From transcriptome to biological function: environmental stress in an ectothermic vertebrate, the coral reef fish Pomacentrus moluccensis

Background
Our understanding of the importance of transcriptional regulation for biological function is continuously improving. We still know, however, comparatively little about how environmentally induced stress affects gene expression in vertebrates, and the consistency of transcriptional stress responses to different types of environmental stress. In this study, we used a multi-stressor approach to identify components of a common stress response as well as components unique to different types of environmental stress. We exposed individuals of the coral reef fish Pomacentrus moluccensis to hypoxic, hyposmotic, cold and heat shock and measured the responses of approximately 16,000 genes in liver. We also compared winter and summer responses to heat shock to examine the capacity for such responses to vary with acclimation to different ambient temperatures.
Results
We identified a series of gene functions that were involved in all stress responses examined here, suggesting some common effects of stress on biological function. These common responses were achieved by the regulation of largely independent sets of genes; the responses of individual genes varied greatly across different stress types. In response to heat exposure over five days, a total of 324 gene loci were differentially expressed. Many heat-responsive genes had functions associated with protein turnover, metabolism, and the response to oxidative stress. We were also able to identify groups of co-regulated genes, the genes within which shared similar functions.
Conclusion
This is the first environmental genomic study to measure gene regulation in response to different environmental stressors in a natural population of a warm-adapted ectothermic vertebrate. We have shown that different types of environmental stress induce expression changes in genes with similar gene functions, but that the responses of individual genes vary between stress types. The functions of heat-responsive genes suggest that prolonged heat exposure leads to oxidative stress and protein damage, a challenge of the immune system, and the re-allocation of energy sources. This study hence offers insight into the effects of environmental stress on biological function and sheds light on the expected sensitivity of coral reef fishes to elevated temperatures in the future.

Heterologous microarray experiments used to identify the early gene response to heat stress in a coral reef fish

Coral reef fishes are expected to experience rising sea surface temperatures due to climate change. How well tropical reef fishes will respond to these increased temperatures and which genes are important in the response to elevated temperatures is not known. Microarray technology provides a powerful tool for gene discovery studies, but the development of microarrays for individual species can be expensive and time-consuming. In this study, we tested the suitability of a Danio rerio oligonucleotide microarray for application in a species with few genomic resources, the coral reef fish Pomacentrus moluccensis. Results from a comparative genomic hybridization experiment and direct sequence comparisons indicate that for most genes there is considerable sequence similarity between the two species, suggesting that the D. rerio array is useful for genomic studies of P. moluccensis. We employed this heterologous microarray approach to characterize the early transcriptional response to heat stress in P. moluccensis. A total of 111 gene loci, many of which are involved in protein processing, transcription, and cell growth, showed significant changes in transcript abundance following exposure to elevated temperatures. Changes in transcript abundance were validated for a selection of candidate genes using quantitative real-time polymerase chain reaction. This study demonstrates that heterologous microarrays can be successfully employed to study species for which specific microarrays have not yet been developed, and so have the potential to greatly enhance the utility of microarray technology to the field of environmental and functional genomics.

Small within-day increases in temperature affects boldness and alters personality in coral reef fish

Consistent individual differences in behaviour, termed personality, are common in animal populations and can constrain their responses to ecological and environmental variation, such as temperature. Here, we show for the first time that normal within-daytime fluctuations in temperature of less than 3°C have large effects on personality for two species of juvenile coral reef fish in both observational and manipulative experiments. On average, individual scores on three personality traits (PTs), activity, boldness and aggressiveness, increased from 2.5- to sixfold as a function of temperature. However, whereas most individuals became more active, aggressive and bold across temperature contexts (were plastic), others did not; this changed the individual rank order across temperatures and thus altered personality. In addition, correlations between PTs were consistent across temperature contexts, e.g. fish that were active at a given temperature also tended to be both bold and aggressive. These results (i) highlight the importance of very carefully controlling for temperature when studying behavioural variation among and within individuals and (ii) suggest that individual differences in energy metabolism may contribute to animal personality, given that temperature has large direct effects on metabolic rates in ectotherms.

