Cytogenetic studies in green mussel, Perna viridis (Mytiloida: Pteriomorphia), from West Coast of India

Chromosomes of Perna viridis were characterized by karyotype analysis, C-banding and nucleolus organizer regions (NORs). The diploid chromosome number was confirmed as 30 and the karyotype is composed of ten metacentric and five submetacentric chromosome pairs. Constitutive heterochromatin blocks were found on all chromosomal pairs except chromosomal pair 15, which showed uniform staining throughout the entire chromosomes. Silver staining revealed nucleolus organizer regions on telomeric region of four chromosomal pairs, viz. 1, 3, 7 and 11. This is the first comprehensive study undertaken on chromosomes of Perna viridis.     

Spermatocyte chromosome banding studies inBuccinulum corneum (Prosobranchia: Neogastropoda): Variation in silver-NOR banding pattern

Diploid number (2n=72), and haploid number (n=36) forBuccinulum corneum (L. 1758) collected from the Gulf of Palermo in December 1987 were determined. A simple method to obtain nucleolar organizer regions (NOR), and constitutive heterochromatin regions (C-bands) of chromosomes ofB. corneum is described. Analyses of silver-stained chromosome preparations ofB. corneum suggest that a within-individual variability in NOR-banding pattern is present in each of the five specimens analysed.     

Karotypic characterization of turbot (Scophthalmus maximus) with conventional,fluorochrome and restriction endonuclease-banding techniques

A karyotypic analysis was carried out in turbot (Scophthalmus maximus) using conventional banding techniques, fluorochromes and restriction endonucleasebanding. The standard karyotype of turbot is 2n=44 chromosomes, with 48 chromosome arms. Nucleolar organizer regions, which were localized in the short arm of Chromosome Pair No. 3, showed C-and chromomycin A₃-positive staining. C-bands were mainly located at the centromeres, but were also present in some interstitial and telomeric locations. No differences were revealed among constitutive heterochromatin bands after treatment of fixed chromosomes with fluorochrome staining and restriction endonucleases. Digestion with DdeI restriction enzyme produced a pattern of interstitial banding which suggested some degree of differentiation along the chromosome arms in turbot. Neither variation in chromosome number of arm number nor polymorphism in nucleolar organizer regions was revealed in the turbot analyzed.     

Karyotypic characterization of the great sturgeon, Huso huso, by multiple staining techniques and fluorescent in situ hybridization

A karyotype analysis by several staining techniques was carried out on the great sturgeon, Huso huso (Linnaeus, 1758). The karyotype (2n = 118 ± 2) was composed of 42 pairs of meta-/submetacentric chromosomes and 17 pairs of acrocentrics/microchromosomes. Constitutive heterochromatin was mainly located at the centromeric regions of the acrocentric chromosomes. The biarmed chromosomes showed weak C-bands. Fluorescent staining with GC-specific chromomycin A₃ showed clearly recognizable fluorescent regions, whereas a more uniform base composition was revealed by the AT-specific 4,6-diamidino-2 phenylindole. After Ag-staining, nucleolar organizer regions could be observed on the short arms of two medium-sized submetacentrics and on two acrocentrics. Digoxigenated 28S and 5S rDNA probes, prepared from Acipenser naccarii DNA and hybridized to metaphase chromosomes, showed signals on six and two chromosomes, respectively. The telomeric sequence (TTAGGG) _n detected by FISH was located at both ends of each chromosome. Results are discussed in relation to karyotype organization and evolution in sturgeons. Received: 1 April 1998 / Accepted: 21 July 1998     

Karyotype, nucleolus organiser regions and constitutive heterochromatin in Ostrea angasi (Molluscae: Bivalvia): evidence of taxonomic relationships within the Ostreidae

Chromosome preparations from gill tissue of the Australian flat oyster Ostrea angasi Sowerby were studied with conventional Giemsa staining, C-banding and silver staining techniques. A diploid complement of 2n = 20 was observed, consisting of five metacentric, three submetacentric and two subtelocentric pairs. Constitutive heterochromatin was distributed as large centromeric blocks in Chromosome Pairs 3, 6, 8, 9 and 10. The nucleolus organizer regions (NORs) were located terminally on long arms of Chromosome Pairs 9 and 10. This allowed the identification of homologous chromosomes in submetacentric and subtelocentric pairs. Intraspecific variability in NOR pattern as revealed by differences in the number of silver-stained NORs (Ag-NORs) per cell was found to be very common. Comparison of the patterns of karyotype, C-band and Ag-NORs between species of the larviparous oysters for which data have been published demonstrate that the chromosomal structure of the endemic Australian and New Zealand species O. angasi shows little similarity to the Southern Hemisphere oysters O. (Eostrea)puelchana Orbigny and Tiostrea chilensis (Philippi) and the Indo-West Pacific oyster O. denselamellosa, but very high resemblance to the European species O. edulis. Received: 12 August 1996 / Accepted: 16 September 1996     

