Toward a Holistic Understanding and Models of Nonphotochemical Quenching Effects on In Vivo Fluorometry of Chlorophyll a in Coastal Waters.

Nonphotochemical quenching (NPQ) is known to depress in vivo fluorescence (IVF) of chlorophyll a (Chla) in aquatic environments, which makes it difficult to interpret the hour-to-hour variations in Chla measured by in situ fluorometers. We hypothesized that ratios between quenched and unquenched IVF are a function of both NPQ and photochemical quenching. In this study, two diatom model species Thalassiosira pseudonana (CCMP1335) and Thalassiosira weissflogii (CCMP1047) incubated under a sinusoidal light:dark cycle were studied; IVF was recorded continuously, and Chla and photo-physiological variables were measured seven times a day. The maximal decline in Chla-specific IVF (IVFB ) attributable to quenching was 50% under the experimental settings. An NPQ and photochemical quenching-based modeling equation exhibited a better match to the measured IVFB than equations representing the sole NPQ effect. Photochemical quenching induced by measuring light beam varied substantially during the day, and the part of the model for this process is excitation intensity-dependent (which is differed between models of in situ fluorometers, implying no straightforward method to correct Chla for all instrument models, instrument-specific parameterization is required). The forms of the IVFB -light relationship are discussed as well. The findings foster a holistic understanding of NPQ effects on in vivo Chla fluorometry.

Quantification and Valuation of Nitrogen Removal Services Provided by Commercial Shellfish Aquaculture at the Subwatershed Scale.

Eutrophication is a global environmental challenge, and diverse watershed nitrogen sources require multifaceted management approaches. Shellfish aquaculture removes nitrogen, but the extent and value of this ecosystem service have not been well-characterized at the local scale. A novel approach was employed to quantify and value nitrogen reduction services provided by the shellfish aquaculture industry to a municipality. Cultivated hard clam and eastern oyster nitrogen removal in Greenwich Bay, Connecticut, was valued using the replacement cost methodology and allocated by municipal nitrogen source. Using the preferred analysis allocating replacement costs by nitrogen source, aquaculture-based removal of 14 006 kg nitrogen was valued at $2.3-5.8 (2.3-6.4€) million year-1. This nitrogen removal represents 9% of the total annual Greenwich-specific nitrogen load, 16% of the combined nonpoint sources, 38% of the fertilizer sources, 51% of the septic sources, 98% of the atmospheric deposition to the watershed, or 184% of the atmospheric deposition to the embayments that discharge to Greenwich Bay. Our approach is transferable to other coastal watersheds pursuing nitrogen reduction goals, both with and without established shellfish aquaculture. It provides context for decisions related to watershed nitrogen management expenditures and suggests a strategy to comprehensively evaluate mechanisms to achieve nitrogen reduction targets.

Energetic response of Atlantic surfclam Spisula solidissima to ocean acidification.

In this study, we assessed the Atlantic surfclam (Spisula solidissima) energy budget under different ocean acidification conditions (OA). During 12 weeks, 126 individuals were maintained at three different ρCO2 concentrations. Every two weeks, individuals were sampled for physiological measurements and scope for growth (SFG). In the high ρCO2 treatment, clearance rate decreased and excretion rate increased relative to the low ρCO2 treatment, resulting in reduced SFG. Moreover, oxygen:nitrogen (O:N) excretion ratio dropped, suggesting that a switch in metabolic strategy occurred. The medium ρCO2 treatment had no significant effects upon SFG; however, metabolic loss increased, suggesting a rise in energy expenditure. In addition, a significant increase in food selection efficiency was observed in the medium treatment, which could be a compensatory reaction to the metabolic over-costs. Results showed that surfclams are particularly sensitive to OA; however, the different compensatory mechanisms observed indicate that they are capable of some temporary resilience.

Design and Use of an Apparatus for Quantifying Bivalve Suspension Feeding at Sea.

