Pectinatella magnifica (Leidy, 1851) is a colonial fresh-water organism from phylum Bryozoa , recently invasive in many areas in Europe and other parts of the world
A colony of P. magnifica is formed by a layer of zooids, living on a self-produced jelly blob ranging in weight from a few grams to 10 s of kilograms. Similar to other bryozoans, P. magnifica is a filter feeders. They feed mainly on micro plankton and detritus . These organisms reproduce, hibernate, and spread through asexual particles, statoblasts. P. magnifica is native to the area east of the Mississippi River, from Ontario to Florida. Its first occurrence recorded outside North America was in Western Europe, in Bille River near Hamburg in 1883 (e.g., ). During the 20th century, this species gradually spread across the Elbe river into Germany, Czech Republic, and Poland [4,5]. In France, it was recorded occurring in the area called Franche-Comte in 1994 [5,6]. At present, it occurs also in the Netherlands (its occurrence in the Netherlands was first reported in 2003), in the Rhine basin in the area between Luxembourg and Germany, in Austria, Romania and Turkey , Hungary , and on the island of Corsica . The newest records of presence are published in Japan and the Korean peninsula [10,11].
The spread in slowly flowing streams is certainly significantly conditioned by the water course . Other possible modes could be spreading thanks to zoochory (statoblasts) on feathers of water birds , unharmed statoblasts in the content of stomach in some fish species or water birds . Important for spreading can be human activities . The view of Borg  is exceptional in that it does not exclude its cosmopolitan origin.
In related marine bryozoans, the specific bioactive compounds, bryostatins, were identified . They primarily have an anticancer effect [16,17]. Bryostatins belong to the class of alkaloids ; furthermore, some isoquinolines, sterols, and some carbohydrates with a heteroatom in structure (nitrophenols or disulfides) were also found in bryozoans. Some of them possess the antibacterial and/or cytotoxic activity [18,19,20]. Except that prevents cell division, some of these metabolites have caused dermatic allergy and have shown antihelmintic activity . Bryostatins are considered to be important promising pharmaceutical substances .
Microbial symbionts (e.g., bacteria, cyanobacteria, algae) of bryozoans represent a significant source of potential bioactive compounds [22,23]. For example, bryostatins are produced by the bacterial symbiont Candidatus Endobugula sertula, which is present in all life stages of bryozoan Bugula neritina . Also, the antimicrobial activity of extracts from marine and freshwater bryozoans including Pectinatella magnifica have been demonstrated [18,25,26,27].
It is assumed that the biomass of P. magnifica could contain biologically active substances. Therefore, it is important to study this issue, as well as the composition, the quantity, and activity of microbiota of bryozoan colonies. The main aim of our work was to evaluate in vitro toxicity (Section 2.2) and antimicrobial activity of various extracts prepared from P. magnifica (Section 2.3). Further, we analyzed the elementary composition of lyophilized P. magnifica gel (Section 2.1) and determined toxins of cyanobacteria related to occurrence of P. magnifica (Section 2.4).
2.1. Elemental Analysis of P. magnifica Gel
The P. magnifica sample for CHN elemental analysis was obtained from a collection of colonies on the pond “Hejtman” in 2014. The gel was mechanically separated from zooids and lyophilized. Elementary analysis showed the composition as 40.0% C, 6.4% H, and 8.7% N.
2.2. Cytotoxicity of Extracts
At all five tested extracts, the cytotoxicity was evaluated as a Relative cytotoxicity (Figure 1), relative to control values (vehicle treated groups). Treatment with P. magnifica extracts led to significant toxic effects according to  (Table 1) on THP-1 cells, as LD50 values were assessed to be <1000 μg/mL. Toxicity expressed as LD50 derived from a dose-response curve of the following P. magnifica extracts increased as follows: PM5 (aqueous portion, 250 μg/mL) > PM2 (hexane portion, 75 μg/mL) > PM3 (chloroform portion, 40 μg/mL) > PM4 (ethyl acetate portion, 31 μg/mL) > PM1 (methanolic extract, 29 μg/mL).
