Flow cytometric characterization of the hemocytes of sea hares from tidal pools in Jeju Island off the south coast of Korea.

The members in the family Aplysidae known as sea hares play a crucial role as a grazer in small tidal pools or shallow subtidal hard bottoms. Like other marine gastropods, hemocyte types and functions of sea hares are limitedly known. This study identified and characterized the hemocytes of common Aplysia species Aplysia kurodai, A. juliana, and A. oculifera in rocky tidal pools in Jeju Island off the south coast of Korea using flow cytometry and light microscopy. The flow cytometry identified three different hemocytes in the hemolymph of the three sea hare species: granulocytes, hyalinocytes, and blast-like cells. The granulocytes exhibited pseudopodia on the cell surface and granules in the cytoplasm. Morphology of the hyalinocyte was similar to that of the granulocytes, while they lack cytoplasmic granules. The blast-like cells were small and round, with very thin cytoplasm. The hyalinocytes were the most abundant in the hemolymph, accounting for 89.8-92.6% of the total hemocytes. Flow cytometry indicated that the granulocytes and blast-like cells were less than 5.6% and 5.4% of the total hemocyte populations. Flow cytometry also revealed that the granulocytes and hyalinocytes are engaged in cellular defensive activities such as intra-cellular lysosomal content, phagocytosis, and ROS production. The mean lysosomal contents of the granulocytes (0.4 × 105-0.2 × 105 A U.) were 2-3 times higher than that of hyalinocytes (0.2 × 105-0.6 × 105 A U.). In addition, the ROS production of the granulocytes (0.98 × 106-1.95 × 106 A U.) was about twice higher than that of the hyalinocytes (0.62 × 106-1.14 × 106 A U.). Of the three species of sea hares, the granulocytes showed comparatively higher phagocytosis capacity (70.4-92.3%) than that of the hyalinocytes (34.8-46.0%). Flow cytometry and microscopy indicated that the hemocyte types and their functions were identical, regardless of the species.

Hyperspectral interference tomography of nacre.

Structural characterization of biologically formed materials is essential for understanding biological phenomena and their enviro-nment, and for generating new bio-inspired engineering concepts. For example, nacre-the inner lining of some mollusk shells-encodes local environmental conditions throughout its formation and has exceptional strength due to its nanoscale brick-and-mortar structure. This layered structure, comprising alternating transparent aragonite (CaCO3) tablets and thinner organic polymer layers, also results in stunning interference colors. Existing methods of structural characterization of nacre rely on some form of cross-sectional analysis, such as scanning or transmission electron microscopy or polarization-dependent imaging contrast (PIC) mapping. However, these techniques are destructive and too time- and resource-intensive to analyze large sample areas. Here, we present an all-optical, rapid, and nondestructive imaging technique-hyperspectral interference tomography (HIT)-to spatially map the structural parameters of nacre and other disordered layered materials. We combined hyperspectral imaging with optical-interference modeling to infer the mean tablet thickness and its disorder in nacre across entire mollusk shells from red and rainbow abalone (Haliotis rufescens and Haliotis iris) at various stages of development. We observed that in red abalone, unexpectedly, nacre tablet thickness decreases with age of the mollusk, despite roughly similar appearance of nacre at all ages and positions in the shell. Our rapid, inexpensive, and nondestructive method can be readily applied to in-field studies.

Functional morphology of the sperm-containing chambers of the sea slug Okenia polycerelloides in the context of sexual selection.

