CAROTENOPROTEINS IN INVERTEBRATES

True carotenoproteins are defined as complexes in which a carotenoid and a protein are in stoichiometric combination. Very few carotenoproteins have been purified, but their presence in extracts may often be presumed from spectroscopic evidence. Carotenoids have been found in stoichiometric association with simple proteins, lipoproteins and glycoproteins. Many lipoproteins also occur in which carotenoids form part of the lipid prosthetic group. Although the relationship between the carotenoid and the remainder of the complex may in these cases not appear to be stoichiometric, it often shows some evidence of selectivity. Carotenoproteins are widely distributed among the invertebrate phyla. They are found mainly in exoskeleton or ectoderm and in eggs and ovaries. Their isolation by standard techniques of protein chemistry is facilitated by their characteristic spectral properties. The ease with which carotenoid prosthetic groups are removed from apoproteins suggests that covalent linkages are not involved in the complex formation. The union of carotenoid with apoprotein has been shown, in at least two cases, to stabilize the carotenoid against photo-oxidation and the protein against denaturative changes of tertiary structure. In crustacyanin, the carapace pigment of the lobster, there is clear evidence that the carotenoid is responsible for establishing the very complex quaternary structure. Only astaxanthin, astaxanthin esters and canthaxanthin have been unequivocally identified as carotenoid prosthetic groups of true carotenoproteins. Experiments in the specificity of interaction of a series of carotenoids with the apoprotein of crustacyanin have indicated the requirement of one or both of the 4- and 4′-keto groups of the carotenoid for interaction with the protein. It is suggested that, at least in this case, the carotenoid-protein interaction might occur between carotenoid keto groups and basic residues of the protein in such a way as to provide a lock on the tertiary configuration. Carotenoproteins may participate in a variety of functions including protective coloration, photosensitivity, electron transport and enzymic activity. They appear, furthermore, to play some part in development.     

Resonance Raman study of reconstituted carotenoproteins incorporating astaxanthin and 15,15'-didehydroastaxanthin

Two reconstituted carotenoproteins have been studied by resonance Raman spectroscopy. They were prepared from the apoprotein of the Asterias rubens carotenoprotein, asteriarubin and either astaxanthin or 15,15'-didehydroastaxanthin. Spectral properties of dehydrocarotenoids are first discussed. The spectral properties of the complexes are compared to those of the free carotenoids and of other carotenoproteins containing astaxanthin, and possible protein-carotenoid interactions are discussed. Greater delocalisation of the pi-electron system in the central part of the polyene chain, and the role of lateral methyl groups in binding is emphasised.     

Astaxanthin binding and structural stability of the apple snail carotenoprotein ovorubin

Ovorubin (OR) is the major perivitellin of the eggs of Pomacea canaliculata. The astaxanthin (ASX) binding and structural stability of OR were investigated by fluorescence spectroscopy and circular dichroism (CD). The apo-OR (without astaxanthin) shows a single, high affinity binding site for ASX (K(D)=0.5 microM). The quenching of tryptophan fluorescence by ASX indicates that about 22% are near the carotenoid-binding site in a non-polar environment, as indicated by tryptophan resonance energy transfer to the ligand. Secondary structure (alpha+beta) was virtually not affected by cofactor removal. Holo-OR shows unusually high thermal stability. The removal of ASX does not affect the thermal or chemical stability of the quaternary structure. In conclusion, although subtle changes were observed, ASX is not essential for OR stability, unlike most invertebrate carotenoproteins. This supports the idea that OR plays an important physiological role in the storage, transport and protection of carotenoids during snail embryogenesis.     

Animal carotenoids—carotenoproteins from hydrocorals

• 1.1. The carotenoid complexes isolated from six corals of the order Hydrocorallina (Stylaster roseus, S. elegans, S. sanguineus, Distichopora coccinea, D. nitida and D. violacea) have been re-examined.
• 2.2. The yellow to violet colours of the calcareous skeletons of these hydrocorals are evoked by carotenoproteins containing astaxanthin (1) and zeaxanthin (2).
• 3.3. A preliminary characterization of these new carotenoproteins is reported.

