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The budding process in Tethya citrina Sarà & Melone (Porifera, Demospongiae) and the incidence of post-buds in sponge population maintenance
Journal of Experimental Marine Biology and Ecology 389 (2010) 93–100
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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e
The budding process in Tethya citrina Sarà & Melone (Porifera, Demospongiae) and the incidence of post-buds in sponge population maintenance Frine Cardone a, Elda Gaino b, Giuseppe Corriero a,⁎ a b
Dipartimento di Biologia Animale e Ambientale, Università degli Studi di Bari, Via Orabona 4, 70125, Bari, Italy Dipartimento di Biologia Cellulare e Ambientale, Università degli Studi di Perugia, Via Elce di Sotto, 06123, Perugia, Italy
a r t i c l e
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Article history: Received 12 November 2009 Received in revised form 23 March 2010 Accepted 24 March 2010 Keywords: Asexual reproduction Mediterranean Sea Post-bud growth Tethya citrina
a b s t r a c t The relevance of the budding process in a population of Tethya citrina inhabiting an unpredictable environment subjected to frequent anoxic crisis (Mar Piccolo di Taranto) is described, and new data about the fate of the buds after their release are provided. At the individual level, for the first time in natural conditions, it has been demonstrated that several budding events follow each other, alternating with short periods (1–2 months) of rest. At the population level, budding proved to be a continuous process, being detected throughout the 13 months of study, with a short summer decrease both in the frequency of budding specimens and bud density. This trend was markedly different from that reported for conspecific populations inhabiting stable environments, in which budding is a seasonal process. It is hypothesized that the sudden and unpredictable variations of the ecological conditions could be responsible for the onset of repeated budding events, thus stressing the role of environmental factors in affecting the asexual reproductive pattern. T. citrina buds, here investigated, tend to detach from the adult sponge within 7/15 days of their differentiation, a feature coherent with experimental data carried out under laboratory conditions. A significant relationship between the size/age of the post-buds and the onset of the asexual reproduction has been proved: in particular, the first budding event was observed in nine-month-old sponges, measuring about 7 mm in diameter. As reported for other demosponges, in T. citrina post-buds the growth rate was positively related to the water temperature, with a significant increase during the summer. In addition, a negative effect of the budding process on the sponge growth, is hypothesized for a post-bud subjected to repeated budding events. Growth rate, however, proved highly variable. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The life-cycles of the marine sponges, as well as those of most benthic animals, are usually less well known, owing to the real difficulties involved in carrying out investigations on some key aspects such as the biology of the larva, recruitment, metamorphosis and further growth in its natural environment. In contrast, asexual bodies produced by some families of sponges, constitute valid material for tracing their fate from the cradle to the grave, because of the low dispersal potentiality and structural organization of these elements that, bypassing metamorphosis, allows growth and functionality to be achieved in a short time. Budding is one of the mechanisms of sponge asexual reproduction. Buds are small spherical asexual bodies, generally brought to the extremity of a stalk that emerges from the surface of the mother sponge.
⁎ Corresponding author. Tel./fax: + 39 080 5443357. E-mail address: [email protected] (G. Corriero). 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2010.03.012
Budding has been widely observed in Tetractinomorpha, Demospongiae, and particularly in the genus Tethya. From the bulk of data, it has emerged that there is a high level of heterogeneity in terms of the time scale in cytological and skeletal organization of buds and the development stage reached at their detachment, thereby proving that their differentiation tends to be a species-specific process (Gaino et al., 2006). In T. seychellensis, buds differentiate choanocyte chambers before their release from the parent sponge, a feature suggesting that, at settlement, these elements can act as a functional young sponge (Gaino et al., 2009). In contrast, in T. aurantium and T. citrina the morphogenetic processes leading to the completion of the aquiferous system take place after bud detachment (Gaino et al., 2006). Despite the effects of this heterochrony, a recent study suggests a spatiotemporal morphogenetic pattern sequence that can be considered common in almost all sponge species able to generate buds (Hammel et al., 2009). Whereas some papers have been devoted to investigate bud production at the population level (Ayling, 1980; Corriero et al., 1996, 1998; Chen et al., 1997), nothing is known about the onset of the budding process or on its duration at the individual level. In addition,
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even if the ecological role performed by asexual reproduction has been stressed by many authors (Stone, 1970; Ayling, 1980; Battershill and Bergquist, 1990; Wulff, 1991, 1995; Corriero et al., 1996, 1998; Maldonado and Uriz, 1999; Ereskovsky and Tokina, 2007), the fate of the buds after their release, here including growth, ability to produce new asexual elements and mortality, is currently unknown. Only one paper, referred to asexual sponge fragments, reports the high survival of such elements after their detachment, with an initial rapid growth, thus suggesting that asexual fragments are responsible for successful recruitment (Wulff, 1991). Consequently, the scenario is very incomplete and more studies are needed to understand the actual role of the budding process in maintenance of sponge populations. We present here the results of a long term study aimed to add more information on the budding process in T. citrina, and to provide new data on the fate of the buds after their release, by monitoring them in natural conditions. 2. Methods 2.1. Species studied Tethya citrina Sarà and Melone, 1965 is a small demosponge, spherical in shape and soft in consistency. Small irregular tubercles emerge from its external surface. Its colour varies from grey to yellowish (Sarà and Melone, 1965). It inhabits rocky substrates from the surface to approximately a hundred metres in depth (Sarà, 1987). The sponge body consists of a thin cortical layer surrounding the choanosome where choanocyte chambers occur. The skeleton is made of strongiloxeas (235–1400 μm in length × 7.5–24 μm in thickness) arranged in radial bundles. Additional megasters (spherasters and spheroxyasters 18–64.8 μm in diameter) and micrasters (oxyasters, chiasters and tylasters 7.5–14.5 μm in diameter) also occur (Sarà and Melone, 1965; Bavestrello et al., 1992). T. citrina is a gonochoric oviparous species (Scalera-Liaci et al., 1971; Gaino et al., 1987; Corriero et al., 1996). Its sexual cycle displays a seasonal pattern, where oogenesis occurs from late spring to autumn (Scalera-Liaci et al., 1971; Corriero et al., 1996), and spermatogenesis from August to September (Scalera-Liaci et al., 1971). 2.2. Study site The Mar Piccolo di Taranto is an inner sea located at the north of the Gulf of Taranto (Ionian Sea). It consists of two distinct basins, the innermost of them receives the inflow from some small streams. The studied area is located in this innermost basin (latitude N 40°29′ 07′′, longitude E 17°16′ 39′′), and consists of a pile of calcareous stones, variable in size and shape, which are periodically subjected to long emersion periods, and to the hydrometric sizigial variations of the sea level. The pile of stones is situated on a silt bottom, often subjected to anoxic crises due to the deposition of large amounts of plant detritus. The calcareous stones are colonized by a rich sessile filter feeder fauna with prevalence of demosponges (Hymeniacidon perlevis, Suberites carnosus, Terpios fugax, Tethya citrina), calcareous sponges (Sycon sp., Paraleucilla magna), sabellids (Branchiomma luctuosum), serpulids (Hydroides sp.), bivalves (Mitylus galloprovincialis and Ostrea edulis), bryozoans and ascidians (some encrusting colonial didemnidae, Microcosmus sp. and Styela plicata). Among the demosponges, Tethya citrina inhabits the shaded side of the stones, where it can reach a density of more than 100 specimens per square metre of substrate (Cardone et al., unpublished data). Water temperature varies from 8.4 °C (December) to 26.8 °C (August) (Caroppo and Cardellicchio, 1995). Salinity values are around 36‰ (Caroppo and Cardellicchio, 1995). Locally, however, continental inflows may determine a marked drop in salinity (25– 30‰) (Corriero et al., unpublished data). Dissolved oxygen shows