Sea slugs are interesting models to study post-copulatory sexual selection in simultaneous hermaphrodites due to the enormous variation of their reproductive systems. However, the knowledge of the functional morphology of their reproductive system is limited to few species, and it is rarely discussed in the context of sexual selection theory. In this study, we investigated the functional morphology of the sperm-containing chambers (i.e., ampulla, seminal receptacle, and bursa copulatrix) of the reproductive system of Okenia polycerelloides (Ortea & Bouchet, 1983), based on light, confocal, and electron microscopy. Although the morphology of the ampulla is similar to other species, indicating that it is a site for autosperm storage, we found some sperm facing the ampullar epithelium, a feature commonly regarded as characteristic of the seminal receptacle of sea slugs. The seminal receptacle of O. polycerelloides showed secretory activity and contained sperm with distribution and orientation suggestive of stratification of allosperm from distinct mating events, a feature that would affect sperm competition. The bursa copulatrix had epithelial cells with secretory and absorptive characteristics, and contained degraded sperm and yolk granules within its lumen. Comparative analyses of the contents of each organ demonstrated that sperm digestion occurs in the bursa copulatrix and affects sperm heads first, changing their morphology from slender and curved to shorter and ellipsoid before complete lysis. Although digestion and absorption of surplus sperm are currently the main hypothesized functions for the bursa copulatrix, its role in cryptic female choice should not be ruled out. The close structural connection between the seminal receptacle and bursa copulatrix, as well as their muscular walls, would enable control over the fate of the sperm received in each mating event, that is, storage or digestion.

New data on spermatogenic cyst formation and cellular composition of the testis in a marine gastropod, Littorina saxatilis.

Knowledge of the testis structure is important for gastropod taxonomy and phylogeny, particularly for the comparative analysis of sympatric Littorina species. Observing fresh tissue and squashing fixed tissue with gradually increasing pressure, we have recently described a peculiar type of cystic spermatogenesis, rare in mollusks. It has not been documented in most mollusks until now. The testis of adult males consists of numerous lobules filled with multicellular cysts containing germline cells at different stages of differentiation. Each cyst is formed by one cyst cell of somatic origin. Here, we provide evidence for the existence of two ways of cyst formation in Littorina saxatilis. One of them begins with a goniablast cyst formation; it somewhat resembles cyst formation in Drosophila testes. The second way begins with capture of a free spermatogonium by the polyploid cyst cell which is capable to move along the gonad tissues. This way of cyst formation has not been described previously. Our data expand the understanding of the diversity of spermatogenesis types in invertebrates.

In vitro effects of glyphosate-based herbicides and related adjuvants on primary culture of hemocytes from Haliotis tuberculata.

Glyphosate-based herbicides are among the most produced and widely-used herbicides. Studies have shown that commercial formulations and adjuvants may be more toxic to non-target organisms than the active ingredients alone, but the mechanisms of action of these chemicals remain unclear. The aim of this study was to investigate the in vitro effects of glyphosate, a commercial formulation and adjuvant alone using primary culture of hemocytes from the European abalone Haliotis tuberculata, a commonly farmed shellfish. Glyphosate was found to have negligible effects on viability, phagocytic activities and lysosome stability even with very high doses (i.e. 100 mg L-1). By contrast, greater effects on viability were observed for the commercial formulation and adjuvant alone, with EC50 values of 41.42 mg L-1 and 1.85 mg L-1, respectively. These results demonstrate that the toxic sublethal effects (i.e. phagocytic activity and destabilization of lysosomal membranes) of formulated glyphosate came from adjuvants and suggest they may be related to cell and organelle membrane destabilization.

Enhanced discrimination of basophilic cells on mussel digestive gland tissue sections by means of toluidine-eosin staining.

Changes in the cell type composition of the digestive gland epithelium constitute a common and recognized biological response to stress in mussels. Usually, these changes are identified as alterations in the relative proportion of basophilic cells, determined in tissue sections stained with hematoxylin-eosin (H&E) and measured in terms of volume density of basophilic cells (VvBAS) after stereological quantification. However, the identification and discrimination of basophilic cells may be a difficult issue, even for a trained operator, especially when, in circumstances of environmental stress, basophilic cells lose their basophilia and the perinuclear area of digestive cells gains basophilia. Thus, the present study was aimed at exploring the best available practices (BAPs) to identify and discriminate basophilic cells on tissue sections of mussel digestive gland. In a first step, a thorough screening of potentially suitable staining methods was carried out; the final selection included several trichrome staining methods and some of their variants, as well as toluidine-based stains. Next, the sample processing (fixation/dehydration steps) was optimized. Toluidine-eosin (T&E) staining after fixation in 4% formaldehyde at 4 °C for 24 h was considered the BAP to identify and discriminate basophilic cells in the digestive gland of mussels. Using the mussel Mytilus galloprovincialis as a target organism, this approach was successfully applied to quantify VvBAS values after automated image analysis and compared with the conventional H&E staining in different field and laboratory tests. It is worth noting that VvBAS values were always higher after T&E staining than after H&E staining, apparently because discrimination of basophilic cells was enhanced. Thus, until more data are available, any comparison with VvBAS values obtained in previous studies using H&E staining must be done cautiously. Finally, the T&E staining was successfully used to discriminate basophilic cells in tissue sections of other marine molluscs of ecotoxicological interest, including Mytilus edulis, Mytilus trossulus, Crassostrea gigas and Littorina littorea.