Cytogenetics in the sacoglossan Oxynoe olivacea (Mollusca: Opisthobranchia): karyotype, chromosome banding and fluorescent in situ hybridization

Developing embryos and sexually mature follicles of the male portion of ovotestis proved to be a suitable material as a source of cleaving cells for advanced cytological investigations on the sacoglossan species Oxynoe olivacea Rafinesque, 1819 (Mollusca: Opisthobranchia). O. olivacea has a diploid chromosomal number of 30 made up of 15 pairs of which six are metacentric/submetacentric (M/SM), four subtelocentric (ST) and five on the borderline between SM and ST. Correspondingly, 15 bivalents occur in spermatocytes at Metaphase I. Constitutive heterochromatin is scarce and restricted to small C-bands seen in five pachytene bivalents. The use of combined silver staining and fluorescent in situ hybridization (FISH) with a Paracentrotus lividus (Echinodermata) 4.3 kilobase (kb) rDNA probe (prR14) consisting of sequences from the 3′ end of 18S rDNA to the 3′ end of 26S rDNA, revealed that nucleolus organizer regions (NORs) are situated terminally on one arm of a small metacentric pair. The telomeric (TTAGGG) _n sequence did not hybridize with termini of O. olivacea chromosomes. Received: 14 December 1999 / Accepted: 10 July 2000     

Karyotype differentiation between two species of carangid fishes,genusTrachurus (Perciformes: Carangidae)

A karyological study ofTrachurus trachurus andT. mediterraneus (Perciformes: Carangidae) was conducted by standard, fluorochrome staining (CMA₃, mithramycin, quinacrine mustard, DAPI), C-, Ag-NOR, and Alu-I banding methods. The karyotypes of both species consisted of 2n = 48 chromosomes, but of different FN:T. trachurus possessed a chromosome complement of 2 metacentric and 46 acrocentric elements, fundamental number (FN) = 50 andT. mediterraneus, a chromosome complement of 4 metacentric, 4 submetacentric, 14 subtelocentric and 26 acrocentric chromosomes, FN = 70. In neither of the two taxa investigated were heteromorphic sex chromosomes observed. The nucleolar organizer region was interstitially located on the long arm of the Ist pair of chromosomes in both species, intermediate inT. mediterraneus and subterminal inT. trachurus. Constitutive heterochromatin was found in nearly all centromeric and telomeric regions inT. trachurus; inT. mediterraneus it formed less intense telomeric and centromeric bands and thin interstitial bands on eight chromosome pairs. In addition, the C-positive material reacted differently to the digestion with endonuclease Alu-l in the two species. The results are discussed and compared with karyological data known for other species of Carangidae.     

Cytogenetic characterization of the greater amberjack, Seriola dumerili (Pisces: Carangidae), by different staining techniques and fluorescence in situ hybridization

A cytogenetic analysis by several staining techniques was carried out on specimens of Seriola dumerili from the Tyrrhenian coast of Sicily. The karyotype was found to be composed of 48 chromosomes in all the specimens examined. All chromosomes are subtelo- and acrocentrics, except for one pair of small-sized submetacentric chromosomes. After Ag-staining, nucleolus organizer regions (NORs) can be observed on the short arms of a medium-sized subtelocentric chromosome pair, and after chromomycin A₃-staining a bright fluorescent signal is evident at the same sites. The 18S rDNA mapping by fluorescence in situ hybridization confirms this unique NOR location. The constitutive heterochromatin is mainly restricted to centromeres and the fluorochrome-staining by chromomycin A₃ and 4′,6-diamidino-2-phenylindole revealed a uniform base composition along the chromosomal arms; therefore stock/population-specific marker chromosomes cannot be identified. Such cytogenetic features, however, rule out the presence of morphologically differentiated sex-chromosomes in the greater amberjack. Received: 16 January 1997 / Accepted: 31 January 1997     

