MICROSCOPY RESEARCH AND TECHNIQUE 37:116–135 (1997) Epibiotic Microorganisms on Copepods and Other Marine Crustaceans KEVIN R. CARMAN1* AND FRED C. DOBBS2 1Department 2Department of Zoology and Physiology, Louisiana State University, Baton Rouge, Louisiana 70803-1725 of Oceanography, Old Dominion University, Norfolk, Virginia 23529-0276 KEY WORDS: epibiosis; symbiosis; algae; diatoms; fungi; protozoa; bacteria; Vibrio; review ABSTRACT Although the occurrence of microbial (algal, protozoan, bacterial, and fungal) epibionts on marine crustaceans and other invertebrates has been documented repeatedly, the ecological context and significance of these relationships generally are not well understood. Recently, several studies have examined the population and community ecology of algal and protozoan epibionts on freshwater crustaceans. Even so, the study of microbial epibionts in aquatic environments is still in its infancy. In this review, we summarize associations of microalgae, protozoans, and bacteria with marine crustaceans, especially copepods. We note differences and commonalities across epibiont taxa, consider host-epibiont cycling of nutrients, generate hypotheses relevant to the ecology of the host and the epibiont, and suggest future research opportunities. Microsc. Res. Tech. 37:116–135, 1997. r 1997 Wiley-Liss, Inc. The presence or absence of microorganisms epibiotic on animals may play an important role in the life of the animal. It seems logical that an adequate description of the epibiotic microbiota, at least that on the smaller forms, should become an integral part of the description and study of marine animals. Sieburth (1975) INTRODUCTION It has long been recognized that aquatic microorganisms (algae, protozoans, bacteria, and fungi) in general, and bacteria in particular, have a strong affinity for surfaces (e.g., Cooksey and Wigglesworth-Cooksey, 1995; Costerton et al., 1978; Zobell, 1943). It is more recently evident that surfaces of many, if not most, limnetic, estuarine, and marine metazoans are suitable sites for attachment and growth of epibiotic microorganisms. Indeed, Sieburth (1975) listed the ‘‘epibiotic’’ (literally, ‘‘living on’’) environment as one of five major microbial environments in aquatic systems. He noted that ‘‘animal surfaces . . . especially the chitinous skeleton of crustaceans, appear to be nutrient surfaces that encourage bacterial attachment and growth and undergo extensive colonization.’’ The crustacean component of these associations is variously referred to as the substrate organism, the host, or the basibiont (Wahl, 1989). A larger list of descriptive terms is applied to the microorganisms involved, including ectocommensal, ectosymbiont, epicuticular, epifauna, episymbiont, epizoan, epizoic (and the variant spelling epizooic), epizoite, and microflora. While our choice is to use the words epibiont and epibiotic, we consider all these terms to be legitimate. We argue, however, that whatever term is used, it should not suggest a functional relationship (e.g., mutualist, commensal, or parasite) without evidence of such. When we use the term symbiosis, we mean it in the broadest of contexts (i.e., living together). Although the occurrence of microbial epibionts on marine crustaceans and other invertebrates has been r 1997 WILEY-LISS, INC. documented repeatedly, in general, the ecological context and significance of these relationships are not well understood; papers by Fenchel (1965) and Weissman et al. (1993) represent notable exceptions. In this review, we report and summarize various microbial associations with marine crustaceans, especially copepods. We complement, expand, and update an overview of copepod symbionts (Ho and Perkins, 1985). We note differences and commonalities across epibiont and host taxa, consider host-epibiont cycling of nutrients, generate hypotheses relevant to the benefits and costs of the host and epibiont, and offer suggestions for research opportunities. It is useful to delineate what this paper does not encompass. First, we did not consider epibionts other than those on the crustacean exoskeleton. Arguably, microorganisms on the branchial chambers, the gill filaments, or even on the gut lining, chitinized or not, may be considered to be ‘‘epibiotic,’’ as they are outside the animal in a histological sense. Second, we only once appraised epibionts on crustacean eggs, although such associations may be very important with respect to infection sequences and density-dependent regulation of crustacean populations (e.g., Shields, 1994). Third, we chose only to reference, not to incorporate, informative reviews of epibionts on crustaceans grown in aquaculture operations (e.g., Brock and LeaMaster, 1992; Lightner, 1983; Sindermann, 1990). Fourth, we rarely focused on epibionts associated with freshwater crustaceans, as their algal and protozoan epibionts recently have been a focus of research (e.g., Allen et al., 1993; Gaiser and Bachmann, 1994; Threlkeld et al., 1993). While generally not discussing freshwater forms, *Correspondence to: Kevin R. Carman, Department of Zoology and Physiology, Louisiana State University, Baton Rouge, LA 70803-1725. Received 25 March 1995; Accepted in revised form 14 June 1995 MICROBIAL EPIBIONTS ON MARINE CRUSTACEANS we have gleaned considerable ecological insight from freshwater researchers. To continue, we cover algal, protozoan, and bacterial epibionts of marine crustaceans in this review. A fourth major category of microorganisms, the Fungi, presented little to synthesize. First, most information about fungal symbionts is in the aquaculture-disease literature and was outside our purview. Second, nearly all crustacean-associated fungi develop in the host’s cuticle or in its underlying soft tissue; thus, the fungi are not epi- but endobiotic. Third, fungi that might be considered truly epibiotic are found not on crustacean carapaces but are saprophytic on crustacean eggs, a habitat we consider only once. Finally, given that research with pathogenic forms has been emphasized, we know of very little effort to study less benign fungal-crustacean associations. Reviews of fungal diseases of Crustacea (Johnson, 1983; Unestam, 1973) and a recapitulation of their relevant sections (Ho and Perkins, 1985) describe epidemics, some causing profound mortality, in zooplankton. Additional information about mycoses is available in aquacultural reviews listed above. We begin by separately reviewing associations of algae, protozoans, and bacteria on crustaceans. The sections covering algae and protozoans follow a different format from that used for bacteria, reflective of the profound differences in approaches to studying these different microbial taxa. In the final section of the paper, however, we consider commonalities and differences among these several groups of epibionts. STUDY SITES AND METHODS Although much of the information in this review is from the literature, significant amounts of material, including all electron microscopy images, are from our previously unpublished studies. Our specimens were obtained from four sites. Labidocera sp. was collected from surface water at a eutrophic site in the northern Gulf of Mexico off the coast of Louisiana (within the plume of the Mississippi River) (Carman, 1994). Palaemonetes pugio, Coullana sp., and Pseudostenhelia wellsi were collected from saltmarsh mudflats near the Louisiana Universities Marine Consortium facility in Cocodrie, LA. Zausodes arenicolus and Heteropsyllus nunni were collected from an intertidal sandflat near the Florida State University Marine Laboratory (Carman and Thistle, 1985). Robertsonia sp. was collected from shallow (1 m water depth) patch reefs in Kaneohe Bay, Oahu, Hawaii (Caetta and Dobbs, 1990). Specimens were preserved and processed for scanning electron microscopy as detailed by Felgenhauer (1987). In essence, glutaraldehyde-fixed copepods were dehydrated in a graded series of ethanol, critical-pointdried, mounted on stubs, and coated with gold/palladium. Specimens were examined using a variety of scanning electron microscopes (see Acknowledgments); details are available from the authors. For transmission electron microscopy (TEM), specimens were fixed with 4% glutaraldehyde in 0.1 M sodium cacodylate buffer with 0.35 M sucrose, decalcified with EDTA in fixative, rinsed in buffer, postfixed with 1% OsO4, and dehydrated in a graded ethanol series. Specimens were embedded in LX112, and ultrathin sections were double- 117 stained with uranyl acetate and lead citrate and then examined in a Zeiss 10/A electron microscope. ALGAL EPIBIONTS The term algal is used here in a very broad sense and includes photoautotrophic microorganisms that arguably are protozoans or cyanobacteria. Diatoms, however, account overwhelmingly for the reports of microalgae on marine crustaceans. The apparent dominance of diatoms as epibionts, relative to other algal types, may indeed be the case in marine systems, but we suggest that there is a bias favoring the detection and recognition of these comparatively large, geometrically distinct, silicified forms over smaller, less widely recognized organisms. Diatoms on Copepods Hiromi et al. (1985) summarized a variety of reports concerning diatoms epibiotic on marine copepods (e.g., Gibson, 1978, 1979; Hiromi and Takano, 1983; Jurilj, 1957; Russell and Norris, 1971; Simonsen, 1970). Although about twenty species of epibiotic diatoms have been reported, Hiromi et al. (1985) evaluated the considerable taxonomic confusion involved, determined that there was much synonymy, and concluded that only five diatom species, representing the families Protoraphidaceae and Diatomaceae, are significantly involved in episymbiosis with marine copepods. There is some level of host-symbiont specificity; particular diatoms apparently consort only with certain copepod hosts (Hiromi et al., 1985; Prasad et al., 1989). The most widespread and abundant of these diatoms are Pseudohimantidium pacificum, found principally on corycaeid copepods, and Protoraphis atlantica, associated mainly with pontellid and candacid