Non-lethal method for the preparation of metaphase spreads using cultured mantle tissue from live adult abalone.

Metaphase spread preparation in adult abalone has not been successful, which has restricted the applications of karyotyping-based technologies. Here, we present a non-lethal method to enable preparation of metaphase spreads from live adult abalone using a tissue culture method. Mantle tissue fragments from live adult abalone were cultured in vitro and the cultured cells were used for metaphase spread preparation. To retrieve a sufficient number of proliferating cells required for metaphase spread preparation, at least 14 days of culture was required, and culturing the marginal zone of mantle was more optimal than culturing other areas. Additionally, it was shown that simple medium consisting of basal medium, fetal bovine serum and antibiotics could stimulate cellular proliferation followed by metaphase spread preparation.

Evaluation of lipid profile in different tissues of Japanese abalone Haliotis discus hannai Ino with UPLC-ESI-Q-TOF-MS-based lipidomic study.

Abalone has been farmed commercially for the last few decades, and the breeding and economic value of abalone have gained extensive attention. In this study, the lipid profile of the foot muscles, viscera, and gonads of male and female Japanese abalone Haliotis discus hannai Ino was explored using ultra performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS), to discuss the effect of lipid composition on its nutritional value. Thirty-four species from ten lipid classes including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, lysophosphatidic acid, triacylglycerol, steroids, terpenoids and fatty acids were annotated by MS-DIAL to obtain all fragment ions for precursors. Glycerophospholipids (GPLs) enriched in unsaturated fatty acids were the major components, which accounted for 52-57% of total lipids. Considering the high-level of GPLs, and their importance in maintaining the integrity and functionality of cell membranes, further utilization of inexpensive tissues, such as viscera and gonads is warranted.

Establishment and activity of the D quadrant organizer in the marine gastropod Crepidula fornicata.

During development in metazoan embryos, the fundamental embryonic axes are established by organizing centers that influence the fates of nearby cells. Among the spiralians, a large and diverse branch of protostome metazoans, studies have shown that an organizer sets up the dorsal-ventral axis, which arises from one of the four basic cell quadrants during development (the dorsal, D quadrant). Studies in a few species have also revealed variation in terms of how and when the D quadrant and the organizer are established. In some species the D quadrant is specified conditionally, via cell-cell interactions, while in others it is specified autonomously, via asymmetric cell divisions (such as those involving the formation of polar lobes). The third quartet macromere (3D) typically serves as the spiralian organizer; however, other cells born earlier or later in the D quadrant lineage can serve as the organizer, such as the 2d micromere in the annelid Capitella teleta or the 4d micromere in the mollusc Crepidula fornicata. Here we present work carried out in the snail C. fornicata to show that establishment of a single D quadrant appears to rely on a combination of both autonomous (via inheritance of the polar lobe) and conditional mechanisms (involving induction via the progeny of the first quartet micromeres). Through systematic ablation of cells, we show that D quadrant identity is established between 5th and 6th cleavage stages, as it is in other spiralians that use conditional specification. Subsequently, following the next cell cycle, organizer activity takes place soon after the birth of the 4d micromere. Therefore, unlike the case in other spiralians that use conditional specification, the specification of the D quadrant and the activity of the dorso-ventral organizer are temporally and spatially uncoupled. We also present data on organizer function in naturally-occurring and experimentally-induced twin embryos, which possess multiple D quadrants. We show that supernumerary D quadrants can arise in C. fornicata (either spontaneously or following polar lobe removal); when multiple D quadrants are present these do not exhibit effective organizer activity. We conclude that the polar lobe is not required for D quadrant specification, though it could play a role in effective organizer activity. We also tested whether the inheritance of the small polar lobe by the D quadrant is associated with the ability to laterally inhibit neighboring quadrants by direct contact in order to normally prevent supernumerary organizers from arising. Finally, we discuss the variation of spiralian organizers in a phylogenetic context.

