Proteomic analysis of larvae during development, attachment, and metamorphosis in the fouling barnacle, Balanus amphitrite

The barnacle, Balanus amphitrite, is one of the primary model organisms for rocky-shore ecology studies and biofouling research. This barnacle species has a complex life cycle during which the swimming nauplius molts six times and transforms into a cyprid stage. Cyprids must attach to a surface to metamorphose into a juvenile barnacle. To clarify the overall profile of protein expression during larval development and metamorphosis, 2-DE was used to compare the proteome of the nauplius, the swimming cyprid, the attached cyprid, and the metamorphosed cyprid. The proteome of the swimming cyprid was distinctly different from that of other life stages and had about 400 spots. The proteomes of the attached and metamorphosed cyprids were similar with respect to major proteins but had significantly lower numbers of spots compared to that of swimming larval stages. Obviously, synthesis of most proteins from swimming cyprids was switched off after attachment and metamorphosis. Our advanced MS analysis (MALDI-TOF/TOF MS/MS) allowed us to identify the proteins that were differentially and abundantly expressed in the swimming cyprid. These proteins included signal transduction proteins (adenylate cyclase and calmodulin) and juvenile hormone binding proteins. In summary, for the first time, we have analyzed the global protein expression pattern of fouling marine invertebrate larvae during metamorphosis. Our study provides new insights into the mechanisms of barnacle larval metamorphosis and also provides a foundation for exploring novel targets for antifouling treatments.     

Go with the flow: tidal import and export of larvae from semi-enclosed bays

There is a growing body of evidence that suggests the effective functioning of marine reserves is dependent on the dispersal and recruitment of larvae. Enhanced production inside reserves is predicted to lead to a net larval export and increased settlement and recruitment outside reserve boundaries. However, larval retention in bays is also well documented. Since bays are increasingly being used as reserve areas, planktonic larvae of benthic marine invertebrates were sampled from two semi-enclosed marine reserves during flood and ebb tides to determine whether these bays are acting as net exporters of larvae. Neither reserve was a net importer or exporter of species richness, larval abundance or diversity, although one reserve showed a small export of species richness during the hours of darkness. Both reserves balanced the net import of some species with a net export of others, which was generally related to adult or larval abundance, although exceptions were found in one reserve. Significant effects of light were found, with the net import or export of some species occurring exclusively during either the hours of daylight or darkness. An increased understanding of larval sink-source dynamics in bays is essential for ensuring their effective use as marine reserves to meet specific conservation needs. Guest editors: J. Davenport, G. Burnell, T. Cross, M. Emmerson, R. McAllen, R. Ramsay & E. Rogan Challenges to Marine Ecosystems     

Benthic assemblages and temperature effects on Paracentrotus lividus and Arbacia lixula larvae and settlement

Recruitment is a principal controlling factor in population dynamics of marine species. In marine invertebrates with a planktonic larval stage, such as echinoids, recruitment is assured by larval supply, settlement and juvenile survival. Larval supply and juvenile survival are affected by a wide range of factors, including temperature, presence of predators, quality and quantity of food. Echinoid larval settlement is mainly conditioned by the finding of a suitable substrate to metamorphose. The sea urchins Arbacia lixula and Paracentrotus lividus are considered key species of the Mediterranean infralittoral rocky shores. At high densities, the grazing activity of both species can produce and maintain barren grounds, a particular habitat condition characterized by extremely low cover values of erect algae with high presence of naked substrates and encrusting corallinales, poor in biodiversity and ecosystem functions. We tested the role of different settlement substrates on the metamorphosis competent larvae of the two species. Furthermore, from our larval rearing trails we were able to identify strong temperature effects on larval development of the two species. P. lividus and A. lixula larvae have been reared at 18 °C but for the second species it was necessary to use higher temperatures (22 °C) to perform settlement experiments, as in the 18 °C set all larvae died in the first week. Both species larvae have been fed Cricosphaera elongata. Metamorphosis of competent larvae has been induced using different substrates: naked stones, Lithophyllum incrustans, Stypocaulon scoparium, Corallina elongata, turf forming algae and Posidonia oceanica. For each species, two larval batches were used for settlement experiments; for each larval batch two replicates/substrates were set up. No differences in the rate of metamorphosis on any of the tested substrates were observed for P. lividus, while A. lixula showed to prefer naked stones and encrusting coralline algae Considering that A. lixula population growth may trigger barren extension on rocky shores, this may lead to a positive feedback between barren extension and A. lixula population density. Furthermore, our results suggest that the predicted rise in seawater temperature may favor A. lixula larval survival and inhibit P. lividus. Combining information on temperature tolerance with other sources of information for these species in the Mediterranean, it is possible to develop a conceptual model of the interaction between the two species and the alternative state of their habitats.     