     

Evolution of proteins of macroglobulin family

The first representatives of proteins of the macroglobulin family appeared 500–700 million years ago. At present representatives of this family have been revealed in crustaceans, molluscs, fish, amphibians, reptiles, ticks, insects, birds, and mammals, the macroglobulin family in blood of some species being represented simultaneously by several proteins that have different molecular weight and partly duplicate functions of each other. In different species, they are present as monomers, dimers, or tetramers. The distinguishing feature of each protein subunit is the presence of a “trap” with cyclic thioether on the bottom and of a sufficiently large hydrophobic area. All representatives are able to form complexes with different regulatory substances through covalent or hydrophobic bonds, which allows them to perform a wide range of regulatory functions. The ancient origin, evolutionary conservatism, widespread presence, and a diversity of regulatory functions permit proteins of the macroglobulin family to be considered as the main regulatory biomolecules of organism fluid media.     

Challenging the model for induction of metallothionein gene expression

Metallothioneins (MTs) are low-molecular-weight, cysteine-rich metal-binding proteins found in a wide variety of organisms including bacteria, fungi and all eukaryotic plant and animal species. MTs bind essential and non-essential heavy metals. In mammalian cells MT genes are highly inducible by many heavy metals including Zn, Cd, Hg, and Cu. Aquatic systems are contaminated by different pollutants, including metals, as a result of man's activities. Bivalve molluscs are known to accumulate high concentrations of heavy metals in their tissue and are widely used as bioindicators for pollution in marine and freshwater environments, with MTs frequently used as a valuable marker of metal contamination. We here describe the MT isoform gene expression patterns of marine and freshwater molluscs and fish species after Cd or Zn contamination. Contamination was carried out at a river site polluted by a zinc ore extraction plant or in the laboratory at low, environmentally relevant metal concentrations. A comparison for each species based on the accumulated MT protein levels often shows discrepancies between gene expression and protein level. In addition, several differences observed in the pattern of MT gene expression between mollusc and mammalian species enable us to discuss and challenge a model for the induction of MT gene expression.     

The distribution and incidence of larval trematodes in the freshwater fauna of the Wentloog level, South Wales

The Wentloog level, in South Wales, is a low-lying coastal plain dissected by a network of drainage channels. The latter contain a rich aquatic invertebrate fauna which harbour a wide range of larval digenetic trematodes. Few of the host species examined were not infected with larval trematodes. Miracidia together with their associated sporocysts, rediae and cercariae were more host specific than metacercariae but many of the miracidia which developed in pulmonate molluscs could do so in more than one genus. Metacercarial host specificity is less rigid, and usually species are only restricted to members of one phylum although several were found which could develop in two or more phyla. Most larval stages showed some host preference when they occurred in several host species and this was attributed to ecological or physiological factors or to mechanical barriers. Seasonal variation of infection rate occurs in all larval stages: infection with developing cercariae showed peaks during June and August, but metacercarial infection rates varied according to the host. In molluscs there may be two peaks of infection with metacercariae (as in the cercarial stages), but for many arthropods infections show a gradual increase through the year. The factors mainly responsible for these seasonal changes are considered to be the life-cycle of the intermediate hosts and the environmental temperature, the latter acting on the rate of development of the trematodes. Rainfall, and patterns of egg production of adult trematodes in the definitive hosts are also important.     

Endocrine-related reproductive effects in molluscs

Research on endocrine disruption has been a major topic of the past decade. Although most studies concentrated on vertebrate species, invertebrates are now gaining more attention. In particular, data on molluscs is increasing. One of the best-documented and more relevant examples of endocrine disruption is the imposex phenomenon affecting some gastropod species. But the increasing interest is also due to the fact that molluscs, especially bivalves, are good bioindicators used for decades in environmental studies and that progress have been made in the understanding of the physiology and endocrinology of some mollusc species. Recent results suggest that molluscs can be adversely affected by compounds that alter their reproduction and that vertebrate-type sex-steroids metabolism or mechanism of action could be involved in these effects. Nevertheless, the endocrine system of molluscs appears to be dissimilar in many aspects to those of vertebrates and sex-steroids might not have the same importance in all mollusc species. This diversity constitutes an important opportunity to examine and understand new and alternative mechanisms for endocrine disruption.     