copepods (Hiromi et al., 1985). Although calanoid and cyclopoid copepods have been most commonly reported as having diatom epibionts, harpacticoid copepods, both pelagic (Gibson, 1978, 1979; Hiromi and Takano, 1983) and benthic (Carman, 1989), also serve as substrates. In general, diatoms attach to the copepod’s exoskeleton using stalks or pads (Fig. 1A–D). Both are mucilaginous secretions, with the distinction being in the length of the attachment structure; pads are shorter (Hoagland et al., 1993). In contrast to such attachments, one of us (K.R.C.) has found diatoms having prostrate attachments (Hoagland et al., 1993) to the exoskeleton of Coullana sp., a harpacticoid copepod formerly referred to as Scottolana canadensis in literature from Louisiana studies (Fig. 1E). Morphologies of such diatoms are typical of epipelic forms (Round, 1971). Russell and Norris (1971) found that attachment sites of Pseudohimantidium pacificum varied systematically between male and female copepods, with a much higher abundance of diatoms on the second antennae of males and on the carapace and thoracic segments of females. While males had diatoms on their genital and anal segments, Russell and Norris (1971) considered that ‘‘spermatophores and egg masses in these areas probably interfere(d) with diatom attachment’’ to females. From these assessments, they proposed that diatoms were transferred during ‘‘copulatory or noncopulatory clasping’’ of the copepods. Gibson (1978, 1979) concurred and expanded on the premise that distribution patterns of Pseudohimantidium pacificum 118 K.R. CARMAN AND F.C. DOBBS Fig. 1. Epibiotic diatoms on marine copepods. A: Colony of diatoms near the genital pore of a calanoid copepod, Labidocera sp. Bar 5 50 µm. B: Higher magnification of cell-cell articulation indicated by arrow b in A. Bar 5 1 µm. C: Higher magnification of attachment to copepod surface indicated by arrow c in A. Bar 5 5 µm. D: Stalked diatoms attached in the urosome-mesosome intersegmental region of Labidocera sp. Bar 5 50 µm. E: Naviculoid diatoms with prostrate attachment on the dorsal surface of the urosome of Coullana sp. Bar 5 20 µm. MICROBIAL EPIBIONTS ON MARINE CRUSTACEANS and Protoraphis atlantica on copepods are related to the hosts’ mating behavior. Patterns were more specific on calanoids than on harpacticoids, presumably a result of the different mating habits of these two groups. On the other hand, Hiromi and Takano (1983) and Hiromi et al. (1985) reported the opposite result; distributions of diatoms were more discrete on harpacticoid than on calanoid copepods. There is little information concerning the abundance of diatoms on marine copepods, and the range is great. Russell and Norris (1971) reported seven to nine cells individual21 of Pseudohimantidium pacificum on Corycaeus affinis. Gibson (1979) considered Labidocera aestiva to be ‘‘heavily infested’’ when it harbored .1,000 cells of Protoraphis atlantica individual21. Even this seemingly high value is small compared to a freshwater association in which the cladoceran Daphnia harbored up to 2.3 3 104 diatoms (Synedra cyclopum) individual21 (Allen et al., 1993). Fewer data exist concerning the incidence of epibiotic diatoms on copepods. The only population-level study is that of Russell and Norris (1971), who found up to 55–82% of a population of Corycaeus affinis to have Pseudohimantidium pacificum as epibionts. Diatoms on Other Crustaceans Few reports exist of diatoms epibiotic on marine crustaceans other than copepods. Sieburth (1975) presented scanning electron micrographs of diatoms Licmophora, Cocconeis, and Synedra on the body of an amphipod, Caprella grahamii. McClatchie et al. (1990) found that up to 50–70% of a euphasiid population, Nyctiphanes australis, harbored high abundances of epibiotic diatoms (Licmophora sp.). The researchers’ concern was that the chlorophyll content of the epibionts might introduce a source of error into copepod grazing rates based on gut fluorescence or radioisotopes. Indeed, heavily infested individuals yielded results that overestimated their actual grazing rates by a factor of two to five. Other Algae In their discussion of the diatom, Pseudohimantidium pacificum, living on the copepod, Corycaeus affinis, Russell and Norris (1971) reported a ‘‘brown algal filament’’ and ‘‘isolated brown algal cells’’ as other epibionts. They gave no further information. Møhlenberg and Kaas (1990) reported an infestation of Colacium vesiculosum, a freshwater euglenoid, on four species of calanoids (Acartia sp., Centropages hamatus, Temora longicornis, and Pseudocalanus elongatus). Some copepods had algal cell densities sufficient to register fluorescent pigment values 25 times greater than uninfested individuals. (The above-mentioned concern of McClatchie et al. (1990) with respect to grazing rates certainly would apply in this case!) That a ‘‘freshwater’’ euglenoid was involved was thought to be related to low salinity, high temperature, and large numbers of copepods. Shelton (1974) described a filamentous cyanobacterium, up to 240 µm in length, living on field and laboratory populations of an estuarine shrimp, Crangon crangon. The filaments were located on the shrimps’ chemoreceptor setae, but Shelton tested for and found no evidence of impaired chemoreception. A congeneric 119 but truly marine shrimp, C. allmani, harbored no cyanobacteria. Epibiotic cyanobacteria are common in freshwater crustaceans, but we know of only one other report for their occurrence on marine crustaceans, a gammarid amphipod (Fenchel, 1965). Discussion Although diatoms appear to be the dominant microalgae on marine copepods and other crustaceans, we again caution that a sampling bias may favor their discovery and reporting. In low-salinity waters, reports by Shelton (1974) and Møhlenberg and Kaas (1990) suggest that microalgae other than diatoms may prevail as photoautotrophic epibionts of crustaceans. Previous investigators have considered the ecological significance of marine copepod-diatom associations. The most informative references (Gibson, 1979; Hiromi et al., 1985; Ikeda, 1977; Russell and Norris, 1971) collectively review previous marine literature, observations, and speculations concerning the effects of the epibionts on the copepods’ swimming, respiration, and mating success. In addition, Ikeda (1977) and Gibson (1979) considered the potential nutritional benefits accrued by the epibionts. Here we synthesize these particulars and contribute our own thoughts. These ideas may well be applied to other microbial epibionts, and, indeed, we will subsequently refer back to these points. Given the relatively large size of diatoms, however, we predict that any size-related effects of epibionts on their host will be more pronounced with diatoms than with fungi, protozoans, or bacteria. Klevenhusen (1933, cited in Russell and Norris, 1971) first suggested that epibiotic diatoms might increase the buoyancy of copepods, presumably lessening energy requirements for swimming. While this idea has been echoed repeatedly, we know of no experiments testing the hypothesis. In the opposite sense, Russell and Morris (1971) suggested that diatoms might hinder the action of the copepods’ swimming legs. Gibson (1979) mentioned that it was unknown whether swimming speed was affected by epibionts. Were such the case, however, then the potential for predation on the host certainly would certainly increase, as has been suggested in freshwater studies (e.g., Willey et al., 1990). Furthermore, diatoms may not only negatively affect swimming speed but increase the host’s visibility to predators (see Willey et al., 1993, and references therein). Physiological studies seem well suited to provide insight into short-term, nonlethal ramifications of epibiont loads on copepods. We know of only one study, however, in which diatom-infested, marine copepods were tested. Ikeda (1977) used animals so heavily infested they appeared to be ‘‘covered with rags’’ and found no significant differences in respiration rate or excretion of ammonia and inorganic phosphate relative to noninfested copepods. In a freshwater study, Allen et al. (1993) also found no difference in respiration rates between diatom-infested and diatom-free cladocerans, Daphnia. There was an increased mortality of diatominfested hosts, however, and Allen et al. (1993) considered physiological stress to be the causative factor. We are unaware of any marine copepod-feeding studies in which diatom epibionts have been noted, much less considered as a variable, but if gut-fluorescence meth- 120 K.R. CARMAN AND F.C. DOBBS ods are to be used, investigators should keep in mind the potential for overestimation of grazing rates (McClatchie et al., 1990). Ikeda (1977) suggested that epibiotic diatoms would encounter ‘‘rich nutrients just excreted’’ by their hosts. Without being specific about a mechanism, Gibson (1979) wrote of the benefit of a ‘‘moving host continually replenishing (the diatoms’) nutrient supply.’’ Indeed, we presume that through excretion the host copepod provides to its diatom epibionts a heightened availability of nutrients and dissolved organic matter (DOM). Copepods also release nutrients and DOM into the surrounding water during so-called ‘‘sloppy feeding’’ (Conover, 1966; Dagg, 1974). In moving to areas in which concentrations of its own food are greater, the copepod would transport diatom epibionts to the enriched environment. Finally, the host’s swimming would result in a thinner, hydrodynamic-boundary layer about itself, thus facilitating the diffusion of dissolved constituents from the ambient water to epibiotic diatoms. The idea of nutrient provision to epibiotic diatoms seems to be well grounded conceptually. It is intriguing, therefore, to recall the only pertinent data; Ikeda (1977) found no difference in the excretion of ammonia and inorganic phosphate between copepods heavily infested and not infested with diatoms. If the epibionts were indeed sequestering nutrients excreted by their hosts, then an infestation-related difference presumably would have emerged. With respect to nutrient transfer from diatom to copepod, there is no record of hosts feeding on algae attached to their exoskeleton. Trophic level transfers of much greater importance concern predation on the host-symbiont association. Gibson (1979) did not know ‘‘whether predators . . . make use of the additional energy and nutrient supply afforded by the attached diatoms.’’ In concluding their review article, Hiromi et al. (1985) wondered why ‘‘epizoic diatoms seem to attach exclusively to pelagic copepods.’’ In fact, such diatoms occur on Coullana sp., a harpacticoid copepod with a semiplanktonic lifestyle (Fig. 1E). Therefore, epibiosis is not associated absolutely with pelagic copepods. It may be significant, however, that we have not observed diatoms on Pseudostenhelia nunni, a benthic harpacticoid that cooccurs in Louisiana with Coullana sp. Among several individuals of various harpacticoids (Zausodes arenicolus, Halicyclops coulli, Heteropsyllus nunni, Leptastacus constrictus) from a Florida sandflat, we detected stalked diatoms on only one individual of H. nunni. Furthermore, we have not found epibiotic diatoms on benthic copepods from Hawaii. This general phenomenon may occur simply because diatoms associated with benthic copepods may not be well exposed to light. Indeed, Coullana lives in sediment burrows and makes excursions into the water column only at night (Decho, 1986; Radziejewska, personal communication), as do many other benthic copepods (Walters, 1991). Thus, it is not apparent that epibiotic diatoms on Coullana would gain any advantage in terms of exposure to light. Their presence on the copepod, then, must involve another factor(s), which we presume is related to provision of nutrients from host to epibiont. PROTOZOAN EPIBIONTS Protozoan epibionts of crustaceans appear to be predominantly members of the phylum Ciliophora, of the subclasses Hypostomata, Suctoria, Hymenostomata, Peritricha, and Spirotricha (Corliss, 1979; classification follows that of Corliss, 1979). To a lesser extent, other groups of protozoans reported as epibiotic on crustaceans include foraminiferans, naked rhizopods, and choanoflagellates. Ellobiopsids also have been reported as epibionts, and, given that they have for decades eluded successful phylogenetic classification, we simply follow the lead of earlier researchers (e.g., Johnson, 1983) and include them here. Protozoans on Copepods Among ciliophorans, members of the subclasses Suctoria and Peritricha have been most widely reported as epibionts on marine copepods (Table 1). Their levels of infestation vary from 0–100% of the individuals in the copepod populations under study. In general, however, it seems that low levels of infestation either are unnoticed or unreported. The number of epibionts per copepod ranges over two orders of magnitude: 1–20 (Marshall and Orr, 1955); 25–200 (Herman and Mihursky, 1964); 1–250 (Sherman and Schaner, 1965); 1–2 (Humes and Ho, 1968); 1–50 (Herman et al., 1971); 1–80 (Sieburth et al., 1976); 1–8 (Bowman, 1977); and 0–160 (Weissman et al., 1993). Infestation sometimes is specific for the copepod’s sex (Marshall and Orr, 1955) or developmental stage (Conover, 1956; Marshall and Orr, 1955; Turner et al., 1979); at other times neither factor applies (sex: Herman and Mihursky, 1964; developmental stage: Herman and Mihursky, 1964; Herman et al., 1971; Weissman et al., 1993). Some researchers have concluded that infestation may be more predictably related to the relative sizes of the copepod host and the ciliate epibiont than to other factors (Herman et al., 1971; Sherman and Schaner, 1965; Weissman et al., 1993). Some protozoan epibionts locate themselves specifically on certain parts of the copepod, (e.g., Ophyrodendron sp. on the caudal ramus of Lichomolgus singularipes [Humes and Ho, 1968]). Sieburth et al. (1976) found suctorians ‘‘in a band along the waterline’’ of surface-dwelling pontellid copepods. Other epibionts attach in a more catholic manner (e.g., Weissman et al. (1993) reported Rhabdostyla on all sides of the cephalothorax and on the urosome of Acartia hudsonica). In the three studies that report long-term ($1 year) data, infestation was strongly seasonal (Nagasawa, 1988; Sherman and Schaner, 1965; Weissman et al., 1993). In the latter two studies, the presence of the epibionts was strongly correlated with the seasonal presence of the copepods. Such correlation is consonant with the high level of specificity epibionts exhibit for the host; other cooccurring copepods rarely carry ciliates that heavily infest the principal host (Herman and Mihursky, 1964; Nagasawa, 1988; Sherman and Schaner, 1965; Turner et al., 1979). In addition to the subclasses Suctoria and Peritricha, apostome ciliates (subclass Hypostomata) have long been known to be epibionts on copepods (e.g., Chatton and Lwoff, 1935) and range in their symbiotic relation- 121 MICROBIAL EPIBIONTS ON MARINE CRUSTACEANS TABLE 1. Ciliophorans epibiotic on marine copepods1 Subclass Peritricha Taxon/Taxa Rhabdostyla sp. Epistylus sp. Zoothamnium sp. Zoothamnium alterans Zoothamium sp. Vorticella sp. Myoschiston centropagidarum Cothurnia sp. (recurva?) Vaginicolidae Suctoria Ephelota gemmipara Paracineta sp. Thecacineta inclusa, T. cothurnoides Lecanophrya sp. Hypostomata Dendrosomides lucicutiae Ophryodendron sp. Vampyrophrya pelagica Vampyrophyra pelagica, Gymnodinoides sp. Hymenostomata 1See Uronema sp. Copepod Reference Acartia hudsonica Acartia tonsa Acartia tonsa, A. clausi Acartia tonsa Centropages abdominalis, Acartia omorii Corycaeus affinis Eurytemora affinis, Acartia tonsa Paronychocamptus curticaudatus, Esola typhlops Stenocaris pygmea, Ameira parvula Scottolana, Amphiascus, Typhlamphiascus, Orthopsyllus, Enhydrosoma Pontella scutifer Metridia Stenocaris pygmea, Cylindropsyllus laevis, Evansula incerta, Paraleptastacus espinulatus, Amphiascoides nanus, Heterolaophonte littoralis Parathalestris intermedia, Nitocra spinipes Lucicutia gaussae, L. flavicornis Lichomolgus singularipes Centropages hamatus, C. typicus, Acartia tonsa, A. longiremis, Corycaeus sp., Eurytemora sp., Labidocera aestiva, Eucalanus sp., Paracalanus sp., Oncaea minuta Parathalestris intermedia, Rhynchothalestris rufocincta, Paramphiascopsis longirostris, Pseudonychocamptus proximus, Heterolaophonte minuta Huntemannia jadensis Weissman et al., 1993 Turner et al., 1979 Herman and Mihursky, 1964 Sieburth et al., 1976 Nagasawa, 1986a Russell and Norris, 1971 Hirche, 1974 Hockin, 1984 Herman et al., 1971 Sieburth et al., 1976 Sherman and Schaner, 1965 Hockin, 1984 Hockin, 1984 Bowman, 1977 Humes and Ho, 1968 Grimes and Bradbury, 1992 Hockin, 1984 Hockin, 1984 Sherman and Schaner (1965, their Table 1) for a summary of earlier reports. ship from exuviotrophic ectocommensals to parasites (Bradbury, 1994; Grimes and Bradbury, 1992). Despite their complex life cycles and difficulties in their identification (based upon the morphology of their feeding stage, the vegetative trophont), the apostomes’ interactions with copepods are comparatively well understood in some cases. In particular, histophagous species may be found encysted on appendages of their hosts. Should the copepod be injured or ingested by an invertebrate predator, the apostomes excyst and the trophonts utilize the copepod’s soft tissues for nutrition. Once fed, the apostomes encyst and divide, and the next generation swims forth seeking copepods. The histophage Vampyrophrya pelagica, found on at least seven genera of copepods (Table 1), has been well studied in this regard (Grimes and Bradbury, 1992). Although a second host is not required for Vampyrophyra to complete its life cycle, the second host (a predator of copepods) benefits the apostome either by injuring the copepod and initiating the single-host life cycle or by killing and ingesting the copepod, continuing the two-host life cycle (Grimes and Bradbury, 1992). Hockin (1984) noted that only surface-dwelling or epiphytic benthic copepods were infested with apostomes, not interstitial ones. He considered this point potentially important for the ciliophorans’ completion of their life history via the second host; that is, he considered predation a less likely fate for interstitial copepods. Additional information on this intriguing group of ciliophorans is found in Bradbury (1994). In addition to ciliophoran epibionts, we report here finding choanoflagellates on Coullana sp., a marine harpacticoid copepod cultured by Prof. Darcy Lonsdale (Fig. 2C). It seems reasonable to assume that many other kinds of flagellates are epibiotic on copepods but are unreported as yet. Finally, whether they are protozoans or not, three species of ellobiopsids (genus Ellobiopsis) have been reported from many species of pelagic marine copepods (Table 1 in Shields, 1994). Flagellated spores of Ellobiopsis settle onto setae of their host’s buccal appendages. There they metamorphose into trophomeres and extrude a root-like organelle through the copepod’s cuticle, presumably to absorb nutrients (Shields, 1994). Penetration causes at least localized damage to host exoskeleton and underlying muscle; in addition, infection may reduce fecundity or even cause parasitic castration (Shields, 1994). Typically, only one or two trophomeres mature simultaneously, but each parasite may have a volume up to 7.5% that of its host; Wickstead (1963) hypothesized that the parasites’ nutritional demands might cause the host to starve. Given its location on the host, Ellobiopsis may also utilize food particles not ingested by the copepod. Additional details about Ellobiopsis biology are related by Marshall and Orr (1955), Ho and Perkins (1985), and Shields (1994). Protozoans on Other Crustaceans In their annotated inventory of ciliate epibionts on decapods, Sprague and Couch (1971) listed 23 (possibly 122 K.R. CARMAN AND F.C. DOBBS Fig. 2. Epibiotic protozoa on marine crustaceans. A: Peritrich ciliate Lagenophrys sp. completely covering the surface of a Palaemonetes pugio egg. Bar 5 100 µm. B: Higher magnification of a Lagenophrys sp. individual. Bar 5 20 µm. C: Epibiotic choanoflagellate on Coullana