Habitat utilization by juvenile hawksbill turtles (Eretmochelys imbricata, Linnaeus, 1766) around a shallow water coral reef

Contemporary studies of sea turtle diving behaviour are generally based upon sophisticated techniques such as the attachment of time depth recorders. However, if the risks of misinterpretation are to be minimized, it is essential that electronic data are analysed in the light of first-hand observations. To this aim, we set out to make observations of juvenile hawksbill turtles (Eretmochelys imbricata, Linnaeus, 1766) foraging and resting in a shallow water coral reef habitat around the granitic Seychelles (4°'S, 55°'E). Data were collected from six study sites characterized by a shallow reef plateau (<5 m) and a flat sandy area at the base of the reef face (<10 m). Observation data were categorized into the following behaviours: (1) stationary foraging; (2) active foraging; (3) resting; and (4) assisted resting. Central to this investigation was the development of a technique for accurately estimating the size of sea turtles in situ based upon previously tested techniques for reef fishes. This revealed that through calibration, the curved carapace length (CCL) of marine turtles can be consistently estimated to within 10 cm of their actual size. Although rudimentary, this has advantages for assessing the residency or absence of specific life history stages from particular environments. Indeed, our data supported previous claims that following the reproductive season, adult hawksbills in the region may move away from the nesting beaches to alternative foraging grounds whilst immature turtles (following the pelagic juvenile stage) may opt to reside in areas close to their natal beaches. With regards to habitat utilization, juvenile hawksbills displayed an alternating pattern of short, shallow foraging dives followed by deeper, longer resting dives. These findings are consistent with previous electronic studies of free-range diving in this species and suggest that the maximization of resting duration may be an important factor driving this behaviour.

Using otolith microchemistry and shape to assess the habitat value of oil structures for reef fish

Over 7500 oil and gas structures (e.g. oil platforms) are installed in offshore waters worldwide and many will require decommissioning within the next two decades. The decision to remove such structures or turn them into reefs (i.e. 'rigs-to-reefs') hinges on the habitat value they provide, yet this can rarely be determined because the residency of mobile species is difficult to establish. Here, we test a novel solution to this problem for reef fishes; the use of otolith (earstone) properties to identify oil structures of residence. We compare the otolith microchemistry and otolith shape of a site-attached coral reef fish (Pseudanthias rubrizonatus) among four oil structures (depth 82-135 m, separated by 9.7-84.2 km) on Australia's North West Shelf to determine if populations developed distinct otolith properties during their residency. Microchemical signatures obtained from the otolith edge using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) differed among oil structures, driven by elements Sr, Ba and Mn, and to a lesser extent Mg and Fe. A combination of microchemical data from the otolith edge and elliptical Fourier (shape) descriptors allowed allocation of individuals to their 'home' structure with moderate accuracy (overall allocation accuracy: 63.3%, range: 45.5-78.1%), despite lower allocation accuracies for each otolith property in isolation (microchemistry: 47.5%, otolith shape: 45%). Site-specific microchemical signatures were also stable enough through time to distinguish populations during 3 separate time periods, suggesting that residence histories could be recreated by targeting previous growth zones in the otolith. Our results indicate that reef fish can develop unique otolith properties during their residency on oil structures which may be useful for assessing the habitat value of individual structures. The approach outlined here may also be useful for determining the residency of reef fish on artificial reefs, which would assist productivity assessments of these habitats.

Asexual reproduction does not produce clonal populations of the brooding coral Pocillopora damicornis on the Great Barrier Reef, Australia

We have investigated the relationship between genotypic diversity, the mode of production of brooded larvae and disturbance in a range of reef habitats, in order to resolve the disparity between the reproductive mode and population structure reported for the brooding coral Pocillopora damicornis. Within 14 sites across six habitats, the ratio of the observed (G o) to the expected (G e) genotypic diversity ranged from 69 to 100% of that expected for random mating. At three other sites in two habitats the G o /G e ranged from 35 to 53%. Two of these sites were recently bleached, suggesting that asexual recruitment may be favoured after disturbance. Nevertheless, our data suggest that brooded larvae, from each of five habitats surveyed, were asexually produced. While clonal recruitment may be important in disturbed habitats, the lack of clonality detected, both in this and earlier surveys of 40 other sites, implies that a disturbance is normally insufficient to explain this species’ continued investment in clonal reproduction.

Mating system variation in the hermaphroditic brooding coral, Seriatopora hystrix

Self-compatible, hermaphroditic marine invertebrates have the potential to self-fertilize in the absence of mates or under sperm-limited conditions, and outcross when sperm is available from a variety of males. Hence, many hermaphroditic marine invertebrates may have evolved mixed-mating systems that involve facultative self-fertilization. Such mixed-mating strategies are well documented for plants but have rarely been investigated in animals. Here, I use allozyme markers to make estimates of selfing from population surveys of reef slope and reef flat sites, and contrast this with direct estimates of selfing from progeny-array analysis, for the brooding coral Seriatopora hystrix. Consistent heterozygote deficits previously reported for S. hystrix suggests that inbreeding (including the extreme of selfing) may be common in this species. I detected significant levels of inbreeding within populations (FIS=0.48) and small but significant differentiation among all sites (FST=0.04). I detected no significant differentiation among habitats (FHT=0.009) though among site differentiation did occur within the reef slope habitat (FSH=0.06), but not within the reef flat habitat (FSH=0.015). My direct estimates of outcrossing for six colonies and their progeny from a single reef flat site revealed an intermediate value (tm (±s.d.)=0.53±0.20). Inbreeding coefficients calculated from progeny arrays (Fe=0.31) were similar to indirect estimates based on adult genotype frequencies for that site (FIS=0.38). This study confirms that the mating system of this brooding coral is potentially variable, with both outcrossing and selfing.