wide seasonal variability: from May to October the oxygen deficit can reach marked levels which are responsible for distrophic events (Caroppo and Cardellicchio, 1995; Alabiso et al., 1997). 2.3. Sampling methods To estimate the relevance of asexual reproduction, ten adult specimens of Tethya citrina were randomly collected monthly (June 2007–June 2008; n = 130), and the total number of buds for each specimen was counted under the stereomicroscope. Moreover, the maximum and minimum diameters were measured both for the collected adult specimens and for 20 buds randomly selected from each specimen to obtain a mean estimation of their volume. During the period of intensified bud production (November 2007), sample collection consisted of weekly pictures of ten labeled adult sponge specimens, in order to evaluate the progression of the budding process up to bud release. The surface of these sponges was photographed using a digicam Canon 400 D, EF-S 60 mm f 2.8 Macro, appropriately set up on a metal support, in such a way that, over time, pictures were taken at the same angle and distance. Data were plotted on a PC and elaborated using the AUTOCAD 2007 software. In particular, for each of the ten labeled specimens, the total number of buds was counted weekly with the aim of identifying the newly produced and detached buds. Post-bud growth and death were assessed for ten labeled young specimens using the same methodology applied to the adult sponges. The asexual origin of these sponges was certified by the lack of sexual reproduction in the studied population at the onset of the monitoring (Cardone et al., unpublished data). The post-buds were monitored monthly over 13 months (June 2007–June 2008); a single post-bud was observed for an additional year (June 2006–May 2007). Photos were collected by applying the same photographic technique described above for each labeled specimen; from each picture, we recorded occurrence of budding on the sponge surface and maximum and minimum diameters of the labeled post-buds. The mean diameter of the post-buds was then calculated, and the specific growth rate (SGR) was estimated, as follows: SGR = ðloge md2 − loge md1 Þ = ðt2 −t1 Þ × 100; where loge are the natural logarithms, md2 and md1 the average diameter at the two different times, and (t2−t1) the time interval in months. During the study period, the water temperature and salinity were measured monthly at 50 cm in depth, using a multi parametric probe (OCEAN SEVEN 316 Plus). Differences in growth of post-buds at monthly time intervals were statistically analyzed by Kruskal–Wallis test (SPSS® 12.0.1). The relationship between the size of the post-buds and the first event of bud production was analyzed using the Sperman correlation coefficient; the same statistical analysis was used to test the relationships between water temperature, salinity and post-bud growth rate (SPSS® 12.0.1). 3. Results In budding specimens of T. citrina, buds sprout out from the sponge cortical layer as small bodies (Fig. 1a), varying in size and extension, because they are produced asynchronously. Buds are connected to the parent by a stalk that in fully differentiated buds greatly enlarges at the apex, giving rise to spiculated bodies. Buds grow gradually and some of them bend towards the substrate, in such a way that their apical region firmly adheres to it before their final detachment (Fig. 1b). Frequently, the stalk breaks off releasing buds, even though their diffusion is limited and they tend to colonise the areas close to the mother sponge. A total of 5207 buds have been counted, from 130
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Fig. 1. T. citrina. a: Budding specimen from the Mar Piccolo di Taranto (bar = 1 cm). b: Buds attached to the substrate (bar = 1 mm).
adult specimens (14.7 ± 3.8 mm in mean diameter) examined. The total number of budding adult specimens counted over 13 months of study was equal to 100 (77%). Specimens with buds occurred throughout the year, with peaks in frequency values (100%) recorded in September, October and from February to April, whereas in December and January the frequency of specimens with buds was remarkably reduced (Fig. 2a). The peaks, expressing the density of the buds (about 40/cm2) produced by the adult sponge, almost coincide with those showing the frequency of the budding specimens (Fig. 2a, b). The mean bud size varied from 0.32 ± 0.1 (August) to 0.56 ± 0.18 mm (February), showing the lowest values in parallel with those of the bud density (Fig. 2b). The reproductive effort (expressed as percent of: total volume of buds/ total volume of parental tissue) was maximum in the winter period, with a peak in November (1%) and a minimum in the spring and summer months. The analysis of the budding process, carried out with weekly frequency on ten labeled specimens, showed that 1 to 2 weeks after their appearance most of the buds had detached from the sponge surface (Fig. 3). Sometimes, instead of detaching from the mother sponge buds remained connected by means of their stalk that bent toward the substrate and finally the bud adhered to it. This mechanism allowed the adult sponge to incorporate the bud tissues, thereby widening its extension on the substrate (Fig. 4). Monthly investigations carried out on the growth and morphological changes of ten settled buds, showed that in 13 months they reached a mean size of 7.1 ± 1.4 mm in diameter, starting from 1.2 ±
0.2 mm (Figs. 5–7). At the onset of settlement, the post-buds formed an encrusting layer, which gradually modified giving rise to a spherical shape. During the following growth, the outline of the young sponges tended to vary from fairly round to more irregularshaped individuals. This trend was typically observed in all the considered post-buds, as became evident by comparing the relationship between the minimum and maximum diameters of each of the specimens. Sometimes, the close proximity of the post-buds promoted their fusion into larger sponges, as occurred for specimens 3 and 6, during the first month of growth, and for specimens 2 with an unmonitored post-bud, after 10 months of monitoring. Specimen 5 was overgrown by a colonial ascidian, which did not kill it, but prevented further observations (Fig. 5). This process of monitoring allowed us to ascertain the minimum size at which, in turn, the postbuds were able to differentiate buds (Figs. 5 and 6): in more than 60% of the ten monitored post-buds the first budding event occurred when the sponge size (diameter) varied from 6.4 to 6.9 mm. In the monitored post-buds, the first event of bud production was significantly correlated with the size of the post-buds (rs = 0.46; P b 0.01) (Fig. 5). No mortality was observed in the monitored post-buds throughout the 13 months of study. However in the following year a persistent anoxic event (November 2008) destroyed most of the benthic assemblage from the study area, killing 70% of the monitored postbuds. Significant differences in growth and growth rate were observed in the ten monitored post-buds (H = 24.3; P b 0.01 and H = 84.5; P b 0.01 respectively). The average value of the specific growth rate was 0.5 ± 0.1 mm/month (13 months). The growth rate showed a seasonal pattern. The maximum average specific growth rate (1.9 ± 0.3) was measured in July, the lowest (0.03 ± 0.09), in January. The growth rate was positively related with water temperature (rs = 0.42; P b 0.01), but not with salinity. Indeed, during summer the highest temperature values paralleled those of post-bud growth rate (Fig. 8). When comparing the growing curve of an individual followed for 25 months with that dealing with the 10 specimens, it emerged that the growth tended to decrease after the initial year (Fig. 9). 4. Discussion
Fig. 2. T. citrina. a: Monthly frequency (%) of specimens with buds. b: Monthly variation of bud density and size (± standard deviation).