Phylogenetic relationships among superfamilies of Neritimorpha (Mollusca: Gastropoda).

Despite the extraordinary morphological and ecological diversity of Neritimorpha, few studies have focused on the phylogenetic relationships of this lineage of gastropods, which includes four extant superfamilies: Neritopsoidea, Hydrocenoidea, Helicinoidea, and Neritoidea. Here, the nucleotide sequences of the complete mitochondrial genomes of Georissa bangueyensis (Hydrocenoidea), Neritina usnea (Neritoidea), and Pleuropoma jana (Helicinoidea) and the nearly complete mt genomes of Titiscania sp. (Neritopsoidea) and Theodoxus fluviatilis (Neritoidea) were determined. Phylogenetic reconstructions using probabilistic methods were based on mitochondrial (13 protein coding genes and two ribosomal rRNA genes), nuclear (partial 28S rRNA, 18S rRNA, actin, and histone H3 genes) and combined sequence data sets. All phylogenetic analyses except one converged on a single, highly supported tree in which Neritopsoidea was recovered as the sister group of a clade including Helicinoidea as the sister group of Hydrocenoidea and Neritoidea. This topology agrees with the fossil record and supports at least three independent invasions of land by neritimorph snails. The mitochondrial genomes of Titiscania sp., G. bangueyensis, N. usnea, and T. fluviatilis share the same gene organization previously described for Nerita mt genomes whereas that of P. jana has undergone major rearrangements. We sequenced about half of the mitochondrial genome of another species of Helicinoidea, Viana regina, and confirmed that this species shares the highly derived gene order of P. jana.

The central pattern generator underlying swimming in Dendronotus iris: a simple half-center network oscillator with a twist.

The nudibranch mollusc, Dendronotus iris, swims by rhythmically flexing its body from left to right. We identified a bilaterally represented interneuron, Si3, that provides strong excitatory drive to the previously identified Si2, forming a half-center oscillator, which functions as the central pattern generator (CPG) underlying swimming. As with Si2, Si3 inhibited its contralateral counterpart and exhibited rhythmic bursts in left-right alternation during the swim motor pattern. Si3 burst almost synchronously with the contralateral Si2 and was coactive with the efferent impulse activity in the contralateral body wall nerve. Perturbation of bursting in either Si3 or Si2 by current injection halted or phase-shifted the swim motor pattern, suggesting that they are both critical CPG members. Neither Si2 nor Si3 exhibited endogenous bursting properties when activated alone; activation of all four neurons was necessary to initiate and maintain the swim motor pattern. Si3 made a strong excitatory synapse onto the contralateral Si2 to which it is also electrically coupled. When Si3 was firing tonically but not exhibiting bursting, artificial enhancement of the Si3-to-Si2 synapse using dynamic clamp caused all four neurons to burst. In contrast, negation of the Si3-to-Si2 synapse by dynamic clamp blocked ongoing swim motor patterns. Together, these results suggest that the Dendronotus swim CPG is organized as a "twisted" half-center oscillator in which each "half" is composed of two excitatory-coupled neurons from both sides of the brain, each of which inhibits its contralateral counterpart. Consisting of only four neurons, this is perhaps the simplest known network oscillator for locomotion.

Octopaminergic system in the central nervous system of the terrestrial slug Limax.

