Purification and characterization of vitellin from the estuarine mysid Neomysis integer (Crustacea; Mysidacea)

Invertebrates account for roughly 95% of all animals, yet surprisingly, little effort has been invested to understand their value in signaling potential environmental endocrine disruption. There has been, however, much recent attention on vitellogenin induction in egg-laying invertebrates and vertebrates as indicators of exposure to estrogenic xenobiotics. Mysid shrimp (Crustacea: Mysidacea) have been put forward by several researchers and regulatory bodies (e.g., US-EPA) as suitable test organisms for the evaluation of environmental endocrine disruption. In view of developing sensitive assays to study endocrine disruption in the estuarine mysid Neomysis integer, we isolated and characterized vitellin, the major yolk protein in eggs. Vitellin was purified using gel filtration and characterized by electrophoresis using different staining procedures. Specific (as shown by Western blotting) polyclonal antibodies were produced in rabbit against the purified vitellin of N. integer. These antisera will be used to develop immunoassays to study vitellogenesis in mysids and to detect potential stimulatory or inhibitory effects of endocrine disruptors on the production of vitellin.     

Fine structure of the statocyst sensilla of the mysid shrimp Neomysis integer (Leach, 1814) (Crustacea,Mysidacea)

The fine structure of the statocyst sensilla of Neomysis integer was investigated. The statocyst contains about 35 sensilla, which are composed of two bipolar sensory cells, nine enveloping cells, and a seta. The sensory cells consist of an axon, a perikaryon, and a dendrite. The dendrite contains a proximal segment with a ciliary rootlet and at least one basal body, and a distal segment with a ciliary axoneme (9 × 2 + 0) at its base. The distal segment extends along the peripheral wall of the seta and is in close contact with the wall of the hair shaft. The enveloping cells surround the proximal and distal segments of the dendrite. The innermost enveloping cell contains a scolopale rod. It surrounds the receptor lymph cavity and secretes flocculent material into this cavity. From the tip of the cell a dendritic sheath, which encloses the distal segment of the dendrite, emerges. A peculiar feature of the second enveloping cell is the presence of a scolopale-like rod, which is more slender and less pronounced than in the first enveloping cell. The seta consists of three parts: a socket, a tubular midpart, and a gutter-like apical part, the tip of which penetrates into the statolith. The seta shows over its full length a bilaterally symmetrical axis that is coplanar with the plane in which the seta is bent toward the statolith. The structure of the seta and the position of the distal segments provide morphological evidence for directional sensitivity of the sensilla and for the magnitude of shear on the setal wall being an adequate stimulus.     

Intersexuality in the mysid Neomysis integer

Intersexuality is described in Neomysis integer (Leach), apparently the first occurrence among the Mysidacea. Seven out of 13,700 specimens examined were masculinized females possessing elongated fourth pleopods, a male secondary sexual characteristic. Each masculinized female appeared to have reproduced as a normal female; there was no evidence of testicular tissue.     

Phylogeography of an estuarine mysid, Neomysis integer (Crustacea, Mysida), along the north-east Atlantic coasts

Aim The brackish water mysid, Neomysis integer, is one of the most common mysid species along the coasts of the north‐east Atlantic. In the present study, the phylogeographical patterns were examined throughout the distribution range of N. integer. In particular, the latitudinal trends in genetic diversity and the distribution of genetic variation were examined in order to elucidate the imprints of the Pleistocene glaciations. Location North‐east Atlantic coasts from the Baltic Sea to the south of Spain. Methods A total of 461 specimens from 11 populations were analysed by means of single‐stranded conformation polymorphism analysis combined with DNA sequencing of a fragment of the mitochondrial cytochrome c oxidase I gene. The genetic structure was examined by using a progression of phylogenetic, demographic and population genetic analyses to elucidate not only the geographical structure, but also the evolutionary history producing that structure. Results The levels of genetic diversity were relatively uniform throughout the distribution range, with the exception of a decline at the northern and southern edges of distribution. A high heterogeneity was observed between the populations analysed (global Φ_ST = 0.787). This is caused by the disparate distribution of the cytochrome oxidase I haplotypes, with several population‐specific haplotypes. A clear genetic break (2.4% sequence divergence) occurred between the southernmost Guadalquivir population and all other populations. Main conclusions The present study corroborates the expectations of the genetic patterns typically observed in an estuarine species. The within‐population variability was low, whereas a significant (moderate to high) divergence was observed between populations. Phylogeographical analysis revealed that northern populations within the English Channel, North Sea and Baltic Sea are characterized by several widespread haplotypes, while the Irish population and all sites south of the Bay of Biscay consist solely of unique haplotypes. This pattern, combined with the relative high levels of genetic diversity, could be indicative for the presence of a glacial refugium in the English Channel region. Under this scenario N. integer must have survived the Last Glacial Maximum in the palaeoriver system present in that region.     