The relevance of the budding process of Tethya citrina is investigated in a population inhabiting the Mar Piccolo di Taranto, a very unpredictable environment subject to frequent anoxic crisis (Vatova, 1972; Caroppo and Cardellicchio, 1995; Alabiso et al., 1997). The monitored specimens grew on the rocky substrates of the uppermost fringe of the infralittoral zone (sensu Pérès and Picard, 1964), where they are seasonally subjected to air exposure periods concomitantly to the hydrometric sizigial variations of the sea level.
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Fig. 3. T. citrina. Weekly monitoring (November 2007) of buds number produced and released from ten labeled adult specimens. All buds that were not possible to monitor during the time of the study were classified as undetermined; □ undetermined buds first production of buds second production third production fourth production.
This field study has provided new data on the budding process investigated under natural conditions, confirming the key role of asexual reproduction for maintenance and dispersal employed by some sponge species, mainly in habitats affected by remarkable fluctuations in environmental parameters. According to the literature, T. citrina reproduces asexually, by elaboration of buds (Connes, 1968; Scalera-Liaci et al., 1971; Corriero et al., 1996; Gaino et al., 2006). At the individual level, for the first time in natural conditions, our data show that several budding events follow each other, alternating with short rest periods (1–2 months). This contrasts with observations carried out under experimental conditions, where, usually, after the bud detachment, no new buds were produced by the same reared specimen of T. citrina (Cardone et al., 2008). Several bud-bearing specimens of Thenea abyssorum kept alive in the laboratory for several months were unable to release buds (Witte, 1996), thus emphasizing that the role of the budding process could be interpreted only by carrying out investigations in natural conditions.
At the population level, budding is a continuous process; in fact, buds were detected throughout the period of study, with a short decrease both in the frequency of budding specimens and bud density during the late summer. This trend markedly differs from that reported for the conspecific population from the Stagnone di Marsala (North-west Sicily). There, T. citrina inhabits a shallow (1 m in depth) but stable habitat (the rhyzomes of Posidonia oceanica, a phanerogam not tolerant to strong variations in environmental conditions), and displays a clear budding seasonality (October–January) (Corriero et al., 1996). Highest bud production in the autumn and winter months has been also reported by Connes (1968) for mixed populations of T. citrina and T. aurantium inhabiting deep environments usually subjected to moderate variations in hydrological parameters (detritic bottoms, 80 to 100 m in depth). At Taranto, the density of buds on the sponge surface is extraordinarily high (up to 40 buds/cm² in October), in comparison with that reported by Corriero et al. (1996) for the Marsala population (maximum value = 2.6 bud/cm²). On the contrary, the mean size of
Fig. 4. T. citrina. A specimen monitored for four weeks: a: some buds still connected by the stalk to the mother sponge attached to the substrate; b, c, d: the adult sponge incorporates the bud tissue widening its extension on the substrate (bar = 1 cm).
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Fig. 5. T. citrina. Monthly variation of mean diameter (± standard deviation) and minimum/maximum diameter ratio in ten post-buds monitored for 13 months. *: budding events.
the buds is much lower at Taranto (0.5 mm in diameter), than at Marsala (0.85 mm) (Gaino et al., 2006). In other words, the population of T. citrina from the Mar Piccolo di Taranto produces
numerous small buds throughout the year, whereas at Marsala asexual reproduction occurs only in autumn–winter, with the elaboration of a few relatively large elements. Such differing
Fig. 6. T. citrina. Monthly variation of mean diameter (± standard deviation) and minimum/maximum diameter ratio in a post-bud monitored for 25 months. *: budding events.
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Fig. 7. T. citrina. Growth phases of a post-bud recorded at different time intervals after settlement: a: 1 month (arrow indicates the position of the sponge on the substrate); b: 4 months; c: 6 months; d: 9 months, during its first budding event. a: (bar = 2 mm); b, c, d: (bar = 5 mm).
reproductive strategies could be due to the different environmental conditions characterizing the two sites. The mechanisms able to control the onset, duration and progression of the reproductive events still remain unclear in Porifera. Connes (1968), Simpson (1980) and Pronzato et al. (1993) put forward the hypothesis of a combined action of exogenous and endogenous factors affecting gamete and asexual element differentiation. In addition, several authors emphasise the effects that variations of the hydrological parameters can have on the reproduction of numerous poriferans (Simpson, 1968, 1984; Diaz, 1973; Fell, 1976, 1993; Corriero et al., 1996, 1998; Ereskovsky, 2000; Meroz-Fine et al., 2005). The here hypothesized relationship between environmental stress and bud production is coherent with the observation that, under laboratory conditions, the budding process in some sponges may be stimulated by injury and/or stress (Connes, 1967; Saller, 1990). According to Cardone et al. (2008), in T.
Fig. 8. T. citrina. Growth performance (specific growth rate in percentage) (± standard error) of ten post-buds monitored for13 months, with seasonal variations of water temperature and salinity.
citrina budding can also be experimentally induced by drastic variations in the hydrological parameters. At Taranto, therefore, the sudden and unpredictable variations of the ecological conditions could be responsible for the onset of repeated budding events. The sponge buds, with a reduced dispersal ability, represent a source of new recruits increasing the stock of the original population, which can balance its high mortality through the clonal process that gives inception to a continuous turn-over. Under environmental stresses, budding would ensure a better opportunity for survival. Such a hypothetical scenario is coherent with the high mortality that affected the benthic fauna in the year that followed the present study. With regard to population dynamics, the present investigation allowed the growth pattern of the post-buds and the onset of budding process to be described. In T. citrina post-buds the budding process starts when the sponge is about nine-months old and measures about 7 mm in diameter. The observations carried out in natural conditions could be regarded as a relevant step in widening knowledge on the budding process, by providing data on the relationship between the size/age of specimens and the onset of asexual reproduction. Research into sponge growth has revealed a seasonal increase in growth concomitantly with the water temperature increase (Barthel, 1986; Garrabou and Zabala, 2001; Page et al., 2005; De Caralt et al., 2007). According to the literature, in T. citrina post-buds the growth rate is positively related to the water temperature, with a significant increase during the summer. However, growth rate has proved to be highly variable. The high variability in growth is a well known problem among researchers who have experimented with sponge cultivation. Verdenal and Vacelet (1985) and Corriero et al. (2004) observed marked differences in the growth of fragments of Spongia
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Fig. 9. T. citrina. Growth curves in ten post-buds monitored for 13 months and in a post-bud monitored for 25 months (± standard deviation).