2.3. Antimicrobial Activity of Extracts
Determined MICs of P. magnifica extract are showed in Table 2. Only methanolic extract (PM1), hexane (PM2), and chloroform portions (PM3) possessed antibacterial effect against some tested bacteria in the range of MICs from 0.5 to 10 mg/mL. The Gram-positive bacteria were more sensitive to PM1-3 extracts than the Gram-negative. From the Gram-negatives only the growth of Listeria monocytogenes ATCC 7644 was inhibited by PM1-3 extracts at MICs 10 mg/mL. The most susceptible bacterium to all three active fractions was potentially pathogenic Clostridium difficile CCM 3593. In general, the best results were obtained for chloroform portion (PM3) which inhibits the growth of 10 out of 22 tested strains at the lowest MICs. The hexane portion, inhibiting eight strains, was the second most active substance and methanolic extract affected only four bacterial strains. None of tested bacteria were affected by ethyl acetate and aqueous phase and no growth inhibition caused by DMSO (solvent control) was observed in the control.
Culturable aerobic bacteria were found in the P. magnifica colonies in counts of 5.88 ± 0.71 log CFU/g (mean ± S.D., n = 8), the numbers varied between 4.96 and 6.71 log CFU/g. More variable were those bacterial counts obtained after anaerobic cultivation. Viability of anaerobes (including facultative anaerobes) was from 2.30 to 6.52 CFU/g, 4.12 ± 1.11 log CFU/g in average. Forty isolates out of 49 selected for detailed identification were satisfactorily classified by MALDI-TOF MS analysis. No reliable results were obtained in nine cases. In 40 strains, a secure genus and probable species identification with score values of 2.000–2.299 were observed. Aeromonas veronii was found to be the most abundant species (25 strains) in Pectinatella colonies, followed by Aeromonas hydrophila, Aeromonas sorbia (four strains of both species), Sphingomonas pituitosa, and Lactobacillus plantarum (one strain each). Five strains were identified only to the genus level, two strains as Chryseobacterium spp., and two others as Herbaspirillum spp., and one as Pseudomonas spp.
Our comparison between bryozoan colonies-associated assemblages and those occurring outside bryozoan colonies showed us that cyanobacteria and algae formed a conspicuous biomass mainly in old colonies. When compared with plankton and periphyton, algae, and cyanobacteria demanding a higher trophic degree prevailed (coccal greens Desmodesmus spp., Chlorococcum etc.; small diatoms Stephanodiscus hantzschii, Nitszchia cf. palea; and filamentous cyanobacteria Leptolyngbya, Komvophoron, and Phormidium spp.).
2.4. Cyanobacterial Toxins Determination
Cyanobacteria were found in P. magnifica colony gels (typically of genus Pseudanabaena, Komvophoron, Phormidium, and Leptolyngbya) . Their number increases with the colony lifetime as indicated by the inner colony gel color (from red to green). Toxicity of P. magnifica occurrence may come from the cyanobacteria which are known as a source of several hepatotoxins, e.g., microcystins (MCs).
Samples were lyophilized biomass (zooids together with the colony gel) and the surrounding water from the location where the colonies were sampled. The results are summarized in Table 3.
Ecological and functional relationships
This biotope is dominated by sessile, permanently fixed, suspension feeding invertebrates that are, therefore, dependant on water flow to provide: an adequate supply of food and nutrients; gaseous exchange; remove metabolic waste products; prevent accumulation of sediment, and disperse gametes or larvae. The majority of species found in this biotope are adapted to strong water flow, siltation and a degree of sediment scour. Little is known of ecological relationships in circalittoral faunal turf habitats (Hartnoll, 1998) and the following has been inferred from studies of other epifaunal communities (Sebens, 1985; 1986).
- Few plants are found in this biotope but include encrusting coralline algae and occasionally small red algae (Sebens, 1985; Hartnoll, 1998).
- Suspension feeders on bacteria, phytoplankton and organic particulates and detritus include sponges (Polymastia spp. and Esperiopsis fucorum), soft corals and anemones( e.g. Alcyonium digitatum and Metridium dianthus), erect and encrusting bryozoans (e.g. Flustra foliacea, and Bugula spp.), brittlestars (e.g. Ophiothrix fragilis), barnacles (e.g. Balanus balanus), caprellid amphipods, porcelain crabs (e.g. Pisidia longicornis), polychaetes (e.g. Sabella pavonina and Spirobranchus spp.) and sea squirts (e.g. Clavelina lepadiformis). However, the water currents they generate are probably localized , so that they are still dependent on water flow to supply adequate food.