Effect of dietary conjugated linoleic acid isomers on water and glycerol permeability of kidney membranes

Conjugated linoleic acid (CLA) refers to a group of positional and geometrical isomers of linoleic acid in which the double bonds are conjugated. Dietary CLA has been associated with various health benefits although details of its molecular mode of action remain elusive. The effect of CLA supplemented to palm oil-based diets in Wistar rats, as a mixture of both or isolated c9,t11 and t10,c12 isomers, was examined on water and glycerol membrane permeability of kidney proximal tubule. Although water permeability was unaltered, an increase in glycerol permeability was obtained for the group supplemented with CLA mixture, even though the activation energy for glycerol permeation remained high. This effect was correlated with an increased CLA isomeric membrane incorporation for the same dietary group. These results suggest that diet supplementation with CLA mixture, in contrast to its individual isomers, may enhance membrane fluidity subsequently raising kidney glycerol reabsorption.     

Production and supply of larvae as determinants of zonation in a brooding tropical coral

Production and settlement of planktonic larvae of the coral Favia fragum (Esper) were studied. The species is restricted to shallow back- and fore-reef habitats throughout the Caribbean Sea. Adults are in their greatest abundance on the reef-crest and shallow reef slope (<3 m) at Tague Bay, St. Croix, in the US Virgin Islands. Because F. fragum broods larvae that are capable of immediate settlement, this distribution pattern may be due to variation in fecundity among depths. Corals were collected from shallow (1.0 m) and deep depths (10-13 m) and cultured in individual containers exposed to shaded ambient light. Corals from shallow depths had greater fecundity (polyp⁻¹ lunar cycle⁻¹) and were larger than deep corals. To test the hypothesis that fecundity was related to successful fertilization, corals were kept in different densities in an area with sea-grass, where there were no natural adults. Production of larvae 6 months later was not affected by density of adults, possibly due to self-fertilization. Larval choice of habitat was also examined. In the laboratory, twice as many larvae settled on coral rubble fragments collected from depths where adults were common (1.5 and 3 m) than on those from depths where adults were rare (10 m). Larval supply may establish the vertical distribution of adults on St. Croix.     

Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide

Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid‐phase IEF sample prefractionation combined with 2‐DE and MALDI‐TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium‐binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required.     

Relation Between Desiccation Sensitivity and Membrane Permeability of Developing Clausena lansium Seeds

Desiccation sensitivity and its relation to membrane permeability of the embryonic axes of the developing wampee (Clausena lansium (Lour.) Skeels) seeds were studied by measuring the changes in electrolyte leakage, germination and vigor index after the embryonic axes were rapidly air-dried to various water contents. During development, the fresh and dry weight per seed reached nearly maximum value at 72 d after anthesis, but the dry weight per embryonic axis continuously increased until 85 d after anthesis. The embryonic axes acquired the full capacity for germination at 58 d after anthesis and their vigor index continuously rose up from 51 to 92 d after anthesis. The electrolyte leakage of the developing the embryonic axes linearly declined to the minimum value at 72 d after anthesis and then went up again. The electrolyte leakage of the embryonic axes was higher than that of the whole seeds at the same time. The immature embryonic axes did not germinate completely, while mild desiccation could improve their viability. Any degree of desiccation decreased the vigor index of the embryonic axes which have reached physiological maturation and the decline of vigor index was corresponded to the increase of electrolyte leakage. According to this experiment, the authors concluded that wampee seeds did not gain desiccation-tolerance which was a characteristic of orthodox seeds during development. High water content was essential for maintaining membrane integrity and stabiligy of matured wampee seeds. The injury of seed viability during dehydration could be estimated by using the electro-conductivity method.     