Les caryotypes de quelques espèces de bivalves et de gastéropodes marins

The karyotypes of the bivalves Ostrea edulis (2 n=20), Crassostrea gigas (2 n=20), Mytilus galloprovincialis (2 n=28) and M. edulis (2 n=28) were obtained from mitotic metaphases of branchial tissue by means of a cellular suspension technique. The occurrence of metacentric, submetacentric and telocentric chromosomes in O. edulis as well as differences in size between Pair 1 and Pair 10 emphasize the cytological interest of this species compared to C. gigas which possesses only metacentric and submetacentric chromosomes. The variation in Pair 2, which is telocentric in M. galloprovincialis and metacencentric in M. edulis, has been observed for the first time and may support taxological separation of these two species. The karyotypes of the mesogastropods Rissoa ventricosa (2 n=32) and Littorina neritoides (2 n=34) were obtained from mitotic metaphases of gonadal tissue by the same technique. Sexual chromosomes were identified in the karyotypes of R. ventricosa. At the beginning of spermatogenesis, the gonad of L. neritoides shows both haploïd and polyploïd meïotic metaphases which correspond to typical (germinal cells) and atypical (nurse cells) forms, respectively.     

Replication,C and Ag-NOR chromosome-banding in two anguilliform fish species

The chromosomes of the Conger conger and Echelus myrus (Anguilliformes: Teleostei), were analysed by conventional (C and Ag-NOR) and by replication-banding techniques. Both species had a diploid number of 38 and one chromosome pair with C-positive nucleolar organizer regions (NOR). These cytogenetic traits are mainly consistent with those recorded for anguilliform species in general. The replication-banding pattern allowed us to identify individual chromosome pairs, to detect different classes of chromatin, and to suggest chromosome evolutive mechanisms in these two species. The results confirm the superiority of replication patterns in resolving longitudinal differentiation of fish chromosomes, that is not generally possible from structural bands.     

Structural chromosomal polymorphism in the dog-whelk Nucella lapillus (Mollusca: Neogastropoda)

On the English and French Channel coasts, the dog-whelk Nucella lapilus (L.) exhibits variation in chromosome number which appears to correlate with the degree of wave action on the shore. The more common, 2n=26 morph is typically found on exposed shores subjected to a high degree of wave action, whereas those with higher chromosome numbers, up to the recorded maximum of 2n=36, are restricted to more sheltered environments. The polymorphism is thought to be Robertsonian in nature, involving centric (centromere) fission or fusion, but detailed analysis of the polymorphism has been restricted by lack of success in labelling individual chromosomes. Using a silver-staining technique for the nucleolar organiser regions (NORs), three pairs of chromosomes, in the basic 2n=26 karyotype, have been positively identified. A series of structural chromosomal rearrangements (pericentric and paracentric inversions) affecting one pair of chromosomes involved in the numerical polymorphism is described. Significant differences exist between populations with respect to this character. These chromosomal rearrangements have the potential to reduce the level of interbreeding between the different types, and may act as isolating mechamisms between breeding groups. Structural chromosomal polymorphism is likely, therefore, to have greater significance in relation to adaptation than simple numerical variation. This finding raises important questions concerning the (cyto)taxonomic status of N. lapillus in different parts of its range.     

Cytogenetic analysis of Epinephelus marginatus (Pisces: Serranidae), with the chromosome localization of the 18S and 5S rRNA genes and of the (TTAGGG)n telomeric sequence

 A cytogenetic analysis was carried out on specimens of Epinephelus marginatus (Lowe, 1834) from three localities in the Mediterranean Sea, to deepen knowledge of the chromosome complement of the species and identify any possible population-specific cytogenetic markers. All specimens had a 2n = 48 acrocentric karyotype with two Ag-, chromomycin A₃- and C-positive NORs (nucleolar organizer regions) in the subcentromeric region of the smallest chromosome pair. The constitutive heterochromatin was distributed centromerically. Except for NORs, neither eu- nor heterochromatin show fluorescence after fluorochrome staining, i.e. there is no localized increase of AT- or GC-rich DNA. In all populations, the (TTAGGG) _n telomeric sequences are restricted to the telomeres, and the 18S and the 5S rDNA clusters are located on different chromosome pairs. In specimens from Sardinia, additional signals after C-banding, Ag-staining and FISH (fluorescence in situ hybridization) with 18S rDNA can be observed in the telomeric region of one or two large-sized chromosomes, classified as No. 2. This suggests that additional and variable NORs could be detected in the species in addition to those on the genus-specific, NOR-bearing Chromosome Pair 24. Received: 20 October 1999 / Accepted: 14 April 2000     

Number and morphology of chromosomes in three geographical populations of Ascidiella aspersa (Ascidiacea)

In contrast with some previous reports, we have found the same number of chromosomes (n=9, 2n=18) for Ascidiella aspersa Müller from the Gulf of Naples, the west coast of Scotland and the Lagoon of Venice. It follows that the hypothesis, that different geographical populations of A. aspersa might possess different chromosome numbers, is no longer tenable. The karyogram of A. aspersa shows 9 pairs of metacentric autosomes. The oocyte bivalents show chiasmata and are characterized by the faintness with which they are stained.This investigation was supported by a grant from C.N.R. for Marine Biology, n. 69.00117 14.43.3.     