- Passive carnivores of zooplankton and other small animals include, hydroids (e.g. Tubularia indivisa and Nemertesia antennina), soft corals (e.g. Alcyonium digitatum), while larger prey are taken by Urticina felina and Metridium dianthus (Hartnoll, 1998).
- Sea urchins (e.g. Echinus esculentus and Psammechinus miliaris) are generalist grazers, removing ascidians, hydroids and bryozoans and potentially removing all epifauna, leaving only encrusting corallines and bedrock. Sea urchins were shown to have an important structuring effect on the community and epifaunal community succession (Sebens, 1985; 1986; Hartnoll, 1998).
- Other grazers include top shells (e.g. Jujubinus miliaris) and Calliostoma zizyphinum, which grazes hydroids, and small crustaceans (e.g. amphipods).
- Specialist predators of hydroids and bryozoans include the nudibranchs (e.g. Janolus cristatus, Doto spp. and Onchidoris spp.) and pycnogonids, (e.g. Achelia echinata), while the nudibranch Tritonia hombergi preys on Alcyonium digitatum, and some polychaetes take hydroids.
- Starfish (e.g. Asterias rubens and Crossaster papposus) are generalist predators feeding on most epifauna, including ascidians.
- Scavengers include polychaetes, small crustaceans such as amphipods, starfish and larger decapods such as hermit crabs (e.g. Pagurus bernhardus) and crabs (e.g. Hyas coarctatus).
- Mobile fish predators are likely to include gobies (e.g. Pomatoschistus spp.), butterfish (Pholis gunnellus), wrasse and eelpout (Zoarces viviparus) feeding mainly on small crustaceans, while species such as flounder (Platichthys flesus) are generalists feeding on ascidians, bryozoans, polychaetes and crustaceans (Sebens, 1985; Hartnoll, 1998)
Intra and interspecific competition occurs for food and space. Filter feeders reduce the concentration of suspended particulates and deplete food to other colonies/individuals downstream (intra and inter specific competition). Sebens (1985, 1986) demonstrated a successional hierarchy, in which larger, massive, thick growing species (e.g. large anemones, soft corals and colonial ascidians) grew over low lying, or encrusting growth forms such as halichondrine sponges, bryozoans, hydroids and encrusting corallines. The epifauna of vertical rock walls became dominated by large massive species, depending on the degree of predation, especially by sea urchins. However, encrusting bryozoans and encrusting corallines may survive overgrowth (Gordon, 1972; Sebens, 1985; Todd & Turner, 1988). In this biotope the degree of sediment scour and siltation probably exerts a controlling factor on the succession (see temporal change below) and is dominated by species tolerant of sediment scour and high water flow.
Seasonal and longer term change
No information on seasonal or temporal change in Flustra dominated communities was found and the following information has been inferred from available studies of subtidal epifaunal communities (Sebens, 1985, 1986; Hartnoll, 1983, 1998).
Some species such as the ascidians Ciona intestinalis and Clavelina lepadiformis are effectively annual while some hydroids an bryozoans, may show annual phases of growth and dormancy or regression. For example, Flustra foliacea becomes dormant in winter, Bugula species die back in winter to dormant holdfasts, while the uprights of Nemertesia antennina die back after 4-5 month and exhibit three generations per year (spring, summer and winter) (see MarLIN reviews; Hughes, 1977; Hayward & Ryland, 1998; Hartnoll, 1998).
Sebens (1985, 1986) described successional community states in the epifauna of vertical rock walls. Clear space was initially colonized by encrusting corallines, rapidly followed by bryozoans, hydroids, amphipods and tube worm mats, halichondrine sponges, small ascidians (e.g. Dendrodoa carnea and Molgula manhattensis), becoming dominated by the ascidian Aplidium spp., or Metridium dianthus or Alcyonium digitatum. High levels of sea urchin predation resulted in removal of the majority of the epifauna leaving encrusting coralline dominated rock. Reduced predation allowed the dominant epifaunal communities to develop, although periodic mortality (through predation or disease) of the dominant species resulted in mixed assemblages or a transition to another assemblage (Sebens, 1985, 1986). Sea urchin predation may play a significant role in freeing space for colonization in this community. Succession will be dependant on species tolerance to silt and sediment scour. For example, the sub-biotope MCR.Flu.Flu is relatively species poor due to high silt levels, while more sponges, ascidians, bryozoans and hydroids occur in increased scour but reduced silt habitats (e.g. MCR.Flu.Hocu or MCR.Flu.Hbys).
