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Characterization of mitotic and meiotic, chromosomesof the vermetid gastropod Dendropoma (Novastoa)Petraeum (Monterosato, 1884) (Mollusca,Caenogastropoda)Roberto Vitturi a , Amalia Pandolfo a , Maria Stella Colomba a , Francesca Damiani a & RenatoChemello aa Institute of Zoology, University of Palermo, Via Archirafi 18, I-90123, Palermo, ItalyVersion of record first published: 05 Mar 2012.

To cite this article: Roberto Vitturi , Amalia Pandolfo , Maria Stella Colomba , Francesca Damiani & Renato Chemello(1997): Characterization of mitotic and meiotic, chromosomes of the vermetid gastropod Dendropoma (Novastoa) Petraeum(Monterosato, 1884) (Mollusca, Caenogastropoda), Ophelia, 46:2, 127-139

To link to this article: http://dx.doi.org/10.1080/00785326.1997.10432579

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OPHELIA 46 (2): 127-139 (May 1997)

CHARACTERIZATION OF MITOTIC AND MEIOTIC,CHROMOSOMES OF THE VERMETID

GASTROPOD DENDROPOMA (NOVASTOA)PETRAEUM (MONTEROSATO, 1884) (MOLLUSCA,

CAENOGASTROPODA)

Roberto Vitturi, Amalia Pandolfo, Maria Stella Colomba,Francesco Damiani & RenataChemello

Institute of Zoology, University of Palermo, Via Archirafi 18,1-90123 Palermo, Italy

ABSTRACT

The vermetid gastropod Dendropoma (Nouastoa) petraeum from the Northwestern coast of Sicilywasinvestigated karyologically. The count of spermatocyte bivalents at diakinesis gave the haploidnumber of this species as n=17. The count of mitotic chromosomes in males, females and earlydeveloping embryos gave the diploid number as 2n=34. Irrespective of sex, the karyotype of D.petraeum consists of 17 homomorphic chromosome pairs, of which 8 are metacentric, 4 subtelo­centric and 5 acrocentric. This species is characterized by the presence of two chromosomes percell involved in nucleolus organization (NOR) and by a low amount of constitutive heterochrom­atin.

Keywords: Mollusca, Vermetidae, Dendropoma; chromosomes, karyotype

INTRODUCTION

The Vermetidae (worm gastropods) are a caenogastropod family, order Nee­taenioglossa (Ponder & Waren 1988) widely distributed in the intertidal zonesof warm-temperate to tropical seas (Keen 1961). Due to adaptation to a sessileexistence, they have a loose coiled shell and produce worm tubes attached tothe substratum.

The Vermetidae have been considered either as. the most highly apomor­phic family among cerithioidean molluscs (Houbrick 1988) or as a separatesuperfamily (Ponder & Waren 1988), this, above all because of the diversity ofsperm morphology (Healy 1988) but also because, unlike other cerithioi­deans, they produce pelagic spermatophores (Hadfield & Hopper 1980). Thelatter is the current hypothesis.

Although abundant in many parts of the world, vermetid species have notbeen fully investigated. As far as we know, existing papers have concentrated

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128 ROBERTO VITTURI ET AL.

on the anatomy, morphology, and physiology of these organisms (e.g. Keen1961, Morton 1965, Barash & Zenziper 1985, Hadfield 1969, 1970). Some ofthese studies include comparative anatomy, systematic position and biogeo­graphic application of two vermetid species inhabiting the intertidal andupper subtidal rocky shores of Sao Miguel, Azores (Bieler 1995), discussion ofthe problem of re-evolution of planktotrophic larvae in the vermetid genusPetaloconchus (Hadfield & Iaea 1989), sibling speciation by life-history diver­gence in Dendropoma (Safriel & Hadfield 1988) and description of reproduc­tive biology of some vermetid genera (Hughes 1979, Hopper 1981, Miloslavich& Penchaszadeh 1992).

Nothing is known of the karyology of the family although this is an impor­tant tool for interspecific comparisons. For this reason a detailed analysis ofmitotic and meiotic chromosomes was undertaken for one member of thisgroup.