officinalis, even when cut from the same specimen, and apparently subjected to the same ecological conditions. A certain degree of growth variability was also observed by Cardone et al. (2008) in postbuds of T. citrina produced by the same specimen under controlled conditions. The differences in growth of the post-buds, apart from the ecological variations occurring in the different microhabitats colonized by each bud, could also be related to the bud organization displayed at the moment of its detachment from the mother sponge (size, functionality, occurrence and quantity of different cellular types, ratio between collagen and cellular component), as suggested by Connes (1968), who hypothesized a different fate for the buds due to differences in cell composition. A recurrent feature is the abrupt drop in the positive growth of the monitored post-buds. This feature is probably due to environmental stress conditions affecting the buds. However, other hypotheses cannot be rejected. According to Wulff (1991) the growth pattern of three tropical demosponges usually subjected to asexual fragmentation, consists of a more or less prolonged phase of linear size increase, terminated by a marked decrease due to fragmentation or mortality of the sponge. Most of our data only concern the initial phase of the sponge life, in which a too short period of budding occurs, and therefore they do not allow evaluation of the impact of budding on the growth of the sponge. However, the monitoring for 25 months of a single specimen of T. citrina seems to indicate a negative effect of the budding process on sponge growth, as revealed by a long period of negative growth associated with repeated budding events. The growth process is associated to marked variations in the shape of young sponges. The first event occurring in the newly released buds is settlement marked by extension and adhesion to the substrate, a process that gives rise to a large thin encrusting formation. In this initial phase of growth, the thickness of the young sponge is greatly reduced, as shown by the high values of the longitudinal axis when compared with those of the vertical one. Therefore, the young sponge has an initial flat shape that changes remarkably through the gradual acquisition of a globular formation during growth. The adhesion to the substrate represents a crucial phase for further development as also observed in post-larvae during metamorphosis. In this regard, asexually and sexually produced elements share similar needs. In particular, it has been shown that larval attachment is due to the secretion of a basal ground matter between the larva and the substrate (Borojevic and Lévi, 1965; Bergquist and Green, 1977; Evans, 1977; Kaye and Reiswig, 1991; Gaino et al., 2007). Therefore, in contrast with larvae, which show various behavioural patterns and preferences in substrate selection (Leys and Degnan, 2002; see review in Maldonado, 2006), buds are not active in such a process and the spicules sprouting out from the bud peripheral border, after having favoured the initial floating of the buds, may be used as a tool for anchoring them to the substrate. In conclusion, the present investigation has supplemented data available in the literature about the sponge budding process and shown that environmental factors are able to deeply affect the reproductive pattern. Tethya citrina, besides its considerable reproductive plasticity (Corriero et al., 1996; Gaino et al., 2006), is able to put into action different reproductive strategies, both sexual and
asexual, by employing the former or the latter according to the environment where the sponge specimens grow. Acknowledgments This work is in memory of Prof. Michele Sarà. The work was financially supported by Italian funds from the Ministero dell'Economia e delle Finanze, Ministero dell'Istruzione, Ministero dell'Università e della Ricerca, Ministero dell'Ambiente e della Tutela del Territorio, and Ministero delle Politiche Agricole e Forestali (Program M.I.C.E.N.A.). [SS] References Alabiso, G., Cannalire, M., Ghionda, D., Milillo, M., Leone, G., Caciorgna, O., 1997. Particulate matter and chemical–physical conditions of an inner sea: the Mar Piccolo in Taranto. A new statistical approach. Mar. Chem. 58, 73–388. Ayling, A.L., 1980. Patterns of sexuality, asexual reproduction and recruitment in some subtidal marine Demospongiae. Biol. Bull. 158, 271–282. Barthel, D., 1986. On the ecophysiology of the sponge Halichondria panicea in Kiel Bight. I. Substrate specificity, growth and reproduction. Mar. Ecol. Progr. Ser. 32, 291–298. Battershill, C.N., Bergquist, P.R., 1990. The influence of storms on asexual reproduction, recruitment, and survivorship of sponges. In: Rützler, K. (Ed.), New Perspectives in Sponge Biology. Smithsonian Institution Press, Washington, D.C., pp. 397–403. Bavestrello, G., Corriero, G., Sarà, M., 1992. Differences between two sympatric species of Tethya (Porifera, Demospongiae) concerning the growth and final form of their megasters. Zool. J. Linn. Soc. 104, 81–87. Bergquist, P.R., Green, C.R., 1977. An ultrastructural study of settlement and metamorphosis in sponge larvae. Cah. Biol. Mar. 18, 289–302. Borojevic, R., Lévi, C., 1965. Morphogenése expérimentale d'une éponge á partir de cellules de la larve nageante dissociée. Z. Zellforsch. Mikrosk. Anat. 68, 57–69. Cardone, F., Nonnis Marzano, C., Spedicato, M.T., Lembo, G., Gaino, E., Corriero, G., 2008. Budding induction in a marine sponge: a cue for cultivation purposes. Proceedings of IFOAM Conference on Organic Aquaculture, Cattolica (RN), pp. 81–85. 18–20 June. Caroppo, C., Cardellicchio, N., 1995. Preliminary study on phytoplankton communities of Mar Piccolo in Taranto (Jonian Sea). Oebalia 21 (2), 61–76. Chen, Y.H., Chen, C.P., Chang, K.H., 1997. Budding cycle and bud morphology of the globe shaped sponge Cynachira australiensis. Zool. Stud. 36 (3), 194–200. Connes, R., 1967. Structure et développement des bourgeons chez l'éponge siliceuse Tethya lyncurium Lamarck. Archs. Zool. Exp. Gèn. 108, 157–195. Connes, R., 1968. Etude histologique, cytologique, et expérimentale de la régénération et de la reproduction asexuée chez Tethya lyncurium Lamark (= T. aurantium Pallas) (Démosponges). P.h. D. thesis, University of Montpellier, France. Corriero, G., Sarà, M., Vaccaro, P., 1996. Sexual and asexual reproduction in two species of Tethya (Porifera: Demospongiae) from a Mediterranean coastal lagoon. Mar. Biol. 126, 175–181. Corriero, G., Scalera-Liaci, L., Nonnis Marzano, C., Gaino, E., 1998. Reproductive strategies of Mycale contarenii (Porifera: Demospongiae). Mar. Biol. 131, 319–327. Corriero, G., Longo, C., Mercurio, M., Nonnis Marzano, C., Lembo, G., Spedicato, M.T., 2004. Rearing performances of Spongia officinalis on suspended ropes off Southern Italian coast (Central Mediterranean Sea). Aquaculture 238, 195–205. De Caralt, S., Uriz, M.J., Wijffels, R.H., 2007. Grazing, differential size-class and survival of a Mediterranean sponge species: Corticium candelabrum (Demospongiae: Homosclerophorida). Mar. Ecol. Progr. Ser. 360, 97–106. Diaz, J.P., 1973. Cycle sexuel de deux démosponges de l'étang de Thau: Suberites massa Nardo et Hymeniacidon caruncula Bowerbank. Bull. Soc. Zool. France 98 (1), 145–156. Ereskovsky, A.V., 2000. Reproduction cycles and strategies of the cold-water sponges Halisarca dujardini (Demospongiae, Halisarcida), Myxilla incrustans and Iophon piceus (Demospongiae, Poecilosclerida) from the White Sea. Biol. Bull. 198, 77–87. Ereskovsky, A.V., Tokina, D.B., 2007. Asexual reproduction of homoscleromorph sponges (Porifera; Homoscleromorpha). Mar. Biol. 151, 425–434. Evans, C.W., 1977. The ultrastructure of larvae from the marine sponge Halichondria moorei Bergquist (Porifera, Demospongiae). Cah. Biol. Mar. 18, 427–433.