Comparative morphology and actuopalaeontology of mysid statoliths (Crustacea,Mysidacea)

Summary A comprehensive and comparative study of the external statolith morphology of the family Mysidae is presented. The study covers 48 species from major systematic groups occupying a large number of habitats in different biogeographical zones of the globe. Statoliths generally show high morphological diversity. The traditional classification scheme of subtaxa and the correlation of statolith characters with segmentation patterns of body appendages suggest that the organic composition and the nearly spherical structure of the statoliths of Boreomysinae and Rhopalophthalminae are plesiomorphic compared with the more complex mineralized statoliths found in all other subfamilies. During ontogenetic development the number of sensorial setae and associated pores and pore groups on the statolith increase with body size and statolith diameter. Although patterns of caudal pores are highly specific for some genera, the high intraspecific variance of pore numbers strongly reduces the diagnostic value of this feature in most species. Statolith characters can be successfully used for identification of subfamilies, tribes, and especially genera. For future palaeontological applications a proper diagnosis of fossil mysid statoliths is essential. Therefore, we provide a key to subfamilies and tribes based exclusively on statolith characters.     

Structure of recent and fossil mysid statoliths (Crustacea,Mysidacea)

Statoliths of 61 Recent species representing all subfamilies of Mysidae were studied with special emphasis on internal structure. In addition 5 samples of fossil statoliths from Miocene deposits were examined. Species of Boreomysinae and Rhopalophthalminae show simple roughly spherical organic statoliths, with setae originating from the sensory cushion and anchored in the statolith with distal branches extending shortly below the surface. All other subfamilies possess mineralized statoliths of greater structural complexity, with differentiation in core and mantle, where each part may consist of up to three layers. Habitus is hemispherical to discoidal. External gross structures are dorsal tegmen, ventral fundus, and the ambitus forming the outer toroidal to semi-toroidal circumference. Setae penetrate the mantle through mineralic canals and insert on the surface of the core. As suggested by congeneric species of Schistomysis, there is no principal structural difference between statoliths mineralized with fluorite compared to vaterite. However, vaterite statoliths tend to be more often of moruloid appearance and are exceptional by showing a central conical hole (the hilum) or a central cavity in certain forms. These structures are typical of fossil calcite statoliths. In vaterite and fluorite statoliths, the mantle shows radially arranged (= spherulitic) crystal aggregates. Such arrangements are badly preserved in fossil calcite statoliths. In large extant statoliths, concentric structures, mainly in the form of superficial striation and/or concentric microstrata, are visible in coexistence with radial aggregates. Stratification is possibly due to stratified deposition of the nonmineralized gland product, while the spherulitic structure is indicative of subsequent radial growth of crystal aggregates. The structure of accessory fluorite statoliths in the statocyst of Mesopodopsis slabberi leads to the hypothesis that mantle material is formed by secretions of the caudal statocyst gland. After demineralization of fluorite, vaterite and calcite statoliths, an organic template remains showing most essential morphological features of the statolith. From this we conclude that the structure of the statolith is (almost) entirely matrix mediated. © 1993 Wiley-Liss, Inc.     

Biogeographical division of antarctic and subantarctic by mysid (Crustacea: Mysidacea) fauna

Based on the distribution of 66 species of Mysidacea (Crustacea), a biogeographical analysis of the cold and temperate waters of the Southern Hemisphere was carried out. The expediency of separate division by pelagic and benthopelagic mysid faunas was supported. The original patterns of the division of Antarctic and subantarctic faunas are given. One biogeographical province and two subprovinces were distinguished by the fauna of pelagic mysids in the areas studied. Two biogeographical provinces and five subprovinces were distinguished by the fauna of benthopelagic mysids in the Antarctic and subantarctic.     