黄皮种子发育过程中脱水敏感性与细胞膜透性的关系

黄皮（Ｃｌａｕｓｅｎａ ｌａｎｓｉｕｍ （Ｌｏｕｒ．） Ｓｋｅｅｌｓ）胚轴与完整种子的发育模式以及发育中电解质渗漏率变化有些不同． 种子生理成熟前、后的胚轴对脱水的反应也不同,前者经轻微脱水可提高萌发率和活力指数,后者不耐任何程度的脱水．活力指数的急剧下降伴随着电解质渗漏率的迅速上升．实验表明,黄皮种子在发育过程中没有形成耐脱水性． 细胞膜透性变化可反映脱水对种子的伤害程度     

Effects of larvae ontogeny, turbidity, and turbulence on prey attack rate and swimming activity of Atlantic herring larvae

The effect of turbulence, light level, and ontogeny on herring larva's attack rate and swimming activity was tested in a previous study. However, during larval seasons (spring and autumn), water clarity is frequently impaired by alga blooms, which also most probably will affect larva feeding rate. Therefore, this study was to investigate the effects of turbidity, turbulence, and ontogeny on the attack rate and swimming activity of herring larvae. By adding diatomaceous earth (DE) to the water, three turbidity levels were established: 0, 35, and 80 Jackson Turbidity Unit [JTU; which coincide with a beam attenuation (c) of 0.1, 2.5, and 4.8 m⁻¹, respectively]. An unfavourable (8×10⁻⁶ W/kg) and a favourable turbulence level (1×10⁻⁶ W/kg) were chosen based on results from the earlier study. The results show that intermediate turbidity (35 JTU) has a positive effect on the attack rate of smaller larvae (20 mm), while high turbidity (80 JTU) has a negative effect on attack rate of all tested larvae size groups. In general, attack rate was lower at the highest turbulence compared to the low level, independent of turbidity level. However, there was one exception, when turbidity was at the highest, the largest larvae (29 mm) seemed to gain from feeding in the highest turbulence level. The overall activity level was higher in the presented study than in the earlier study without turbidity. The favourable turbidity level (35 JTU) coincides with turbidity levels normally found at the equivalent depth during spring and autumn blooms in the area of where the experimental larvae originate. In addition, turbidity's effect on light absorbtion and how it influences the maximum feeding depth of the larva are discussed.     

Crane reproductive physiology and conservation

Some unique features of crane reproduction, management, and conservation are described. Because cranes are sexually monomorphic, sexing is difficult and must be accomplished using behavior, laparoscopy, cloacal examination, genetic techniques, or fecal steroid analysis. Although husbandry techniques for cranes are similar to those used with other nondomestic birds, a number of basic characteristics, such as extreme aggressiveness, imprinting by the crane chick on man, a delayed molt in the immature crane, delayed sexual maturity, and infertility, pose special problems for the propagator. Artificial insemination is a practical solution to crane infertility. Vigorous captive management and propagation efforts must become increasingly important if several endangered crane species are to survive the continuing decline in wild populations. The ultimate goal is the restoration of suitable habitat and sustainable native populations.     

Studies on the interference among larvae of the citrus leaf miner,Phyllocnistis citrella stainton (lepidoptera: Phyllocnistidae)

Observation of the citrus leaf miner, Phyllocnistis citrellaStainton suggested that contacts among larvae frequently occur and either of two larvae encountered is sometimes killed by the interference. A model to describe the competitive interactions in the process of interference was constracted with this species. The distribution of hatchling on an area was considered in the model. The model was based on that of encounters in randomly moving particles in two dimensions. First the time when first death occurs was calculated and n hatchlings were reduced to n−1. Further calculation was made to obtain the time that next death occurs. Such calculation was repeated until the number of larvae was reduced to 1 or the area of mine attained to the completed size. By the simulation based on the data observed on the citrus leaf miner, the numbers of survivors were calculated at different hatchling densities and on different leaves in size. Based on this simulation analysis it was suggested that the following characteristics observed in the present studies enable the maximul number of individuals to become adults.

1. When two hatchlings occur on a leaf, they tend to be separated by the midrib. This implies that female moths tend to reject to lay eggs on the area which has already contained the egg.
2. The larva sometimes avoids the contact immediately before it occurs.
3. The larval mortality caused by a contact is reduced with the larval growth. Since the frequency of contacts increases as the mine is enlarged, the change to high survival prevents the larva from high mortality caused by the increase of contacts.
4. The larval survival does not decrease in spite of the reduction of the area available for mining.