Residency and spatial use by reef sharks of an isolated seamount and its implications for conservation

Although marine protected areas (MPAs) are a common conservation strategy, these areas are often designed with little prior knowledge of the spatial behaviour of the species they are designed to protect. Currently, the Coral Sea area and its seamounts (north-east Australia) are under review to determine if MPAs are warranted. The protection of sharks at these seamounts should be an integral component of conservation plans. Therefore, knowledge on the spatial ecology of sharks at the Coral Sea seamounts is essential for the appropriate implementation of management and conservation plans. Acoustic telemetry was used to determine residency, site fidelity and spatial use of three shark species at Osprey Reef: whitetip reef sharks Triaenodon obesus, grey reef sharks Carcharhinus amblyrhynchos and silvertip sharks Carcharhinus albimarginatus. Most individuals showed year round residency at Osprey Reef, although five of the 49 individuals tagged moved to the neighbouring Shark Reef (∼14 km away) and one grey reef shark completed a round trip of ∼250 km to the Great Barrier Reef. Additionally, individuals of white tip and grey reef sharks showed strong site fidelity to the areas they were tagged, and there was low spatial overlap between groups of sharks tagged at different locations. Spatial use at Osprey Reef by adult sharks is generally restricted to the north-west corner. The high residency and limited spatial use of Osprey Reef suggests that reef sharks would be highly vulnerable to targeted fishing pressure and that MPAs incorporating no-take of sharks would be effective in protecting reef shark populations at Osprey and Shark Reef.

Climate change as an unexpected co-factor promoting coral eating seastar (Acanthaster planci) outbreaks

Coral reefs face a crisis due to local and global anthropogenic stressors. A large proportion of the ~50% coral loss on the Great Barrier Reef has been attributed to outbreaks of the crown-of-thorns-seastar (COTS). A widely assumed cause of primary COTS outbreaks is increased larval survivorship due to higher food availability, linked with anthropogenic runoff . Our experiment using a range of algal food concentrations at three temperatures representing present day average and predicted future increases, demonstrated a strong influence of food concentration on development is modulated by temperature. A 2°C increase in temperature led to a 4.2–4.9 times (at Day 10) or 1.2–1.8 times (Day 17) increase in late development larvae. A model indicated that food was the main driver, but that temperature was an important modulator of development. For instance, at 5000 cells ml−1 food, a 2°C increase may shorten developmental time by 30% and may increase the probability of survival by 240%. The main contribution of temperature is to ‘push’ well-fed larvae faster to settlement. We conclude that warmer sea temperature is an important co-factor promoting COTS outbreaks.

Biochemical responses to ocean acidification contrast between tropical corals with high and low abundances at volcanic carbon dioxide seeps

At two natural volcanic seeps in Papua New Guinea, the partial pressure of carbon dioxide (pCO2) in the seawater is consistent with projections for 2100. Here, the cover of massive scleractinian corals Porites spp. is twice as high at elevated compared with ambient pCO2, while that of branching corals such as Acropora millepora is greater than twofold reduced. To assess the underlying mechanisms for such community shifts under long-term exposure to elevated pCO2, biochemical parameters related to tissue biomass, energy storage, pigmentation, cell protection, and cell damage were compared between Porites spp. and A. millepora from control (mean pHtotal = 8.1, pCO2 = 323 µatm) and CO2 seep sites (mean pHtotal = 7.8, pCO2 = 803 µatm) each at two reefs. In Porites spp., only one of the biochemical parameters investigated (the ratio of photoprotective to light-harvesting pigments) responded to pCO2, while tissue biomass, total lipids, total proteins, and some pigments differed between the two reefs, possibly reflecting differences in food availability. Furthermore, some fatty acids showed pCO2 –reef interactions. In A. millepora, most pigments investigated were reduced at elevated pCO2, while other parameters (e.g. tissue biomass, total proteins, total lipids, protein carbonyls, some fatty acids and pigments) differed between reefs or showed pCO2–reef interactions. Tissue biomass, total lipids, and cell-protective capacities were distinctly higher in Porites spp. than in A. millepora, indicating higher resistance to environmental stress in massive Porites. However, our data suggest that important biochemical measures remain relatively unaffected in these two coral species in response to elevated pCO2 up to 800 µatm, with most responses being smaller than differences between species and locations, and also when compared with responses to other environmental stressors such as ocean warming.