Rapid counting and spectral sorting of live coral larvae using large-particle flow cytometry

Research with coral embryos and larvae often requires laborious manual counting and sorting of individual specimens, usually via microscopy. Because many coral species spawn only once per year during a narrow temporal window, sample processing is a time-limiting step for research on the early life-history stages of corals. Flow cytometry, an automated technique for measuring and sorting particles, cells, and cell-clusters, is a potential solution to this bottleneck. Yet most flow cytometers do not accommodate live organisms of the size of most coral embryos (> 250 µm), and sample processing is often destructive. Here we tested the ability of a large-particle flow cytometer with a gentle pneumatic sorting mechanism to process and spectrally sort live and preserved Montipora capitata coral embryos and larvae. Average survival rates of mechanically-sorted larvae were over 90% and were comparable to those achieved by careful hand-sorting. Preserved eggs and embryos remained intact throughout the sorting process and were successfully sorted based on real-time size and fluorescence detection. In-line bright-field microscopy images were captured for each sample object as it passed through the flow-cell, enabling the identification of early-stage embryos (2-cell to morula stage). Samples were counted and sorted at an average rate of 4 s larva−1 and as high as 0.2 s larva−1 for high-density samples. Results presented here suggest that large-particle flow cytometry has the potential to significantly increase efficiency and accuracy of data collection and sample processing during time-limited coral spawning events, facilitating larger-scale and higher-replication studies with an expanded number of species

Niche specialization of reef-building corals in the mesophotic zone: metabolic trade-offs between divergent Symbiodinium types

The photobiology of two reef corals and the distribution of associated symbiont types were investigated over a depth gradient of 0–60 m at Scott Reef, Western Australia. Pachyseris speciosa hosted mainly the same Symbiodinium C type similar to C3 irrespective of sampling depth. By contrast, Seriatopora hystrix hosted predominantly Symbiodinium type D1a or D1a-like at shallow depths while those in deeper water were dominated by a Symbiodinium C type closely related to C1. The photosynthesis/respiration (P/R) ratio increased consistently with depth at the two sampling times (November 2008 and April 2009) for P. speciosa and in November 2008 only for S. hystrix, suggesting a reduction in metabolic energy expended for every unit of energy obtained from photosynthesis. However, in April 2009, shallow colonies of S. hystrix exhibited decreased P/R ratios down to depths of approximately 23 m, below which the ratio increased towards the maximum depth sampled. This pattern was mirrored by changes in tissue biomass determined as total protein content. The depth of change in the direction of the P/R ratio correlated with a shift from Symbiodinium D to C-dominated colonies. We conclude that while photobiological flexibility is vital for persistence in contrasting light regimes, a shift in Symbiodinium type may also confer a functional advantage albeit at a metabolic cost with increased depth

The propensity of non-concussive and concussive head contacts during elite-level women's rugby league matches : A prospective analysis of over 14,000 tackle events

Objectives Identify the frequency, propensity, and factors related to tackle events which result in contact with the head in elite-level women's rugby league. Design Prospective video analysis study. Methods Video footage from 59 Women's Super League matches were analysed (n = 14,378 tackle events). All tackle events were coded as no head contact or head contact. Other independent variables included: area contacting head, impacted player, concussion outcome, penalty outcome, round of competition, time in match and team standard. Results There were 83.0 ± 20.0 (propensity 304.0/1000 tackle events) head contacts per match. The propensity of head contact was significantly greater for the tackler than ball-carrier (178.5 vs. 125.7/1000 tackle events; incident rate ratio 1.42, 95 % confidence interval 1.34 to 1.50). Head contacts occurring from an arm, shoulder, and head occurred significantly more than any other contact type. The propensity of concussions was 2.7/1000 head contacts. There was no significant influence of team standard or time in match on the propensity of head contacts. Conclusions The observed head contacts can inform interventions, primarily focusing on the tackler not contacting the ball-carrier's head. The tackler's head should also be appropriately positioned to avoid contact with the ball-carrier's knee (highest propensity for concussion). The findings are consistent with other research in men's rugby. Law modifications and/or enforcement (reducing the number of un-penalised head contacts), concurrent with coaching interventions (optimising head placement or reducing the head being contacted) may help minimise head contact risk factors for women's rugby league

Water Contamination Reduces the Tolerance of Coral Larvae to Thermal Stress

Coral reefs are highly susceptible to climate change, with elevated sea surface temperatures (SST) posing one of the main threats to coral survival. Successful recruitment of new colonies is important for the recovery of degraded reefs following mortality events. Coral larvae require relatively uncontaminated substratum on which to metamorphose into sessile polyps, and the increasing pollution of coastal waters therefore constitutes an additional threat to reef resilience. Here we develop and analyse a model of larval metamorphosis success for two common coral species to quantify the interactive effects of water pollution (copper contamination) and SST. We identify thresholds of temperature and pollution that prevent larval metamorphosis, and evaluate synergistic interactions between these stressors. Our analyses show that halving the concentration of Cu can protect corals from the negative effects of a 2–3°C increase in SST. These results demonstrate that effective mitigation of local impacts can reduce negative effects of global stressors

Crustose Coralline Algae and a Cnidarian Neuropeptide Trigger Larval Settlement in Two Coral Reef Sponges