As shellfish aquaculture moves from coastal embayments and estuaries to offshore locations, the need to quantify ecosystem interactions of farmed bivalves (i.e., mussels, oysters, and clams) presents new challenges. Quantitative data on the feeding behavior of suspension-feeding mollusks is necessary to determine important ecosystem interactions of offshore shellfish farms, including their carrying capacity, the competition with the zooplankton community, the availability of trophic resources at different depths, and the deposition to the benthos. The biodeposition method is used to quantify feeding variables in suspension-feeding bivalves in a natural setting and represents a more realistic proxy than laboratory experiments. This method, however, relies upon a stable platform to satisfy the requirements that water flow rates supplied to the shellfish remain constant and the bivalves are undisturbed. A flow-through device and process for using the biodeposition method to quantify the feeding of bivalve mollusks were modified from a land-based format for shipboard use by building a two-dimensional gimbal table around the device. Planimeter data reveal a minimal pitch and yaw of the chambers containing the test shellfish despite boat motion, the flow rates within the chambers remain constant, and operators are able to collect the biodeposits (feces and pseudofeces) with sufficient consistency to obtain accurate measurements of the bivalve clearance, filtration, selection, ingestion, rejection, and absorption at offshore shellfish aquaculture sites.

Cultivation of the Ribbed Mussel (Geukensia demissa) for Nutrient Bioextraction in an Urban Estuary.

Shellfish aquaculture is gaining acceptance as a tool to reduce nutrient over enrichment in coastal and estuarine ecosystems through the feeding activity of the animals and assimilation of filtered particles in shellfish tissues. This ecosystem service, provided by the ribbed mussel (Geukensia demissa), was studied in animals suspended from a commercial mussel raft in the urban Bronx River Estuary, NY, in waters closed to shellfish harvest due to bacterial contamination. Naturally occurring populations of ribbed mussels were observed to be healthy and resilient in this highly urbanized environment. Furthermore, mussels grown suspended in the water column contained substantially lower concentrations of heavy metals and organic contaminants in their tissues than blue mussels (Mytilus edulis) collected at a nearby benthic site. Spat collection efforts from shore and within the water column were unsuccessful; this was identified as a key bottleneck to future large-scale implementation. Filtration experiments indicated that a fully stocked G. demissa raft would clear an average 1.2 × 107 L of Bronx River Estuary water daily, removing 160 kg of particulate matter from the water column, of which 12 kg would be absorbed into mussel digestive systems. At harvest, 62.6 kg of nitrogen would be sequestered in mussel tissue and shell. These values compare favorably to other resource management recovery methods targeting agricultural and stormwater nitrogen sources.

Bioconversion of (13)C-labeled microalgal phytosterols to cholesterol by the Northern Bay scallop, Argopecten irradians irradians.

Bivalve mollusks lack de novo cholesterol biosynthesis capabilities and therefore rely upon dietary sources of sterols for rapid growth. Microalgae that constitute the main source of nutrition for suspension-feeding bivalves contain a diverse array of phytosterols, in most cases lacking cholesterol. Rapid growth of bivalves on microalgal diets with no cholesterol implies that some phytosterols can satisfy the dietary requirement for cholesterol through metabolic conversion to cholesterol, but such metabolic pathways have not been rigorously demonstrated. In the present study, stable isotope-labeled phytosterols were used to supplement a unialgal diet of Rhodomonas sp. and their biological transformation to cholesterol within scallop tissues was determined using (13)C-NMR spectroscopy. Scallops efficiently dealkylated ∆(5) C29 (24-ethyl) sterols to cholesterol, and the only C28 sterol that was dealkylated efficiently possessed the 24(28)-double bond. Non-metabolized dietary phytosterols accumulated in the soft tissues. Observed formation of ∆(5,7) sterols (provitamin D) from ∆(5) sterols may represent initiation of steroid hormone (possibly ecdysone) biosynthesis. These findings provide a key component necessary for formulation of nutritionally complete microalgal diets for hatchery production of seed for molluscan aquaculture.

Physiological and pathological changes in the eastern oyster Crassostrea virginica infested with the trematode Bucephalus sp. and exposed to the toxic dinoflagellate Alexandrium fundyense.

