Lethal and sub-lethal effects of phototrophic endoliths ... - Springer Link

Marine Biology (1999) 135: 497±503

Ó Springer-Verlag 1999

S. Kaehler á C. D. McQuaid

Lethal and sub-lethal effects of phototrophic endoliths attacking the shell of the intertidal mussel Perna perna

Received: 26 January 1999 / Accepted: 17 August 1999

Abstract Animals that bore into calcareous material can cause considerable damage to molluscan shells. In contrast, smaller microbial phototrophic endoliths have until recently been thought of as relatively benign. Phototrophic endoliths (primarily cyanobacteria) infest the shells of 50 to 80% of midshore populations of the mussel Perna perna (L.) in South Africa. This infestation causes clearly visible shell degradation, and we record here ecologically important lethal and sub-lethal e€ects (e.g. changes in growth and reproductive output) of the endoliths on their mussel hosts. Endolith infestation reduced the strength of shells signi®cantly and also affected shell growth. In situ marking of shells, using the ¯uorochrome calcein, showed that infested and non-infested mussels increased in shell length at the same rate. However, the rate of increase in shell thickness (associated with shell repair) was signi®cantly faster in infested than in uninfested individuals. This increase in the rate of shell thickening was not sucient to compensate for rapid endolith-induced shell degradation and, around the site of adductor muscle attachment, infested shells were thinner than their uninfested counterparts. The shells of 18% of recently dead mussels had holes induced by endolith erosion. This e€ect was highly size dependent, and the proportion of mortality due to endoliths rose to almost 50% for the largest mussels. The re-routing of energy due to shell repair had important sub-lethal e€ects on the reproductive rates of mussels. During the reproductive period, mean dried ¯esh mass for large (>70 mm), non-infested P. perna was substantially higher than for infested individuals. This difference was almost entirely due to di€erences in gonad mass, which was approximately 100% higher for noninfested mussels. We conclude that, by attacking the Communicated by O. Kinne, Oldendorf/Luhe S. Kaehler (&) á C.D. McQuaid Rhodes University, Department of Zoology and Entomology, P.O. Box 94, Grahamstown 6140, Republic of South Africa

shell, phototrophic endoliths reduce both the longevity and reproductive output of large mussels on the midshore.

Introduction While a number of studies have reported on endolithinduced biodegradation of carbonate skeletal materials, severe damage to host organisms is usually restricted to the bioerosive activity of invertebrate or fungal borers (e.g. Alderman and Jones 1967; Kent 1981; Thangavelu and Sanjeevaraj 1988; Ambariyanto and Seed 1991; Hook and Golubic 1993; Mao Che et al. 1996). Due to light limitation within the substratum, phototrophic endoliths are generally thought to penetrate only the uppermost layer of the carbonate and thus to be incapable of causing severe structural damage (see review by Laukner 1983). Only recently, has it been realised that occasionally the boring activity of photosynthetic endoliths may be sucient to cause extensive damage to living organisms (Raghukumar et al. 1988, 1991; Webb and KorruÃbel 1994; Kaehler 1999). Investigations of endolith-infested mussel and oyster shells at Goa (India), and of mussels on the west and south coasts of South Africa have shown that severe shell damage may occur in the absence of heterotrophic shell borers (Raghukumar et al. 1991; Webb and KorruÃbel 1994; Kaehler 1999). In these studies, the boring activity of endolithic cyanobacteria and/or microalgae resulted in the degradation of the structural integrity of shells, which in some cases resulted in bivalve mortality. No quantitative information is available, however, on the frequency of mortalities induced by these endoliths. Neither is it known whether infestations by phototrophic endoliths may have other indirect e€ects on bivalves. Investigations of large shell-boring invertebrates have reported a number of possible secondary e€ects on host organisms. Although di€erent methodologies have produced con¯icting results, several studies have

498

reported a decline in bivalve condition with increasing infestation intensity (Kent 1979; Tkachuk 1988; Ambariyanto and Seed 1991; Wargo and Ford 1993). Other e€ects reported by these authors include an increase in shell thickness, a reduction in mantle tissues and a possible decline in the fecundity of infested bivalves. To our knowledge, no such e€ects have ever been linked to the boring activity of phototrophic endoliths. This is surprising, as recent studies have shown that cyanobacterial and microalgal endoliths may infest a high proportion of total shell surface and may result in severe shell degradation (Webb and KorruÃbel 1994; Kaehler 1999). It is likely, therefore, that as in the case of larger invertebrate borers, bivalves attacked by phototrophic endoliths will protect themselves by regenerating damaged parts of the shell. The process of molluscan shell repair, however, is energetically costly (Kent 1979; Geller 1990) and may result not only in increased shell mass but possibly also in a reduction of somatic and reproductive growth. On the south coast of South Africa, the dominant rocky shore mussel Perna perna exhibits a high incidence of infestation by cyanobacterial endoliths towards its upper distributional limit (Kaehler 1999). Endolithinduced mortalities have previously been recorded, but no quantitative information is available either on the frequency of such mortalities or on possible secondary e€ects of the phototrophic borers. The present study initially investigates the proportion of total P. perna mortality attributable to endolith activity and then goes on to study the e€ects of endoliths on a number of shell, somatic and reproductive parameters. The latter were investigated by directly comparing infested and uninfested (clean) bivalves collected from the same sites.