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Fell, P.E., 1976. The reproduction of Haliclona loosanoffi and its apparent relationship to water temperature. Biol. Bull. 150, 200–210. Fell, P.E., 1993. Porifera: asexual propagation and reproductive strategies. In: Adyodi, K.G., Adyodi, R.G. (Eds.), Reproductive biology of Invertebrates, vol. VI. Oxford and I.B.H. Publishing, New Delhi, pp. 1–44. Gaino, E., Burlando, B., Buffa, P., Sarà, M., 1987. Ultrastructural study of the mature egg of Tethya citrina Sarà and Melone (Porifera, Demospongiae). Gamete Res. 16, 259–265. Gaino, E., Scalera-Liaci, L., Sciscioli, M., Corriero, G., 2006. Investigation of the budding process in Tethya citrina and Tethya aurantium (Porifera, Demospongiae). Zoomorphology 125, 87–97. Gaino, E., Baldacconi, R., Corriero, G., 2007. Post-larval development of the commercial sponge Spongia officinalis L. (Porifera, Demospongiae). Tissue Cell 39, 325–334. Gaino, E., Mercurio, M., Sciscioli, M., Corriero, G., 2009. Choanocyte chambers in unreleased buds of Tethya seychellensis (Wright, 1881) (Porifera, Demospongiae). Ital. J. Zool. 76 (1), 64–69. Garrabou, J., Zabala, M., 2001. Growth dynamics in four Mediterranean demosponges. Estuar. Coast. Shelf Sci. 52, 293–303. Hammel, J.U., Herzen, J., Beckmann, F., 2009. Sponge budding is a spatiotemporal morphological patterning process: insights from synchrotron radiation-based xray microtomography into the asexual reproduction of Tethya wilhelma. Front. Zool. 6, 19. Kaye, H.R., Reiswig, H.M., 1991. Sexual reproduction in four Carribean commercial sponges. I. Reproductive cycles and spermatogenesis. Invertebr. Reprod. Dev. 19, 1–11. Leys, S.P., Degnan, B.M., 2002. Embryogenesis and metamorphosis in a haplosclerid demosponge: gastrulation and transdifferentiation of larval ciliated cells to choanocytes. Invertebr. Biol. 121, 171–189. Maldonado, M., 2006. The ecology of sponge larva. Can. J. Zool. 84, 175–194. Maldonado, M., Uriz, M.J., 1999. Sexual propagation by sponge fragments. Nature 398, 476. Meroz-Fine, E., Shefer, S., Ilan, M., 2005. Changes in morphology and physiology of an East Mediterranean sponge in different habitats. Mar. Biol. 147, 243–250. Page, M.J., Northcote, P.T., Webb, V.L., Mackey, S., Handley, S.J., 2005. Aquaculture trials for the production of biologically active metabolites in the New Zealand sponge Mycale hentscheli (Demospongiae: Poecilosclerida). Aquaculture 250, 256–269.
Pérès, J.M., Picard, J., 1964. Nouveau Manuel de bionomie bentique de le Mer Méditerranée. Rec. Trav. Sta. Mar. Endoume 31 (47), 5–137. Pronzato, R., Manconi, R., Corriero, G., 1993. Biorhythm and environmental control in the life history of Ephydatia fluviatilis (Demospongiae, Spongillidae). Boll. Zool. 60, 63–67. Saller, U., 1990. Formation and construction of asexual buds of the freshwater sponge Radiospongilla cerebellata (Porifera, Spongillidae). Zoomorphology 109, 295–301. Sarà, M., 1987. A study on the genus Tethya (Porifera, Demospongiae) and new perspectives in sponge systematics. In: Vacelet, J., Boury-Esnault, N. (Eds.), Taxonomy of Porifera. Springer, Berlin Heidelberg New York, pp. 205–225. Sarà, M., Melone, N., 1965. Una nuova specie del genere Tethya, T. citrina sp. n. dal Mediterraneo (Porifera Demospongiae). In: Atti Soc. peloritana Sci. Fis. Mat. Nat. 11, 123–138. Scalera-Liaci, L., Sciscioli, M., Papa, O., Lepore, E., 1971. Raffronto tra i cicli sessuali di Tethya aurantium (Pallas) Gray e Tethya citrina Sarà & Melone (Porifera, Hadromerina): analisi Statistica. In: Atti Soc. peloritana. Sci. Fis. Mat. Nat. 17, 287–298. Simpson, T.L., 1968. The biology of the marine sponge Microciona prolifera (Ellis and Solander). II. Temperature-related, annual change in functional and reproductive elements with a description of larval metamorphosis. J. Exp. Mar. Biol. Ecol. 2, 252–277. Simpson, T.L., 1980. Reproductive processes in sponges: a critical evaluation of current data and views. Invertebr. Reprod. Dev. 2, 251–269. Simpson, T.L., 1984. The cell biology of sponges. Springer-Verlag, New York. Stone, A.R., 1970. Growth and reproduction of Hymeniacidon perleve (Montagu) (Porifera) in Langstone Harbour, Hampshire. J. Zool. Lond. 161, 443–459. Vatova, A., 1972. Osservazioni chimico fisiche periodiche nel Mar Grande e nel Mar Piccolo di Taranto (1962–1969). Boll. Pesca Piscic. Idrobiol. 27 (1), 43–79. Verdenal, B., Vacelet, J., 1985. Sponge culture on vertical ropes in the Northwestern Mediterranean Sea. In: Rützler, K. (Ed.), New Perspectives in Sponge Biology. Smithsonian Institution Press, Washington, D.C., pp. 416–424. Witte, U., 1996. Seasonal reproduction in deep sea sponges — triggered by vertical particle flux? Mar. Biol. 124, 571–581. Wulff, J.L., 1991. Asexual fragmentation, genotype success, and population dynamics of erect branching sponges. J. Exp. Mar. Biol. Ecol. 149, 227–247. Wulff, J.L., 1995. Effects of a hurricane on survival and orientation of large erect coral reef sponges. Coral Reefs 14, 55–61.
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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e
The budding process in Tethya citrina Sarà & Melone (Porifera, Demospongiae) and the incidence of post-buds in sponge population maintenance Frine Cardone a, Elda Gaino b, Giuseppe Corriero a,⁎ a b
Dipartimento di Biologia Animale e Ambientale, Università degli Studi di Bari, Via Orabona 4, 70125, Bari, Italy Dipartimento di Biologia Cellulare e Ambientale, Università degli Studi di Perugia, Via Elce di Sotto, 06123, Perugia, Italy
a r t i c l e
i n f o
Article history: Received 12 November 2009 Received in revised form 23 March 2010 Accepted 24 March 2010 Keywords: Asexual reproduction Mediterranean Sea Post-bud growth Tethya citrina
a b s t r a c t The relevance of the budding process in a population of Tethya citrina inhabiting an unpredictable environment subjected to frequent anoxic crisis (Mar Piccolo di Taranto) is described, and new data about the fate of the buds after their release are provided. At the individual level, for the first time in natural conditions, it has been demonstrated that several budding events follow each other, alternating with short periods (1–2 months) of rest. At the population level, budding proved to be a continuous process, being detected throughout the 13 months of study, with a short summer decrease both in the frequency of budding specimens and bud density. This trend was markedly different from that reported for conspecific populations inhabiting stable environments, in which budding is a seasonal process. It is hypothesized that the sudden and unpredictable variations of the ecological conditions could be responsible for the onset of repeated budding events, thus stressing the role of environmental factors in affecting the asexual reproductive pattern. T. citrina buds, here investigated, tend to detach from the adult sponge within 7/15 days of their differentiation, a feature coherent with experimental data carried out under laboratory conditions. A significant relationship between the size/age of the post-buds and the onset of the asexual reproduction has been proved: in particular, the first budding event was observed in nine-month-old sponges, measuring about 7 mm in diameter. As reported for other demosponges, in T. citrina post-buds the growth rate was positively related to the water temperature, with a significant increase during the summer. In addition, a negative effect of the budding process on the sponge growth, is hypothesized for a post-bud subjected to repeated budding events. Growth rate, however, proved highly variable. © 2010 Elsevier B.V. All rights reserved.
