Endobacterial morphotypes in nudibranch cerata tips: a SEM analysis

Abstract

The SEM investigation of nudibranch cerata material exhibits endobacterial morphotypes found in 12 out of 13 species tested: Aeolidia papillosa, Berghia caerulescens, Coryphella brownii, Coryphella lineata, Coryphella verrucosa, Cuthona amoena, Facelina coronata, Flabellina pedata, Dendronotus frondosus, Doto coronata, Tritonia plebeia and Janolus cristatus. Endobacteria could not be detected inside Tritonia hombergi. Endobacterial morphology found inside nudibranch species was compared to bacterial morphotypes detected earlier in tentacles of cnidarian species. SEM micrographs show endobacterial analogy among nudibranch species, but also similarity to cnidarian endobacteria investigated earlier. Of course, morphological data of microbes do not allow their identification. However, since most of these nudibranch species prey on cnidaria, it cannot be excluded that many of the endobacteria detected inside nudibranch species may originate from their cnidarian prey. Our previous data describing genetic affiliation of endobacteria from nudibranchian and cnidarian species support this assumption. Dominant coccoid endobacteria mostly exhibit smooth surface and are tightly packed as aggregates and/or wrapped in envelopes. Such bacterial aggregate type has been described previously in tentacles of the cnidarian species Sagartia elegans. Similar coccoid bacteria, lacking envelopes were also found in other nudibranch species. A different type of coccoid bacteria, characterized by a rough surface, was detected inside cerata of the nudibranch species Berghia caerulescens, and surprisingly, inside tentacles of the cnidarian species Tubularia indivisa. In contrast to cnidarian endobacteria, rod-shaped microorganisms are largely absent in nudibranch cerata.

Introduction

Microbial activity is present in a wide range of habitats and has even been found in extreme environments (Staley and Gosink 1999; Brinkmeyer et al. 2003; La Duc et al. 2007). Life often is made possible by symbiosis, usually between specialized bacteria and various eukaryotic partners, which demonstrates the prominent relevance of such interactions. Well known is the interaction between man and trillions of symbiotic bacteria inside the intestinal tract (Xu et al. 2007). Similarly established is the vital microbial role of cellulose fermentation inside the ruminant gut system harbouring many bacterial symbionts (Leschine 1995). Efficient N₂ fixation (nitrogenase production) generated by Rhizobium spp. provides legumes with the needed nitrogen (Fischer 1994). Fascinating microbial bioluminescence observed in eukaryotic marine organisms may make lanterns of anglerfish glowing and illuminates eye regions of octopus species (Case et al. 1977; Nealson and Hastings 1979; Ruby and McFall-Ngai 1992). The manifold helpful microbial activities emphasize the importance of symbiotic life strategies; only these wondrous abilities may allow for the survival of the eukaryotic partners. Unique and prominent is the symbiotic function in evolution such as mitochondrial ATP production, serving the entire eukaryotic world with essential endobacterial ‘power units’ (Cray et al. 1999; Dyall et al. 2004).

Certainly, literature provides various examples of symbiotic life inside marine organisms (Deming and Colwell 1982; Palincsar et al. 1989; Thuesen and Kogure 1989; Burnett and McKenzie 1997; Cary et al. 1997; Althoff et al. 1998; Ivanova et al. 2003; Kurahashi and Yokoto 2006). However, these observations are often too sketchy, and interaction mechanisms between prokaryotic and eukaryotic marine life are considered as black boxes. This particularly applies to the unknown physico-chemical conditions inside eukaryotic hosts and to endobacterial physiological activities (e.g. pathogenic traits, Schuett and Doepke 2010). Information on the structure and function of these endobacterial communities is mostly nonexistent. Moreover, realistic imagination of dimensions and spatial images of the detailed structures are unclear.