Karyology of the toadfish Porichthys plectrodon (Jordan and Gilbert, 1882) (Batrachoididae) from Margarita Island,Venezuela

This paper reports the results of cytogenetic analyses carried out on Porichthys plectrodon using conventional Giemsa staining, C-banding and silver staining techniques. A diploid chromosome count of 2n=44 was observed, consisting of 8 metacentric, 10 submetacentric, 6 subtelocentric and 20 acrocentric chromosomes. Differences in length made it possible to identify homologous chromosomes within the metacentric group. Constitutive heterochromatin was distributed as large pericentromeric blocks in pairs 1 and 2, while the rest of the chromosomes were marked in centromeric regions, some more conspicuously than others. One pair of small-sized acrocentric NOR-bearing chromosomes (21) was identified by the nucleolar regions located terminally on their short arms.Communicated by P.W. Sammarco, Chauvin     

Karyotype analysis of the sea urchinParacentrotus lividus (Echinodermata): evidence for a heteromorphic chromosome sex mechanism

A consistent diploid number of 2n = 36 was determined for the sea urchinParacentrotus lividus from the Gulf of Palermo by analysis of mitotic chromosomes of both early developing embryos and male gonads. The haploid numbern = 18 was determined by counts of spermatocyte bivalents at diakinesis. A heteromorphic chromosome sex mechanism of the XY type is likely present in this species. This is indicated by the occurrence of a chromosomal pair, pair No. 2, which is heteromorphic in both morphology and size in about 50% of the mitotic figures (metaphases and anaphases) of einbryos. In addition, heteromorphism of the same pair of chromosomes occurred during spermatogonial metaphases in the five male specimens investigated here. The detection of a low chromosome number (2n = 36) compared to other echinoids (2n = 42 to 44), a heteromorphic chromosome sex system and the involvement of three chromosome pairs in nucleolar organization (NORs) provide evidence of the specialization of theP. lividus karyotype.     

Karyotype and sex determination in <Emphasis Type="Italic">Dinophilus gyrociliatus</Emphasis> (Polychaeta: Dinophilidae)

The study of the male and female karyotypes of the polychaete Dinophilus gyrociliatus was performed with the aim of ascertaining the diploid number of chromosomes and confirming the existence of karyological differences between the two sexes. Our results evidenced that the female karyotype of D. gyrociliatus consists of 2n=24 chromosomes, while the male karyotype has 2n=23 chromosomes. X chromosomes are subtelocentric and much bigger than the autosomes. The size of the D. gyrociliatus chromosomes, which in the female embryos vary in length from little more than 2.8 wm to less than 0.7 wm, are relatively small by comparison with those of other polychaetes. The existence of maternal and environmental factors able to influence the sex ratio of D. gyrociliatus and chromosomal differences between the sexes confirms that: (1) sex determination is chromosomal and syngamic of the XX-X0 type and (2) control of the sex ratio is progamic and depends on genetic and environmental factors.     

Cytogenetic analysis of Oedalechilus labeo (Pisces: Mugilidae), with a report of NOR variability

 A cytogenetic analysis by several staining techniques was carried out on Oedalechilus labeo specimens from the west coast of Italy, in order to extend the karyological knowledge on Mediterranean mullets. The karyotype of the species is composed of 46 acrocentric and 2 subtelocentric chromosomes. Constitutive heterochromatin was found to be restricted to the centromeres. Fluorochrome staining revealed a uniform base composition along the chromosome arms. Nucleolus organizer regions (NORs) are located on the short arms of chromosome pair 9 and can be either homomorphic or quite heteromorphic in size. In some specimens chromomycin A₃-staining and fluorescence in situ hybridization with 18S rDNA revealed a third additional and inactive NOR on the short arms of a medium-sized chromosome. This is the first report of NOR variability in Mugilidae, a family generally characterized by a quite conservative karyotype. Heterochromatin composition and NOR location differentiate O. labeo from a cytotaxonomical point of view from the Liza species studied, Liza being considered the genus from which the genus Oedalechilus derived. Received: 10 April 1999 / Accepted: 21 October 1999     