1. Introduction The life-cycles of the marine sponges, as well as those of most benthic animals, are usually less well known, owing to the real difficulties involved in carrying out investigations on some key aspects such as the biology of the larva, recruitment, metamorphosis and further growth in its natural environment. In contrast, asexual bodies produced by some families of sponges, constitute valid material for tracing their fate from the cradle to the grave, because of the low dispersal potentiality and structural organization of these elements that, bypassing metamorphosis, allows growth and functionality to be achieved in a short time. Budding is one of the mechanisms of sponge asexual reproduction. Buds are small spherical asexual bodies, generally brought to the extremity of a stalk that emerges from the surface of the mother sponge.
⁎ Corresponding author. Tel./fax: + 39 080 5443357. E-mail address: [email protected] (G. Corriero). 0022-0981/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2010.03.012
Budding has been widely observed in Tetractinomorpha, Demospongiae, and particularly in the genus Tethya. From the bulk of data, it has emerged that there is a high level of heterogeneity in terms of the time scale in cytological and skeletal organization of buds and the development stage reached at their detachment, thereby proving that their differentiation tends to be a species-specific process (Gaino et al., 2006). In T. seychellensis, buds differentiate choanocyte chambers before their release from the parent sponge, a feature suggesting that, at settlement, these elements can act as a functional young sponge (Gaino et al., 2009). In contrast, in T. aurantium and T. citrina the morphogenetic processes leading to the completion of the aquiferous system take place after bud detachment (Gaino et al., 2006). Despite the effects of this heterochrony, a recent study suggests a spatiotemporal morphogenetic pattern sequence that can be considered common in almost all sponge species able to generate buds (Hammel et al., 2009). Whereas some papers have been devoted to investigate bud production at the population level (Ayling, 1980; Corriero et al., 1996, 1998; Chen et al., 1997), nothing is known about the onset of the budding process or on its duration at the individual level. In addition,
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even if the ecological role performed by asexual reproduction has been stressed by many authors (Stone, 1970; Ayling, 1980; Battershill and Bergquist, 1990; Wulff, 1991, 1995; Corriero et al., 1996, 1998; Maldonado and Uriz, 1999; Ereskovsky and Tokina, 2007), the fate of the buds after their release, here including growth, ability to produce new asexual elements and mortality, is currently unknown. Only one paper, referred to asexual sponge fragments, reports the high survival of such elements after their detachment, with an initial rapid growth, thus suggesting that asexual fragments are responsible for successful recruitment (Wulff, 1991). Consequently, the scenario is very incomplete and more studies are needed to understand the actual role of the budding process in maintenance of sponge populations. We present here the results of a long term study aimed to add more information on the budding process in T. citrina, and to provide new data on the fate of the buds after their release, by monitoring them in natural conditions. 2. Methods 2.1. Species studied Tethya citrina Sarà and Melone, 1965 is a small demosponge, spherical in shape and soft in consistency. Small irregular tubercles emerge from its external surface. Its colour varies from grey to yellowish (Sarà and Melone, 1965). It inhabits rocky substrates from the surface to approximately a hundred metres in depth (Sarà, 1987). The sponge body consists of a thin cortical layer surrounding the choanosome where choanocyte chambers occur. The skeleton is made of strongiloxeas (235–1400 μm in length × 7.5–24 μm in thickness) arranged in radial bundles. Additional megasters (spherasters and spheroxyasters 18–64.8 μm in diameter) and micrasters (oxyasters, chiasters and tylasters 7.5–14.5 μm in diameter) also occur (Sarà and Melone, 1965; Bavestrello et al., 1992). T. citrina is a gonochoric oviparous species (Scalera-Liaci et al., 1971; Gaino et al., 1987; Corriero et al., 1996). Its sexual cycle displays a seasonal pattern, where oogenesis occurs from late spring to autumn (Scalera-Liaci et al., 1971; Corriero et al., 1996), and spermatogenesis from August to September (Scalera-Liaci et al., 1971). 2.2. Study site The Mar Piccolo di Taranto is an inner sea located at the north of the Gulf of Taranto (Ionian Sea). It consists of two distinct basins, the innermost of them receives the inflow from some small streams. The studied area is located in this innermost basin (latitude N 40°29′ 07′′, longitude E 17°16′ 39′′), and consists of a pile of calcareous stones, variable in size and shape, which are periodically subjected to long emersion periods, and to the hydrometric sizigial variations of the sea level. The pile of stones is situated on a silt bottom, often subjected to anoxic crises due to the deposition of large amounts of plant detritus. The calcareous stones are colonized by a rich sessile filter feeder fauna with prevalence of demosponges (Hymeniacidon perlevis, Suberites carnosus, Terpios fugax, Tethya citrina), calcareous sponges (Sycon sp., Paraleucilla magna), sabellids (Branchiomma luctuosum), serpulids (Hydroides sp.), bivalves (Mitylus galloprovincialis and Ostrea edulis), bryozoans and ascidians (some encrusting colonial didemnidae, Microcosmus sp. and Styela plicata). Among the demosponges, Tethya citrina inhabits the shaded side of the stones, where it can reach a density of more than 100 specimens per square metre of substrate (Cardone et al., unpublished data). Water temperature varies from 8.4 °C (December) to 26.8 °C (August) (Caroppo and Cardellicchio, 1995). Salinity values are around 36‰ (Caroppo and Cardellicchio, 1995). Locally, however, continental inflows may determine a marked drop in salinity (25– 30‰) (Corriero et al., unpublished data). Dissolved oxygen shows
wide seasonal variability: from May to October the oxygen deficit can reach marked levels which are responsible for distrophic events (Caroppo and Cardellicchio, 1995; Alabiso et al., 1997). 2.3. Sampling methods To estimate the relevance of asexual reproduction, ten adult specimens of Tethya citrina were randomly collected monthly (June 2007–June 2008; n = 130), and the total number of buds for each specimen was counted under the stereomicroscope. Moreover, the maximum and minimum diameters were measured both for the collected adult specimens and for 20 buds randomly selected from each specimen to obtain a mean estimation of their volume. During the period of intensified bud production (November 2007), sample collection consisted of weekly pictures of ten labeled adult sponge specimens, in order to evaluate the progression of the budding process up to bud release. The surface of these sponges was photographed using a digicam Canon 400 D, EF-S 60 mm f 2.8 Macro, appropriately set up on a metal support, in such a way that, over time, pictures were taken at the same angle and distance. Data were plotted on a PC and elaborated using the AUTOCAD 2007 software. In particular, for each of the ten labeled specimens, the total number of buds was counted weekly with the aim of identifying the newly produced and detached buds. Post-bud growth and death were assessed for ten labeled young specimens using the same methodology applied to the adult sponges. The asexual origin of these sponges was certified by the lack of sexual reproduction in the studied population at the onset of the monitoring (Cardone et al., unpublished data). The post-buds were monitored monthly over 13 months (June 2007–June 2008); a single post-bud was observed for an additional year (June 2006–May 2007). Photos were collected by applying the same photographic technique described above for each labeled specimen; from each picture, we recorded occurrence of budding on the sponge surface and maximum and minimum diameters of the labeled post-buds. The mean diameter of the post-buds was then calculated, and the specific growth rate (SGR) was estimated, as follows: SGR = ðloge md2 − loge md1 Þ = ðt2 −t1 Þ × 100; where loge are the natural logarithms, md2 and md1 the average diameter at the two different times, and (t2−t1) the time interval in months. During the study period, the water temperature and salinity were measured monthly at 50 cm in depth, using a multi parametric probe (OCEAN SEVEN 316 Plus). Differences in growth of post-buds at monthly time intervals were statistically analyzed by Kruskal–Wallis test (SPSS® 12.0.1). The relationship between the size of the post-buds and the first event of bud production was analyzed using the Sperman correlation coefficient; the same statistical analysis was used to test the relationships between water temperature, salinity and post-bud growth rate (SPSS® 12.0.1). 3. Results In budding specimens of T. citrina, buds sprout out from the sponge cortical layer as small bodies (Fig. 1a), varying in size and extension, because they are produced asynchronously. Buds are connected to the parent by a stalk that in fully differentiated buds greatly enlarges at the apex, giving rise to spiculated bodies. Buds grow gradually and some of them bend towards the substrate, in such a way that their apical region firmly adheres to it before their final detachment (Fig. 1b). Frequently, the stalk breaks off releasing buds, even though their diffusion is limited and they tend to colonise the areas close to the mother sponge. A total of 5207 buds have been counted, from 130
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Fig. 1. T. citrina. a: Budding specimen from the Mar Piccolo di Taranto (bar = 1 cm). b: Buds attached to the substrate (bar = 1 mm).