sp. Bar 5 1 µm. 24) genera of epibiotic ciliates distributed among ten families across four orders: Holotrichia, Peritrichia, Spirotrichia, and Suctoria (classification follows that of their paper). This tally does not include protozoans living in the branchial chambers or on the gill filaments of their hosts. The peritrich genus Lagenophyrs comprised an intriguing component of their inventory; its speciation was particularly compelling. Sprague and Couch (1971) listed 52 species within the genus, 22 of which exhibited strong host specificity within the Decapoda. Subsequently, Felgenhauer (1982) reported Lagenophrys as an epibiont on a variety of marine and freshwater decapods belonging to the infraorders Anomura, Brachyura, Astacidea, and Caridea. Depending upon the host species, the ciliates are frequently associated with gill lamellae but may be specifically associated with appendages or generally distributed over the exoskeleton of the decapod. One of us (K.R.C.) has observed Lagenophrys sp. on the eggs of Palaemonetes pugio (Fig. 2A,B). More recently, Camacho and Chinchilla (1989) published a taxonomic key to ciliates epibiotic on natant decapods, listing 13 genera distributed among ten families. In a subsequent paper, they demonstrated that seven of these ciliate genera are not randomly distributed on the freshwater shrimp, Macrobrachium rosenbergii, but are positioned in a manner that maximizes the feeding efficiency exhibited by the different protozoans and partitions the resources made available by the host (Granados and Chinchilla, 1990). Other reports are less diverse taxonomically. Euphausiids harbor populations of ciliophorans (McClatchie et al., 1990, and references therein); in particular, an ecological study of Euphausia superba by RakusaSuszczewski and Filcek (1988) provides much pertinent information. Three species of protozoans that attach to the appendages of the krill were examined over time. The three species exhibited site-specific attachment (or at least site-specific proliferation). More than 99% of the krill population was infested, and the degree of infestation was greater in autumn than in spring. From this and their other studies, the researchers suggested that the species composition of the protozoan epibionts might provide a clue to the geographical origin of the krill population. Jones and Khan (1970) found a ciliate, Conidophrys sp. (cf. pilisuctor), attached to the antennules, antennae, and peraeopods of males and females of the isopod Jaera albifrons. Their literature review indicated that this epibiont also occurs on Corophium, Erichthonius, Microdeutopus, two species of Jassa, two species of Gammarus, and Dexamine (amphipods) as well as on Limnoria and Idotea (isopods). The epibiont’s congener, C. guttipotor, is found on Sphaeroma (isopod). In landmark ecological research, Fenchel (1965) studied 25 ciliophoran species associated with five marine species of the amphipod Gammarus. He showed that the distribution of many epibionts was site-specific on the host and classified the epibionts into four feeding groups (suspension feeders, herbivores, carnivores, and parasites). He demonstrated that peritrichs and chonotrichs formed swarming cells in anticipation of their host’s molting. He judged 18 ciliates to be epibionts specific to the genus, found no ciliates in common MICROBIAL EPIBIONTS ON MARINE CRUSTACEANS between freshwater and saltwater gammarids, and determined that the greatest number of ciliate species was associated with brackish-water populations of the amphipods. Fenchel’s detailed study, published over 30 years ago, presaged some of current ecological interest in these associations (e.g., Fenchel, 1987). Ciliophorans certainly are the dominant protozoans on crustaceans, but other phyla within the Protozoa have been reported as well. Smaldon (1974) found a foraminiferan, Neoconorbina sp., on the carapace of a porcelain crab, Pisidia longicornis. Moore (1985) described foraminiferans, Cibicides lobatulus, on the isopod Astacilla longicornis. Although females were host to more foraminiferans than males, Moore believed that size, hence, length of the intermolt period, was the critical factor in determining the level of infestation. His subsequent examination of museum material revealed another foraminiferan, Miliolinella subrotunda, as well as naked agglutinating rhizopods on other specimens of A. longicornis. Finally, members of the taxonomically enigmatic Ellobiopsidae have been reported as epibionts on euphausiids, amphipods, mysids, carideans, and decapods (e.g., Johnson, 1983; Shields, 1994; Vader, 1973). The range of epibiont-host relationships is great. For example, although the life cycle and attachment sites of Ellobiocystis are thought to be similar to Ellobiopsis (see previous section), the former do not penetrate the body of their hosts, principally shrimps and mysids, and Shields (1994) classifies them as commensals. On the other hand, members of the genus Thalassomyces infect a variety of crustaceans and are overtly parasitic. However, the externally visible symptoms of such infection are trophomeres extruded through the host’s cuticle following internal development of the parasite (Shields, 1994); thus, Thalassomyces is not an epibiont in a strict sense. Discussion While there has been speculation for decades about the potential effects of protozoan epibionts on their hosts (and vice versa), experimental approaches have only recently been attempted. Fenchel’s (1987) statement is telling: ‘‘Colonial peritrichs are very common on planktonic copepods, so much so that they could have a significant ecological impact, but this has never been studied.’’ Since then, the freshwater literature has seen a plethora of pertinent publications. The marine literature, by comparison, is paltry. For example, the May 1993 issue of Limnology and Oceanography contained six articles dealing with algal and protozoan epibionts on zooplankton, only one of which dealt with a marine copepod (Weissman et al., 1993). From the viewpoint of population ecology, the most elegant work on marine copepod-epibiont interactions is that of Weissman et al. (1993), who studied infestation of Acartia hudsonica by the peritrich Rhabdostyla sp. They showed that egg production but not hatching success was negatively correlated with the number of epibionts per copepod. Peritrich-infested nauplii had lower rates of survival, but their developmental rates were not affected. The possible population-level ramifications of these effects are intriguing. Weissman et al. (1993) also demonstrated that live copepods bearing epibionts sank significantly slower 123 than uninfested copepods. Their results are opposite those of Herman and Mihursky (1964), who used preserved copepods and determined sinking rates 100–300 times greater. Weissman et al. (1993) proposed that increased drag associated with ciliate infestation creates a ‘‘parachutelike effect.’’ It is not clear, however, whether sinking rates may be extended to swimming rates (i.e., the speed necessary to escape from a predator). Thus, infestation may or may not be disadvantageous to the copepod host with respect to swimming speed. The increased visibility of a highly infested host, however, may result in increased predation, as has been suggested (Willey et al., 1990) and experimentally demonstrated (Willey et al., 1993) in freshwater studies. Another potentially negative effect of the epibiont on its host was suggested by Turner et al. (1979), who found lesions attractive to bacteria at sites where the peritrich, Epistylis, attached to the copepod, Acartia tonsa. The presence of epibiotic bacteria suggested to them microbial utilization of dissolved organic matter (DOM). They hypothesized that attachment of ciliophorans, and the copepods’ subsequent loss of internal body contents, may influence the seasonal change in populations of estuarine copepods. This idea was echoed by Nagasawa (1986a, 1988), who showed bacteria attached to former attachment sites of protozoan epibionts. Thus, provision of DOM and other nutrients to protozoan epibionts may occur not only via excretion, feeding, and transport phenomena, as suggested earlier for attached diatoms, but also through leakage from a histologically compromised carapace. BACTERIAL EPIBIONTS Our review’s format changes with this section on bacteria. First, very nearly all the literature dealing with these microorganisms as symbionts on crustaceans concerns copepods. Thus, there is no organizational gain in segregating the relatively few reports that cover other crustacean taxa. Second, despite a decrease in size of the individual epibiont, when bacteria are abundant on copepods, their metabolic impact on the surrounding water may be biogeochemically profound. Third, and as a consequence of the second, researchers’ questions and approaches differ slightly to substantially from those applied to algal and protozoan epibionts. Incidence and Abundance Table 2 lists reports of epibiotic bacteria on aquatic crustaceans; most involve microscopical studies of bacterial epibionts. A few (Carli et al., 1993; Davis and Sizemore, 1982; Gil-Turnes et al., 1989; Huq et al., 1984; Sochard et al., 1979) involve the use of cultural techniques. Only two studies deal with freshwater crustaceans (Holland and Hergenrader, 1981; Nagasawa, 1988), reflecting the greater interest shown by marine researchers in copepod-bacterial associations. Nagasawa has been a prolific contributor to the literature and in her review (Nagasawa, 1989) found that the ‘‘incidence of copepods with bacteria’’ (ICWB) from all available reports varied from 0–100%. Nagasawa (1986b) reported that ICWB was unrelated to the abundance of free-living bacteria and suggested that bacterial colonization of copepods may involve the 124 K.R. CARMAN AND F.C. DOBBS TABLE 2. Reports of epibiotic bacteria on crustaceans1 Host Copepods Diaptomus sp., D. nevadensis Acartia clausi A. longiremis, A. tonsa, A. spp. A. omerii, A. plumosa A. clausi Pseudocalanus minutus Calanus cristatus Acartia tonsa Labidocera aestiva Pontellopsis