Evaluation of laser scanning confocal microscopy as a method for characterizing reef-building coral tissue thickness and Symbiodiniaceae fluorescence.

Predicting the sensitivity of reef-building corals to disturbance, including bleaching, requires an understanding of physiological responses to stressors, which may be limited by destructive sampling and the capacity of common methodologies to characterize early life history stages. We developed a new methodology using laser scanning confocal microscopy (LSCM) to measure and track the physiological condition of corals. In a thermal stress experiment, we used LSCM to track coral condition during bleaching in adults and juveniles of two species, Montipora capitata and Pocillopora acuta Depth of fluorescence in coral tissues provides a proxy measure of tissue thickness, whereas Symbiodiniaceae population fluorescence relates to both population density and chlorophyll a content. In response to thermal stress, there were significant shifts in tissue thickness and Symbiodiniaceae fluorescence with differences between life stages. This method is particularly well suited for detecting shifts in physiological condition of living corals in laboratory studies, especially in small juvenile colonies.

Elevated pCO2 affects tissue biomass composition, but not calcification, in a reef coral under two light regimes.

Ocean acidification (OA) is predicted to reduce reef coral calcification rates and threaten the long-term growth of coral reefs under climate change. Reduced coral growth at elevated pCO2 may be buffered by sufficiently high irradiances; however, the interactive effects of OA and irradiance on other fundamental aspects of coral physiology, such as the composition and energetics of coral biomass, remain largely unexplored. This study tested the effects of two light treatments (7.5 versus 15.7 mol photons m-2 d-1) at ambient or elevated pCO2 (435 versus 957 µatm) on calcification, photopigment and symbiont densities, biomass reserves (lipids, carbohydrates, proteins), and biomass energy content (kJ) of the reef coral Pocillopora acuta from Kane'ohe Bay, Hawai'i. While pCO2 and light had no effect on either area- or biomass-normalized calcification, tissue lipids gdw-1 and kJ gdw-1 were reduced 15% and 14% at high pCO2, and carbohydrate content increased 15% under high light. The combination of high light and high pCO2 reduced protein biomass (per unit area) by approximately 20%. Thus, under ecologically relevant irradiances, P. acuta in Kane'ohe Bay does not exhibit OA-driven reductions in calcification reported for other corals; however, reductions in tissue lipids, energy content and protein biomass suggest OA induced an energetic deficit and compensatory catabolism of tissue biomass. The null effects of OA on calcification at two irradiances support a growing body of work concluding some reef corals may be able to employ compensatory physiological mechanisms that maintain present-day levels of calcification under OA. However, negative effects of OA on P. acuta biomass composition and energy content may impact the long-term performance and scope for growth of this species in a high pCO2 world.

Integrating structure-from-motion photogrammetry with geospatial software as a novel technique for quantifying 3D ecological characteristics of coral reefs.

The structural complexity of coral reefs plays a major role in the biodiversity, productivity, and overall functionality of reef ecosystems. Conventional metrics with 2-dimensional properties are inadequate for characterization of reef structural complexity. A 3-dimensional (3D) approach can better quantify topography, rugosity and other structural characteristics that play an important role in the ecology of coral reef communities. Structure-from-Motion (SfM) is an emerging low-cost photogrammetric method for high-resolution 3D topographic reconstruction. This study utilized SfM 3D reconstruction software tools to create textured mesh models of a reef at French Frigate Shoals, an atoll in the Northwestern Hawaiian Islands. The reconstructed orthophoto and digital elevation model were then integrated with geospatial software in order to quantify metrics pertaining to 3D complexity. The resulting data provided high-resolution physical properties of coral colonies that were then combined with live cover to accurately characterize the reef as a living structure. The 3D reconstruction of reef structure and complexity can be integrated with other physiological and ecological parameters in future research to develop reliable ecosystem models and improve capacity to monitor changes in the health and function of coral reef ecosystems.

Depth specialization in mesophotic corals (Leptoseris spp.) and associated algal symbionts in Hawai'i.

Corals at the lower limits of mesophotic habitats are likely to have unique photosynthetic adaptations that allow them to persist and dominate in these extreme low light ecosystems. We examined the host-symbiont relationships from the dominant coral genus Leptoseris in mesophotic environments from Hawai'i collected by submersibles across a depth gradient of 65-125 m. Coral and Symbiodinium genotypes were compared with three distinct molecular markers including coral (COX1-1-rRNA intron) and Symbiodinium (COI) mitochondrial markers and nuclear ITS2. The phylogenetic reconstruction clearly resolved five Leptoseris species, including one species (Leptoseris hawaiiensis) exclusively found in deeper habitats (115-125 m). The Symbiodinium mitochondrial marker resolved three unambiguous haplotypes in clade C, which were found at significantly different frequencies between host species and depths, with one haplotype exclusively found at the lower mesophotic extremes (95-125 m). These patterns of host-symbiont depth specialization indicate that there are limits to connectivity between upper and lower mesophotic zones, suggesting that niche specialization plays a critical role in host-symbiont evolution at mesophotic extremes.