In the present paper, characterization of mitotic chromosomes of females,males, and early developing embryos of a population of Dendropoma petraeum(Monterosato, 1884) from the Gulf of Palermo is provided. The morphomet­ric results obtained from specimens of both sexes are reported. As karyologicalanalyses are greatly improved by the use of banding techniques, the chromo­somes of D. petraeum were differentially stained using silver nitrate (NORs) , thefluorochrome nApI (4'-fKliamidino-2-phenylindole) and Giemsa dye for e­banding. The results obtained are discussed and the chromosome number ofD. petraeum is compared to those reported for the superfamily Cerithioidea.

According to Keen (1961), the genus Dendropoma consists of two subgenera:Dendropoma and Nooastoa; which along with Vermetus, Serpulorbis; Petaloconchusand others constitute the Vermetidae family. D. petraeum, described by Keen(1961) as belonging to the Nooastoa; includes aggregate forms, usually encrust­ing rocks of the high intertidal zones of the South Mediterranean.

Along the NW coast of Sicily, D. (Novastoa) petraeum grows at intertidal levelon the calcarenitic abrasion platform. The result of aggregate growing is a bio­construction called "trottoir" which is a few centimetres to some metres wide.

This work was supported by a grant (60%) from the Ministero dell'Universita e delle RicercheScientifiche e Tecnologiche.

MATERIALS AND METHODS

Samples of the vermetid gastropod Dendropoma petraeum, were collected everytwo wk from the high intertidal zone of Cape Gallo (NW Sicily, Italy). Collec­tions were made from November 1994 to October 1995. Three to four samplestaken on a Dendropoma platform were transported to the laboratory at theInstitute of Zoology, University of Palermo in plastic bags with seawater. Fiftyadult specimens of each sample were inspected by stereomicroscope at a mag-

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CHROMOSOMES OF A VERMETID GASTROPOD 129

nification of 20x for the occurrence of sexually mature gonads, or incapsulat­ed early developing embryos carried in the mantle cavity of female specimens.When sexually mature specimens occurred, samples of 10 males and 10females were analyzed, karyologically.

Chromosome preparations were carried out either from mature testes and.ovaries or early developing embryos using the air-drying technique for the for­mer (Vitturi et al. 1991) and squash technique for the latter (Vitturi et al.1983). Testes, ovaries and embryos were collected by breaking the tube-likeshells of these sessile gastropods and removing the gonads or the ovoid cap­sules.

Air-drying chromosome preparations were performed as follows: testes andovaries of animals previously treated in a seawater colchicine (0.01%) solutionfor 12-16 hr were dissected and treated separately in a KCI 0.075 M hypotonicsolution for 20 min. They were then fixed in a methyl alcohol/acetic acid solu­tion (3:1) for at least 15 min. Small pieces of testes and ovaries were crushedseparately with dissecting needles in 1-2 ml of 50% acetic acid. Some drops ofcell suspension were then placed on warm precleaned slides and flame-dried.

Chromosome squash preparations were obtained from early developingembryos dissected from their capsules after incubation in a seawater colchi­cine solution (0.01%) for 6-8 hr, according to the following schedule: 1) wash­ing in freshly prepared 50% acetic acid for 5 min, 2) staining in 50% aceticorcein for about 15 min, 3) washing in 50% acetic acid to remove excess orce­in, and 4) gentle squeezing between the slide and cover of each embryo care­fully deprived of the nurse yolk. Slides were immediately inmersed in an ethylalcohol/acetic acid solution (3:1) to separate the cover from the slide andstained with Giemsa 5% (pH 6.8) for 10-15min.

Silver nitrate staining and C-banding were performed on male air-driedsmears using the methods of Howell & Black (1980) and Sumner (1972),respectively. Chromosomes were classified according to the nomenclature pro­posed by Levan et al. (1964).

Considering the "messy taxonomy" of this group with much alpha-taxonom­ic work still to be done, voucher material numbered 1949 has been depositedin the Museum of the Institute of Zoology, University of Palermo.

