanied by a decrease in the expression of Igf1 and an increase in the expression of Mstn1. As such the expression of both genes can be reliably used to indicate the growth status of barramundi at high temperatures, however, long term control of growth at cool temperatures seems to be under the control of alternate gene pathways, as no significant differences in the expression of these two growth related genes was observed.

To further investigate population differences, the underlying transcriptome profile of barramundi reared over a long term period was examined via Illumina mRNA deep sequencing as a means of determining the major contributing gene categories giving rise to the phenotypic differences in population growth. White muscle tissue from warm and cool adapted barramundi reared for 106 days was sampled and used for pathway expression analysis in conjunction with the phenotypic data collected previously. Gene ontology (GO) analysis revealed enrichment in categories relating to the regulation of peptidase activity as well as microtubule, cytoplasmic and cellular metabolic based processes. Further analysis of the GO category "microtubule based process" with associated genes from the "response to stress" category revealed an apparent reorganisation of cytoskeletal elements in response to an induced cold stress in northern barramundi reared at 22 ºC, when compared with northern barramundi reared at 36 ºC. Between southern barramundi and northern barramundi reared at 36 ºC, an analysis of the "endopeptidase inhibitor activity" GO category, in conjunction with stress genes, indicated a suppression of the immune complement system in southern barramundi, along with an increase in the cellular stress response. As southern populations of barramundi from a cooler environment grew significantly faster at 22 ºC than northern barramundi populations from a warm environment; the results of the present study show that southern populations of barramundi exhibit underlying molecular adaptation to cooler water temperatures, but still retain a tolerance for warm water temperatures. Furthermore, GO profiling has revealed groups of genes that underlie population differences in temperature tolerance as a means to prioritize the analysis of differential gene expression in studies of local adaptation in the future.

The results of this thesis demonstrate the occurrence of local adaptation to environmental temperature amongst Australian populations of barramundi from significantly different environments. Both phenotypic and genetic indicators reveal the hardy and adaptable nature of barramundi to adverse temperatures in accordance with the variable characteristics of estuarine environments. Specifically, barramundi from the northern end of the species distribution range show a greater tolerance to short, but significant spikes, in water temperature compared with barramundi from southern populations. However, gene expression analysis and growth data reveal that southern populations of barramundi show adaptive traits leading to better growth at cooler temperatures when compared with northern populations. The performance of southern barramundi at cool temperatures was accompanied by significant differences in the expression of heat shock genes and other associated stress responsive genes, suggesting that faster induction and higher expression of heat shock genes aids the continuation of growth at cooler temperatures. The expression of nominated growth related genes was only affected at high rearing temperatures and it is therefore likely that growth at cooler temperatures is under the influence of alternate mechanisms, which could prove interesting for future research.

Item ID:	41078
Item Type:	Thesis (PhD)
Keywords:	aquaculture; barramundi; biological adaption; climate; dissociated caudal fin cells; evolutionary biology; fishes; gene expression; gene ontology; genetics; geographical distribution; giant perch; heat-shock proteins; Lates calcarifer; local adaptation; next-gen sequencing; normalization; qrt-PCR; stress response; temperature tolerance; temperature; upper thermal tolerance
Related URLs:	http://researchonline.jcu.edu.au/16654/ http://researchonline.jcu.edu.au/23145/ http://researchonline.jcu.edu.au/33360/
Copyright Information:	Copyright © 2013 James Raymond Newton
Additional Information:	Publications arising from this thesis are available from the Related URLs field. The publications are: Chapter 2.0: Newton, James R., Smith-Keune, Carolyn, and Jerry, Dean R. (2010) Thermal tolerance varies in tropical and sub-tropical populations of barramundi (Lates calcarifer) consistent with local adaptation. Aquaculture, 308 (Supplement 1). S128-S132. Chapter 3.0: Newton, J.R., De Santis, C., and Jerry, D.R. (2012) The gene expression response of the catadromous perciform barramundi Lates calcarifer to an acute heat stress. Journal of Fish Biology, 81 (1). pp. 81-93. Chapter 5.0: Newton, James R., Zenger, Kyall R., and Jerry, Dean R. (2013) Next-generation transcriptome profiling reveals insights into genetic factors contributing to growth differences and temperature adaptation in Australian populations of barramundi (Lates calcarifer). Marine Genomics, 11. pp. 45-52.
Date Deposited:	29 Oct 2015 03:56
FoR Codes:	06 BIOLOGICAL SCIENCES > 0603 Evolutionary Biology > 060303 Biological Adaptation @ 34% 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060405 Gene Expression (incl Microarray and other genome-wide approaches) @ 33% 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060411 Population, Ecological and Evolutionary Genetics @ 33%
SEO Codes:	83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830102 Aquaculture Fin Fish (excl. Tuna) @ 50% 97 EXPANDING KNOWLEDGE > 970106 Expanding Knowledge in the Biological Sciences @ 50%
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