Studies on enzymes associated with calcification of the cuticle of the hermit crab Clibanarius olivaceous

In the anomuran Clibanarius olivaceous Henderson, the cuticle of the cephalic and thoracic regions is calcified, while that of the abdominal region is uncalcified. Histochemical observations have been made on the possible enzymes involved in the process of calcification of cuticle. The calcified regions show the presence of enzymes such as phosphorylase, alkaline phosphatase and carbonic anhydrase. The possible role of phosphorylase in calcification may be production of phosphoric esters, which form the required substrate for alkaline phosphatase. The latter appear to be associated with the production of phosphate ions from phosphoric esters. The carbonic anhydrase may be associated with the production of carbonate ions. The suggestion that these enzymes are involved in the process of calcification is further supported by experimental evidence of inhibition of the action of enzymes by enzyme inhibitors. The inhibition of enzymes leads to reduction in the amount of calcium added to the cuticle.This paper formed part of a thesis approved for the award of Ph. D. degree of the University of Madras.     

On the nature and role of protein constituents of the cuticle of crustaceans in relation to permeability of the cuticle

The chemical composition of the cuticle in regions which are permeable and impermeable to water in 4 crustaceans (Ocypode platytarsis, Metapenaeus monoceros, Emerita asiatica, Ligia exotica) was investigated, with special reference to the protein components. It was found that the cuticle which is permeable lacks the outer epicuticle or the entire epicuticle compared to the cuticle in the impermeable regions. The permeable cuticle is distinguished by the absence of a fuchsinophil protein, which is a precursor of tanning. Permeability of the cuticle is also associated with the presence of a simple protein, positive to the biuret test, and with a protein containing sulphydryl groups. It is considered that the cuticle protein may play a role in conferring permeability properties to the cuticle itself.     

Ultrastructure and histochemistry of the integument of air-breathing polychaetes from mangrove swamps of sumatra

The integument of S.E. Asian air-breathing polychaetes exhibits a variable ultrastructure with regard to cuticle and epithelial cells. The cuticle varies in thickness, contents of regularly arranged collagen fibrils, and structure of the epicuticle. The basal portion of the epithelium is either of smooth outline, or consists of slender processes of the epidermal cells separated by wide intercellular spaces which are invaded by a loose system of glia cells and nerve fibres. Extracellular spaces within the epidermis of the gills of Dendronereides heteropoda are transformed into blood lacunae. The enzyme content of the epithelial cells is relatively low.Supported by the Deutsche Forschungsgemeinschaft (Sto 75/2, We 380/4).     

Ultrastructural study of the exoskeleton of the estuarine crab Rhithropanopeus harrisii: Effect of the insect growth regulator Dimilin® (diflubenzuron) on the formation of the larval cuticle

Ultrastructure of larval cuticle during the molt cycle of the estuarine crab Rhitropanopeus harrisii (Gould) (Crustacea: Brachyura) was studied in control larvae as well as in larvae exposed to 10 ppb of the insect growth regulator Dimilin^® (diflubenzuron). First zoeal larvae were used as test organisms. It has earlier been shown that 10 ppb Dimilin is lethal to zoeal larvae of R. harrisii, and nearly all exposed larvae died during molting to the next stage (Christiansen et al., 1978). Distinct differences in structure of the cuticle were found between the two groups of larvae. Both endocuticle and exocuticle appear to be deformed in Dimilin-treated larvae, whereas formation of epicuticle did not seem to be affected. The results indicate that Dimilin probably inhibits chitin synthesis in crab larvae as shown earlier by several authors for insect larvae.     

Bewertung der Umweltrelevanz von Arzneistoffen

Aim The aim of this work was to investigate, which human and veterinary pharmaceuticals are of high ecological relevance due to the input into the environment as well as with regard to the effects and behaviour in the environment. Furthermore, the state of knowledge concerning the ecologically relevant substances and the need of research was evaluated. Methods A two-step approach considering the criteria quantity of sold pharmaceuticals, concentrations, effects and fate in the environment was developed to identify the pharmaceuticals of potential ecological relevance. In a second step the identified individual substances and groups of substances were considered in more detail concerning metabolism, environmental behaviour and ecological effects. Results In a first step 29 out of approximate 2,700 licensed human pharmaceuticals and seven substances as well as three substance groups used in veterinary medicine were identified as potentially ecologically relevant. The detailed assessment in a second step lead to the conclusion, that of the human pharmaceuticals 9 substances as well as two substance groups concerning the environmental behaviour and seven substances concerning the effects are of high environmental relevance. Concerning the environmental behaviour six out of seven veterinary pharmaceuticals as well as three substance groups and only three substances concerning the ecological effects were classified as relevant for the environment. Discussion By means of the presented method it was possible to limit the scope of a wide spectrum of pharmaceuticals, so that a detailed assessment of only relevant active agents was possible. Nevertheless, it is a precondition that investigations concerning the occurrence, fate and effects of the individual compounds in the environment have been already carried out and published in the literature. Conclusions The method is suitable for a comprehensive assessment of the ecological relevance of pharmaceuticals, but for a lot of human and veterinary pharmaceuticals the data available in the literature are insufficient. Recommendations For a final extensive assessment of the environmental relevance for some of the human and veterinary pharmaceuticals more studies are necessary. Especially data concerning the environmental behaviour in water and soils, data from long-term-studies for the assessment of ecological effects and data concerning metabolites and mixtures of pharmaceutical compounds are lacking.     