In sessile marine invertebrates, larval settlement is fundamental to population maintenance and persistence. Cues contributing to the settlement choices and metamorphosis of larvae have important implications for the success of individuals and populations, but cues mediating larval settlement for many marine invertebrates are largely unknown. This study assessed larval settlement in two common Great Barrier Reef sponges, Coscinoderma matthewsi and Rhopaloeides odorabile, to cues that enhance settlement and metamorphosis in various species of scleractinian coral larvae. Methanol extracts of the crustose coralline algae (CCA), Porolithon onkodes, corresponding to a range of concentrations, were used to determine the settlement responses of sponge larvae. Cnidarian neuropeptides (GLW-amide neuropeptides) were also tested as a settlement cue. Settlement in both sponge species was approximately two-fold higher in response to live chips of CCA and optimum concentrations of CCA extract compared to 0.2 µm filtered sea water controls. Metamorphosis also increased when larvae were exposed to GLW-amide neuropeptides; R. odorabile mean metamorphosis reached 42.0±5.8% compared to 16.0±2.4% in seawater controls and in C. matthewsi mean metamorphosis reached 68.3±5.4% compared to 36.7±3.3% in seawater controls. These results demonstrate the contributing role chemosensory communication plays in the ability of sponge larvae to identify suitable habitat for successful recruitment. It also raises the possibility that larvae from distinct phyla may share signal transduction pathways involved in metamorphosis

Habitat Specialization in Tropical Continental Shelf Demersal Fish Assemblages

The implications of shallow water impacts such as fishing and climate change on fish assemblages are generally considered in isolation from the distribution and abundance of these fish assemblages in adjacent deeper waters. We investigate the abundance and length of demersal fish assemblages across a section of tropical continental shelf at Ningaloo Reef, Western Australia, to identify fish and fish habitat relationships across steep gradients in depth and in different benthic habitat types. The assemblage composition of demersal fish were assessed from baited remote underwater stereo-video samples (n = 304) collected from 16 depth and habitat combinations. Samples were collected across a depth range poorly represented in the literature from the fringing reef lagoon (1–10 m depth), down the fore reef slope to the reef base (10–30 m depth) then across the adjacent continental shelf (30–110 m depth). Multivariate analyses showed that there were distinctive fish assemblages and different sized fish were associated with each habitat/depth category. Species richness, MaxN and diversity declined with depth, while average length and trophic level increased. The assemblage structure, diversity, size and trophic structure of demersal fishes changes from shallow inshore habitats to deeper water habitats. More habitat specialists (unique species per habitat/depth category) were associated with the reef slope and reef base than other habitats, but offshore sponge-dominated habitats and inshore coral-dominated reef also supported unique species. This suggests that marine protected areas in shallow coral-dominated reef habitats may not adequately protect those species whose depth distribution extends beyond shallow habitats, or other significant elements of demersal fish biodiversity. The ontogenetic habitat partitioning which is characteristic of many species, suggests that to maintain entire species life histories it is necessary to protect corridors of connected habitats through which fish can migrate

The effects of Produced Formation Water (PFW) on coral and isolated symbiotic dinoflagellates of coral

There is concern of the effects of Produced Formation Water (PFW, an effluent of the offshore oil and gas industry) on temperate/tropical marine organisms of the North West Shelf (NWS) of Australia. Little is known of the effects of PFW on tropical marine organisms, especially keystone species. Exposing the coral Plesiastrea versipora to a range (3-50% v/v) of PFW from Harriet A oil platform resulted in a reduction in photochemical efficiency of the symbiotic dinoflagellate algae in hospite ( in the coral tissues), assessed as a decrease in the ratio of variable fluorescence (F-v) to maximal fluorescence (F-m) measured using chlorophyll fluorescence techniques. Significant differences were noted at PFW concentrations >12.5% ( v/v). In corals where F-v/F-m was significantly lowered by PFW exposure, significant discolouration of the tissues occurred in a subsequent 4-day observation period. The discolouration ( coral bleaching) was caused by a loss of the symbiotic dinoflagellates from the tissues, a known sublethal stress response of corals. PFW caused a significant decrease in F-v/F-m in symbiotic dinoflagellates freshly isolated from the coral Heliofungia actiniformis at 6.25% PFW, slightly lower than the studies in hospite. Corals exposed to lower PFW concentrations (range 0.1%-10% PFW v/v) for longer periods (8 days) showed no decrease in F-v/F-m, discolouration, loss of symbiotic dinoflagellates or changes in gross photosynthesis or respiration ( measured using O-2 exchange techniques). The study demonstrates minor toxicity of PFW from Harriet A oil platform to corals and their symbiotic algae

Immobilisation of living coral embryos and larvae

Embedding and immobilisation of living cells and microorganisms is used in a variety of research and commercial applications. Here we report the successful extended immobilisation of coral larvae in a low-gelling temperature agarose. Embryos and larvae of five broadcast-spawning Scleractinian species were immobilised in agarose gel and tested in a series of exploratory survival and settlement assays. The optimal developmental stage for immobilisation was after ciliation at approximately 24 hours post-fertilisation, after which, survival of immobilised larvae of all species was nearly 100%. In long-term assays, 50% of Montipora digitata larvae survived immobilised for 89 days. Furthermore, immobilised larvae of multiple species, that were released from the agarose, generally remained capable of settlement. These results demonstrate that the immobilisation of the early life-history stages of corals is possible for a variety of applications in basic and applied science