regalis Pleuromamma sp. Centropages furcatus Tigriopus fulvus Copepods Halicyclops coulli, Zausodes arenicolus, Robertsonia sp. Robertsonia sp. Labidocera sp. Coullana sp. Pseudostenhelia wellsi Porcellidium Decapods Various decapods Callinectes sapidus Paleamon macrodactylus Homarus americanus Pagurus longicarpus Planes minutus Carcinus maenus C. irroratus, Lithodes maio, H. americanus Palaemonetes pugio Other crustacea Caprella grahamii (amphipod) Jassa falcata (amphipod) Limnoria tripunctata (isopod) Boeckosimus afinis (amphipod) Limnoria tripunctata, L. lignorum (isopods), and Chelura terebrans (amphipod) L. lignorum Epibiont Pseudomonas, Bacillus, Acinetobacter, Flavobacterium Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Vibrio, Chromobacterium, Pseudomonas, Cytophaga, or Flavobacterium Vibrio, Pseudomonas, Cytophaga, or Flavobacterium Pseudomonas, Vibrio Pseudomonas Vibrio, Pseudomonas V. alginolytus, Aeromonas spp., Pseudomonas spp. V. cholerae, V. parahaemolyticus Bacteria, rods, filamentous Location Appendages, genital opening, joints Joints, legs, depressions Antennule, ventral side, legs, labrum, body Everywhere Depressions, antennules Antennae, mandible Joints, mandible, urosome Reference Holland and Hergenrader, 1981 Nagasawa et al., 1985 Nagasawa, 1986a Nagasawa, 1986b Nagasawa and Terazaki, 1987 Sochard et al., 1979 Egg sac, body Carli et al., 1993 Egg sacs, mouth, body Legs, mouth, joints Huq et al., 1983, 1984 Carman, 1990 Bacteria, rods, filamentous, cocci Legs, mouth, joints, anus Bacteria, rods, filaments; diatoms Bacteria, filaments, rods; diatoms Bacteria, rods Bacteria Legs, mouth, joints, anus Legs, mouth, joints, anus Legs, mouth, joints Pits on dorsal surface of body Caetta and Dobbs (1990); this study This study This study This study Gharagozlou-Van Ginneken and Bouligand, 1975 Leucothrix Antennules, gills, embryos, carapace, eyes, cephalic region, legs, joints Bauer, 1989a Vibrio cholerae, V. vulnificus, V. parahaemolyticus Alteromonas sp. Leucothrix mucor L. mucor L. mucor L. mucor L. mucor Davis and Sizemore, 1982 Embryos Body, appendages Antennae Pleopods Antennae, pleopods Eggs L. mucor Pleopods, eggs Filamentous bacteria Bacteria Bacteria: rods, cocci, stalked None detected Bacteria, rods Body Body Mouth parts, legs, telson Pleopods and pleotelson Sleeter et al., 1978 Atlas et al., 1982 Boyle and Mitchell, 1978 Aeromonas hydrophila, Pseudomonas, Vibrio Pleopods and pleotelson Boyle and Mitchell, 1981 Gil-Turnes et al., 1989 Sieburth, 1975 Johnson et al., 1971 Sieburth, 1975 1See also Nagasawa (1989) for further listings. When only bacteria is listed as the epibiont, the bacterial taxa were not determined. Where bacterial names are given, they are not necessarily comprehensive but represent those species that were identified. Bacterial morphologies, when reported, are also noted. physiological activity or developmental stage of the host. Carli et al. (1993), however, noted that Vibrio alginolyticus abundance on the copepod Tigriopus fulvus was positively correlated to the abundance of V. alginolyticus in the water but did not consider the possible importance of copepod developmental stage. Copepods shed their carapace (and any associated epibionts) at the transition between each naupliar and copepodite stage and do not molt again after adulthood. Thus, bacterial (and other) epibionts are more likely to accumulate on copepods after the terminal molt has occurred. As such, copepod populations consisting primarily of copepodites should contain relatively fewer epibionts than populations consisting primarily of adults. Epibiotic bacteria have been routinely observed on a variety of crustaceans other than copepods, including isopods, amphipods, and decapods (Table 2). These reports, however, have been largely anecdotal. For example, large accumulations of epibiotic bacteria on isopods and amphipods have been reported from studies designed to examine gut bacteria (Boyle and Mitchell, 1978). Atlas et al. (1982) noted that no epibiotic bacteria could be found on the benthic Arctic amphipod Boeckosimus affinis. Intriguingly, however, other cooccurring amphipod species had ‘‘extensive’’ (Atlas et al., 1982) MICROBIAL EPIBIONTS ON MARINE CRUSTACEANS assemblages of epibiotic bacteria. The reason(s) for this dramatic difference in epibiotic bacteria on cooccurring amphipod species is not known. Also listed in Table 2 are the locations on the host at which bacterial epibionts were reported to be most abundant. This compilation of anecdotal information may be of use in future studies. For example, as discussed below, different Vibrio species show different distribution patterns on copepods. In general, many hosts have epibiotic bacteria that accumulate around the anus, in intersegmental regions, and near mouth parts. These bacteria apparently proliferate in areas where excretion or leakage of organic and inorganic nutrients are greatest; presumably they exploit these resources. Other hosts, however, are characterized by more even distributions of bacteria. Perhaps these latter bacteria merely use the host as a generic site for attachment. It would be interesting to understand the basis for variability in epibiotic bacterial distribution on different host species. Although some investigations have failed to detect epibiotic bacteria, we feel that it is difficult to demonstrate the definitive absence of bacteria, especially in light of their uneven and cryptic distribution. As discussed below, these distribution patterns also have implications for determining the abundance of epibiotic bacteria. Presumably because of the technical difficulties associated with their quantification, only a few investigators have even offered an estimate of the abundance of epibiotic bacteria on crustaceans. Nagasawa (1986c) estimated that adult Acartia (planktonic copepods) had 10–105 epibiotic bacteria. Holland and Hergenrader (1981) estimated epibiotic bacterial abundance on freshwater copepods to be approximately 6 3 105 bacteria per copepod. These estimates were based on scanning electron microscopy (SEM) examination of copepods, but neither Holland and Hergenrader (1981) nor Nagasawa (1986c) provided details of how counts were determined. As noted above, epibiotic bacteria on copepods are generally not evenly distributed over the exoskeleton but are concentrated in particular areas (e.g., Boyle and Mitchell, 1978; Carman, 1990), such as between segments (Fig. 3A,B), around mouthparts (Fig. 3C,D), near the anus (Fig. 3E–I), and on legs (Fig. 3J). Epibiotic bacteria are also frequently present in dense clumps (Fig. 3K), which would be virtually impossible to enumerate unless the clumps were somehow dispersed. Thus, the uneven distribution of epibiotic bacteria makes their quantification with SEM extremely problematic. Further, epibiotic bacteria may be cryptic and not detectable by routine SEM examination. For example, TEM examination of copepod sections (Fig. 3L) and SEM examination of copepods that have had legs and mouth parts removed (Fig. 3M,N) show that epibiotic bacteria are frequently hidden from view. Finally, although many epibiotic bacteria appear to adhere strongly to crustacean surfaces, no attempt has been made to determine the number of bacteria lost as the result of preparing specimens for SEM examination. Based on their determination of colony-forming units (CFU), Sochard et al. (1979) concluded that ‘‘many more bacteria were found associated with copepods than were found to be free-living in the water column,’’ but 125 the absolute amounts of bacteria associated with copepods were not specified. It is also not clear how possible biases of the culturing techniques used could have influenced comparisons between abundances of freeliving vs. epibiotic bacteria. Boyle and Mitchell (1981) used acridine-orange direct counts to estimate that approximately 1.4 3 107 epibiotic bacteria per individual were present on the wood-boring isopod Limnoria lignorum. They also noted that plate counts consistently yielded approximately 100 times fewer bacteria (i.e., 105 ) per isopod. Carli et al. (1993) detected as many as 105 CFU associated with the rockpool harpacticoid copepod Tigriopus fulvus but did not attempt to perform direct counts for comparison. If Carli et al.’s (1993) cultural techniques yielded underestimates of bacterial abundance comparable to those reported by Boyle and Mitchell (1981), the copepods would appear to harbor approximately as many epibiotic bacteria as the much larger Limnoria. Host Specificity: Vibrios Members of the bacterial genus Vibrio seem to have a particularly strong affinity for surfaces, especially under nutrient-limiting conditions (Dawson et al., 1981). Indeed, Hood et al. (1984) noted that the preferred habitat of V. cholerae is ‘‘the epibiotic one in which it lives attached to the substrate.’’ The evidence cited below indicates that vibrios are especially fond of crustacean surfaces. Vibrio harveyi, for example, possesses specific proteins for attachment to chitin (Montgomery and Kirchman, 1993, 1994), but the generality of this attachment mechanism has not been determined. Vibrios have also been shown to possess chitinolytic enzymes and thus may be able to use crustacean exoskeletons as a growth substrate (Bassler et al., 1991). Bacterial degradation of living crustacean exoskeletons has been demonstrated for copepods (Nagasawa, 1987) (Fig. 4), but the identify of these chitindegrading bacteria has not been determined. Bacterial degradation of decapod exoskeleton (e.g., ‘‘shell disease’’) is a commonly associated with various Vibrio spp., especially among animals reared in aquaculture facilities (Sindermann, 1990). Montgomery and Kirchman (1993) demonstrated that enzymes involved in chitin degradation are distinct from proteins involved in attachment to chitin. Even though certain bacteria such as vibrios show an affinity for surfaces, there is little evidence that specific relationships exist between crustacean hosts and their bacterial epibionts. The available indications, however, suggest that the epibiotic bacterial assemblage associated with zooplankton is taxonomically distinct from free-living bacteria. Sochard et al. (1979) used culture techniques to characterize and quantify the bacterial assemblage associated with Acartia tonsa. They found a diverse assemblage of bacteria associated with healthy copepods. These bacteria were primarily associated with external surfaces of copepods and were dominated by members of the genus Vibrio. A. tonsa collected from an estuarine environment (Chesapeake Bay) had a higher incidence of vibrios than either pelagic or laboratory-reared A. tonsa. Huq et al. (1983, 1984), also using culture techniques, found that V. cholerae and V. parahaemolyticus preferentially attach to planktonic copepods relative to other 126 K.R. CARMAN AND F.C. DOBBS Fig. 3A–F. Epibiotic bacteria on marine crustaceans. A: Colony of rod-shaped bacteria with prostate attachment on dorsal mesosome of Pseudostenhelia wellsi (harpacticoid copepod). Note plaque that has formed at intersegmental articulation. Bar 5 20 µm. B: Dense growth of rod-shaped bacteria around the pereopod articulation with the body of a planktonic amphipod. Bar 5 50 µm. C: Ventral view of Zausodes arenicolus (harpacticoid copepod) showing feeding appendages. Bar 5 20 µm. D: Magnified view of region indicated by arrow in C showing colony of rod-shaped bacteria with polar attachment. Bar 5 1 µm. E–I: Epibiotic bacteria associated with the caudal region of copepods. E: Filamentous bacteria on the terminal urosome segment on Robertsonia sp. (harpacticoid). Bar 5 10 µm. F: Higher magnification of filaments shown in E. Note that rod-shaped bacteria with a polar attachment have densely colonized the surface of the filamentous bacteria. Bar 5 1 µm. MICROBIAL EPIBIONTS ON MARINE CRUSTACEANS Fig. 3G–L. G: Caudal rami of Labidocera sp. (calanoid). Bar 5 100 µm. H: Magnified view of region designated by the arrow in G; a colony of rod-shaped bacteria with polar attachment is seen. Bar 5 10 µm. I: Rod-shaped bacteria with prostate attachment on the caudal rami of Robertsonia sp. Bar 5 10 µm. J: Rod-shaped bacteria with polar attachment on the P1 exopod of Zausodes arenicolus. Bar 5 1 µm. 127 K: Rod-shaped bacteria with prostate attachment growing on ventral surface of planktonic amphipod. In the upper left corner of the panel, a large clump of bacteria is attached to the amphipod surface. Bar 5 50 µm. L: TEM image of bacteria attached to cryptic regions around the mouth of Coullana sp. Arrows point to bracketed regions where bacteria were detected. Bar 5 10 µm. 128 K.R. CARMAN AND F.C. DOBBS Fig. 3M,N. M: Filamentous bacteria growing cryptically around the mouth region of Labidocera sp. These filaments were detected only after feeding appendages were dissected from the copepod. Bar 5 10 µm. N: A higher magnification showing cryptic filamentous bacteria attached to copepod exoskeleton. Bar 5 5 µm. Fig. 4. Bacterial degradation of copepod exoskeleton. Regions resembling craters are associated with the site of the polar attachment of these rod-shaped bacteria on Labidocera sp. Bar 5 1 µm. bacteria (e.g., E. coli and Pseudomonas sp.) and suggested that copepods serve as a reservoir for pathogenic species such as V. cholerae O1. This relationship led Huq et al. (1984) to suggest that there was a ‘‘correlation between incidence of planktonic copepods and the distribution of V. cholerae.’’ They also observed in laboratory studies that the presence of live copepods stimulated V. cholerae to grow from densities of 104 ml21 to 108 ml21; when bacteria were incubated with dead instead of live copepods, bacterial abundance increased to only 106 ml21. Live (or dead) copepods did not stimulate similar growth by other bacterial species tested (V. parahaemolyticus or Pseudomonas sp.). Further, Huq et al. (1984) showed that V. cholerae readily attached to copepods and that the attached cells were particularly concentrated in the oral region and on egg sacs of copepods. Very little attachment to copepods was observed for E. coli or Pseudomonas sp. V. parahaemolyticus attached to copepods but was distributed evenly over copepod surfaces and not concentrated in particu- lar areas. It is not known if the lateral attachment of V. parahaemolyticus vs. the polar attachment of V. cholerae (Belas and Colwell, 1982) influences a differential spatial distribution on copepods. Huq et al. (1984) suggested that association of V. cholerae with the oral region and eggs of copepods could both lead to effective means of dispersal; eggs released by copepods would carry with them an inoculum for copepod nauplii when they hatch, and bacteria associated with the oral region could be ingested during normal feeding and dispersed in fecal pellets. Given the apparent strong affinity of V. cholerae for copepods, Huq et al. (1984) suggested that seasonal variability in V. cholerae abundance may be closely linked with variability in the abundance of copepods. The different distributions of V. parahaemolyticus and V. cholerae on copepods observed by Huq et al. (1984) may also reflect different nutritional requirements. If V. cholerae is stimulated by copepod exudates, then living around the oral region would provide an optimal proximity to material released from ‘‘sloppy feeding’’ (see discussion of algal epibionts) as well as to excretions of liquid waste through the maxillary pores. V. parahaemolyticus, which is apparently not stimulated by copepod exudates, may simply use copepods as a surface for attachment, and thus their attachment sites are nonspecific. It would also be interesting to contrast these Vibrio species in terms of their ability to degrade chitin. If V. cholerae derives its nutrition from living copepods, one might predict that it would attach to copepod surfaces but not degrade the exoskeleton. V. parahaemolyticus, which does not seem to be stimulated by copepod exudates, may exploit the nutrients available in the copepod exoskeleton. Kaneko and Colwell (1973, 1975) presented evidence that V. parahaemolyticus overwintered in sediment and then attached to the exterior surfaces of zooplankton where they proliferated. They noted that more than 80% of the environmental isolates of V. parahaemolyticus were from zooplankton dominated by copepods. Huq et al. (1990) used fluorescent antibodies (FA) to detect V. cholerae O1 in Bangladesh waters. V. cholerae MICROBIAL EPIBIONTS ON MARINE CRUSTACEANS O1 were detected in 64% of plankton samples, whereas cultural methods led to the detection of V. cholerae O1 in only 0.3% of plankton samples. V. cholerae O1 was detected on zooplankton with FA, and its occurrence was ‘‘most extensive’’ (Huq et al., 1990) on copepods. These observations compelled Huq et al. (1990) to suggest that V. cholerae attach to plankton to withstand environmental shifts in temperature, salinity, or nutrients and during this period were nonculturable and thus undetectable by conventional microbiological techniques. Dietrich et al. (1984) also speculated that V. cholerae may attach to the surface of the blue crab Callinectes sapidus as a survival mechanism. Carli et al. (1993) examined the relationship between the copepod Tigriopus fulvus and Vibrio alginolyticus in tide pools. Using cultural techniques for quantification, they noted a strong affinity of V. alginolyticus for T. fulvus in general and for egg sacs in particular. The abundance of V. alginolyticus on T. fulvus, however, appeared to be a direct response to the abundance of V. alginolyticus in water, and thus the relationship appears to be opportunistic. Boyle and Mitchell (1981) also detected V. alginolyticus (and other Vibrio spp.) on the wood-boring isopod Limnoria lignorum. Thus, there is a substantial body of literature that links the ecology of various Vibrio spp. with that of crustaceans in general and copepods in particular. Much of this evidence is, however, strictly correlative or inferred from laboratory studies. Some field observations, however, do not support the hypothesis that the ecology of all vibrios is closely linked to zooplankton (or other crustaceans). Venkateswaran et al. (1989) serotyped environmental isolates from Japanese coastal waters and detected no seasonal relationship between V. cholerae and zooplankton. Further, although Tamplin et al. (1990) found that V. cholerae preferentially attached to live vs. dead copepods in the laboratory, they were unable to detect V. cholerae associated with zooplankton collected from Bangladesh waters. Clearly, more work is needed to understand the significance of species-specific relationships between bacteria and crustaceans. Host Specificity: Nonvibrios The rationale for studying Vibrio clearly has its basis in public-health microbiology. From the standpoint of pure microbial ecology, it would be good to understand the association of other bacterial types with their crustaceans hosts. For example, micrometer-scale diversity along the spermatophore of a harpacticoid copepod is illustrated in Figure 5A–D, in which a variety of bacterial morphologies is distributed along a gradient extending approximately 90 µm. How many bacterial species are include in this assemblage? How are the species interacting with one another (i.e., do they compete for limiting resources)? What is the level of host specificity, if any? In one of the few studies of epibiotic bacteria not focused on pathogenic vibrios, Boyle and Mitchell (1981) found that the most abundant bacterium living on the exoskeleton of Limnoria lignorum was Aeromonas hydrophila, which is, intriguingly, another known pathogen to humans and other vertebrates. Venkateswaran et al. (1989) noted that several serotypes of Salmonella sp. appeared to be found exclusively on zooplankton in the coastal waters of Japan. 