Distribution,structure and importance of the cephalic dorsal hump,a new sensory organ in calanoid copepods

The occurrence, external morphology and internal ultrastructure of a cephalic integumental organ in calanoid copepods were studied, using the specimens from the Pacific, Indian and Atlantic Oceans. This organ is located on the dorsoanterior surface of the cephalosome, and a name, cephalic dorsal hump (CDH) is proposed. Externally, it usually has two pores, anterior and apical, a dorsal plate, and a thin cuticle along the sides. CDH is found only in the male of Calanidae, Megacalanidae, Mecynoceridae and Paracalanidae, and showed some variation between species or species groups both in size and shape. Transmission electron microscopy (TEM) on the specimens from Sagami Bay, Central Japan, revealed that the CDH of Paracalanus parvus and Calanus sinicus consists of two dermal glands and a receptor, which is assumed to be chemosensory. A comparison of the distributions of CDH and prehensile fifth legs of male calanoid copepods suggests that it plays an important role in mate recognition.     

Experimental ecology of the temperate scleractinian coral Astrangia danae

The combined effects of temperature, light and symbiont density on the metabolic rate and calcification of the temperate coral Astrangia danae were studied experimentally using colonies containing different concentrations of zooxanthellae. After acclimation to five temperatures between 6.5° and 27°C, and incubation at three light levels and in darkness, respiration and photosynthesis were measured and corrected for rates due to commensals alone. Calcification rates were regressed on zooxanthellae concentration and production in order to define “symbiotic” and “non-symbiotic” averages, and the enhancement of calcification by symbiotic interactions in the polyps. Respiration by the polyparium varied less with temperature between 11.5° and 23°C than that of the commensals, suggesting physiological acclimation by the coral tissue. In-vivo zooxanthellae photosynthesis increased linearly with temperature and was near its maximum at 400 μEin m^?2 s^?1, but the photosynthesis of the endolithic algae of the corallum varied little between 11.5° and 27°C. Calcification at any given temperature was near its maximum at 40 μEin m^?2 s^?1 in both symbiotic and non-symbiotic corals. CaCO₃ deposition increased linearly with temperature in non-symbiotic colonies and in symbiotic colonies incubated in the dark. In symbiotic colonies, calcification in the light increased above these basic rates as temperature rose above 15°C. Below 15°C, symbiotic interactions failed to stimulate calcification, apparently due both to a lowering of zooxanthellae photosynthesis and to a decrease in the enhancing effect of any given level of primary production.     

Histological and histochemical changes in the gills of the yellowtail Seriola quinqueradiata exposed to the Raphidphycean flagellate Chattonella marina

The gills of the yellowtail Seriola quinqueradiata, exposed to Chattonella marina a red tide species, were examined histologically and histochemically. The pavement cells of primary lamellae were swollen in the exposed specimens. This presumably indicates that C. marina contains undetermined toxic substances. The number of mucous cells containing mucous decreased in proportion to the duration of exposure. The loss of the mucous substance in the cells is probably due to the ichthyotoxic stimulus caused by C. marina. A decrease of carbonic anhydrase activity in the secondary lamellae was confirmed, when mucous substances were lost in most mucous cells. Since the carbonic anhydrase usually exists in the epithelia of the secondary lamellae, it was presumably resolved by C. marina. The decrease of the carbonic anhydrase activity may cause certain physiological disadvantages to the fish exposed to C. marina.     