The terrestrial slug Limax can learn to avoid the odor of some food (e.g., carrot juice) by the simultaneous presentation of an aversive stimulus (e.g., bitterness of quinidine). This type of associative memory critically depends on the higher olfactory center, the procerebrum in the central nervous system. The modulation of the local field potential (LFP) oscillation recorded on the procerebrum has been thought to reflect the information processing of the odor that elicits the behavioral change, such as avoidance of the aversively learned odor or approaching an attractive food's odor. Here we focused on octopamine, an important neuromodulator involved in learning and memory in invertebrates, and considered to be the invertebrate equivalent of noradrenaline. We identified a few octopaminergic neurons in the subesophageal and buccal ganglia, and a larger number near the procerebrum in the cerebral ganglia, using immunohistochmical staining and in situ hybridization of tyramine ß-hydroxylase, an octopamine-synthesizing enzyme. Application of octopamine reduced the frequency of LFP oscillation in a dose-dependent manner, and this effect was inhibited by preincubation with phentolamine. High-performance liquid chromatography analysis revealed the presence of octopamine, noradrenaline, and adrenaline in the central nervous system. Unexpectedly, noradrenaline and adrenaline both accelerated the LFP oscillation, in contrast to octopamine. Our results suggest that octopamine and noradrenaline have distinct functions in olfactory information processing, in spite of their structural similarity. J. Comp. Neurol. 524:3849-3864, 2016. © 2016 Wiley Periodicals, Inc.

Distribution of histaminergic neurons and their modulatory effects on oscillatory activity in the olfactory center of the terrestrial slug Limax.

Terrestrial mollusks can form an odor aversion memory following the simultaneous presentation of a food odor and an aversive stimulus. The local field potential oscillation recorded on the surface of the procerebrum (PC; the higher olfactory center) exhibits a frequency change in response to the detection of a learned odor; such a change is thus considered to reflect the internal state of the brain during memory recall. Thus far, dopamine and serotonin have been demonstrated to change the oscillatory frequency. Other monoamines, however, have not yet been studied. In the present study, we investigated the possible involvement of histamine (HA). Immunohistochemical staining of HA and in situ hybridization against histidine decarboxylase revealed the location of the cell bodies of HAergic neurons in all ganglia of the brain. The majority of them were located at the medial aspect of the pedal ganglia, and the cerebral ganglia also contained numerous HAergic neurons in their posterior regions. The neuropil layers of the PC received HAergic innervation from the neurons in the cerebral ganglion, as well as from a few neurons located in the dorsomedial part of the cell mass layer of the PC. The HAergic fibers, however, innervated spatially limited regions of the PC, and seemed to affect a small fraction of the PC neurons. HA exerted accelerating effects on the LFP oscillation in a dose-dependent manner, and this effect was suppressed by an H2 blocker, cimetidine. Our results support the involvement of HA in the functioning of the PC.

Innovative application of classic and newer techniques for the characterization of haemocytes in the New Zealand black-footed abalone (Haliotis iris).

Haemocytes play an important role in innate immune responses within invertebrate organisms. However, identification and quantification of different types of haemocytes can be extremely challenging, and has led to numerous inconsistencies and misinterpretations within the literature. As a step to rectify this issue, we present a comprehensive and detailed approach to characterize haemocytes using a combination of classical (cytochemical and phagocytosis assays with optical microscopy) and novel (flow cytometry with Sysmex XN-1000 and Muse(®) Cell analyser) techniques. The Sysmex XN-1000 is an innovative fluorescent flow cytometric analyser that can effectively detect, identify and count haemocytes, while the Muse(®) Cell analyser provides accurate and rapid haemocyte cell counts and viability. To illustrate this approach, we present the first report on morphological and functional features of New Zealand black-footed abalone (Haliotis iris) haemocyte cells. Two types of haemocytes were identified in this study, including type I (monocyte-like) and type II (lymphocyte-like) cells. Granular cells, which have been reported in other molluscan species, were not detected in H. iris. Cell types were categorized based on shape, size, internal structures and function. The lymphocyte-like haemocytes were the most abundant hemocytes in the haemolymph samples, and they had large nuclei and basic cytoplasms. Monocyte-like cells generally were larger cells compared to lymphocyte-like cells, and had low nucleus-cytoplasm ratios. Monocyte-like cells showed higher phagocytic activity when encountering Zymosan A particles compared to lymphocyte-like cells. The present study provides a comprehensive and accurate new approach to identify and quantify haemocyte cells for future comparative studies on the immune system of abalone and other molluscan species.