Long term studies of fixed quadrats in epifaunal communities demonstrated that while seasonal and annual changes occurred, subtidal faunal turf communities were relatively stable, becoming more stable with increasing depth and substratum stability (i.e. bedrock and large boulders rather than small rocks) (Osman, 1977; Hartnoll, 1998). Many of the faunal turf are long-lived, e.g. 6 -12 years in Flustra foliacea, 5-8 years in Ascidia mentula, over 20 years in Alcyonium digitatum, 8-16 years in Echinus esculentus and probably many hydroids (Stebbing, 1971a; Gili & Hughes, 1995; Hartnoll, 1998).
Habitat structure and complexity
- The bedrock is covered by a layer of encrusting corallines, and encrusting bryozoans, overgrown by dominant erect bryozoans and hydroids (e.g. Flustra foliacea, Bugula species, Nemertesia antennina, Thuiaria thuja) interspersed with encrusting sponges (e.g. Polymastia spp.), ascidians (e.g. Dendrodoa grossularia), Alcyonium digitatum and Urticina felina. The dominance by Flustra foliacea and other erect bryozoans and hydroids and ascidians forms a faunal turf over the substratum.
- The faunal turf provides interstices and refuges for a variety of small organisms such as nemerteans, polychaetes, and amphipods, while the erect species provide substrata for caprellid amphipods, which use them as 'platforms' to suspension feed.
- The erect bryozoans and hydroids support a variety of epizoics that use them as substratum and in some cases affect their growth rates. For example, Flustra foliacea supported 25 species of bryozoan, 5 hydroid species, some sessile polychaetes, barnacles, lamellibranchs and tunicates (Stebbing, 1971b). The bryozoans Bugulina flabellata, Crisia spp. and Scrupocellaria spp. were major epizoics. Scrupocellaria spp. settled preferentially on the youngest, distal, portions of the frond, possibly to elevate their branches into faster flowing water (Stebbing, 1971b). Similarly, Alcyonidium parasiticum is epizoic on hydroid stems or the bryozoan Cellaria spp. and the sponge Esperiopsis fucorum may grow on the stem of Tubularia species or on the test of ascidians.
- Mobile species include decapods crustaceans such as shrimp, crabs and lobsters, sea urchins, starfish and fish.
- Gobies, shannies and butterfish probably utilize available rock ledges and crevices, while large species such as flounder and cod probably feed over a wide area.
- Pockets of sediment that accumulate between boulders or in crevices (where present) may support benthic infaunal species such as Mya truncata and Sabella pavonina.
- The rock may support the rock boring bivalve Hiatella arctica.
- The biotope may show spatial variation in community complexity and exhibit a mosaic of different species patches (Hartnoll, 1998), due to colonization of areas recently cleared by predation, disease or physical disturbance in the process of re-colonization. The upper edges or boulders or rocky outcrops, most directly in water flow, tend to exhibit the most species rich and abundance faunal turfs, while species richness decreases with proximity to the sediment/ rock interface, which favours species such as the sponges Polymastia spp. or the anemone Urticina felina. Areas subject to increased scour or vertical surfaces tend to be dominated by tube worms such as Spirobranchus triqueter (Stebbing, 1971b, Eggleston, 1972b; Sebens, 1985, 1986; Connor et al., 1997a; Brazier et al., 1998; Hartnoll, 1998).
- Periodic disturbance of the community due to physical disturbance by storms, extreme scour, or fluctuations in predation, especially by sea urchins, may encourage species richness by preventing dominance by a few species (Osman, 1977; Sebens, 1985, 1986; Hartnoll, 1998).
Circalittoral faunal turf biotopes are primarily secondary producers. Food in the form of phytoplankton, zooplankton and organic particulates from the water column together with detritus and abraded macroalgal particulates from shallow water ecosystems are supplied by water currents and converted into faunal biomass. Their secondary production supplies higher trophic levels such as mobile predators (e.g. fish) and scavengers (e.g. starfish and crabs) and the wider ecosystem in the form of detritus (e.g. dead bodies and faeces). In addition, reproductive products (sperm, eggs, and larvae) also contribute to the zooplankton (Hartnoll, 1998). However, no estimates of faunal turf productivity were found.