Epithelial immunity in a marine invertebrate: a cytolytic activity from a cuticular secretion of the American horseshoe crab, <Emphasis Type="Italic">Limulus polyphemus</Emphasis>

The cuticle of the American horseshoe crab, Limulus polyphemus, is largely free of the macroscopic epibionts that rapidly colonize most solid surfaces exposed to sea water. Here, we describe a viscous surface secretion that coats the carapace of the horseshoe crab. We report methods for stimulating production of the substance, identify hemolytic and liposome-permeabilizing activities and provide a partial biochemical characterization. We propose that this secretion functions as an anti-fouling agent protecting the chitinized epithelium of the cuticle from colonization by deleterious epibionts.     

Schadstoffbelastete Erzeugnisse im Verbraucherbereich: Wird REACH zu Verbesserungen führen?

Aim and Background Currently, the protection from hazardous substances occurring in consumer articles is often insufficient. In this paper, we discuss whether this situation will improve relevantly in the course of the implementation of the new European regulation on chemical substances (REACH). Changes under REACH may be heterogeneous. Therefore, differential effects depending on the tonnage as placed on the market, on the amount of substances contained in an article, and depending on timelines and exemptions for meeting the legal requirements, are discussed in this paper, together with a presentation of some uses of substances serving as examples. Discussion and Conclusions If substances are adequately registered, all identified and supported uses including their use in articles should be assessed. Therefore, where the full implementation of the registration conditions applies a substantial improvement may be expected due to REACH. Usually, producers or importers of articles are not required to register substances contained in these articles. Instead, the manufacturer or importer of the respective substance is usually responsible for registration, if exposure to the substance cannot be excluded and emission of the substance from an article is not the intended purpose. This may differ for substances intentionally released from articles. Additional obligations for substances not intentionally released mainly apply to substances of very high concern (SVHC). For the latter, the producer or importer of articles has to notify the European Chemicals Agency (ECHA) on the occurrence of the respective SVHC in articles. This information is important, especially because substances in imported articles may not be registered in advance. Authorisation within REACH is a very far reaching and effective tool of the legislation. However, authorisation is not required for SVHC imported as components of articles. Therefore, one main effect intended by REACH, i.?e. the substitution of SVHC, may possibly be undermined by importers of SVHC in articles. Restrictions, as specified in Annex XVII of REACH, offer a powerful opportunity to improve health and environmental safety with respect to hazardous substances. We support an extended understanding of what is defined as a “substance of concern” in the context of restrictions. This definition should be broader than just what is currently covered by the term SVHC. Consequences of restrictions may equally apply to European producers and importers of articles from outside of Europe, which contain such substances of concern. Another instrument currently closely linked to SVHC (in its more restricted definition), is the obligation of the producer or importer of articles to provide information according to article 33 (REACH). The downstream recipient of an article has to be informed on the content of SVHC in this article and how its safe use is ensured. Warehouses, wholesalers and other traders may therefore be aware of the SVHC contained in articles and may look for substitutes without such ingredients. Also, consumers may request this information on SVHC (just provided on demand) and are thus enabled to decide to buy other articles with less hazardous properties.     

Chemical characterization of contact semiochemicals for host-recognition and host-acceptance by the specialist parasitoid <Emphasis Type="Italic">Cotesia plutellae</Emphasis> (Kurdjumov)

Summary. Cotesia plutellae is a specialist parasitoid of Plutella xylostella. This specificity is potentially under the control of several factors before and after oviposition. Thereby, the stimuli that lead female parasitoids to host locations and to oviposition, might be at the basis of the specificity. We explore here the response of C. plutellae females exposed to host cuticular lipids. A total cuticular lipid extract of host caterpillars was fractionated into a hydrocarbon fraction and a non-hydrocarbon fraction. Neither fraction alone had any effect on oviposition behaviour in C. plutellae but the hydrocarbon fraction alone did seem to have a positive effect on the rate of antennal contact by the females. To induce oviposition behaviour, both fractions were necessary and reflect cooperation between at least one compound in each fraction. Identification of cuticular lipids shows that hydrocarbons were dominant (77%). Non-hydrocarbon compounds were mainly represented by 15-nonacosanone (18% of the total lipid extract). This ketone is rare in insect cuticle lipids and is thought to originate from the cabbage epicuticle where it is dominant with n-C₂₉ and 14- and 15-nonacosanol also found among the cuticular lipids of the host caterpillar.     