Evolutionary regularities of somatic polyploidy manifestation in salivary glands of gastropod molluscs. I. Subclasses Cyclobranchia and Scutibranchia

By means of histological and cytochemical methods, including DNA cytophotometry, the salivary glands of 11 species of molluscs of two old gastropod subclasses--Cyclobranchia and Scutibranchia (limpets)--have been investigated. In spite of some anatomical differences, the glandular epithelium of investigated molluscs includes functionally similar cell types: granular cells (with glycoproteid granular inclusions), mucocytes-I (that include sulfatic acid mucopolysaccharides), mucocytes-II (that include neutral and acid polysaccharides and proteins) and also the epithelial ciliated cells. Data of experiments on starvation and synchronous feeding of molluscs testify that all described cell types are independent. According to DNA cytophotometry data, the glandular cell nuclei are diploid in the main; only small part of the nuclei, varying in different species from 0.5 to 5.0%, displayed tetraploid DNA mass. A conclusion is made that in the oldest subclasses of gastropods (Cyclobranchia and Scutibranchia) somatic polyploidy, as a factor of tissue growth in salivary glands, is actually absent.     

Structural characteristics of hen egg ovalbumin expressed in yeast Pichia pastoris

The recombinant ovalbumin (OVA) produced in yeast Pichia pastoris was purified from the culture medium by anion exchange chromatography, and its structural characteristics were compared with those of hen egg OVA, mainly from the point of view of posttranslational modification. The expressed OVA consisted of two molecular species immmunoreactive with antibody for hen egg OVA. The two molecular species, 45 and 47 kDa in molecular size, were thought to correspond to mono-glycosylated form and di-glycosylated form respectively. The non-glycosylated form was not produced in the system. The other posttranslational modifications (N-terminal acetylation and phosphorylation) observed in hen egg OVA were not detected in either of the molecular species. The two recombinant proteins displayed almost exactly the same circular dichroism and intrinsic tryptophan fluorescence spectra as hen egg OVA. The melting temperature, Tm, which was determined from the thermal unfolding curve, was almost identical in the two recombinant proteins, despite the difference in glycosylation levels, while it decreased by about 2.5 degrees C as compared with that of hen egg OVA (77.3 degrees C). These data indicate that the additional glycosylation to Asn-311 in the recombinant protein does not affect protein conformation or thermostability.     

Correlated evolutionary divergence of egg size and a mitochondrial protein across the Isthmus of Panama

An explicit assumption of studies that employ a mitochondrial DNA (mtDNA) molecular clock is that mtDNA evolves independently of morphology. Here we report a very strong correlation between egg size divergence and cytochrome c oxidase-1 (CO1) amino acid sequence divergence among sister species of bivalve molluscs separated by the Central American Isthmus (i.e., "geminate" species). Analyses of the molecular data reveal that CO1 sequences likely did not diverge as a function of time or evolve in response to positive natural selection. Given that an excess of CO1 amino acid polymorphism exists within species (as expected if most mutations are only slightly deleterious), a third hypothesis is that reductions in effective population size could simultaneously increase the fixation rate of nearly neutral mtDNA polymorphisms and in some way also facilitate egg size evolution. The remarkable strength of the relationship between egg size and CO1 amino acid sequence demonstrates that, even in the absence of an obvious functional relationship or clock-like evolution, the amounts of molecular and morphological change can be tightly correlated, and therefore may reflect common processes. Accordingly, the assumption that the evolutionary divergence of molecules and morphology are independent must always be carefully examined.     