The present paper resumes previous reports on endobacteria in nudibranch and cnidarian species (Schuett et al. 2007; Schuett and Doepke 2010; Doepke et al. 2011) and provides a first comparative scanning microscopic study on symbiotic endobacterial morphotypes detected in the tentacles of cnidarian and in cerata of selected nudibranch species.

Materials and methods

Nudibranch cerata and cnidarian tentacles were collected in July 2008 and 2009 by divers from waters around the Orkney Islands and the Outer Hebrides. Cnidarian tentacles and nudibranch cerata material were fixed in glutaraldehyde. Ethanol replaced sea water, which was exchanged later by amyl acetate. After critical point drying in liquid CO₂, sample material was mounted on SEM stabs. A first Au-coating (sputter) and the clipping off cerata or tentacle tip regions were followed by a second Au-coating. This procedure allowed an insight into the inner tissue structure and their endobacteria. Micrograph documentation of epidermis regions was conducted by using SEM Hitachi S-800. Complete details are given in Doepke et al. (2011).

Results

This first scanning microscopic documentation shows a wide distribution of different characteristic endobacterial morphotypes in nudibranch cerata (Table 1). The data do not claim for completeness; however, the investigation enables a tentative overview of the morphological diversity of endobacteria in nudibranch cerata tips. These results were compared to those on endobacterial aggregates and dispersed bacteria previously detected in tentacle material of the cnidarians Metridium senile, Sagartia elegans and Tubularia indivisa (Table 2) collected from Scottish diving locations (Schuett et al. 2007; Schuett and Doepke 2010; Doepke et al. 2011).

Table 1 Overview of endobiotic bacteria and their aggregates within the epidermic tip region of cerata from 12 nudibranch species

Table 2 Similarity of bacterial shapes detected in cnidarian tentacles and in cerata of nudibranch predators

Endobacteria

Twelve of 13 nudibranch species examined harboured endobacteria inside the cerata epidermis, which corresponds to earlier findings of Doepke et al. (2011). No endobacteria could be detected inside of Tritonia hombergi. A characteristic spectrum of streptococcus-like bacteria with smooth surface represented the majority of the endobacteria found.

Oval-shaped bacteria were less frequent. These microorganisms occurred as two types of tightly packed bacterial aggregates. The first type was covered by envelopes (Janolus cristatus)and the second exhibited no sheath (Janolus cristatus, Berghia caerulescens). Distinct similarities but also differences between endobacteria in nudibranch cerata and cnidarian tentacles were noticed.

Bacterial aggregates

Aggregates with envelopes harboured two bacterial types of mainly coccoid forms with smooth surface and oval forms. The streptococcus-like bacteria display a wide range of diameters between 0.5 and 1.5 μm, which may account for bacterial division and growing stages inside the envelopes. The envelopes are extremely thin, and the spherical shapes of the tightly packed endobacteria are visible through the envelopes (Fig. 1). These characteristic aggregates were detected in most of the nudibranch species studied (Aeolidia papillosa, Berghia caerulescens, Coryphella brownii (Fig. 2), Coryphella verrucosa, Facelina coronata (Fig. 3), Flabellina pedata, Janolus cristatus). Interestingly, this type of aggregate containing the typical coccoid endobacteria was also found in tentacles of the cnidarian species Sagartia elegans (Fig. 4). Uncommon large coccoid bacteria of 2–4 μm were detected exclusively in Coryphella brownii and Dendronotus frondosus.

Fig. 1

Thin envelope of aggregate from Berghia caerulescens; single coccoid bacterium visible (arrow)

Fig. 2

Aggregate (Ø ca. 23 μm) with envelope (arrow) originating from cerata of nudibranch Coryphella brownii containing streptococcus-like bacteria (Ø ca. 0.5–2.5 µm) with smooth surface. Thread may derive from fired cnidocyst (arrow)

Fig. 3

Aggregate (Ø ca. 5 μm) with envelope collected from cerata of nudibranch Facelina coronata harbouring coccoid bacteria with smooth surface of ca. 0.5 μm diameter

Fig. 4

Streptococcus-like bacteria (Ø ca. 1.3 μm) inside tentacle of cnidarian species Sagartia elegans, located in a cavern, no envelope

Only Janolus cristatus carried a type of oval bacteria covered by envelopes (Fig. 5). This particular shape has been observed in cerata sample material collected in 2008. The image shows additional coccoid bacteria with smooth surface, tightly packed in aggregates and covered by envelopes as described above.