Potential Response to Selection of HSP70 as a Component of Innate Immunity in the Abalone Haliotis rufescens.

Assessing components of the immune system may reflect disease resistance. In some invertebrates, heat shock proteins (HSPs) are immune effectors and have been described as potent activators of the innate immune response. Several diseases have become a threat to abalone farming worldwide; therefore, increasing disease resistance is considered to be a long-term goal for breeding programs. A trait will respond to selection only if it is determined partially by additive genetic variation. The aim of this study was to estimate the heritability (h2) and the additive genetic coefficient of variation (CVA) of HSP70 as a component of innate immunity of the abalone Haliotis rufescens, in order to assess its potential response to selection. These genetic components were estimated for the variations in the intracellular (in haemocytes) and extracellular (serum) protein levels of HSP70 in response to an immunostimulant agent in 60 full-sib families of H. rufescens. Levels of HSP70 were measured twice in the same individuals, first when they were young and again when they were pre-harvest adults, to estimate the repeatability (R), the h2 and the potential response to selection of these traits at these life stages. High HSP70 levels were observed in abalones subjected to immunostimulation in both the intracellular and extracellular haemolymph fractions. This is the first time that changes in serum levels of HSP70 have been reported in response to an immune challenge in molluscs. HSP70 levels in both fractions and at both ages showed low h2 and R, with values that were not significantly different from zero. However, HSP70 induced levels had a CVA of 13.3-16.2% in young adults and of 2.7-8.1% in pre-harvest adults. Thus, despite its low h2, HSP70 synthesis in response to an immune challenge in red abalone has the potential to evolve through selection because of its large phenotypic variation and the presence of additive genetic variance, especially in young animals.

IGFBP7 promotes hemocyte proliferation in small abalone Haliotis diversicolor, proved by dsRNA and cap mRNA exposure.

Insulin-like growth factor binding protein 7 (IGFBP7) binds IGFs with a low affinity, but in contrast, recognizes insulin with a high affinity. Many studies show that IGFBP7 involves several cellular processes of vertebrates and functions as a tumor suppressor gene in different tumors. However, the function of IGFBP7 in invertebrates is unclear. In this research, we studied the function of IGFBP7 in the proliferation of small abalone Haliotis diversicolor hemocytes by exposure to dsRNA or cap mRNA of saIGFBP7. We found that exposure to dsRNA or cap mRNA of saIGFBP7 could significantly affect the mRNA and protein expression of IGFBP7 in cultured small abalone hemocytes (p<0.05). There was a significant increase in hemocyte density and the number of adherent hemocytes after exposure to cap mRNA of saIGFBP7 (p<0.05). Similarly, exposure to dsRNA of saIGFBP7 could significantly decrease the hemocyte density and the number of adherent hemocytes (p<0.05). These findings suggest that IGFBP7 increases hemocyte growth. It is the first time to report the effect of IGFBP7 on the proliferation of marine invertebrate cells.

Genotoxic potency of mercuric chloride in gill cells of marine gastropod Planaxis sulcatus using comet assay.

In vivo and in vitro exposures were used to investigate the genotoxicity of mercuric chloride (HgCl2) to the marine snail, Planaxis sulcatus. The comet assay protocol was validated on gill cells exposed in vitro to hydrogen peroxide (H2O2, 0-50 µM). Snails were exposed in vivo for 96 h to HgCl2 (10, 20, 50, and 100 µg/l). Our results showed significant concentration-dependent increase in the tail DNA (TDNA) and olive tail moment (OTM) in exposed snails for all doses compared with controls. In vitro exposure to HgCl2 (10-100 µg/l) resulted in significantly higher values for TDNA at all concentrations. Our results showed that DNA damage increased in the gill cell with increasing exposure time. This study demonstrates the usefulness of comet assay for detection of DNA damage after exposure to HgCl2 and the sensitivity of marine snail P. sulcatus as a good candidate species for metal pollution.

