Edmunds, Richard C. (2009) Evidence for thermal adaptation among geographically, genetically and thermally distinct populations of the Australian barramundi, Lates calcarifer (Bloch 1790): a multi-level approach. PhD thesis, James Cook University.

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Abstract

The ability of tropical fish to adapt and/or acclimate to their native thermal habitat is of biological interest, especially in the face of climate change. Evidence for this can be sought at the phenotypic, genotypic, transcript abundance and/or deduced protein sequence level. In this thesis a multi-level approach is employed to examine the question of thermal adaptation in a tropical fish. I use the Australian barramundi (Lates calcarifer) as a target species because genetically distinct populations occur throughout their tropical distribution range. The phenotype of L. calcarifer from two such divergent populations was investigated for a response to two ecologically relevant stressors, namely temperature and swimming challenge. Subsequently investigation of genotype, transcript abundance and deduced protein sequence of the candidate gene lactate dehydrogenase-B (ldh-b) in two discrete Australian L. calcarifer populations were carried out. This candidate gene was chosen because of its known metabolic (aerobic) function and previous association with thermal adaptation and swimming performance in the temperate fish Fundulus heteroclitus. This thesis presents novel evidence for local adaptation of tropical Australian L. calcarifer to their native thermal habitats using a comparative and integrative multi-level approach, as follows:

Chapter 2 investigated at the organismal level whether juveniles (n = approx. 300) from two genetically discrete Australian L. calcarifer populations (Darwin, NT: 12° S, 130° E and Bowen, QLD: 20° S, 148° E; herein referred to as northern and southern, respectively) possess differences in mortality rate, growth rate and exhaustive swimming capacity following 28 day acclimation to both cold- (20°C) and heat-stress (35°C) conditions. This chapter uses swimming challenge as an ecologically relevant aerobic stress, applied in the presence and absence of thermal stress, to establish physiological or metabolic differences between these discrete L. calcarifer populations. Among-population differences were consistent with local thermal adaptation, with southern juveniles from the cooler climatic region exhibiting significantly higher performance than their northern counterparts under cold-stress (20°C) conditions (F₁, ₁₂ = 18.023; p = 0.001). Conversely, northern populations from the warmer climatic region performed significantly better than their southern counterparts under heat-stress (35°C) conditions (F₁, ₁₂ = 13.948; p = 0.003). Mortality rates differed significantly from expectation at the two temperature stress conditions, with cold-stress resulting in significantly reduced mortality (1.4 %) for the southern population (p < 0.05) and heat-stress resulting in significantly increased mortality for both populations (53.2 and 17.6 % respectively), but particularly for the southern population (p < 0.02). These phenotypic differences indicate that discrete L. calcarifer populations may be locally adapted to their native thermal regime across their tropical Australian distribution and provides the basis for subsequent investigations of genotype, transcript abundance and deduced amino acid sequences for the candidate gene lactate dehydrogenase-b (ldh-b).

Chapter 3 investigates the ldh-b genotype in Lates calcarifer and congeneric Lates niloticus and compares the nucleotide (exons and introns) and deduced amino acid sequence of the ldh-b locus among these two tropical fishes. Ldh-b was 5,004 and 3,527 bp in length in L. calcarifer and L. niloticus, respectively, with coding regions comprising 1,005 bp in both species. A high level of sequence homology existed between species for both coding and non-coding regions (> 97% homology), corresponding to a 98.5% amino acid sequence homology. All six known functional sites within the encoded protein sequence (LDH-B) were conserved between the two Lates species. This chapter also identifies putative regulatory motifs and elements embedded within non-coding (intron) regions including 10 simple sequence repeat (SSR) motifs and 30 putative microRNA elements (miRNAs).. Five single nucleotide polymorphisms (SNPs) were also identified within putative miRNA regions. This ldh-b characterization provided valuable sequence for the subsequent population genetics and transcript abundance studies.

Chapter 4 uses a population approach to assess the level of nucleotide (exons and introns) and deduced amino acid sequence variation in the ldh-b locus both among and within Australian L. calcarifer populations from different thermal regimes (n = 8). We identified a high homology of nucleotide and amino acid sequences among discrete populations lending further support to a hypothesis for selective constraint acting on the ldh-b locus in this tropical fish (Chapter 6). Interestingly, we found that the southernmost population sampled (Gladstone, Queensland, 23°S 151°E) differed significantly (p < 0.05) from all the other six populations screened with F(ST) values ranging from 0.12 to 0.30, making it the ideal target population for future research on this thermally sensitive species. This chapter also reveals the presence of several intronic SNPs which may impact several overlapping putative miRNA elements and may ultimately influence the expression of the ldh-b locus itself or other loci of the transcriptome.