Parasitism of Molluscs by Nematodes: Types of Associations and Evolutionary Trends

Although there are no confirmed fossil records of mollusc parasitic nematodes, diverse associations of more than 108 described nematode species with slugs and snails provide a fertile ground for speculation of how mollusc parasitism evolved in nematodes. Current phylogenic resolution suggests that molluscs have been independently acquired as hosts on a number of occasions. However, molluscs are significant as hosts for only two major groups of nematodes: as intermediate hosts for metastrongyloids and as definitive hosts for a number of rhabditids. Of the 61 species of nematodes known to use molluscs as intermediate hosts, 49 belong to Metastrongyloidea (Order Strongylida); of the 47 species of nematodes that use molluscs as definitive hosts, 33 belong to the Order Rhabditida. Recent phylogenetic hypotheses have been unable to resolve whether metastrongyloids are sister taxa to those rhabditids that use molluscs as definitive hosts. Although most rhabditid nematodes have been reported not to kill their mollusc hosts prior to their reproduction, some species are pathogenic. In fact, infective juveniles of Phasmarhabditis hermaphrodita vector a lethal bacterium into the slug host in which they reproduce. This life cycle is remarkably similar to the entomopathogenic nematodes in the families Steinernematidae and Heterorhabditidae. Also, the discoveries of Alloionema and Pellioditis in slugs are interesting, as these species have been speculated to represent the ancestral forms of the entomopathogenic nematodes. Development of the infective stage appears to be an important step toward the acquisition of molluscs as definitive hosts, and the association with specific bacteria may have arisen in conjunction with the evolution of necromeny.     

Astaxanthin binding to solubilized muscle proteins of Atlantic salmon (Salmo salar L.), haddock (Melanogrammus aeglefinus L.) and Atlantic halibut (Hippoglossus hippoglossus L.)

A study was conducted to compare astaxanthin binding ability of solubilized muscle proteins of Atlantic salmon (Salmo salar L.), haddock (Melanogrammus aeglefinus L.) and Atlantic halibut (Hippoglossus hippoglossus L.). Muscle proteins of juvenile Atlantic salmon, haddock and halibut were solubilized by sequential extraction of muscle tissue using low ionic strength solutions. Electrophoretic protein profiles of the six solubilized fractions from these species were similar. Each solubilized fraction from the three species was examined for its relative astaxanthin binding capacity. The amount of bound astaxanthin was significantly different (P < 0.05) among the six fractions of each species. Significant differences in astaxanthin binding were only found for fractions A and E among the species. The amount of bound astaxanthin in various fractions of each species showed a good correlation (R² = 0.80–0.92) with the ANS (8-anilino-1-naphthalenesulfonate) fluorescence intensity of those fractions. The pattern and extent of astaxanthin binding to the muscle proteins of juvenile salmon, haddock and halibut is comparable to that reported previously for adult Atlantic salmon [Saha, M.R., Ross, N.W., Gill, T.A., Olsen, R.E., Lall, S.P., 2005. Development of a method to assess binding of astaxanthin to Atlantic salmon S. salar L. muscle proteins. Aquacult. Res. 36, 336–343.]. These combined observations suggest that the carotenoid binding capacity of the muscle proteins of salmon is not the limiting factor in the deposition of carotenoid in their flesh.     

Protamines and related proteins from spermatozoa of molluscs. Characterization and molecular weight determination by gel electrophoresis

The spermatozoa of most species of molluscs contain a mixture of proteins with compositions related to those of histones and protamines. The various components present in the spermatozoa of Cryptochiton stellerii, Donax trunculus, Mactra corallina and Gibbula divaricata have been isolated and characterized. The results obtained, taken together with those previously found (Subirana, J.A., Cozcolluela, C., Palau, J. and Unzeta, M. (1973) biochim. Biophys. Acta 317, 364--379), show that in all the molluscs studied one or more arginine-rich components are present. The molecular weight of these proteins varies vary much in different species and is usually much greater than in conventional fish protamines. Conventional histones, as well as lysine-rich proteins of low molecular weight, have also been found in ripe spermatozoa of several species. The molecular weights have been estimated by gel electrophoresis, using polymerized iridine as a standard.     