adult specimens (14.7 ± 3.8 mm in mean diameter) examined. The total number of budding adult specimens counted over 13 months of study was equal to 100 (77%). Specimens with buds occurred throughout the year, with peaks in frequency values (100%) recorded in September, October and from February to April, whereas in December and January the frequency of specimens with buds was remarkably reduced (Fig. 2a). The peaks, expressing the density of the buds (about 40/cm2) produced by the adult sponge, almost coincide with those showing the frequency of the budding specimens (Fig. 2a, b). The mean bud size varied from 0.32 ± 0.1 (August) to 0.56 ± 0.18 mm (February), showing the lowest values in parallel with those of the bud density (Fig. 2b). The reproductive effort (expressed as percent of: total volume of buds/ total volume of parental tissue) was maximum in the winter period, with a peak in November (1%) and a minimum in the spring and summer months. The analysis of the budding process, carried out with weekly frequency on ten labeled specimens, showed that 1 to 2 weeks after their appearance most of the buds had detached from the sponge surface (Fig. 3). Sometimes, instead of detaching from the mother sponge buds remained connected by means of their stalk that bent toward the substrate and finally the bud adhered to it. This mechanism allowed the adult sponge to incorporate the bud tissues, thereby widening its extension on the substrate (Fig. 4). Monthly investigations carried out on the growth and morphological changes of ten settled buds, showed that in 13 months they reached a mean size of 7.1 ± 1.4 mm in diameter, starting from 1.2 ±
0.2 mm (Figs. 5–7). At the onset of settlement, the post-buds formed an encrusting layer, which gradually modified giving rise to a spherical shape. During the following growth, the outline of the young sponges tended to vary from fairly round to more irregularshaped individuals. This trend was typically observed in all the considered post-buds, as became evident by comparing the relationship between the minimum and maximum diameters of each of the specimens. Sometimes, the close proximity of the post-buds promoted their fusion into larger sponges, as occurred for specimens 3 and 6, during the first month of growth, and for specimens 2 with an unmonitored post-bud, after 10 months of monitoring. Specimen 5 was overgrown by a colonial ascidian, which did not kill it, but prevented further observations (Fig. 5). This process of monitoring allowed us to ascertain the minimum size at which, in turn, the postbuds were able to differentiate buds (Figs. 5 and 6): in more than 60% of the ten monitored post-buds the first budding event occurred when the sponge size (diameter) varied from 6.4 to 6.9 mm. In the monitored post-buds, the first event of bud production was significantly correlated with the size of the post-buds (rs = 0.46; P b 0.01) (Fig. 5). No mortality was observed in the monitored post-buds throughout the 13 months of study. However in the following year a persistent anoxic event (November 2008) destroyed most of the benthic assemblage from the study area, killing 70% of the monitored postbuds. Significant differences in growth and growth rate were observed in the ten monitored post-buds (H = 24.3; P b 0.01 and H = 84.5; P b 0.01 respectively). The average value of the specific growth rate was 0.5 ± 0.1 mm/month (13 months). The growth rate showed a seasonal pattern. The maximum average specific growth rate (1.9 ± 0.3) was measured in July, the lowest (0.03 ± 0.09), in January. The growth rate was positively related with water temperature (rs = 0.42; P b 0.01), but not with salinity. Indeed, during summer the highest temperature values paralleled those of post-bud growth rate (Fig. 8). When comparing the growing curve of an individual followed for 25 months with that dealing with the 10 specimens, it emerged that the growth tended to decrease after the initial year (Fig. 9). 4. Discussion
Fig. 2. T. citrina. a: Monthly frequency (%) of specimens with buds. b: Monthly variation of bud density and size (± standard deviation).
The relevance of the budding process of Tethya citrina is investigated in a population inhabiting the Mar Piccolo di Taranto, a very unpredictable environment subject to frequent anoxic crisis (Vatova, 1972; Caroppo and Cardellicchio, 1995; Alabiso et al., 1997). The monitored specimens grew on the rocky substrates of the uppermost fringe of the infralittoral zone (sensu Pérès and Picard, 1964), where they are seasonally subjected to air exposure periods concomitantly to the hydrometric sizigial variations of the sea level.
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Fig. 3. T. citrina. Weekly monitoring (November 2007) of buds number produced and released from ten labeled adult specimens. All buds that were not possible to monitor during the time of the study were classified as undetermined; □ undetermined buds first production of buds second production third production fourth production.
This field study has provided new data on the budding process investigated under natural conditions, confirming the key role of asexual reproduction for maintenance and dispersal employed by some sponge species, mainly in habitats affected by remarkable fluctuations in environmental parameters. According to the literature, T. citrina reproduces asexually, by elaboration of buds (Connes, 1968; Scalera-Liaci et al., 1971; Corriero et al., 1996; Gaino et al., 2006). At the individual level, for the first time in natural conditions, our data show that several budding events follow each other, alternating with short rest periods (1–2 months). This contrasts with observations carried out under experimental conditions, where, usually, after the bud detachment, no new buds were produced by the same reared specimen of T. citrina (Cardone et al., 2008). Several bud-bearing specimens of Thenea abyssorum kept alive in the laboratory for several months were unable to release buds (Witte, 1996), thus emphasizing that the role of the budding process could be interpreted only by carrying out investigations in natural conditions.