Development of antennal sensilla during moulting inNeomysis integer (Leach) (Crustacea,Mysidacea)

Summary The sensilla are associated with 6 enveloping cells. The innermost enveloping cell (e 1) secretes the dendritic sheath (=thecogen cell). All other enveloping cells are involved in the formation of the outer cuticular apparatus in secreting the cuticle of a definite region of the new hair shaft.The development of the new sensilla begins when an exuvial space expands between old cuticle and epithelium. The newly forming hair shafts lie folded back in an invagination of the epidermal tissue. Only a distal shaft part projects into the free exuvial space. The cuticle of the distal and middle shaft region is secreted by the three middle enveloping cells (e 2–e 4) (=trichogen cells), which are arranged around the dendritic sheath.The wall of the cylinder, in which the distal shaft is situated, is formed by the cuticle of the future proximal shaft region. It is secreted by the outer enveloping cells (e 5 and e 6). Furthermore, both enveloping cells form the hair socket (=trichogen-tormogen cells).The outer dendritic segments encased within a dendritic sheath run up through the newly formed hair shaft and continue to the old cuticular apparatus. The connection between sensory cells and old hair shaft is maintained until ecdysis. On ecdysis the old cuticle is shed and the newly formed shaft of the sensillum is everted like the invaginated finger of a glove. The dendritic sheath and the outer dendritic segments break off at the tip of the new hair shaft. Morphologically this moulting process ensures that the sensitivity of the receptors is maintained until ecdysis.The internal organization of the sensory cells shows no striking changes during the moulting cycle. An increased number of vesicles is accumulated distally within the inner dendritic segments and distributed throughout the outer segments of the dendrites. The cytoplasmic feature of the enveloping cells indicates that synthesis and release of substances for the cuticular apparatus of the new sensillum take place.     

Doxomysis algoaensis, a new mysid species (Crustacea: Mysidacea) from Algoa Bay (South Africa)

Doxomysis algoaensis sp.nov. is described from Algoa Bay, South Africa where it is common in nearshore marine waters just beyond the breaker line. D. algoaensis sp.nov. is morphologically similar to D. australiensis, but can readily be distinguished by the shape and armature of the telson and the length of the exopod of the fourth male pleopod. The apical cleft is one fifth the telson length in the former species and one third the length in D. australiensis. The telson apex on each side of the cleft is also armed with five and four stout spines in the two species, respectively. In D. australiensis, the exopod of the fourth male pleopod is almost three times the length of the endopod; in D. algoaensis sp.nov., the exopod is only slightly longer than the endopod. Other distinctive features of D. algoaensis sp.nov. include the maxillary palp, which is only slightly broader than long and the greater number of spines on the endopod of the uropod.     

A new freshwater mysid from the Amazon, with a reassessment of Surinamysis Bowman (Crustacea: Mysidacea)

A new species of mysis, Surinamysis robertsonae , is described from the plankton of the Lago Calado, a whitewater lake in the Amazon Basin. This is only the second freshwater mysid to be described from the Amazon. Surinamysis Bowman, 1977, comprising S. robertsonae, S. americana and S. merista , is raised to full generic rank on the grounds of telson morphology (compared to the other species of the Antromysis group) and the flagelliform process of the male antennule.     

The biogeographical division of the Arctic and North Atlantic by the mysid (Crustacea: Mysidacea) fauna

The biogeographical analysis of Arctic and North Atlantic waters north of 30°N is based upon the distribution of 150 Mysidacea (Crustacea) species. The reasons for biogeographical divisions conducted independently by faunae of pelagic and benthopelagic mysids are adduced. The original schemes of the Arctic and North Atlantic division are proposed. Using the fauna of pelagic mysids, one biogeographical realm, one province and one transitional zone are designated in cold and temperate waters. Using the fauna of benthopelagic mysids, one biogeographical realm and four provinces are designated in those waters.     

Spelaeomysis cochinensis,a new mysid (Crustacea: Mysidacea) from a prawn culture field in Cochin,India

Spelaeomysis cochinensis sp. nov., the eighth species of the genus is described from a prawn culture field in Cochin, India. It is characterised by a pair of eye plates with few ommatidia and the fully spiny border of the telson.     

A quantitative enzyme-linked immunosorbent assay for Dermatophagoides pteronyssinus-specific immunoglobulin G

A monoclonal mouse anti-human IgG was used to develop an enzyme-linked immunosorbent assay (ELISA) for the measurement of Dermatophagoides pteronyssinus (DP)-specific IgG in human sera. This monoclonal antibody (HG2-25) binds to all subclasses of IgG but not to IgA, IgM, or IgE. For the assay, the DP antigen is coated firmly on polystyrene beads through physical adsorption and any leakable antigen is washed off. The assay gives satisfactory reproducibility and parallelism of the dilution curves. Using 0.1% human serum albumin as a substitute for the DP-specific IgG preabsorbed diluent gave extremely low backgrounds and high sensitivity. Horseradish peroxidase-labeled HG2-25 prepared with the optimum degree of conjugation and free of polymerized conjugates gave responses fairly proportional to the doses. This ELISA gives a satisfactory recovery and is not affected by nonspecific IgG levels in human sera.     