RESULTS

As are other vermetids, D. petraeum is a gonochoristic species with no obvioussexual dimorphism, hence, the sex of each individual could be identified onlyfrom the gonad. Sexually mature testes were found from November-December1994 to May:June 1995, while ovaries appeared at the beginning of summer inJune. Testes, which resembled distinct lobes, were closely connected to thedigestive gland and yellow in colour. The ovary, found in the same position asthe testes, was creamy in colour and structurally compact. Encapsulated

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130 ROBERTO VITTURI ET AL.

Males25 I I

l A20

] IS~'0 10:;;z

Fig. 1. Distribution of the chromosomenumbers in Dendropoma (Nooastoa) pe­traeum; A) in males; B) in females; and

C) in early developing embryos.

:L32 33 34 35 36 40 50 60 68

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embryos at different stages of development occurred in females from June toOctober 1995. In Vennetus sp. and Dendropoma corrodens these stages have beenclassified in order of development from egg to hatching as I. II, III and IV(Miloslavich & Penchaszadeh 1992). In D. petraeum we found stages of develop­ment corresponding to egg. embryo. early veliger and hatching juvenile too.The best results of chromosome analysis were obtained using early veliger lar­vae. Despite the colchicine treatment, metaphase spreads occurred only occa­sionally so many specimens had to be inspected to gather spreads for morpho­metric analyses.

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CHROMOSOMES OF A VERMETID GASTROPOD 131

2-~,;::

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ploid spread.'.~

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132 ROBERTO VITTURl ET AL.

3

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Fig. 3. Giemsa stained male karyotype of Dendropoma petraeum.

Distribution of diploid chromosome numbers in a total of 97 metaphases(Fig. lA-C) obtained from males (Fig. 2a), females (Fig. 2b) and early develop­ing embryos (Fig. 2c) showed that D. petraeum had a diploid count of 34 chro­mosomes. In males some tetraploid spreads were also found (Fig. 2d).

For each sex, five sets of chromosomes were arranged in pairs according tosize, centromere position and general morphological appearance (Fig. 3, onemale karyotype is represented). Average karyograms obtained from male andfemale specimens (Fig. 4) showed that irrespective of sex, D. petraeum pos­sessed 17 homomorphic chromosome pairs recognized as autosomes of which8 were metacentric, 4 subtelocentric and 5 acrocentric.

Spermatocyte bivalents were observed at the stage of pachytene (Fig. 5) anddiakinesis (Fig. 6) . Pachytene chromosomes showed a chromomeric structurewhich gave these bivalents a transverse banding appearance. Homologuescould not be distinguished from each other due to their tight pairing. Countof the bivalents at diakinesis gave n=17 in 176 out of 185 spreads. In the othernine spreads, which did not yield a modal count, there wasa lower number ofchromosomes. The values of the nine spreads were explained as the result ofloss during preparation or overlap. Diakinetic bivalents had cross- and rod­morphologies. Some elements were condensed into two deeply stained andclosely connected sub-spherical bodies. Dimensions of these bivalents rangedfrom about 3.5 }lm to 1.4 }lID.

Silver nitrate staining of spermatogonial rnetaphases showed the presence ofminute Ag signals terminally located on the short arms of two small-sized sub­telocentric chromosomes. These elements were assigned to the same pair (PairNo. 13) due to their resemblance in morphology and size (Fig. 7). Analysis ofinterphase nuclei revealed one or two nucleoli per nucleus in the proportion

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CHROMOSOMES OF A VERMETID GASTROPOD 133

Fig. 4 Karyograms (a and c) and Stan­dard Deviation (b and d) in both sexesof Dendropoma petraeum each based onthe arm ratio and absolute length val­ues of the chromosomes of five meta­phase spreads, a) in males (a and b);.

and c) in females (c and d).

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Chromosome pairs

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Chromosome pairs

of 46% and 54%, respectively (Fig. 8). At pachytene, one evident NOR (nucle­olus organizer region) was terminally located on a small-sized bivalent (Fig. 9).Small silver positive signals, presumably involving the centromeric region,were visible in all bivalents at diakinesis (Fig. 10).