Materials and methods Study site On the south coast of South Africa, the rocky shore mussel Perna perna (L.) occurs from the low to mid-shore. All studies and collections were carried out within 20 cm of the upper-most vertical limit of its distribution, where mussels rarely exceeded 80 mm in length. Three sites were used along 25 km of wave-exposed coastline in the Bathurst and Peddie districts (33°30¢N; 27°00¢E). The sites were Rufanes, Riet River and Waterloo Bay and corresponded to Sites 5, 9 and 20, respectively, in Kaehler (1999). Mussel cover was high but patchy at all sites and endolith infestation incidence ranged from 50 to 80%. Along this coastline endolith infestation by cyanobacteria (e.g. Plectonema terebrans, Mastigocoleus testarum, Hyella caespitosa and Pleurocapsa sp.) frequently results in shell degradation and ultimately mussel mortality (for details see Kaehler 1999). Contribution to mortality Mussel mortality was investigated by clearing three 1 m2 quadrats of all dead (gaping during low tide) mussels. Collections were carried out at Riet River during January 1998 and at all three sites during January 1999 (n ˆ 12 quadrats). In the laboratory, shells were sized (shell length) and grouped according to their condition.

In order to ensure that only recent mortalities were included in the surveys, only shells with a shiny inner nacreous layer were used. The insides of mussel shells became heavily fouled within a month after death and all such shells were discarded. Phototrophic endoliths cause a distinctive discoloration, dissolution and ®nally fracturing of the shell around the site of adductor muscle attachment (for details see Kaehler 1999). All shells exhibiting these distinctive ``fracture holes'' were assumed to have died from endolith activity. As heterotrophic endoliths are rare at the study sites and restricted to the structurally sound umbonal region of the shell, all such mortalities were attributed to cyanobacterial borers (see also Kaehler 1999). Shell strength In order to quantify the e€ect of endoliths on the structural integrity of the shells, the force required to penetrate 50 infested and 50 clean shells was determined. An Instron 4301 Universal Materials Testing Instrument was used to exert an increasing force onto a 10 mm2 area of the shell, at the site of adductor muscle attachment. The instrument then determined the force required to penetrate the shells. The adductor muscle scar is the most common site for naturally occurring holes as here a high abundance of the large cavity-forming cyanobacterium Pleurocapsa sp. coincides with an area of reduced shell repair and increased mechanical stress (for details see Kaehler 1999). Due to the small area of applied force, the resulting measurements should be interpreted only as relative and not absolute estimates of shell strength. Shell growth parameters Shell growth in 36 infested and 36 clean mussels was determined by injecting the ¯uorochrome calcein (Sigma, Chem. Abstracts ID No. 1461-15-0; 125 mg l)1) into the mantle cavity of individual mussels and using the resulting ¯uorescent band to estimate growth from time of injection (for details see Kaehler and McQuaid 1999). Mussels were injected at Waterloo Bay in February 1998 and growth was determined 30 d after treatment. Once recovered, treated shells were sectioned sagitally and observed through an Olympus ¯uorescent microscope exciting at 460 to 490 nm (U-MWIB Cube). Growth was then determined with a micrometer, by measuring the distance between the ¯uorescent mark and the growing edge of the shell and related to initial shell length (determined with vernier calipers). At a later stage the same technique was used to measure shell thickness and increase in shell thickness around the site of adductor muscle attachment for 27 and 30 infested and clean mussels, respectively. Mussel condition In order to investigate the e€ects of endolith infestation on mussel condition, dry mass of both shell and ¯esh (i.e. total mass±shell± byssus) was determined in mussels from Riet River. Initially, during June 1997, a large sample size of n ˆ 236 mussels was used. In order to minimise destructive sampling of the protected brown mussel, smaller sample sizes of 35 infested and 35 clean individuals were used thereafter. This sample size was found to be adequate to display signi®cant di€erences in shell or ¯esh mass, as they occurred. Collections were made twice during the main reproductive period, shortly before spawning (January 1998, 1999) and twice during times of the year when the majority of mussels were nonreproductive (June 1997 and November 1998). Dry mass was determined after desiccation for 24 h at 50 °C. As ¯esh mass di€ered greatly between infested and clean individuals during the reproductive season, a further collection was made in January 1999 to study the contribution of gonad mass to total ¯esh mass. For this study a total of 20 infested and 20 clean female mussels were dissected and the gonads were removed. Dry gonad and somatic mass was then determined for each individual.