Ocean acidification reduces biomineralization-related gene expression in the sea urchin, Hemicentrotus pulcherrimus

Although recent studies have demonstrated that calcification in a wide range of marine organisms is profoundly affected by CO₂-induced ocean acidification, the mechanism of this phenomenon is still unclear. To clarify the effects of ocean acidification on the calcification process at the molecular level, we evaluated the expression of three biomineralization-related genes in the sea urchin Hemicentrotus pulcherrimus exposed under control, 1,000, and 2,000?ppm CO₂ from egg to pluteus larval stage. We found that the expression of the gene msp130, which is proposed to transport Ca²⁺ to the calcification site, is suppressed by increased CO₂ at pluteus larval stage. Meanwhile, expression of the spicule protein matrix genes SM30 and SM50 was apparently not affected. The results suggest that the combined effects of ocean acidification on the expression of skeletogenesis-related genes as well as the change in seawater carbonate chemistry affect the biomineralization ability of sea urchins.     

Preliminary observations on the circulatory system in eggs and early larvae of some teleostean fishes

Observations on the pattern of blood circulation in eggs and early larvae of Ambassis, Mugil, Dorosoma and Thrissocles species have been made. In the initial stages, the tubular heart lies on the left side, near the cephalic region. The anterior end of the heart lies near the snout. The posterior end is attached to a point mid-way between the orbits and auditory vesicles; the position of the anterior end is greatly influenced by the size of yolk. Initially, the tubular heart and dorsal aorta lie in a line. As a result of yolk absorption, the anterior end of the tubular heart gradually sinks to a position posterior to the attached end. Liquified yolk is circulated through the heart in early stages; blood corpuscles become apparent only in larvae. The heart-beat, which is initially irregular, gradually becomes regular as development progresses. In the initial stages the blood flows to the caudal and not to the cephalic region. The blood vessels are narrow, and the oval-shaped blood corpuscles must therefore pass through individually, with their long axes parallel to the passage. A definite capillary system is not evident. The blood vessels can dilate and contract when required.     

Effect of natural aquatic humic substances on the photodegradation of bisphenol A

The photochemical degradation of bisphenol A (BPA) was studied in the presence of natural humic substances from different origins under simulated solar irradiation. BPA underwent insignificant direct photolysis in neutral water, but rapid photosensitized degradation in four humic substances solutions via pseudo-first-order reaction occurred. The photo-degradation rate of BPA was insensitive to the different initial BPA concentrations and was inhibited in aerated solution compared with the deoxygenated medium. The reactive oxygen species (ROS) such as ·OH and ¹O₂ produced from excitation of humic substances under irradiation was determined from the quenching kinetic experiment using molecular probe. The five main intermediate photoproducts of BPA in Nordic lake fulvic acid (NOFA) were tentatively identified using gas chromatography/mass spectrometer (GC/MS). Based on the identification of ROS and the analysis of photoproduct formation, the possible phototransformation pathways of BPA were proposed, involving the direct photolysis due to the energy transfer from the triplet state humic substance (³HS*) to BPA molecules and hydroxyl radical addition and oxidation as well.     

Effect of temperature and temperature adaptation on calcification rate in the hermatypic coral Pocillopora damicornis

Using ⁴⁵Ca incorporation into the coral skeleton as a measure of calcification rate, the effect of temperature on clacification rate was studied in the hermatypic coral Pocillopora damicornis. Both immediate and long-term (adaptation) effects were investigated. Temperature has a marked effect on rate — an effect that varies depending on the temperature history of the coral (i.e., temperature adaptation occurs). P. damicornis showed both 27° and 31°C temperature optima, one or the other being dominant depending on the natural water temperature to which the coral was adapted. The two optimum temperatures may indicate two isoenzymes or two alternate metabolic pathways involved in the calcification process.     

An electron-microscope investigation into the cuticle and associated tissues of the operculum of some marine serpulids

The fine structure of the opercula of Spirobis borealis Daudin and S. pusilloides Bush, and the primary and secondary operculum of S. granulatus (L) have been examined by electron microscopy. In different regions, the epithelium of the operculum exhibits variations in thickness and fine structure which correspond to transitions from peduncular to opercular wall to opercular plate epithelia. Externally, the peduncle and walls of the operculum are covered by a cuticle which is composed of several layers of fibrils/fibres embedded in an amorphous matrix. It is regularly traversed by microvilli from underlying cells. At the operculum rim, the cuticle changes so that its appearance is thick and solid. It continues across the roof of the operculum as a thin layer overlying a calcareous plate. The secondary operculum of S. granulatus differs from those of the other species in that an internal calcareous plate and solid cuticle line the roof of the operculum. It differs further in possessing an opercular pore. The possibilities are discussed of the cuticle fibrils/fibres of the peduncle and the opercular wall being collagenous in nature, and later being transformed to a solid tanned structure overlying a calcareous plate. The modes of secretion of the cuticle and calcareous plate by underlying cells are also discussed.     