After Cbanding, evident Cpositive blocks, probably centromerical, werepresent in two bivalents at pachytene (Fig. 11).

Counterstaining of chromosomes by means of the fluorochrome DAPI gavea uniform fluorescence in both diakinesis bivalents (Fig. 12) and spermatogo­nial chromosomes (Fig. 13).

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134 ROBERTO VITTURI ET AL.

'1:.

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Figs 5-6. Dendropoma petraeum. 5. Giemsa stained pachytene chromosomes in male gonads. 6.Giemsa stained spermatocyte bivalents at diakinesis.

DISCUSSION

The modal diploid number of chromosomes of Dendropoma petraeum was foundto be 2n=34 whatever the sex of specimens examined. This value was con­firmed by count of spermatocyte bivalents at diakinesis (n=17). As no karyo-

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Figs 7-lD. Dendropoma peuaeum. 7. Mitotic chromosomes of pair No. 13 : Giemsa stained (g); andsilver stained (n). 8. Silver stained interphase nuclei. 9. Silver stained pachytene chromosomesshowing one NOR (arrow). 10. Silver stained spermatocyte diakinetic bivalents with small Ag sig-

nals in all chromosomes.

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•-!.•

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CHROMOSOMES OF A VERMETID GASTROPOD

..

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135

-.>~;!f.' . c "; . " ,',. '~;\ ~ i1:.f.1;~ :: ~:V.: , ~,.•~. "i:,' ' ;' -"t;.....;~";.::" ::,' !fD.:'> .. { d ', · .' :·~t1.c ?:" .. . , .,;;,,$. ~: ... oJ: ,~ ~ ', . .. . -c

~~i~'~ ~.: c·~\;~~~~;~:~~:'~',~: .~;~':.,.~., · · ~:.,;1;;::;,;"Figs 11-13. Dendropoma petraeum. 11. C-banded pachytene chromosomes in male gonads (arrowsindicate evident C-bands). 12. DAPI stained spermatocyte diakinesis bivalents. 13. DAPI stained

spermatogonial chromosomes at metaphase.

logical information is available for Vermetoidea, we will compare the chromo­some number of D. petraeum with that of other species of Cerithioidea. Hap­loid values of 16, 18, 19, 35-36, 38-39, 45-47 and 60 have been found in Thiari­dae , n=7, 12, 13,14,18 and 20 in Pleuroceridae, n=16 in Turritellidae and n=18in Batillariidae, Potamididae and Cerithiidae (Patterson 1969, Davis 1972, Vit­turi & Catalano 1984). All haploid numbers summarized here differ from then= 17 of D. petraeum.

As the detailed morphometric analysis of mitotic chromosomes showed onlythe 17 homomorphic chromosome pairs in both sexes, we may exclude thepresence of heteromorphic sex chromosomes of the XY, ZW or XO type in D.petraeum. The absence of differentiated sex chromosomes in molluscs was for a

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136 ROBERTO VITTURI ET AL.

long time considered an established fact (White 1973), but different sex chro­mosome systems have recently been reported in species of Prosobranchia. AnXYsex-determining mechanism has, for example, been described in the meso­gastropod Rissoa ventricosa (see Thiriot-Quievreux & Ayraud 1982) and theneogastropod Fasciolaria lignaria (see Vitturi et al. 1995a). A male chromosomesex mechanism of the XO type is present in the family Neritidae (cfr. Komatsu& Inaba 1982, Komatsu 1983, Nakamura 1983, Vitturi & Catalano 1988) andseems to characterize two geographically separated populations of the meso­gastropod Melaraphe (Littorina) neritoides (Vitturi et al. 1988; Vitturi et al.1995b). Moreover, a multiple chromosome sex system of the X1X2Yo-/IX1X2X29 type has been described for the heteropod Pterotrachea hippocampus(Vitturi et al. 1993).