499 Data analyses

Shell strength

All statistical comparisons between infested and clean organisms were carried out by analyses of covariance using length as the covariate (STATISTICA for Windows R. 5.1.). Log transformations prior to analyses were utilised where indicated in ®gure legends. Statistics are presented with the ®gures.

Applying force to the shell surface may result in two types of shell damage. The blunt nail did not penetrate 98% of structurally sound (clean) shells but caused them to shatter into several pieces when intense pressure was applied (Fig. 2). Only 38% of infested shells exhibited this type of shell damage. Instead, the majority of infested shells (62%) were punctured (holed) at force applications of approximately one-third of those required to shatter shells (Fig. 2). The majority of, but not all, infested shells were therefore severely weaker than clean (uninfested) shells.

Results Contribution to mortality A total of 686 recently dead mussels was collected from twelve 1 m2 quadrats. Of these shells, 17.9% exhibited large endolith-induced fracture holes (Fig. 1) which the mussels could not have survived for long. While there was some variation in frequency of holes amongst the samples, the proportion of mortalities attributed to endolith activity increased with shell size at all sites. The majority of large individuals >70 mm were heavily infested and more than 40% of these exhibited fracture holes. In contrast, fewer of the smaller mussels had holes and none were observed in shells 70 mm) was almost 80% greater than that of infested mussels. As signi®cant differences in ¯esh mass occurred only during the reproductive periods, a further study investigated the contribution of gonad and somatic mass to total ¯esh mass. This study revealed that during January 1999, di€erences in total ¯esh mass were due solely to gonad mass (Fig. 7A). The reproductive output (gonad mass) of clean mussels was 100% higher than that of infested mussels, while there was no signi®cant di€erence in the mass of somatic tissues (Fig. 7B).

Discussion

Fig. 4 Perna perna. A Increase in thickness (associated with shell repair) and B actual thickness of infested and clean mussel shells. Growth and thickness were determined around the site of adductor muscle attachment. Analyses of covariance showed that infested shells grew in thickness signi®cantly more but were signi®cantly thinner than uninfested individuals

The activity of phototrophic endoliths of the brown mussel Perna perna resulted in severe shell weakening and ultimately the mortality of a large number of adult mussels. In addition to these direct e€ects of shell dissolution, however, it was found that the presence of endoliths also resulted in a marked increase in shell regeneration and a reduction in the reproductive output of host organisms. Early descriptive studies of phototrophic endoliths date back for more than a century (reviewed by Golubic et al. 1975). It is only in recent years, however, that their destructive and potentially lethal e€ects on bivalves have come to light (Webb and KorruÃbel 1994; Kaehler 1999). In the present study it was shown that at the upper distributional limit of mussels, 17.9% of recent mortalities bore large endolith-induced fracture holes. As large holes usually occur at the adductor muscle scar and result in the detachment of the muscle from the shell, these mortalities were attributed to endolith activity. Our estimates of the proportion of mortality due to endoliths do not take into account individuals that are entirely removed from the mussel bed by large predators such as birds, ®sh and octopuses (Smale and Buchan

501

Fig. 6 Perna perna. Dry shell mass (left panels) and dry ¯esh mass (right panels) of infested and clean mussels during four time periods. Analyses of covariance on log-transformed data showed that during the reproductive periods (January 1997 and 1998) ¯esh mass was signi®cantly lower in infested than in clean mussels. Conversely, during periods of reduced reproduction, shell mass was greater in infested than in uninfested individuals (*signi®cant di€erence in dry mass between clean and infested mussels)

1981). Nor do these estimates allow for mortality through intraspeci®c competition for space, though this is likely to be of greater importance lower on the shore. Consequently our data may overestimate the importance of endolith-induced mortality. Despite this, it is clear