Adaptations to barnacle fouling in the mangroves <Emphasis Type="Italic">Kandelia obovata</Emphasis> and <Emphasis Type="Italic">Aegiceras corniculatum</Emphasis>

Mangrove trees planted for restoration or rehabilitation often fail to show satisfactory survivals. Although it is generally assumed that barnacle fouling is a factor in mangrove mortality, the sub-lethal response of mangroves to this fouling is not known. In the present study, we compare the leaf morphology (stomata density, leaf thickness, percentage thickness of palisade layer and number of hypodermal layers), lenticel density and the plant fitness (number of flowers or droppers per tree) between mangroves fouled by the acorn barnacle Fistulobalanus albicostatus (the fouled group, number of barnacles ~ 120 individuals per tree) and non-fouled mangroves (the control group). We observed 55 Kandelia obovata from three locations, two in Hong Kong (n = 35, fouled 25 and control 10) and one location in Taiwan (n = 20, fouled 10 and control 10). Aegiceras corniculatum (n = 45) was observed from two sites in Hong Kong, one on the east coast (n = 20, 10/10) and one on the west coast (n = 25, 10/15). In seedlings (K. obovata n = 10, 5/5), fouling density did not affect leaf morphology. In mature trees, however, leaves from fouled trees had significantly higher density of stomata and stem lenticel, a lower percentage thickness of leaf palisade layer and more layers of lower hypodermis in the leaves, probably for compensating the impeded gaseous exchange functions of the lenticels. The number of droppers and flowers on fouled mangroves was significantly lower. It is possible that after compensating for respiratory stress, the plant had less energy available for reproduction. Although there was no obvious barnacle-induced mortality in this study, barnacles appear to be related to a reduction in plant fitness. This may induce sufficient stress that over time fouled mangroves cannot sustain viable populations.     

Use of blubber levels of progesterone to determine pregnancy in free-ranging live cetaceans

We tested the possibility of measuring progesterone levels in bubbler samples collected from free-ranging live bottlenose dolphins (n = 11) and long-finned pilot whales (n = 2) as a tool to evaluate the pregnancy status of individuals. Samples were collected during January 2004 and September–October 2005 in the Strait of Gibraltar and Gulf of Cadiz and used for sex-determination by genetic methods (skin samples) and for quantification of progesterone levels by enzyme immunoassay. Photo-identification tracking of females after taking the biopsy was used to observe the presence of newborns and, in this way, to determine if the female was pregnant at the time of sampling. Mean progesterone levels from pregnant bottlenose dolphins (n = 2) were around 9 times higher than those from non-pregnant females (n = 9), with no overlap between concentration ranges demonstrating that this method could constitute an effective tool for determining pregnancy in wild populations of bottlenose dolphins and other cetacean species.     

Chemical consequences of relocation of the soft coral Lobophytum compactum and its placement in contact with the red alga Plocamium hamatum

Seven colonies of Lobophytum compactum Tixier-Durivault, 1956, which produce isolobophytolide as the major secondary metabolite, were selected from a fringing reef in the Pelorus Channel, Palm Island Group (18°34S; 146°29E), North Queensland, Australia. In September 1991, they were sectioned to afford two portions which were relocated to a grid, and a significant part of the parent colony which was left in place. The aim of the experiment was to determine the effect of relocation and contact with a toxic alga on the secondary metabolite content of a soft coral. A significant increase in the concentration of isolobophytolide was observed for all relocated colonies (n=14, p=0.001) compared to the non-relocated control colonies. This decreased after 2 mo, and was not significantly different from that of the non-relocated control colonies (n=14, p=0.881). After 1 mo, Plocamium hamatum J. Agardh plants were placed in direct contact with 50% of the relocated colonies. All soft-coral colonies in contact with the alga (n=7), showed tissue necrosis on the parts in direct contact with the alga after a further 2 wk. Tissues of the relocated control colonies (n=7), and those portions of treated colonies which were not in direct contact with the alga, were not affected. The parts of the colonies in contact with the alga showed a significant decrease in lipid content over time (n=7, p=0.001) and also a decrease in the concentration of the diterpene isolobophytolide (n=7, p=0.001). The effects of P. hamatum on the soft coral were essentially restricted to contract necrosis; chemical variations in the affected tissue were the outcomes of this necrosis. These results indicate that stress due to relocation is a more important factor in the variation of isolobophytolide levels in the soft coral L. compactum than is contact with the alga P. hamatum.