Cytogenetic analysis and chromosomal characteristics of the polymorphic 18S rDNA of Haliotis discus hannai from Fujian, China.

We report on novel chromosomal characteristics of Haliotis discus hannai from a breeding population at Fujian, China. The karyotypes of H. discus hannai we obtained from an abalone farm include a common type 2n = 36 = 10M + 8SM (82%) and two rare types 2n = 36 = 11M + 7SM (14%) and 2n = 36 = 10M + 7SM + 1ST (4%). The results of silver staining showed that the NORs of H. discus hannai were usually located terminally on the long arms of chromosome pairs 14 and 17, NORs were also sometimes located terminally on the short arms of other chromosomes, either metacentric or submetacentric pairs. The number of Ag-nucleoli ranged from 2 to 8, and the mean number was 3.61 ± 0.93. Among the scored interphase cells, 41% had 3 detectable nucleoli and 37% had 4 nucleoli. The 18S rDNA FISH result is the first report of the location of 18S rDNA genes in H. discus hannai. The 18S rDNA locations were highly polymorphic in this species. Copies of the gene were observed in the terminal of long or/and short arms of submetacentric or/and metacentric chromosomes. Using FISH with probe for vertebrate-like telomeric sequences (CCCTAA)3 displayed positive green FITC signals at telomere regions of all analyzed chromosome types. We found about 7% of chromosomes had breaks in prophase. A special form of nucleolus not previously described from H. discus hannai was observed in some interphase cells. It consists of many small silver-stained nucleoli gathered together to form a larger nucleolus and may correspond to prenucleolar bodies.

Does a short-term exposure to cadmium chloride affects haemocyte parameters of the marine gastropod Haliotis tuberculata?

In this study, a model based on primary cultured haemocytes from the gastropod mollusc Haliotis tuberculata was established to investigate the effects of cadmium chloride in vitro. Cells were exposed for 24 h to CdCl2 concentrations of 0, 1 and 100 µg ml(-1). The effects of cadmium on haemocyte parameters were investigated using morphological, spectrophotometric and flow cytometry analysis. Results showed that cadmium has no significant effects on cell viability and phagocytotic activity under the tested conditions. However, haemocytes became more rounded after cadmium exposure, which could explain the significant decrease of cell area beginning at 1 µg ml(-1) of CdCl2.

Distribution and characterization of rhogocyte cell types in the mantle tissue of Haliotis laevigata.

Molluscan rhogocytes are known to be the only cells able to synthesize hemocyanin that is one of the largest respiratory proteins in nature. However, investigation of rhogocyte cells in vitro is limited due to difficulty in isolating and establishing marine cell culture. The aim of this study was to investigate the nature and distribution of rhogocyte cells of Haliotis laevigata in the mantle tissue with respect to the expression of the two known isoforms of hemocyanin. Rhogocyte cells were identified using immunofluorescence-fluorescence in situ hybridization (IF-FISH) that involved simultaneous staining of localized hemocyanin by a polyclonal antibody while the mRNA was hybridized with FISH probes. The distribution of rhogocyte cells was demonstrated using flow cytometry, followed by cell sorting with fluorescence-activated cell sorter (FACS) and confocal microscope imaging for further characterization. Our results suggested that the mantle tissue is dominated by two distinct populations of rhogocyte cells that synthesize hemocyanin type 1. Observation with confocal microscopy of both populations revealed hemocyanin localization in the periphery of the cell membrane. Cell population with higher antibody signal had irregular and elongated cell morphology with punctate mRNA probe signals. The second population with lower antibody signal had ovoid morphology and wide distribution of mRNA probe signals. We suggest that these populations represent two distinct phases of hemocyanin biosynthesis of a single isoform, which is closely related to Haliotis tuberculata type 1 hemocyanin (HtH1). The knowledge acquired in this study enhances the understanding of the biology of rhogocyte cells and biosynthesis of hemocyanin.