     

Animal reproduction and physiology: from basis to application

<正>Animal reproduction and physiology is one of the traditional subjects in biology, and also one of the most rapidly developing fields because it is related to human food     

Coral larvae under ocean acidification: survival, metabolism, and metamorphosis

Ocean acidification may negatively impact the early life stages of some marine invertebrates including corals. Although reduced growth of juvenile corals in acidified seawater has been reported, coral larvae have been reported to demonstrate some level of tolerance to reduced pH. We hypothesize that the observed tolerance of coral larvae to low pH may be partly explained by reduced metabolic rates in acidified seawater because both calcifying and non-calcifying marine invertebrates could show metabolic depression under reduced pH in order to enhance their survival. In this study, after 3-d and 7-d exposure to three different pH levels (8.0, 7.6, and 7.3), we found that the oxygen consumption of Acropora digitifera larvae tended to be suppressed with reduced pH, although a statistically significant difference was not observed between pH conditions. Larval metamorphosis was also observed, confirming that successful recruitment is impaired when metamorphosis is disrupted, despite larval survival. Results also showed that the metamorphosis rate significantly decreased under acidified seawater conditions after both short (2 h) and long (7 d) term exposure. These results imply that acidified seawater impacts larval physiology, suggesting that suppressed metabolism and metamorphosis may alter the dispersal potential of larvae and subsequently reduce the resilience of coral communities in the near future as the ocean pH decreases.     

The relative importance of reproduction and survival for the conservation of two dolphin populations

It has been proposed that in slow‐growing vertebrate populations survival generally has a greater influence on population growth than reproduction. Despite many studies cautioning against such generalizations for conservation, wildlife management for slow‐growing populations still often focuses on perturbing survival without careful evaluation as to whether those changes are likely or feasible. Here, we evaluate the relative importance of reproduction and survival for the conservation of two bottlenose dolphin (Tursiops cf aduncus) populations: a large, apparently stable population and a smaller one that is forecast to decline. We also assessed the feasibility and effectiveness of wildlife management objectives aimed at boosting either reproduction or survival. Consistent with other analytically based elasticity studies, survival had the greatest effect on population trajectories when altering vital rates by equal proportions. However, the findings of our alternative analytical approaches are in stark contrast to commonly used proportional sensitivity analyses and suggest that reproduction is considerably more important. We show that

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Effects of small nonpolar molecules on membrane compressibility and permeability: A theoretical study of the effects of anesthetic gases

We explore from a theoretical perspective the effects of small nonpolar molecules, such as anesthetic gases, on membrane compressibility and permeability. As a model system we expand a previously proposed generalization of Nagle's model for biomembrane phase transitions. In this model anesthetic gases alter membrane compressibility, causing profound changes in membrane permeability. Anesthetics either increase or decrease membrane permeability, depending on whether the membrane lipid is originally in the solid or melted state, or in a two-phase region. These changes are reversed by high pressure, in agreement with experimental results. Anesthetic-induced changes in compressibility are predicted to inhibit fusion of phospholipid vesicles to each other and to planar bilayers, and thus might be expected to inhibit the fusion of presynaptic vesicles with the presynaptic nerve membrane. This work provides a detailed molecular theory for many of the effects of anesthetic gases on both synapse and axon, and provides a coherent framework for understanding diverse experimental results.     

Factors influencing initial larval settlement: temporal, spatial and surface molecular components

The impact of initial surface chemistry on settlement of barnacle, bryozoan, and hydroid larvae was assessed. Temporal (on a scale of weeks to months) and fine scale spatial (centimeter to meter) variation in settlement were quantified. Four arrays of silanized glass surfaces, deployed at ≈2-wk intervals, were monitored after 1 and 3 days of immersion. Settlement of all larval types exhibited strong temporal variation. There was a 25, 22- and 18-fold difference between the highest and lowest Day 1 settlement for barnacles, bryozoans and hydroids, respectively. Bryozoan and hydroid settlement was spatially variable, barnacle settlement was not. Barnacle and bryozoan settlement was influenced by inttial surface chemistry, hydroid settlement was not. For barnacles, there was a 2-fold difference between total settlement over the four arrays on untreated glass and diphenyl-silanized surfaces; for bryozoans, there was a 51-fold difference, and in the opposite direction. There was a negative correlation between barnacle and bryozoan settlement with respect to surface. This result was independent of the presence of the other species. Bryozoan settlement was also spatially and temporally quantified in a separate, single surface 56-h array, and total bryozoan settlement ·h⁻¹ was found to be correlated with light intensity. Patterns of initial colonization were strongly influenced by surface chemistry and spatial temporal variation in larval supply. These effects on initial colonization may influence subsequent community development.     