Protein expression and genetic structure of the coral Porites lobata in an environmentally extreme Samoan back reef: does host genotype limit phenotypic plasticity?

The degree to which coral reef ecosystems will be impacted by global climate change depends on regional and local differences in corals' susceptibility and resilience to environmental stressors. Here, we present data from a reciprocal transplant experiment using the common reef building coral Porites lobata between a highly fluctuating back reef environment that reaches stressful daily extremes, and a more stable, neighbouring forereef. Protein biomarker analyses assessing physiological contributions to stress resistance showed evidence for both fixed and environmental influence on biomarker response. Fixed influences were strongest for ubiquitin-conjugated proteins with consistently higher levels found in back reef source colonies both pre and post-transplant when compared with their forereef conspecifics. Additionally, genetic comparisons of back reef and forereef populations revealed significant population structure of both the nuclear ribosomal and mitochondrial genomes of the coral host (F(ST) = 0.146 P < 0.0001, F(ST) = 0.335 P < 0.0001 for rDNA and mtDNA, respectively), whereas algal endosymbiont populations were genetically indistinguishable between the two sites. We propose that the genotype of the coral host may drive limitations to the physiological responses of these corals when faced with new environmental conditions. This result is important in understanding genotypic and environmental interactions in the coral algal symbiosis and how corals may respond to future environmental changes.

The isolation of a Distal-less gene fragment from two molluscs.

The transcription factor, Distal-less, and its orthologues (e.g. DLL/Dlx) are best known for their roles in proximo-distal and nervous system patterning as well as for their use in inferring the evolution of limbs and other proximo-distal structures. However, evolutionary applications to non-model taxa have primarily employed a polyclonal antibody, and sequence data are generally lacking. We have designed a degenerate PCR primer set specific to the recovery of a Distal-less gene fragment, and present sequence data for two bivalve molluscs, Crassostrea gigas and Nutricola sp. These sequences represent the first Distal-less homologs isolated from lophotrochozoan taxa.

Molluscan engrailed expression, serial organization, and shell evolution.

Whether the serial features found in some molluscs are ancestral or derived is considered controversial. Here, in situ hybridization and antibody studies show iterated engrailed-gene expression in transverse rows of ectodermal cells bounding plate field development and spicule formation in the chiton, Lepidochitona cavema, as well as in cells surrounding the valves and in the early development of the shell hinge in the clam, Transennella tantilla. Ectodermal expression of engrailed is associated with skeletogenesis across a range of bilaterian phyla, suggesting a single evolutionary origin of invertebrate skeletons. The shared ancestry of bilaterian-invertebrate skeletons may help explain the sudden appearance of shelly fossils in the Cambrian. Our interpretation departs from the consideration of canonical metameres or segments as units of evolutionary analysis. In this interpretation, the shared ancestry of engrailed-gene function in the terminal/posterior addition of serially repeated elements during development explains the iterative expression of engrailed genes in a range of metazoan body plans.

Free amino acids exhibit anthozoan "host factor" activity: they induce the release of photosynthate from symbiotic dinoflagellates in vitro.

Reef-building corals and other tropical anthozoans harbor endosymbiotic dinoflagellates. It is now recognized that the dinoflagellates are fundamental to the biology of their hosts, and their carbon and nitrogen metabolisms are linked in important ways. Unlike free living species, growth of symbiotic dinoflagellates is unbalanced and a substantial fraction of the carbon fixed daily by symbiont photosynthesis is released and used by the host for respiration and growth. Release of fixed carbon as low molecular weight compounds by freshly isolated symbiotic dinoflagellates is evoked by a factor (i.e., a chemical agent) present in a homogenate of host tissue. We have identified this "host factor" in the Hawaiian coral Pocillopora damicornis as a set of free amino acids. Synthetic amino acid mixtures, based on the measured free amino acid pools of P. damicornis tissues, not only elicit the selective release of 14C-labeled photosynthetic products from isolated symbiotic dinoflagellates but also enhance total 14CO2 fixation.

Temperature Stress Causes Host Cell Detachment in Symbiotic Cnidarians: Implications for Coral Bleaching.

During the past decade, acute and chronic bleaching of tropical reef corals has occurred with increasing frequency and scale. Bleaching, i.e., the loss of pigment and the decrease in population density of symbiotic dinoflagellates (zooxanthellae), is often correlated with an increase or decrease in sea surface temperature. Because little is known of the cellular events concomitant with thermal bleaching, we have investigated the mechanism of release of zooxanthellae by the tropical sea anemone Aiptasia pulchella and the reef coral Pocillopora damicornis in response to cold and heat stress. Both species released intact host endoderm cells containing zooxanthellae. The majority of the released host cells were viable, but they soon disintegrated in the seawater leaving behind isolated zooxanthellae. The detachment and release of intact host cells suggests that thermal stress causes host cell adhesion dysfunction in these cnidarians. Knowledge of the cellular entity released by the host during bleaching provides insight into both the underlying release mechanism and the way in which natural environmental stresses evoke a bleaching response.