Mutants with aberrant numbers of gametic cells shed new light on old questions

In contrast to animals, plant gametes form in distinct haploid generations, termed gametophytes. The female gametophyte of Arabidopsis consists of two gametic cells, the egg and central cell, which are flanked by accessory cells. The gametic cells differ with respect to morphology, molecular attributes and, importantly, their fate: whereas the egg cell, upon fertilisation, gives rise to the embryo, the central cell forms the endosperm. To ensure correct endosperm formation, not only the egg cell but also the central cell has to fuse with a sperm cell. The respective sperm cell pair is delivered by a single pollen tube. In some plant species, the two male gametes appear to express a different bias towards the female gametes. Such a preference consequently determines their respective contribution to either embryo or endosperm development. In Arabidopsis and many other species the sperm cells are indistinguishable and it has been discussed whether they possess an inherent preference for either of the female gametes. The recent isolation of mutants that form an aberrant number of either male or female gametes stimulates discussion, albeit with different results. Furthermore, some data indicate that the central cell is competent to initiate endosperm formation without a paternal contribution. These data support the theory that the endosperm is of gametophytic rather than sporophytic origin.     

The late Cenozoic ampullariidae (mollusca,gastropoda) of the Albertine Rift Valley (Uganda-Zaire)

The Albertine Rift Valley (Uganda-Zaire) contains vast sedimentary sequences of late Cenozoic age. They were deposited in an extensive riftlake, Lake Obweruka, which existed from c. 8 Ma to 2.5 Ma and was comparable in size and depth to the present L. Tanganyika.Many freshwater molluscs that occur in these lacustrine deposits are characterised by their aberrant shell morphology, their extreme ornamentation and general form, making them resemble marine species. This convergence, rare in freshwater molluscs, is called thalassoidism and extreme ornamentation in marine as well as in freshwater molluscs is considered to be the result of a gradual process of prey/predator coevolution.In the present paper the Albertine representants of the ampullariid genera Lanistes and Pila, most of which are new to science, are taxonomically described and their phylogenetic relation, based upon apomorphic characters, is given. In addition the evolutionary history of these freshwater snails in the basin has been reconstructed.In the pre-riftlake environment 3 species of Lanistes occurred, with no special shell adaptations against predation. After the formation of a riftlake, 2 of these, colonising the new lacustrine ecospace, changed morphologically and radiated. The 3 derived lines show minor adaptations against predation. After the extinction of the dominant Lanistes species group around 6 Ma, the sole surviving lacustrine Lanistes suddenly radiates, the ancestral line persisting next to the 3 new daughter lines. This second morphological shift is spectacular as it produces shells with distinct thalassoid features. All the Lanistes species of L. Obweruka became extinct during a cataclysmic event around 4.5 Ma. Populations of the genus Pila colonised lacustrine habitats after this event, the derived form also showing striking thalassoid characters. There is no doubt that the intense morphological change occurred during a brief period, geologically speaking. The degree of morphological change in molluscs appears hence not to be linked with time. After the sudden radiation all lineages remain morphologically stable until they became extinct c. 1 Ma later. This pattern corresponds to the punctuated equilibrium model. Other groups (viviparids, thiarids) show more gradual changes.     

Change of carotenoid composition in crabs during embryogenesis

Changes of the qualitative and quantitative compositions of carotenoids are studied at various development stages of the external hard roe, determined based on color differences, for the species C. opilio, P. camtschaticus, and P. platypus. It has been revealed that the major carotenoids of the new egg are astaxanthin and β-carotene. Intermediate products of transformation of β-carotene into astaxanthin are identified: echinenone, canthaxanthine, and phenicoxanthine. The carotenoid content per embryo for the new hard roe of C. opilio (the orange egg) amounted to 22.7 ng, of P. camtschaticus and P. platypus (the violet egg)—to 49.2 and 23.3 ng, respectively. In the hard roe at the later development stage (the brown egg) the carotenoid content was decreased to 13.1 ng in C. opilio and to 20.1 ng in P. camtschaticus. Development of embryos is accompanied by accumulation of esterified carotenoids and a decrease of β-carotene and astaxanthine concentrations in all studied species.     