Regular distribution of larvae and resource monopolization in the seed beetle Bruchidius dorsalis infesting seeds of the Japanese honey locust Gleditsia japonica

Larvae of the wild multivoltine bruchid seed beetle Bruchidius dorsalis infest dry mature seeds of the Japanese honey locust Gleditsia japonica. The frequency distribution of B. dorsalis larvae per seed was examined in the laboratory. First, preliminary observation showed that the first-instar B. dorsalis larva crawled from an eggshell and traveled in excess of 25 cm before boring into a seed. Larvae prefer boring into noninfested rather than infested seeds (indicated by a previous emergence hole). When a larva occasionally bored into a previously infested seed, it could emerge as an adult in spite of decreased body weight. Second, we conducted experiment 1 to investigate the frequency distributions of the number of boring holes per seed; results showed significantly regular distributions (a smaller variance than Poisson) in which most seeds had been bored by one larva. Third, we performed experiment 2 to examine the relationship between the initial number of larvae boring into one seed and the number of emerged adults and their body weight from a given seed. When we increased the ratio of egg density to seed abundance by experimental manipulation, multiple larvae (up to ten) could bore into one seed. However, only one adult emerged from each of about 85% seeds with multiple borings. When one adult emerged, the adult body weight was constant, independent of the initial number of larvae boring into the seed. Two adults occasionally emerged from one seed with a large difference in their body sizes. Even in this case, the larger adult was as heavy as that in a single emergence, although the other was significantly smaller (about 60%) than the normal size. These results show severe contest-type competition among B. dorsalis larvae within one seed. The winner often monopolizes almost all the resources even though a part of the seed resource still remains. Received: September 17, 2001 / Accepted: October 5, 2001     

Heligmosomoides polygyrus: Simple recovery of post-infective larvae from mouse intestines

Mice infected orally with third-stage larvae of Heligmosomoides polygyrus were killed at various times after infection. Their small intestines were removed, tied at each end and incubated at 37 C in dilute culture medium. When intestines were taken from mice infected for a period of between 1 and 7 days, a number of developing larvae comprising up to 20% of the infective dose emerged within 60 min through the intestinal wall into the medium. The recovery of emergent larvae was highest using intestines from mice infected 36 to 120 hr previously. The proportion of parasites emerging from the intestines of 48-hr-infected mice was similar for doses of 100 to 2400 larvae. Significantly fewer larvae emerged from the intestines of mice resistant to reinfection and challenged with third-stage larvae 36–72 hr before necropsy.     

Survival dynamics of scleractinian coral larvae and implications for dispersal

Survival of pelagic marine larvae is an important determinant of dispersal potential. Despite this, few estimates of larval survival are available. For scleractinian corals, few studies of larval survival are long enough to provide accurate estimates of longevity. Moreover, changes in mortality rates during larval life, expected on theoretical grounds, have implications for the degree of connectivity among reefs and have not been quantified for any coral species. This study quantified the survival of larvae from five broadcast-spawning scleractinian corals (Acropora latistella, Favia pallida, Pectinia paeonia, Goniastrea aspera, and Montastraea magnistellata) to estimate larval longevity, and to test for changes in mortality rates as larvae age. Maximum lifespans ranged from 195 to 244 d. These longevities substantially exceed those documented previously for coral larvae that lack zooxanthellae, and they exceed predictions based on metabolic rates prevailing early in larval life. In addition, larval mortality rates exhibited strong patterns of variation throughout the larval stage. Three periods were identified in four species: high initial rates of mortality; followed by a low, approximately constant rate of mortality; and finally, progressively increasing mortality after approximately 100 d. The lifetimes observed in this study suggest that the potential for long-distance dispersal may be substantially greater than previously thought. Indeed, detection of increasing mortality rates late in life suggests that energy reserves do not reach critically low levels until approximately 100 d after spawning. Conversely, increased mortality rates early in life decrease the likelihood that larvae transported away from their natal reef will survive to reach nearby reefs, and thus decrease connectivity at regional scales. These results show how variation in larval survivorship with age may help to explain the seeming paradox of high genetic structure at metapopulation scales, coupled with the maintenance of extensive geographic ranges observed in many coral species. Communicated by Environment Editor Prof. van Woesik.