Effects of experimental exposure to Alexandrium fundyense, a Paralytic Shellfish Toxin (PST) producer known to affect bivalve physiological condition, upon eastern oysters, Crassostrea virginica with a variable natural infestation of the digenetic trematode Bucephalus sp. were determined. After a three-week exposure to cultured A. fundyense or to a control algal treatment with a non-toxic dinoflagellate, adult oysters were assessed for a suite of variables: histopathological condition, hematological variables (total and differential hemocyte counts, morphology), hemocyte functions (Reactive Oxygen Species (ROS) production and mitochondrial membrane potential), and expression in gills of genes involved in immune responses and cellular protection (MnSOD, CAT, GPX, MT-IV, galectin CvGal) or suspected to be (Dominin, Segon). By comparing individual oysters infested heavily with Bucephalus sp. and uninfested individuals, we found altered gonad and digestive gland tissue and an inflammatory response (increased hemocyte concentration in circulating hemolymph and hemocyte infiltrations in tissues) associated with trematode infestation. Exposure to A. fundyense led to a higher weighted prevalence of infection by the protozoan parasite Perkinsus marinus, responsible for Dermo disease. Additionally, exposure to A. fundyense in trematode-infested oysters was associated with the highest prevalence of P. marinus infection. These observations suggest that the development of P. marinus infection was advanced by A. fundyense exposure, and that, in trematode-infested oysters, P. marinus risk of infection was higher when exposed to A. fundyense. These effects were associated with suppression of the inflammatory response to trematode infestation by A. fundyense exposure. Additionally, the combination of trematode infestation and A. fundyense exposure caused degeneration of adductor muscle fibers, suggesting alteration of valve movements and catch state, which could increase susceptibility to predation. Altogether, these results suggest that exposure of trematode-infested oysters to A. fundyense can lead to overall physiological weakness that decrease oyster defense mechanisms.

Modification of cell wall properties in lettuce improves shelf life.

It is proposed that post-harvest longevity and appearance of salad crops is closely linked to pre-harvest leaf morphology (cell and leaf size) and biophysical structure (leaf strength). Transgenic lettuce plants (Lactuca sativa cv. Valeria) were produced in which the production of the cell wall-modifying enzyme xyloglucan endotransglucosylase/hydrolase (XTH) was down-regulated by antisense inhibition. Independently transformed lines were shown to have multiple members of the LsXTH gene family down-regulated in mature leaves of 6-week-old plants and during the course of shelf life. Consequently, xyloglucan endotransglucosylase (XET) enzyme activity and action were down-regulated in the cell walls of these leaves and it was established that leaf area and fresh weight were decreased while leaf strength was increased in the transgenic lines. Membrane permeability was reduced towards the end of shelf life in the transgenic lines relative to the controls and bacteria were evident inside the leaves of control plants only. Most importantly, an extended shelf-life of transgenic lines was observed relative to the non-transgenic control plants. These data illustrate the potential for engineering cell wall traits for improving quality and longevity of salad crops using either genetic modification directly, or by using markers associated with XTH genes to inform a commercial breeding programme.

QTLs for shelf life in lettuce co-locate with those for leaf biophysical properties but not with those for leaf developmental traits.

Developmental and biophysical leaf characteristics that influence post-harvest shelf life in lettuce, an important leafy crop, have been examined. The traits were studied using 60 informative F9 recombinant inbed lines (RILs) derived from a cross between cultivated lettuce (Lactuca sativa cv. Salinas) and wild lettuce (L. serriola acc. UC96US23). Quantitative trait loci (QTLs) for shelf life co-located most closely with those for leaf biophysical properties such as plasticity, elasticity, and breakstrength, suggesting that these are appropriate targets for molecular breeding for improved shelf life. Significant correlations were found between shelf life and leaf size, leaf weight, leaf chlorophyll content, leaf stomatal index, and epidermal cell number per leaf, indicating that these pre-harvest leaf development traits confer post-harvest properties. By studying the population in two contrasting environments in northern and southern Europe, the genotype by environment interaction effects of the QTLs relevant to leaf development and shelf life were assessed. In total, 107 QTLs, distributed on all nine linkage groups, were detected from the 29 traits. Only five QTLs were common in both environments. Several areas where many QTLs co-located (hotspots) on the genome were identified, with relatively little overlap between developmental hotspots and those relating to shelf life. However, QTLs for leaf biophysical properties (breakstrength, plasticity, and elasticity) and cell area correlated well with shelf life, confirming that the ideal ideotype lettuce should have small cells with strong cell walls. The identification of QTLs for leaf development, strength, and longevity will lead to a better understanding of processability at a genetic and cellular level, and allow the improvement of salad leaf quality through marker-assisted breeding.

