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

Perception Test: A Diagnostic Benchmark for Multimodal Video Models

We propose a novel multimodal video benchmark - the Perception Test - to evaluate the perception and reasoning skills of pre-trained multimodal models (e.g. Flamingo, BEiT-3, or GPT-4). Compared to existing benchmarks that focus on computational tasks (e.g. classification, detection or tracking), the Perception Test focuses on skills (Memory, Abstraction, Physics, Semantics) and types of reasoning (descriptive, explanatory, predictive, counterfactual) across video, audio, and text modalities, to provide a comprehensive and efficient evaluation tool. The benchmark probes pre-trained models for their transfer capabilities, in a zero-shot / few-shot or limited finetuning regime. For these purposes, the Perception Test introduces 11.6k real-world videos, 23s average length, designed to show perceptually interesting situations, filmed by around 100 participants worldwide. The videos are densely annotated with six types of labels (multiple-choice and grounded video question-answers, object and point tracks, temporal action and sound segments), enabling both language and non-language evaluations. The fine-tuning and validation splits of the benchmark are publicly available (CC-BY license), in addition to a challenge server with a held-out test split. Human baseline results compared to state-of-the-art video QA models show a significant gap in performance (91.4% vs 43.6%), suggesting that there is significant room for improvement in multimodal video understanding. Dataset, baselines code, and challenge server are available at https://github.com/deepmind/perception_testComment: 25 pages, 11 figure

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

Induction of Larval Metamorphosis of the Coral Acropora millepora by Tetrabromopyrrole Isolated from a Pseudoalteromonas Bacterium

The induction of larval attachment and metamorphosis of benthic marine invertebrates is widely considered to rely on habitat specific cues. While microbial biofilms on marine hard substrates have received considerable attention as specific signals for a wide and phylogenetically diverse array of marine invertebrates, the presumed chemical settlement signals produced by the bacteria have to date not been characterized. Here we isolated and fully characterized the first chemical signal from bacteria that induced larval metamorphosis of acroporid coral larvae (Acropora millepora). The metamorphic cue was identified as tetrabromopyrrole (TBP) in four bacterial Pseudoalteromonas strains among a culture library of 225 isolates obtained from the crustose coralline algae Neogoniolithon fosliei and Hydrolithon onkodes. Coral planulae transformed into fully developed polyps within 6 h, but only a small proportion of these polyps attached to the substratum. The biofilm cell density of the four bacterial strains had no influence on the ratio of attached vs. non-attached polyps. Larval bioassays with ethanolic extracts of the bacterial isolates, as well as synthetic TBP resulted in consistent responses of coral planulae to various doses of TBP. The lowest bacterial density of one of the Pseudoalteromonas strains which induced metamorphosis was 7,000 cells mm−2 in laboratory assays, which is on the order of 0.1 –1% of the total numbers of bacteria typically found on such surfaces. These results, in which an actual cue from bacteria has been characterized for the first time, contribute significantly towards understanding the complex process of acroporid coral larval settlement mediated through epibiotic microbial biofilms on crustose coralline algae

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

Genetic systems and hereditary structures of reef corals

Processes involved in the generation of new coral colonies were investigated with a view to elucidating their consequences for population genetic structure. \ud \ud Electrophoretic and histocompatibility methods of estimating clonal structure in populations were evaluated. Spatial patterns of graft acceptance and rejection were different for two species of Montipora on a patch reef in Kaneohe Bay, Hawaii. Electrophoretically distinct tissues were capable of fusing in both species, indicating that clonal identity is not nescessarily inferred by acceptance of grafts. Tissue fusion between graft pairs of dissimilar genotypes occurred in 18 of 40 cases for M. dilatata and in 3 of 7 cases for M. verrucosa. In the majority of these allogeneic fusions, the donor and recipient colonies had one allele in common at the single polymorphic locus assayed. In one case, fusion occurred between genotypes which shared no alleles. The specificity of the self-recognition response, although different for the two species, was constant through time. It is suggested that self-recognition systems have species-specific levels of genotypic discrimination. A greater understanding of the genetics of invertebrate immunology is required before histocompatibility criteria can be used independently to assess population genetic structure with confidence.\ud \ud The mode and timing of sexual reproduction was investigated in the genus Montipora. During late spring and summer M. verrucosa, M. dilatata, M. verrili, M. studeri and AL flabellata were studied in Kaneohe Bay, Hawaii. Two populations of M. digitata were studied in the central region of the Great Barrier Reef, Australia. All species were simultaneous hermaphrodites, bearing male and female gonads in the same polyp. In each case, there were four male and four female mesenteries arranged as alternating pairs. An annual gametogenic cycle was apparent for M. digitata and inferred for the Hawaiian species. Oocytes mature over approximately six months, exhibiting a steady increase in mean diameter. The most rapid increase in oocyte size occurs in the last month prior to spawning. Testes may also be present at the onset of oocyte development, although almost all development takes place in the final two months prior to spawning. Spawning, which followed a lunar rhythm, was observed for the first time in M. digitata, M. verrucosa and M. dilatata. These species packaged eggs and sperm into a single cluster in each polyp. The gamete clusters, which were shed through the mouth, were highly buoyant and broke up on the surface of the sea. Fertilization and development were external. \ud \ud The mating patterns of four species which broadcast gametes were compared. All species were simultaneous hermaphrodites, from the central region of the Great Barrier Reef, which shed eggs and sperm into the water during brief annual spawning events. Gametes were collected from colonies which had been isolated in separate containers and combined in controlled fertilization trials. Montipora digitata cross-fertilized exclusively. Acropora tenuis, Goniastrea aspera and G. favulus-were capable of self-fertilization, but to varying degrees. In all species, cross-fertilization was the dominant mating pattern. Studies of gamete viability indicated that cross-fertilizations were possible at least 6 hours after spawning, although fertilization rates suggest that most fertilization will occur within 3 hours at high gamete concentrations. \ud \ud Embryogenesis and larval development were studied in detail in the broadcast spawners Montipora digitata, Acropora pulchra, Lobophyllia hemprichii and Favites abdita. Similar developmental patterns and rates were observed in all species. Cleavage was initiated 1.5 to 2.5 hours after spawning. Cell division proceeded by progressive furrow formation. Hollow blastulae were formed within 5 to 6 hours. Endoderm formation appeared to be by delamination after the blastulae had flattened into a disk shape. All species were mobile after 24 hours. Highly mobile planulae were formed after 48 hours. Earliest settlement was after 3 days by Acropora pulchra. The remaining 3 species first settled on day 4. Approximately 45 % of L. hemprichii and F. abdita larvae settled on day 4. In all species the majority of larvae had settled within 7 days. These results suggest that broadcast spawning species have less probability of settling on the parental reef than brooders. \ud \ud Rapidly dividing embryonic tissue was used to investigated coral cytogenetics. A comparison of chromosome number and morphology was made for 4 species of broadcast spawning corals. All species were diploid, with 28 (n = 14) mostly metacentric chromosomes. Differences between the species were largely restricted to alterations in the position of the centromeres on particular chromosomes. Fusion and fission type chromosomal rearrangements, or large quantitatative changes in DNA do not appear to have occurred in the evolution of these broadcast spawning species