129 The occurrence of the filamentous bacterium Leucothrix mucor is reported to be ‘‘widespread on benthic crustacea’’ (Johnson et al., 1971). L. mucor can also be found on algae, other invertebrates, and nonorganismal surfaces (Sieburth, 1975). Thus, while L. mucor may have an affinity for crustacean surfaces, its occurrence is certainly not limited to crustaceans. Size of Epibiotic Bacteria Bacteria attached to particles or aggregates such as marine snow tend to be larger than free-living bacteria (e.g., Iriberri et al., 1987; Simon, 1987). Alldredge and Gotschalk (1990) found that the average bacterial cell volume on various types of marine snow ranged from 0.11–0.21 µm3, which was greater than that of freeliving bacteria by a factor of 1.1–1.9. Similarly, Iriberri et al. (1987) found that attached bacteria (0.102 µm3 cell21 ) were about twice as large as free-living bacteria (0.047 µm3 cell21 ). The volumes of epibiotic bacteria on copepods examined in this study were determined from SEM micrographs. Rods on benthic copepods (0.177 6 0.124 µm3 cell21; mean 6 1 s.d.) were larger than those on planktonic copepods (Labidocera sp.; 0.125 6 0.069 µm3 cell21 ) but not significantly so. Cell biovolumes of filamentous epibiotic bacteria were fiveto tenfold higher than those of rod-shaped epibiotic bacteria, and filamentous bacteria on benthic copepods (1.189 6 0.432 µm3 cell21 ) were significantly larger than those on Labidocera sp. (0.501 6 0.167 µm3 cell21 ). Overall, the biovolumes of epibiotic bacteria (this study) are considerably larger than biovolumes of freeliving or attached bacteria (e.g., Alldredge and Gotschalk, 1990; Iriberri et al., 1987). The differences in size may in fact be somewhat underestimated, as Fuhrman (1981) suggested that cell volumes determined with SEM are significantly less than those determined using acridine-orange staining (the method commonly used in the above-cited references). Larger bacteria are generally considered to be an indication of nutritionally favorable conditions (Morita, 1982). Our data indicate that epibiotic bacteria are considerably larger than free-living bacteria or bacteria associated with nonliving aggregates. As discussed below, we hypothesize that the larger size of these bacteria is a consequence of the nutritionally favorable conditions that they experience. Metabolic Relationships There is at least anecdotal evidence that epibiotic bacteria associated with crustaceans are metabolically quite active (Anderson and Stephens, 1969; Carman, 1989, 1990). Enhanced activity by epibiotic bacteria has also been reported for bacteria associated with the external surfaces of hydrothermal-vent invertebrates (de Angelis et al., 1991). Boyle and Mitchell (1981) suggested that epibiotic bacteria on Limnoria could exploit organic carbon and inorganic nitrogen released by the isopods. As noted above, planktonic bacteria attached to nonliving particles are frequently larger and/or have higher metabolic rates than free-living bacteria. The contribution of attached bacteria to overall bacterial production, activity, or biomass, however, is highly variable (range 0–100%; see literature review by Iriberri et al. ). In a broad sense, zooplankton could be considered as a form of ‘‘suspended (non-sinking) particulate material,’’ 130 K.R. CARMAN AND F.C. DOBBS Fig. 5. Diversity of epibiotic bacteria. A: A gradient of bacterial morphologies is found associated with a spermatophore attached to a female Robertsonia sp. Bar 5 10 µm. B: At one end of the spermatophore (designated by arrow b in A), large cocci predominate. Bar 5 5 µm. C: Small filamentous forms become relatively more abundant in the region designated by arrow c in A. Bar 5 5 µm. D: At the opposite end (arrow d in A), the small filamentous forms are numerically dominant, and a colony of large filaments is present. Bar 5 5 µm. Karl et al. (1988) with which a large fraction of bacterial activity may be associated. Many water-column procedures for studying microbial activity, however, include pre- or postscreening through mesh to eliminate larger animals (e.g., Chin-Leo and Kirchman, 1990; Karl et al., 1988; Kirchman and Mitchell, 1982; Simon, 1987). Prescreening would remove from consideration any effects of epibiotic bacteria, and postscreening would sequester those effects with the trapped invertebrates. In any case, explicit investigations of activity by bacteria associated with the surfaces of marine invertebrates are lacking. Jumars et al. (1989) proposed that byproducts of grazing are the principal mechanisms by which dissolved organic carbon (DOC) is transferred to bacteria, and many authors have noted that bacterial activity and production is enhanced in the presence of copepods (e.g., Carman, 1994; Copping and Lorenzen, 1980; Eppley et al., 1981; Mopper and Lindroth, 1982; Peduzzi and Herndl, 1992). In addition to DOC, crustaceans excrete large and relatively constant quantities of NH41 (e.g., Gardner and Paffenhöfer, 1982) that may provide an important source of N for marine bacteria (Wheeler and Kirchman, 1986). As an alternative strategy to their association with fecal pellets, bacteria might better exploit nutrients liberated by zooplankton by maintaining themselves in close spatial proximity to the grazer. As opposed to the ephemeral and finite resources available from fecal pellets, grazers would represent a continuous source (i.e., a ‘‘sustained gradient’’ [Azam and Ammerman, 1984]) of nutrition for bacteria that would persist for many bacterial generation times. Relatively fast swimming speeds of zooplankton, however, would prevent motile bacteria from efficiently exploiting microzones of enhanced nutrients that surround zooplankton (Jackson, 1987; Purcell, 1977). Alternatively, bacteria could exploit zooplankton exudates by becoming physically attached to grazers. The distribution of bacteria on both planktonic and benthic copepods is consistent with this hypothesis. Bacteria are typically abundant in the anal region (Fig. 3E–I) and around mouth parts (Fig. 3C,D) and intersegmental areas (Fig. 3A) where release of nutrients should be highest. Carman (1994) has determined that, at an abundance of 0.1 copepod ml21, epibiotic bacteria on Labidocera sp. account for approximately 20% of the total bacterial activity in surface-water samples from the MICROBIAL EPIBIONTS ON MARINE CRUSTACEANS Gulf of Mexico. Further, Carman concluded that liberation of amino acids and NH41 by Labidocera significantly stimulated activity by free-living bacteria. Collectively, the available information indicates that bacteria-crustacean interactions could be significant to the ‘‘microbial loop’’ and biogeochemical cycles in general. These relationships undoubtedly vary spatially and temporally in association with events such as phytoplankton blooms and zooplankton (e.g., euphasiids) swarms. Effects on Substrate Organism The evolutionary significance of epibiotic microbes is well illustrated by the fact that many decapod (Bauer, 1989a) and stomatopod (Bauer, 1989b) crustaceans have sophisticated appendages and grooming behaviors designed specifically for removing such epibionts. Bauer (1989a) provided an excellent review of this topic, noting that grooming behavior and well-developed grooming appendages are essentially limited to natant decapods, on which macroscopic fouling is rare. Significant accumulations of epibiotic microbes could severely hinder swimming by natant decapods. Reptant decapods, such as lobsters and brachyuran crabs, do not have specialized grooming appendages and are frequently observed to have macroscopic fouling by filamentous bacteria such as Leucothrix. Grooming behavior among smaller crustaceans, such as copepods, has not been reported, and they do not appear to have the specialized appendages necessary for this behavior. Although planktonic copepods are much smaller than natant decapods (and therefore exposed to a hydrodynamic regime dominated by viscous forces), heavy fouling from filamentous organisms would also increase drag and potentially be very detrimental. It is interesting to note that filamentous epibiotic bacteria have not been previously reported on planktonic copepods from freshwater or marine environments (Table 1). We have observed, however, high abundances of filamentous bacteria on the planktonic copepod Labidocera sp. (Fig. 3M) but only at the bases of its feeding appendages and legs, where they presumably would contribute little to the surface drag of the animal. Conversely, epibiotic filamentous bacteria are relatively common among benthic copepods living in siliceous sand (Fig. 6A), carbonate sand (Figs. 3E, 6B), or mud (Fig. 6C,D). Indeed, one of us has observed 389 out of 777 (50%) adult female Coullana sp. fouled with epibiotic filamentous bacteria detectable under low magnification (3100, light microscopy) (Carman, unpublished) (see Fig. 6C). Conversely, filamentous bacteria were never detected with light microscopy on a cooccurring species, Pseudostenhelia wellsi. P. wellsi was, however, found to be commonly colonized with nonfilamentous bacteria (Fig. 3A). We hypothesize that, like decapods, filamentous bacteria are less detrimental to benthic copepods than to planktonic forms. The above argument notwithstanding, the influence of epibiotic bacteria on copepods is not well understood. Holland and Hergenrader (1981) suggested that a mutualistic relationship occurred between freshwater calanoid copepods and their bacterial epibionts. They proposed that copepod exoskeletons provide a nutrientrich substrate upon which epibiotic bacteria can flourish; copepods, in turn, may benefit from bacterial degradation of organic material that might otherwise 131 accumulate on copepod surfaces. Nagasawa (1987) observed pits and scars on copepod exoskeletons that were clearly the result of activity by chitinoclastic epibiotic bacteria and thus proposed that epibiotic bacteria were parasitic on marine calanoid copepods (Acartia spp.). We have also observed evidence of chitinoclastic epibiotic bacteria on the planktonic copepod Labidocera sp. (Fig. 4). Other negative effects, such as interference with mobility or gas exchange (Bauer, 1989b) could also accrue from epibiotic bacteria. Hypotheses of mutualistic or parasitic relationships between copepods and epibiotic bacteria have not been explicitly tested. There are no data from the literature to assess Holland and Hergenrader’s (1981) hypothesis of mutualism. With regards to