Genetic and physiological characterization of tomato cv. Micro-Tom.

Based on its compact habit, Micro-Tom, a dwarf cultivar of tomato (Solanum lycopersicum L.), has been proposed as a preferred variety to carry out molecular research in tomato. This cultivar, however, is poorly characterized. It is shown here that Micro-Tom has mutations in the SELF-PRUNING (SP) and DWARF (D) genes. In addition to this, it is also shown that Micro-Tom harbours at least two independently segregating resistance loci to the plant pathogen Cladosporium fulvum. The presence of the self-pruning mutation in Micro-Tom, that generates a determinate phenotype, was confirmed by crossing and sequence analysis. It was also found that Micro-Tom has a mutation in the DWARF gene (d) that leads to mis-splicing and production of at least two shorter mRNAs. The d mutation is predicted to generate truncated DWARF protein. The d sequence defect co-segregates with dark-green and rugose leaves, characteristics of brassinosteroid biosynthesis mutants. Micro-Tom also carries at least another mutation producing internode length reduction that affects plant height but not active gibberellin (GA) levels, which were similar in dwarf and tall Micro-TomxSeverianin segregants. GAs and brassinosteroids act synergistically in Micro-Tom, and the response to GA depends on brassinosteroids because the elongation of internodes was at least six times higher when GA(3) was applied simultaneously with brassinolide. A novel variety, Micro-0 that is fully susceptible to C. fulvum and almost as dwarf as Micro-Tom, has been generated from the cross of Cf0xMicro-Tom. This line represents a valuable resource for future analysis of Cf resistance genes through breeding or transformation.

Variable leucine-rich repeats of tomato disease resistance genes Cf-2 and Cf-5 determine specificity.

SUMMARY The tomato Cf-2 and Cf-5 genes confer race specific resistance to infection by the leaf mould pathogen Cladosporium fulvum. The encoded proteins induce a defence response upon recognition of the fungal Avr2 and Avr5 determinants, respectively. Each resistance protein is comprised largely of leucine-rich repeats (LRRs) and the specificity of recognition is thought to occur through a particular domain. We have investigated this further using domain swaps between Cf-2 and Cf-5. Engineered chimeric genes containing portions of Cf-2 and Cf-5 were expressed and shown to be functional. The results clearly show that the specificity for the particular avirulence determinant is restricted to a region of each gene that encodes a subset of LRRs containing the highest level of intergenic variability. In addition, two non-functional mutants of Cf-5 were characterized and their significance discussed.

A tomato cysteine protease required for Cf-2-dependent disease resistance and suppression of autonecrosis.

Little is known of how plant disease resistance (R) proteins recognize pathogens and activate plant defenses. Rcr3 is specifically required for the function of Cf-2, a Lycopersicon pimpinellifolium gene bred into cultivated tomato (Lycopersicon esculentum) for resistance to Cladosporium fulvum. Rcr3 encodes a secreted papain-like cysteine endoprotease. Genetic analysis shows Rcr3 is allelic to the L. pimpinellifolium Ne gene, which suppresses the Cf-2-dependent autonecrosis conditioned by its L. esculentum allele, ne (necrosis). Rcr3 alleles from these two species encode proteins that differ by only seven amino acids. Possible roles of Rcr3 in Cf-2-dependent defense and autonecrosis are discussed.