Rapid staining for the sperm penetration assay using a bisbenzimide dye.

Our rapid staining method, accomplished by adding the vital fluorochrome bisbenzimide (Hoechst 33342) directly into the sperm droplet before fertilization, eases visual interpretation of sperm penetration and decondensation. For both within- and between-experiment comparisons, inclusion of bisbenzimide did not influence the overall SPA score or the progress of sperm-egg interaction. These results suggest that bisbenzimide can be used routinely to facilitate scoring of the SPA.

Neuropsychological studies on adolescents with phenylketonuria returned to phenylalanine-restricted diets.

Nine adolescents with phenylketonuria (PKU), who had been on unrestricted diets for 2 to 11 years, underwent serial neuropsychological testing over two consecutive 4- to 5-week periods during which each was maintained on a low-phe diet supplemented in a triple-blind fashion either with L-phe (high phe) or L-alanine (low phe). Assignment to the initial condition was done randomly, and the alternate condition was substituted at the end of the first 4- to 5-week period. In 6 of 7 subjects with PKU, baseline median choice reaction times (RTs) were slower than those of controls matched for age, sex, handedness, and Full-Scale IQ (WISC-R). A highly significant improvement occurred during the low-phe phases of the study. Results suggest that adolescents with PKU on unrestricted diets have a neuropsychological deficit that is out of proportion to their overall intellectual handicap. Moreover, this deficit appears to be at least partly reversible by return to dietary phe restriction despite years of hyperphenylalaninemia.

Single-case study of left cerebral hemispherectomy: development in the first five years of life.

A prospective single-case study of an infant who underwent surgical removal of the entire left cerebral hemisphere at 6 months of age is presented to address, in part, the issue of cerebral hemispheric specialization of function. Serial developmental assessments were made using standardized clinical instruments. A preoperative assessment revealed normal development while postoperative assessments generally revealed delayed development but with a normal rate of maturation. These results are discussed in relation to the issues of early functional specialization of the cerebral cortex and functional plasticity following early brain trauma. In particular, the evidence is interpreted as suggesting that the effect of left hemispherectomy early in life was effectively to delay normal development, and that no specific impairment of either linguistic-communicative or visual-motor/visual-spatial development was apparent.

Experience with adolescents with phenylketonuria returned to phenylalanine-restricted diets.

In order to test the feasibility of returning older children with classical phenylketonuria (PKU) to therapeutically and nutritionally adequate phenylalanine-restricted diets after 8 to 13 years of unrestricted diets, 7 adolescents (13 to 19 years old) of normal or near-normal intelligence with classical PKU were returned to phenylalanine-limited diets for periods of 8 to 10 weeks. During a 4- to 5-week period when the phenylalanine-restricted diet was supplemented with added L-phenylalanine to mimic pretreatment conditions, plasma phenylalanine levels were 1,327 +/- 282 microM on total phenylalanine intakes of 2,794 +/- 248 mg/day (55.3 +/- 11.5 mg/kg/day). During a similar period of dietary phenylalanine restriction, plasma phenylalanine levels were successfully maintained at 713 +/- 266 microM on dietary intakes of 655 +/- 210 mg/day (12.6 +/- 4.3 mg/kg/day). On the basis of 3-day diet records, the intakes of total protein, energy, calcium, phosphorus, iron, vitamins D, E, and A, ascorbic acid, thiamin, riboflavin, niacin, folacin, and vitamin B-12 over the entire 8- to 10-week study period were adequate by the standards of the Recommended Nutrient Intakes (RNI) for Canada, where the study was undertaken. Intakes of magnesium and zinc were significantly lower than the RNI. Although the return to nutritionally and therapeutically adequate dietary phenylalanine restriction was judged to be successful in each case, the amount and intensity of re-education and reinforcement required to maintain compliance was much greater than anticipated.

Interpretation of the Personality Inventory for Children-Revised (PIC-R): influence of cognitive impairment.

The Personality Inventory for Children-Revised (PIC-R) is a parent-report measure of social-emotional adjustment for children 3 to 16 years of age. The present study examined the clinical value of the PIC-R by exploring the impact of age and developmental status on PIC-R profiles, particularly on the Psychosis (PSY) scale, within a sample of preschool and school-age children referred for assessment of suspected cognitive impairment and learning problems. Compared to school-age children, significantly more preschoolers obtained elevated scores on the Psychosis scale; those preschoolers with cognitive impairments were even more likely to obtain significantly elevated Psychosis scale scores, despite the absence of diagnosed childhood psychosis. No significant relationship was found between Psychosis scale elevations and cognitive impairment in the school-age group. The PIC-R was moderately accurate in identifying cognitively impaired preschoolers but only minimally accurate in identifying cognitively impaired school-age children.