502

that endoliths are a signi®cant cause of mortality. This is especially true for large mussels (see also Webb and KorruÃbel 1994) for which endoliths were responsible for almost 50% of observed mortality. Greater shell damage in larger mussels may be linked directly to the infestation intensity of shells, which also increased with shell length. Small mussels are generally free of endoliths and become colonised only once the protective periostracum has been worn away. Thereafter, a succession of cyanobacterial species invade the shells, resulting in increasing degradation with shell age, especially around the site of adductor muscle attachment (Kaehler 1999). In the present study, the majority of heavily infested shells (62%) exhibited signs of severe shell weakening. When force was applied to the area of adductor muscle attachment, these shells punctured at less than half the force required to damage uninfested shells (see also Webb and KorruÃbel 1994). Most clean shells (98%) and some infested shells (38%), however, were structurally more sound and could not be punctured. Instead, these shells shattered into several pieces, but only when much greater force was applied. It seems, therefore, that not all shells are equally a€ected by endoliths and that some heavily infested individuals are not suciently weakened to make them prone to puncturing. A number of studies have remarked on the ability of molluscs to regenerate damaged parts of the shell

Fig. 7 Perna perna. A Gonad dry mass and B somatic dry mass of infested and clean mussels during January 1999. Analyses of covariance on log-transformed data show a signi®cantly greater gonad mass in clean than in infested individuals, while no di€erences were exhibited between the somatic mass of infested and uninfested mussels

through the deposition of new shell material (reviewed by Wilbur 1964; Watanabe 1983). In the present study, growth of the inner shell layer (increase in shell thickness) was signi®cantly greater in infested than uninfested shells. Furthermore, it was the thinnest (most damaged) shells that exhibited the greatest amount of secondary shell deposition. It is likely, therefore, that such regeneration occurred primarily as a repair mechanism cued by shell damage, though some reduced shell thickening also occurred in a few young uninfested shells. This suggests, that in small mussels naturally occurring shell deposition takes place until an optimum thickness is reached, irrespective of the presence or absence of endoliths. Beyond 40 mm in length, however, no clean mussels exhibited an increase in thickness, while infested mussels continued to regenerate their shells. Di€erences in the growth of the inner shell layer were not mirrored by growth in shell length. There was no signi®cant di€erence in the increase in shell length between infested and clean mussels. Nevertheless, in infested mussels, growth of the inner shell layer was not solely restricted to the site of greatest shell damage (see also Waage 1952). In individuals that exhibited regeneration, calcein ¯uorescence showed that new growth occurred throughout much of the apical half of the inner shell. In most cases this growth was not sucient to stem the disintegration of the shell around the site of adductor muscle attachment. As a result, infested shells were signi®cantly thinner close to the adductor muscle scar than were clean shells. Overall, however, deposition rates were far greater in infested than in clean shells and the shell mass of infested mussels was never lower than that of uninfested mussels. As endoliths cause a steady dissolution and erosion of the shell surface, infested mussels must, therefore, have invested heavily in shell repair. A number of studies have suggested that the process of molluscan shell repair is energetically costly (Davis and Hillman 1971; Kent 1979; Geller 1990; Ambariyanto and Seed 1991). Geller (1990), who showed that the repair of shell damage was faster in fed molluscs than in starved individuals, provided the most direct evidence of this. As a result, it has been argued that the energetic cost of shell repair may reduce the energy available for somatic and possibly reproductive growth. In the present study, it was shown that infested mussels regenerate shell material at a much greater rate than do clean mussels and that this does indeed have important consequences. Endoliths seem to have a strong e€ect on the allocation of energy in mussels. During non-reproductive periods, infested mussels had signi®cantly heavier shells than clean individuals, re¯ecting large amounts of deposition of material during shell repair. However, during the reproductive period, this di€erence was not apparent but instead infested mussels had a signi®cantly lower ¯esh mass. This di€erence in ¯esh mass was solely due to a failure of the gonad to develop to the same extent as in clean individuals. This suggests that channelling energy into reproduction limits the ability of the mussel to re-

503

generate fully parts of the shell that are eroded by endoliths. It also suggests that, had infested mussels channelled energy into reproduction at normal levels, shell collapse could have resulted because insucient energy would have been available for shell repair. Apparently, the need to invest heavily in reproduction is outweighed by the more urgent need to maintain the shell. While it is known that large heterotrophic endoliths can cause reductions in ¯esh weight and condition in bivalves (Kent 1979; Tkachuk 1988; Ambariyanto and Seed 1991; Wargo and Ford 1993), this is the ®rst record of serious non-lethal e€ects due to phototrophic endoliths. Indeed the e€ects we recorded are likely to have important ecological consequences. Large mussels were most susceptible to mortality through endolith-induced shell collapse. In addition, these large mussels, which are responsible for the bulk of gamete output from the population, also su€ered the greatest decrease in gonad tissue due to endolith infestation. Cyanobacterial endoliths are common on South African shores and may locally infest up to 100% of large midshore mussels (Webb and KorruÃbel 1994; Kaehler 1999). Through their in¯uence on the mortality and energetics of the largest size classes they are likely to have important negative e€ects on the reproductive output and ultimately the stability of midshore mussel populations. Acknowledgements We would like to thank the Leather Industry Research Institute (LIRI) for the use of their materials testing instrument. This work was funded by the Joint Research Committee of Rhodes University and the National Research Foundation (NRF) and was supported by the South African Network for Coastal and Oceanic Research (SANCOR).