Cellular energy allocation in the estuarine mysid shrimp Neomysis integer (Crustacea: Mysidacea) following tributyltin exposure

Recently, we described the cellular energy allocation (CEA) methodology to asses the effects of abiotic stress on the energy metabolism of the estuarine crustacean Neomysis integer (Crustacea: Mysidacea) [J. Exp. Mar. Biol. Ecol. 279 (2002) 61]. This short-term assay is based on the biochemical assessment of changes in the energy reserves (total carbohydrate, protein and lipid content) and the energy consumption (electron transport activity), and has been shown to be predictive of effects at the population level in daphnids [J. Aquat. Ecosyst. Stress Recovery 6 (1997) 43]. In the present study, the CEA methodology was evaluated using adult N. integer exposed for 96 h to the antifoulant tributyltinchloride (TBTCl). From a range-finding experiment with juvenile N. integer, a 96-h LC50 of 164 ng TBTCl/l was calculated. The energy metabolism of N. integer, as summarized by the CEA, was significantly altered by TBTCl exposure. Mysids exposed to 10, 100 and 1000 ng TBTCl/l consumed less energy and had lower respiration rates (in 10 and 1000 ng TBTCl/l treatments) than the control, resulting in a lower CEA. These changes at the cellular level occurred at environmentally relevant concentrations of the toxicant TBTCl which were an order of magnitude lower than reported effect concentrations for scope for growth in other marine invertebrates.     

A quantitative enzyme-linked immunosorbent assay for bovine herpesvirus type 1 (BHV-1) antibody

A quantitative enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure antibody to bovine herpesvirus type 1 (BHV-1) in cattle sera. The optical density produced from a single dilution of test serum was compared with a standard curve and the results were read and printed out from a computer interfaced to a multichannel ELISA reader. The printed results were expressed in ELISA units. The ELISA results obtained on 370 cattle sera were compared with those of the serum neutralisation test (SNT). An agreement of 90.5% was obtained when reciprocal SNT titres equal to or greater than 4 and IgG ELISA units equal to or greater than 50 were taken as indicative of a specific reaction. Of the 370 sera, 35 gave discrepant results of which 21 were SNT positive/IgG ELISA negative and 14 were SNT negative/IgG ELISA positive. When the SNT positive sera negative in the IgG ELISA were tested in an IgM ELISA, 19 were found to be positive. Thus, when the IgG and IgM ELISA results were combined the overall agreement between the ELISA and SNT increased to 95.7%. The IgG ELISA had a sensitivity of 82.4% and specificity of 94.4% relative to the SNT, whereas the combined IgG and IgM ELISA results gave a sensitivity and specificity of 98.3% and 94.4% respectively. There was a good positive correlation between the two tests (r = 0.86).     

Optimality assessment in the enzyme-linked immunosorbent assay (ELISA)

An optimality criterion is proposed for evaluating the precision of alternative designs in the enzyme-linked immunosorbent assay. Assay profiles are represented as four-parameter logistic functions with parameter estimation based on either a weighted nonlinear regression or a simple nonlinear regression after a logarithmic transformation. Assay design changes are characterized in terms of their effects on parameters in the four-parameter logistic model. General optimality results are derived for the variance of relative potency estimates in routine assay applications.     

Development of recombinant antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of skatole

The occurrence of boar taint and the European Commission recommendation to discontinue the surgical castration of pigs by the year 2018 creates an urgent need for new analytical methods that are simple, affordable, and suitable for field testing. We describe the generation and engineering of a skatole-specific antibody derived from a synthetic antibody library and the development of ELISA for its detection. The immunoassay is capable of detecting skatole with IC50 of 222 μg L⁻¹, which is within the analytical threshold level suggested for skatole, and with low cross-reactivity interference from other indolic compounds.     

An enzyme-linked immunosorbent assay (ELISA) for plasma cortisol

A direct ELISA for plasma cortisol is described which is carried out in a standard 96 well microtitre plate. In this ELISA cortisol-thyroglobulin conjugate is immobilised to the microtitre plate and competes with cortisol in the standard or plasma sample for antibody binding sites. Following washing the rabbit cortisol antibody bound to immobilised cortisol is incubated with peroxidase labelled goat antirabbit IgG. Following further washing o-phenylenediamine is added, colour developed, and the plate read at 492 nm on a standard ELISA plate reader. This ELISA shows good agreement with RIA and its sensitivity, specificity and precision allow its use in the routine steroid laboratory.