Tetraploid spreads are found occasionally in the male gonads of D. petraeum(Fig. 1A). 4n spreads have previously been described in the testes of the meso­gastropod Melaraphe (Littorina) neritoides from Villefranche-sur-Mer (Thiriot­Quievreux & Ayraud 1982). It has been suggested for Melaraphe that a tetra­ploid complement might characterize testicular cells with a trophic function,this may also be true in D. petraeum. In fact it is unlikely that the tetraploidyoriginated from the action of colchicine as this substance produced no 4nspreads in early developing embyos or in females.

Silver staining consistently shows Ag positive signals in two small-sized sub­telocentric chromosomes of spermatogonial metaphases. The involvement innucleolus organization of a single chromosome pair is confirmed by the find­ing of one NOR carrier pachytene bivalent and by the presence of a maximumnumber of 2 nucleoli in interphase nuclei (Goldman et al. 1983, Thode et al.1983, Vitturi et al. 1991).

Small Ag granules could be seen in all spermatocyte bivalents at diakinesis.This phenomenon is not new in molluscs. Diffuse NOR activity has previouslybeen observed in metaphase-I chromosomes of the mesogastropods Buccino­lum corneum (see Vitturi & Catalano 1990) and Pterotrachea hippocampus (seeVitturi et al. 1993), in the snail Helicella virgata (see Vitturi et al. 1991) and inthe slug Milax nigricans (Vitturi 1992). It was also reported in Kalothermesflavi­collis (Isoptera, Kalotermitidae) (Goldoni & Fontana 1991), Pelagia noctiluca(Coelenterata, Scyphomedusae) (Vitturi et al. 1994) and in some species ofscarab beetle (Vitturi et al. 1995c). We exclude that such a finding may beattributed to technical variation, due to the constancy of the result obtained.

Although this phenomenon remains to be explained, the visualization offunctionally active NORs in all diakinetic bivalents may imply that a potentialnucleolus activity is present in all chromosomes in D. petraeum. In turn, thiswould imply the presence of rDNA in the centromeric regions of all chromo­somes of this species. Alternatively, diffuse silver positivity might be due to thepresence of silver proteins, identified in mammals mainly as nucleolin or C23(Hernandez-Verdun 1991, Hernandez-Verdun et al. 1993) these may not be

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CHROMOSOMES OF A VERMETID GASTROPOD 137

directly related to the presence of rDNA. In fact, it is known that these pro­teins, which are multifunctional components of the nucleolus, remain partlyassociated with rDNA while others are scattered from the region of synthesisthroughout the cytoplasm. Some nucleolar proteins are also known to migratefrom the nucleolus to the chromosomes during the transition between inter-­phase and mitosis. Therefore, similar proteins may perhaps be responsible forthe diffuse silver positivity detected in male diakinetic bivalents of the specieshere investigated. This feature suggests that further investigation with in situhybridization techniques (FISH) using ribosomal probes to determine the realDNA composition of the NORs in these vermetids should prove useful.

Only a very small amount of constitutive heterochromatin occurs in D.petraeum. Moreover, this heterochromatin does not seem to be particularly richin A+T base composition, since the fluorochrome DAPI, which is specific forA+T rich DNA, and generally stains heterochromatic segments brightly(Schweizer 1976, Sumner 1990), reacts homogeneously in all parts of thegenome of the species under study.

From the results of this study it is possible to affirm that the vermetid speciesinvestigated here does not possess differentiated sex chromosomes. This spe­cies shows a rather asymmetrical karyotype (including both mono-armed andbi-armed chromosomes), a small amount of constitutive heterochromatin, andtwo chromosomes per cell involved in nucleolus organization. According toOhno (1970) the absence of a chromosome sex system may be a primitivecharacter. Similarly, it has been maintained that asymmetrical karyotypes mayidentify primitive organisms in most animal groups (Colombera & Lazzaretto­Colombera 1978), while the presence ofa single pair of NOR-bearing chromo­somes is plesiomorphic in most invertebrate (Thiriot-Quievreux & Insua 1992,Vitturi et al. 1995a, Sella et al. 1995), and vertebrate (e.g. Schmid 1978, Arne­miya & Gold 1990) species.

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