Chapter 5 investigates transcript abundance by quantifying the magnitude of variation in hepatic ldh-b transcripts among the two sampled populations (Darwin, NT and Bowen, QLD) following 28 day acclimations to cold-stress (20°C), heat-stress (35°C) and native control temperature (25 or 30°C) treatments. Ldh-b transcript abundance was also quantified in individuals subjected to swimming stress in control temperatures and in the presence of cold- or heat-stress, following acclimation to these temperatures. This chapter uses L. calcarifer-specific qRT-PCR primers and Sybr GreenER fluorescence based assays to quantify ldh-b transcript abundance. Fish from the southern and northern populations exhibited a significant increase in hepatic ldh-b transcript abundance (F₅, ₄₁ = 6.459; p < 0.001 and F₅, ₃₉ = 3.866; p = 0.006 respectively) in response to swimming stress at their respective native culturing temperatures (25°C and 30°C respectively) (i.e. in the absence of thermal stress). Fish from both populations exhibited a significant increase in hepatic ldh-b transcript abundance following heat-stress (35°C) acclimation compared to controls. Fish from the southern, but not the northern, population possessed significantly higher abundance of hepatic ldh-b transcripts following cold-stress (20°C) acclimation compared to controls. These observations suggest that the southern population possesses a unique transcriptional response to that of the northern population and that southern fish may be evolutionarily accustomed to greater variance in seasonal temperatures.

Chapter 6 uses an analytical approach, in lieu of empirical protein data, to identify evidence of selective constraint acting on ldh-b in two tropical congeneric Lates (L. calcarifer and L. niloticus) and Plectropomus (P. leopardus and P. laevis) species compared to one temperate congeneric species pair (Fundulus). In regard to both coding nucleotide (ldh-b) and amino acid (LDH-B) sequences, pairwise and phylogenetic comparisons identify that the two tropical species pairs are more homologous to one another (94 – 96%) than either is to the temperate species pair (90 – 92%). Observed pairwise species differences between coding and deduced amino acid sequences do not occur at known functional residues. In summary, this analytical approach identifies that selective constraint appears to be acting on both coding nucleotide and deduced amino acid sequences of this candidate gene.

Chapter 7 synthesizes the combined results of the comparative and integrative multi-level approach used in this thesis and presents novel ideas for future studies which would expand on the biological insight generated by this thesis and could provided further evidence for local adaptation of Australian L. calcarifer to their native tropical habitats.

Item ID:	29298
Item Type:	Thesis (PhD)
Keywords:	thermal adaptation; functional genomics; candidate gene approach; barramundi; Lates calcarifer
Related URLs:	http://eprints.jcu.edu.au/5518/ http://eprints.jcu.edu.au/10555/ http://eprints.jcu.edu.au/11838/
Additional Information:	Publications arising from this thesis are available from the Related URLs field. The publications are: Chapter 3: Edmunds, Richard C., van Herwerden, Lynne, Smith-Keune, Carolyn, and Jerry, Dean R. (2009) Comparative characterization of a temperature responsive gene (lactate dehydrogenase-B, ldh-b) in two congeneric tropical fish, Lates calcarifer and Lates niloticus. International Journal of Biological Sciences, 5 (6). pp. 558-569. Appendix: Edmunds, Richard C., Hillersøy, Grethe, Momigliano, Paolo, and Van Herwerden, Lynne (2009) Classic approach revitalizes genomics: complete characterization of a candidate gene for thermal adaptation in two coral reef fishes. Marine Genomics, 2 (3-4). pp. 215-222. Appendix: Edmunds, Richard C., Van Herwerden, Lynne, and Fulton, Christopher J. (2010) Population-specific locomotor phenotypes are displayed by barramundi, Lates calcarifer, in response to thermal stress. Canadian Journal of Fisheries and Aquatic Sciences, 67 (7). pp. 1068-1074.
Date Deposited:	29 Sep 2013 22:48
FoR Codes:	06 BIOLOGICAL SCIENCES > 0602 Ecology > 060205 Marine and Estuarine Ecology (incl Marine Ichthyology) @ 33% 06 BIOLOGICAL SCIENCES > 0603 Evolutionary Biology > 060303 Biological Adaptation @ 33% 06 BIOLOGICAL SCIENCES > 0604 Genetics > 060411 Population, Ecological and Evolutionary Genetics @ 34%
SEO Codes:	83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8398 Environmentally Sustainable Animal Production > 839899 Environmentally Sustainable Animal Production not elsewhere classified @ 50% 83 ANIMAL PRODUCTION AND ANIMAL PRIMARY PRODUCTS > 8301 Fisheries - Aquaculture > 830102 Aquaculture Fin Fish (excl. Tuna) @ 50%
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