Temperature, Food Availability, and the Development of Marine Invertebrate Larvae

SYNOPSIS. Marine invertebrates develop in waters that extendfrom the poles to the equator, experiencing the full range ofenvironmental temperature and food conditions. How selectionhas modified their development under the influence of thesetwo factors has been a matter of debate. In this paper we arguethat the primary influence on developmental rate is temperature,while other factors such as food availability are much lessimportant. From existing literature we demonstrate that (1)developmental rates of both lecithotrophic and planktotrophicasteroids decrease in a similar way from the tropics to thepoles, as they do also in other groups of invertebrates (echinoids,molluscs, crustaceans), and (2) rates of development at anyone temperature cluster around the function describing the effectof temperature, without any relationship to egg size, suggestingthat developmental rates are near the maximum for a given temperatureregardless of other variables such as nutrition. We also investigatedthe response of development to temperature in four species ofplanktotrophic asteroids, one tropical, one temperate, two polar.There was limited temperature compensation among these fourspecies, but little or no apparent ability to compensate forthe retarding effects of reduced temperature within species.Arrhenius analysis of the data suggests that Q₁₀ values forthe upper region of each species' tolerance range are approximately2, indicating that enzyme-based reactions have evolved to beclosely integrated with uncatalyzed, temperature-dependent,physicalchemical processes. Values of Q₁₀ at lower regions ofthe tolerance range, on the other hand, range between 9.5 and14.7, indicative of abrupt temperature-dependent shifts in reactionequilibria, or in the organization of macromolecules and membranes.We conclude that temperature itself, rather than egg size, food,or other variables, best explains observed latitudinal differencesin developmental rates in marine invertebrates.     

方斑东风螺单孢子虫病的研究

文章对方斑东风螺(Babylonia areolata Link, 1807)单孢子虫病进行了首次报道, 对其病理学特征、流行季节和诊断方法进行了研究。结果认为方斑东风螺被单孢子虫侵袭后, 吻管、足肌、肠、消化腺、鳃、胃和肝等器官引起一系列病变。病理组织切片观察到肠上皮黏膜组织细胞布满该虫营养体和各发育期的多核质体, 造成肠结缔组织呈现炎症反应。寄生部位病灶肿胀、混浊、坏死、崩解。       

A Test of the Higher-Taxon Approach in the Identification of Candidate Sites for Marine Reserves

Alternatives to species-level identification have been advocated as one solution to the problem of selecting marine reserves with limited information on the distribution of marine biodiversity.This study evaluated the effects on selection of candidate sites for marine reserves from using the higher-taxon approach as a surrogate for species-level identification of intertidal molluscs and rocky reef fishes. These effects were evaluated by determining the percentage of species included in candidate reserves identified from genus-, family- and order-level data by a complementarity-based reserve selection algorithm, and by testing for correlations between the irreplaceability values of locations. Candidate reserves identified from genus- and family-level data of intertidal molluscs included a similar percentage of all species as the reserves identified from species-level data. Candidate reserves selected from genus- and family-level data of rocky reef fishes included, respectively, 3–7% and 14–23% fewer species than reserves selected from species-level data. When the reserve identification process was constrained by a practical planning limit (a maximum of 20% locations able to be reserved) the reserves selected from genus- and family-level data of intertidal molluscs, and genus-level data of rocky reef fishes, included a similar percentage of species as the reserves identified from species-level data. Irreplaceability values of locations for species, genera and families of intertidal molluscs were highly correlated, and irreplaceability values of locations for species and genera of rocky reef fishes were highly correlated. This study suggests that genus- and family-level data for intertidal molluscs, and genus-level data for rocky reef fishes, are suitable surrogates for species in the identification of candidate sites for marine reserves.