Ecological and physiological differences between two colour morphs of the coral Pocillopora damicornis

Pocillopora damicornis (Linnaeus) is a ubiquitous branching coral found throughout the Indo-Pacific region. Like many other species of coral, P. damicornis displays a large range of morphologies. At One Tree Island, it occurs as two distinct morphs that are easily distinguished by the presence or absence of pink pigmentation. The two colour morphs of P. damicornis were found to differ in their distribution and abundance in the One Tree Island Lagoon. The brown morph was more abudant than the pink morph in the shallows (<1 m),whereas the pink morph was more abundant at deeper sites (>3 m). The two morphs also differed physiologically. The brown morph tended to have a greater calcification rate than the pink morph, regardless of environmental conditions. However, the difference in the calcification rate between the two morphs became non-significant under shaded conditions (5% full sunlight), indicating some degree of physiological plasticity of the morphs. The pink colour in P. damicornis was due to a hydrophilic pigment with a major peak absorbance at 560 nm. The expression of pink pigment had both genetic and phenotypic components. The brown morph has a reduced genetic capacity to express the pigment relative to the pink morph. On the other hand, pigment expression could be induced by light in the pink morph. Although genetic differences ultimately determine the differences between the two morphs of P. damicornis, the extent of pigment expression is under some degree of environmental influence.     

Effects of feeding and light intensity on the response of the coral Porites rus to ocean acidification

Recently, it has been suggested that there are conditions under which some coral species appear to be resistant to the effects of ocean acidification. To test if such resistance can be explained by environmental factors such as light and food availability, the present study investigated the effect of 3 feeding regimes crossed with 2 light levels on the response of the coral Porites rus to 2 levels of pCO₂ at 28 °C. After 1, 2, and 3 weeks of incubation under experimental conditions, none of the factors—including pCO₂—significantly affected area-normalized calcification and biomass-normalized calcification. Biomass also was unaffected during the first 2 weeks, but after 3 weeks, corals that were fed had more biomass per unit area than starved corals. These results suggest that P. rus is resistant to short-term exposure to high pCO₂, regardless of food availability and light intensity. P. rus might therefore represent a model system for exploring the genetic basis of tolerance to OA.     

Ocean acidification and temperature rise: effects on calcification during early development of the cuttlefish Sepia officinalis

This study investigated the effects of seawater pH (i.e., 8.10, 7.85 and 7.60) and temperature (16 and 19 °C) on (a) the abiotic conditions in the fluid surrounding the embryo (viz. the perivitelline fluid), (b) growth, development and (c) cuttlebone calcification of embryonic and juvenile stages of the cephalopod Sepia officinalis. Egg swelling increased in response to acidification or warming, leading to an increase in egg surface while the interactive effects suggested a limited plasticity of the swelling modulation. Embryos experienced elevated pCO₂ conditions in the perivitelline fluid (>3-fold higher pCO₂ than that of ambient seawater), rendering the medium under-saturated even under ambient conditions. The growth of both embryos and juveniles was unaffected by pH, whereas ⁴⁵Ca incorporation in cuttlebone increased significantly with decreasing pH at both temperatures. This phenomenon of hypercalcification is limited to only a number of animals but does not guarantee functional performance and calls for better mechanistic understanding of calcification processes.     

Biomineralisation during operculum regeneration in the polychaete Spirobranchus lamarcki

Formation of calcified biominerals is widespread in marine animals and is often associated with important elements of their biology, such as support and protection. Serpulid polychaetes are relatively understudied examples of biomineralisation despite their prominence in many marine ecosystems. An investigation of calcification in the regenerating opercular plate of the serpulid polychaete Spirobranchus (formerly Pomatoceros) lamarcki was performed using optical microscopy, calcein labelling and powder diffraction analysis. Worms were collected between January 2012 and June 2013 from East Sands beach, St Andrews, Scotland (56.33°N, 2.78°W). The earliest visible signs of calcification were birefringent grains. Later-stage regenerates displayed a complex mixture of calcified structures including grains, round, smooth tiles, and larger tiles with a rugged appearance. The plate matures by the growth and eventual merging of tiles into a contiguous crust. Calcein pulse-chase experiments showed the progression of calcification from the centre towards the edge of the plate, and powder diffraction analysis of three regenerative stages revealed a major shift in mineralogy from a predominantly calcitic to a predominantly aragonitic composition. The mechanisms underlying the shift are currently unknown. These are the first mineralogical data comparing different developmental stages in a serpulid operculum and contribute to the understanding of biomineralisation in this group.