References Alderman DJ, Jones EBG (1967) Shell disease of Ostrea edulis L. Nature, Lond 216: 797±798 Ambariyanto, Seed R (1991) The infestation of Mytilus edulis Linneaus by Polydora ciliata (Johnston) in the Conwy estuary, North Wales. J mollusc Stud 57: 413±424 Davis NM, Hillman RE (1971) The e€ect of arti®cial shell damage on sex determination in oysters. Proc natn Shell®sh Ass 61: p2 Geller JB (1990) Reproductive responses to shell damage by the gastropod Nucella emariginata (Deshayes). J exp mar Biol Ecol 136: 77±87

Golubic S, Perkins RD, Lukas KJ (1975) Boring microorganisms and borings in carbonate substrates. In: Frey RW (ed) The study of trace fossils. Springer-Verlag, Berlin, pp 229±259 Hook JE, Golubic S (1993) Microbial shell destruction in deep-sea mussels, Florida Escarpment. Pubbl Staz zool Napoli (I Mar Ecol) 14: 81±89 Kaehler S (1999) Incidence and distribution of phototrophic shelldegrading endoliths of the brown mussel Perna perna. Mar Biol 135: 505±514 Kaehler S, McQuaid CD (1999) Use of the ¯uorochrome calcein as an in situ growth marker in the brown mussel Perna perna. Mar Biol 133: 455±460 Kent RML (1979) The in¯uence of heavy infestations of Polydora ciliata on the ¯esh content of Mytilus edulis. J mar biol Ass UK 59: 289±297 Kent RML (1981) The e€ect of Polydora ciliata on the shell strength of Mytilus edulis. J Con int Explor Mer 39: 252±255 Laukner G (1983) Diseases of Mollusca: Bivalvia. In: Kinne O (ed) Diseases of marine animals. Biologische Anstalt Helgoland, Hamburg, pp 477±961 Mao Che L, Le Campion-Alsumard T, Boury-Esnault, Payri C, Golubic S, BeÂzac C (1996) Biodegradation of shells of the black pearl oyster, Pinctada margaritifera var. cumingii, by microborers and sponges of French Polynesia. Mar Biol 126: 509±519 Raghukumar C, Pathak S, Chandramohan D (1988) Biodegradation of calcareous shells of the window pane oyster by shellboring cyanobacteria. In: Thompson MF, Sarojini R, Nagabhushanam R (eds) Marine biodeterioration: advanced techniques applicable to Indian Ocean. Oxford and IBH Publishing, New York, pp 692±701 Raghukumar C, Sharma S, Lande V (1991) Distribution and biomass estimation of shell-boring algae in the intertidal at Goa, India. Phycologia 30: 303±309 Smale AM, Buchan PR (1981) Biology of Octopus vulgaris o€ the east coast of South Africa. Mar Biol 65: 1±12 Thangavelu R, Sanjeevaraj PJ (1988) Boring and fouling organisms of the edible oyster Crassostrea madrasensis (Preston) from the Pulicat Lake, South India. J mar biol Ass India 30: 47±53 Tkachuk LP (1988) Vermins of the Black Sea mussel plantations. Ekol Moria 30: 60±64 Waage LE (1952) Quantitative studies of the calcium metabolism in Helix aspersa. J exp Zool 120: 311±342 Wargo RN, Ford SE (1993) The e€ect of shell infestation by Polydora sp. and infection by Haplosporidium nelsoni on the tissue condition of oysters, Crassostrea virginica. Estuaries 16: 229±234 Watanabe N (1983) Shell repair. In: Wilbur KM (ed) The Mollusca. Vol. 4. Academic Press, New York, pp 289±316 Webb SC, KorruÃbel JL (1994) Shell weakening in marine mytilids attributable to blue-green alga, Mastigocoleus sp. (Nostochopsidaceae). J Shell®sh Res 13: 11±17 Wilbur KM (1964) Shell formation and regeneration. In: Wilbur KM, Yonge CM (eds) Physiology of Mollusca. Vol. 1. Academic Press, New York, pp 243±282