Most of the species within this biotope produce short-lived, larvae with relatively poor dispersal capacity, resulting in good local recruitment but poor long range dispersal. Although, the biotope occurs within moderately strong to strong water flow that could remove a large proportion of the reproductive output, most reproductive propagules are probably entrained within the reduced flows within the faunal turf or in turbulent eddies produced by flow over the uneven substratum, resulting in turbulent deposition of propagules locally. Many species are capable of asexual propagation and rapidly colonize space. For example:
- Hydroids are often the first organisms to colonize available space in settlement experiments (Gili & Hughes, 1995). The characteristic hydroids in this biotope (e.g. Abietinaria abietina and Sertularia argentea) lack a medusa stage, releasing planula larvae. Planula larvae swim or crawl for short periods (e.g.
- The brooded, lecithotrophic coronate larvae of many bryozoans (e.g. Flustra foliacea, Securiflustra securifrons, and Bugula species), have a short pelagic life time of several hours to about 12 hours (Ryland, 1976). Flustra foliacea releases larvae in spring (February- April) (Eggleston, 1972a; Hayward & Ryland, 1998), while Bugulina flabellata exhibits two generations per year and releases larvae between April to October (Dyrynda & Ryland, 1982). Recruitment is dependant on the supply of suitable, stable, hard substrata (Eggleston, 1972b; Ryland, 1976; Dyrynda, 1994). In temperate waters most bryozoans species tend to grow rapidly in spring and reproduce maximally in late summer, depending on temperature, day length and the availability of phytoplankton (Ryland, 1970). However, even in the presence of available substratum Ryland (1976) noted that significant recruitment in bryozoans only occurred in the proximity of breeding colonies. For example, Hatcher (1998) reported colonization of slabs, suspended 1 m above the sediment, by Bugula fulva within 363 days while Castric-Fey (1974) noted that Bugulina turbinata, Crisularia plumosa and Bugula calathus did not recruit to settlement plates after ca two years in the subtidal even though present on the surrounding bedrock. Similarly, Keough & Chernoff (1987) noted that Bugula neritina was absent from areas of seagrass bed in Florida even though substantial populations were present
Time for community to reach maturity
No information was found on the development of this biotope and the following has been inferred from studies of similar epifaunal communities (Sebens, 1985, 1986; Hartnoll, 1998).
The recolonization of epifauna on vertical rock walls was investigated by Sebens (1985, 1986). He reported that rapid colonizers such as encrusting corallines, encrusting bryozoans, amphipods and tubeworms recolonized within 1-4 months. Ascidians such as Dendrodoa carnea, Molgula manhattensis and Aplidium spp. achieved significant cover in less than a year, and, together with Halichondria panicea, reached pre-clearance levels of cover after 2 years. A few individuals of Alcyonium digitatum and Metridium dianthus colonized within 4 years (Sebens, 1986) and would probably take longer to reach pre-clearance levels.
Jensen et al. (1994) reported the colonization of an artificial reef in Poole Bay, England. They noted that erect bryozoans, including Crisularia plumosa, began to appear within 6 months, reaching a peak in the following summer, 12 months after the reef was constructed. Similarly, ascidians colonized within a few months e.g. Aplidium spp. Sponges were slow to establish with only a few species present within 6-12 months but beginning to increase in number after 2 years, while anemones were very slow to colonize with only isolated specimens present after 2 years (Jensen et al., 1994.). In addition, Hatcher (1998) reported a diverse mobile epifauna after a years deployment of her settlement panels.
Flustra foliacea occurs in this biotope. New colonies of Flustra foliacea take at least 1 year to develop erect growth and 1-2 years to reach maturity, grow slowly (Stebbing, 1971a; Eggleston, 1972a), and would probably several years to reach high abundance, depending on environmental conditions. Recruitment may be enhanced in areas subject to sediment abrasion, where less tolerant species are removed, making more substratum available for colonization, especially if larval release in spring coincides with the end of winter storms. The wreck of a small coaster (the M.V. Robert) off Lundy became dominated by erect bryozoans, including occasional Flustra foliacea, within 4 years of sinking, when it was first surveyed (Hiscock, 1981).
Overall, encrusting bryozoans, hydroids, and ascidians will probably develop a faunal turf within less than 2 years, and Flustra foliacea can evidently colonize and reach an abundance of occasional (1-5% cover) within 4 years. Slow growing species such as Flustra foliacea and some sponges and anemones, will probably take many years to develop significant cover, so that this biotope may take between 5 -10 years to develop an stable community after disturbance, depending on local conditions.