parasitism, bacterial degradation of copepod exoskeleton would clearly be detrimental to the host (Nagasawa, 1987), but chitinoclastic activity by epibiotic bacteria on copepods appears to be the exception rather than the rule. For example, in only one of Nagasawa’s several papers concerning epibiotic bacteria on copepods (Nagasawa, 1987) did she note the presence of exoskeletal scars from chitinoclastic bacteria. We have examined hundreds of benthic and planktonic copepods, as well as various other crustacean taxa, and have found evidence of bacterial degradation of the exoskeleton only once (Fig. 4). Thus, we hypothesize that epibiotic bacteria are not, as a rule, chitinoclastic. If most epibiotic bacteria benefit from the exploitation of nutrients liberated by living copepods (see Metabolic Relationships above), it would be adaptive for bacteria not to diminish the longevity of their host by degrading their exoskeleton. This consideration would be especially important for the copepod-epibiont relationship since copepods have a terminal molt and cannot continuously replace a damaged exoskeleton. SUGGESTIONS FOR FUTURE RESEARCH The study of epibiont-host relationships in aquatic environments is still in its infancy. The population, community, and biogeochemical implications of epibionthost interactions remain almost entirely unresolved in marine environments and only slightly less so in freshwater regimes. When these relationships are studied, several fundamental points of biology must be first understood, including the epibionts’ species composition, abundance, life histories, attachment mechanisms, and morphologies. There is a spectrum of functionality among the microbial epibionts of marine crustaceans. Some protozoans and diatoms (and bacteria?) use the carapace only as a substrate, without evidence of negative or positive interaction; we consider such a relationship to be commensalism. On the other hand, however, some decapod larvae rely on specific bacteria to prevent fouling by pathogenic fungi (Gil-Turnes and Fenical, 1992; Gil-Turnes et al., 1989). How do decapods attract and maintain the helpful bacteria? They must somehow provide conditions that facilitate the growth of antifungal bacteria while largely excluding others. In these cases there may be a mutualistic relationship between the decapods and bacteria; the benefit accrued to the bacteria, however, has not been demonstrated. The nature of other functional relationships is less clear. As noted previously, we see little evidence that epibiotic bacteria visibly degrade copepod exoskeletons. Nevertheless, it seems quite possible that epibionts in 132 K.R. CARMAN AND F.C. DOBBS Fig. 6. Epibiotic filamentous bacteria on benthic (harpacticoid) copepods. A: Filamentous bacteria on the caudal region of Heteropsyllus nunni. Bar 5 10 µm. B: Filament of cocci attached to Robertsonia sp. Bar 5 1 µm. C: Low magnification view of Coullana sp. dorsal urosome. Thread-like structures are long bacterial filaments. Bar 5 100 µm. D: Higher magnification of bacterial filaments in C. Bar 5 20 µm. general may have some negative effects on their hosts, as has been demonstrated for some freshwater taxa (e.g., Threlkeld et al., 1993). At what point do commensal relationships become parasitic, or at least detrimental (e.g., amensalistic)? Perhaps it is a matter of degree, in which relatively few epibionts do not significantly influence the substrate organism, but larger epibiont loads are deleterious. In such cases, how do organisms unable to groom themselves and having a terminal molt (e.g., copepods) limit their abundance of epibiotic associates? Willey and Threlkeld (1995) reported that protozoan epibiont load is correlated with intermolt duration in freshwater cladocerans, with longer intermolt periods resulting in greater accumulations of epibionts. This phenomenon has not been examined in the marine environment. The relationship between the abundance of bacterial epibionts and crustacean intermolt periods has not been examined in any aquatic environment. The possibility of coevolved, species-specific relationships between epibionts and hosts should be considered (e.g., Fenchel, 1965; Chiavelli et al., 1993). Perhaps the best examples are the apostome protozoans, whose life cycles are very closely associated with those of their hosts. This symbiosis is widespread and easily manipulated in the laboratory (e.g., Grimes and Bradbury, 1992), making it a good model for testing hypotheses. With regard to bacteria, the available evidence suggests that while obligate epibiont-host relationships may be rare, the epibiont bacterial community may nevertheless be distinct from free-living bacteria. Researchers also should be sensitive to spatial and temporal variability in epibiont-host relationships, as variability in host availability may strongly influence population dynamics of epibionts. Again, seasonality and the epibiont dynamics of an entire community have been considered in freshwater systems (Chiavelli et al., 1993) but are virtually unstudied in the marine environment. As a starting point, for example, various pathogenic Vibrio bacteria are known to be highly seasonal in occurrence and to prefer an epibiotic existence on crustaceans. Does the crustacean-bacterial symbiosis function as a vector for potential infection? Are seasonal fluctuations in Vibrio as well as other bacterial and nonbacterial epibionts associated with fluctuations in the availability of zooplankton in general? Are some MICROBIAL EPIBIONTS ON MARINE CRUSTACEANS zooplankton species a ‘‘preferred’’ substrate over others? Do epibionts switch between planktonic and benthic hosts? Molecular techniques (e.g., DeLong et al., 1993) could provide a useful way of determining the unique character and seasonal variability of epibiotic bacterial communities associated with crustaceans. Scientists working in freshwater systems have made significant progress toward understanding the ecological implications of algal and protozoan epibionts as they relate to interactions of the host with its predators. These population- and community-level questions remain largely unexamined in marine systems. In addition to a focus on predation, other approaches, such as physiological studies of hosts and epibionts, will provide information applicable to higher levels of organization. Collectively, knowledge of positive effects and negative consequences—to both epibionts and hosts— may be used to construct predictive, cost-benefit models for populations and communities. Finally, the biogeochemical implications of epibiont associations with copepods and other crustaceans deserve further study. Do epibionts consume disproportionately large amounts of nutrients (organic and inorganic) as they are liberated by their host through various processes? Are there certain environmental conditions that favor existence as an epibiont vs. a free-living microorganism? Do epibionts exhibit physiological traits that allow them to more effectively exploit nutrients made available by the substrate organism (i.e., are they copiotrophs)? Our suggestions for future research on microbial epibionts are by no means exhaustive. We are, however, convinced that epibionts directly or indirectly have a significant influence on aquatic communities, and their study should provide the basis of fruitful and stimulating research. NOTE ADDED IN PROOF Since this manuscript’s acceptance in June 1995, the following articles that are relevant to the topics discussed in this paper have been published. Additional References: Becker, K. (1996) Epibionts on carapaces of some malacostracans from the Gulf of Thailand. J. Crust. Biol., 16:92–104. Dumontet, S., Krovacek, K., Baloda, S.B., Grottoli, R., Pasquale, V., and Vanucci, S. (1996) Ecological relationship between Aeromonas and Vibrio spp. and planktonic copepods in the coastal marine environment in southern Italy. Comp. Immunol. Microbiol. Infect. Dis., 19:245–254. Hansen, B., and Bech, G. (1996) Bacteria associated with a marine planktonic copepod in culture. I. Bacterial genera in seawater, body surface, intestines and fecal pellets and succession during fecal pellet degradation. J. Plankton Res., 18:257–273. Huq, A., Xu, B., Chowdhury, M.A.R., Islam, M.S., Montilla, R., and Colwell, R.R. (1996) A simple filtration method to remove planktonassociated Vibrio cholerae in raw water supplies in developing countries. Appl. Environ. Microbiol., 62:2508–2512. Kirchner, M. (1995) Microbial colonization of copepod body surfaces and chitin degradation in the sea. Helgol. Meeresunters., 49:201– 212. Morado, J.F., and Small, E.B. (1995) Ciliate parasites and related diseases of Crustacea: A review. Rev. Fish. Sci., 4:275–354. Pruzzo, C., Crippa, A., Bertone, S. Pane, L., and Carli, A. (1996) Attachment of Vibrio alginolyticus to chitin mediated by chitinbinding proteins. Microbiology, 142:2181–2186. Svavarsson, J. and Davı́dsdóttir, B. (1995) Cibicides spp. (Protozoa, Foraminifera) as epizoites on the Arctic antenna-brooding Arcturus baffini (Crustacea, Isopoda, Valvifera). Polar Biol., 15:569–574. 133 ACKNOWLEDGMENTS We are grateful to the following for their contributions to the images in this manuscript: Donald McGee and James Cowen (University of Hawaii’s Analytical EM Facility), Tina Carvalho and Marilyn Dunlap (University of Hawaii’s Biological EM Facility), Ron Bouchard and Sharon Matthews (Louisiana State University’s Life Sciences EM Facility), Sandy Silver and Kim Riddle (Florida State University’s EM Facility), Elizabeth Caetta, and Darcy Lonsdale. We appreciate critical comments on an earlier draft of this manuscript by Lisa Drake, Evelyn Gaiser, Barbara Grimes, Darcy Lonsdale, Michael Montgomery, Stephen Threlkeld, and Ruth Willey. This work was supported in part by National Science Foundation grants OCE-90-18599 (Dobbs) and OCE-90-12540 (Lonsdale and Dobbs), a research and training revolving fund award from the University of Hawaii (Dobbs), and a grant from the Louisiana Education Quality Support Fund 1993-95RD-A-03 (Carman). 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