At the population level, budding is a continuous process; in fact, buds were detected throughout the period of study, with a short decrease both in the frequency of budding specimens and bud density during the late summer. This trend markedly differs from that reported for the conspecific population from the Stagnone di Marsala (North-west Sicily). There, T. citrina inhabits a shallow (1 m in depth) but stable habitat (the rhyzomes of Posidonia oceanica, a phanerogam not tolerant to strong variations in environmental conditions), and displays a clear budding seasonality (October–January) (Corriero et al., 1996). Highest bud production in the autumn and winter months has been also reported by Connes (1968) for mixed populations of T. citrina and T. aurantium inhabiting deep environments usually subjected to moderate variations in hydrological parameters (detritic bottoms, 80 to 100 m in depth). At Taranto, the density of buds on the sponge surface is extraordinarily high (up to 40 buds/cm² in October), in comparison with that reported by Corriero et al. (1996) for the Marsala population (maximum value = 2.6 bud/cm²). On the contrary, the mean size of
Fig. 4. T. citrina. A specimen monitored for four weeks: a: some buds still connected by the stalk to the mother sponge attached to the substrate; b, c, d: the adult sponge incorporates the bud tissue widening its extension on the substrate (bar = 1 cm).
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Fig. 5. T. citrina. Monthly variation of mean diameter (± standard deviation) and minimum/maximum diameter ratio in ten post-buds monitored for 13 months. *: budding events.
the buds is much lower at Taranto (0.5 mm in diameter), than at Marsala (0.85 mm) (Gaino et al., 2006). In other words, the population of T. citrina from the Mar Piccolo di Taranto produces
numerous small buds throughout the year, whereas at Marsala asexual reproduction occurs only in autumn–winter, with the elaboration of a few relatively large elements. Such differing
Fig. 6. T. citrina. Monthly variation of mean diameter (± standard deviation) and minimum/maximum diameter ratio in a post-bud monitored for 25 months. *: budding events.
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Fig. 7. T. citrina. Growth phases of a post-bud recorded at different time intervals after settlement: a: 1 month (arrow indicates the position of the sponge on the substrate); b: 4 months; c: 6 months; d: 9 months, during its first budding event. a: (bar = 2 mm); b, c, d: (bar = 5 mm).
reproductive strategies could be due to the different environmental conditions characterizing the two sites. The mechanisms able to control the onset, duration and progression of the reproductive events still remain unclear in Porifera. Connes (1968), Simpson (1980) and Pronzato et al. (1993) put forward the hypothesis of a combined action of exogenous and endogenous factors affecting gamete and asexual element differentiation. In addition, several authors emphasise the effects that variations of the hydrological parameters can have on the reproduction of numerous poriferans (Simpson, 1968, 1984; Diaz, 1973; Fell, 1976, 1993; Corriero et al., 1996, 1998; Ereskovsky, 2000; Meroz-Fine et al., 2005). The here hypothesized relationship between environmental stress and bud production is coherent with the observation that, under laboratory conditions, the budding process in some sponges may be stimulated by injury and/or stress (Connes, 1967; Saller, 1990). According to Cardone et al. (2008), in T.
Fig. 8. T. citrina. Growth performance (specific growth rate in percentage) (± standard error) of ten post-buds monitored for13 months, with seasonal variations of water temperature and salinity.
citrina budding can also be experimentally induced by drastic variations in the hydrological parameters. At Taranto, therefore, the sudden and unpredictable variations of the ecological conditions could be responsible for the onset of repeated budding events. The sponge buds, with a reduced dispersal ability, represent a source of new recruits increasing the stock of the original population, which can balance its high mortality through the clonal process that gives inception to a continuous turn-over. Under environmental stresses, budding would ensure a better opportunity for survival. Such a hypothetical scenario is coherent with the high mortality that affected the benthic fauna in the year that followed the present study. With regard to population dynamics, the present investigation allowed the growth pattern of the post-buds and the onset of budding process to be described. In T. citrina post-buds the budding process starts when the sponge is about nine-months old and measures about 7 mm in diameter. The observations carried out in natural conditions could be regarded as a relevant step in widening knowledge on the budding process, by providing data on the relationship between the size/age of specimens and the onset of asexual reproduction. Research into sponge growth has revealed a seasonal increase in growth concomitantly with the water temperature increase (Barthel, 1986; Garrabou and Zabala, 2001; Page et al., 2005; De Caralt et al., 2007). According to the literature, in T. citrina post-buds the growth rate is positively related to the water temperature, with a significant increase during the summer. However, growth rate has proved to be highly variable. The high variability in growth is a well known problem among researchers who have experimented with sponge cultivation. Verdenal and Vacelet (1985) and Corriero et al. (2004) observed marked differences in the growth of fragments of Spongia
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Fig. 9. T. citrina. Growth curves in ten post-buds monitored for 13 months and in a post-bud monitored for 25 months (± standard deviation).
officinalis, even when cut from the same specimen, and apparently subjected to the same ecological conditions. A certain degree of growth variability was also observed by Cardone et al. (2008) in postbuds of T. citrina produced by the same specimen under controlled conditions. The differences in growth of the post-buds, apart from the ecological variations occurring in the different microhabitats colonized by each bud, could also be related to the bud organization displayed at the moment of its detachment from the mother sponge (size, functionality, occurrence and quantity of different cellular types, ratio between collagen and cellular component), as suggested by Connes (1968), who hypothesized a different fate for the buds due to differences in cell composition. A recurrent feature is the abrupt drop in the positive growth of the monitored post-buds. This feature is probably due to environmental stress conditions affecting the buds. However, other hypotheses cannot be rejected. According to Wulff (1991) the growth pattern of three tropical demosponges usually subjected to asexual fragmentation, consists of a more or less prolonged phase of linear size increase, terminated by a marked decrease due to fragmentation or mortality of the sponge. Most of our data only concern the initial phase of the sponge life, in which a too short period of budding occurs, and therefore they do not allow evaluation of the impact of budding on the growth of the sponge. However, the monitoring for 25 months of a single specimen of T. citrina seems to indicate a negative effect of the budding process on sponge growth, as revealed by a long period of negative growth associated with repeated budding events. The growth process is associated to marked variations in the shape of young sponges. The first event occurring in the newly released buds is settlement marked by extension and adhesion to the substrate, a process that gives rise to a large thin encrusting formation. In this initial phase of growth, the thickness of the young sponge is greatly reduced, as shown by the high values of the longitudinal axis when compared with those of the vertical one. Therefore, the young sponge has an initial flat shape that changes remarkably through the gradual acquisition of a globular formation during growth. The adhesion to the substrate represents a crucial phase for further development as also observed in post-larvae during metamorphosis. In this regard, asexually and sexually produced elements share similar needs. In particular, it has been shown that larval attachment is due to the secretion of a basal ground matter between the larva and the substrate (Borojevic and Lévi, 1965; Bergquist and Green, 1977; Evans, 1977; Kaye and Reiswig, 1991; Gaino et al., 2007). Therefore, in contrast with larvae, which show various behavioural patterns and preferences in substrate selection (Leys and Degnan, 2002; see review in Maldonado, 2006), buds are not active in such a process and the spicules sprouting out from the bud peripheral border, after having favoured the initial floating of the buds, may be used as a tool for anchoring them to the substrate. In conclusion, the present investigation has supplemented data available in the literature about the sponge budding process and shown that environmental factors are able to deeply affect the reproductive pattern. Tethya citrina, besides its considerable reproductive plasticity (Corriero et al., 1996; Gaino et al., 2006), is able to put into action different reproductive strategies, both sexual and
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