Fig. 5

Two different types of aggregates were found in cerata of nudibranch Janolus cristatus. a Aggregate covered with envelope (Ø ca. 5 µm) containing oval microorganisms (Ø ca. 0.8 × 1.6 µm); b aggregates in envelopes (Ø ca. 6 µm) harbouring streptococcus-like bacteria (Ø ca. 1.0 µm)

Apart from the coccoid bacterial majority with smooth surface, Berghia caerulescens moreover harboured spherical bacterial aggregates containing coccoid-shaped forms with rough surface (Fig. 6). Similar organisms with rough coccoid appearance have been detected earlier in Tubularia indivisa; however, these structures were lacking envelopes (Doepke et al. 2011). It cannot be excluded that envelopes were lost during preparation.

Fig. 6

Aggregate (Ø ca. 13 μm) in envelope with tightly packed bacteria (Ø ca. 0.9 μm) with rough surface (arrow), detected in nudibranch cerata of Berghia caeruslescens

Bacterial aggregate sizes and numbers

Each of the cerata was found to harbour approximately 5–20 aggregates. Sizes of the mainly egg-shaped aggregates ranged between 4 and 25 μm. Aggregates may be hidden in caverns (Facelina coronata, Fig. 7). The number of bacteria inside a single envelope was between fifty and some hundreds. In comparison, cnidarian tentacles carry significantly higher numbers of aggregates and endobacteria than those found in the certata of nudibranchia.

Fig. 7

Bunches of cnidocyst threads (arrow) in the vicinity of bacterial aggregates (envelopes cannot be identified). A cavern contains smooth surface streptococcus-like bacteria (Ø ca. 0.7 µm, arrow) detected in cerata of nudibranch Facelina coronata

Specially shaped endobacteria

Coryphella lineata exclusively carried spindle-like bacteria (Fig. 8). Berghia caerulescens showed unusual polymorphic organisms (Fig. 9). These bacterial structures have not been found inside cnidarian tentacle material.

Fig. 8

Aggregates without envelope consisting of spindle-like bacteria (Ø ca. 0.5 × 0.9 μm; arrow); only found inside Coryphella lineata cerata

Fig. 9

Aggregate without envelope consisting of polymorphic bacteria (Ø ca. 1.3 μm, arrow) found in an epidermis cerata cavern of nudibranch Berghia caerulescens

Filaments

Extremely fine fabric structures interconnecting endobacteria have been detected inside cerata material of Coryphella brownii, Coryphella lineata and Flabellina pedata (Fig. 10). Similar structures were detected earlier in tentacles of the cnidarian species Metridium senile (Schuett et al. 2007) and Tubularia indivisa (Doepke et al. 2011).

Fig. 10

Endobacteria detected inside cerata of nudibranch Flabellina pedata (arrow), connected by thin filaments (arrows)

Kleptocnides

For nine out of 13 different cerata samples, the SEM micrographs demonstrated a close association between kleptocnides and endobacteria. Figure 11 shows typical kleptocnides inside of Aeolidia papillosa; these observations agree with earlier findings (Doepke et al. 2011).

Fig. 11

Cnidocysts (arrows) on the surface and/or within the epidermis region of the nudibranch Aeolidida papillosa cerata

Discussion

This paper provides a scanning electron microscopic survey of endobacterial morphotypes living inside cerata tip regions of several nudibranch species. These findings are compared to those on endobacterial morphotypes found earlier in tentacles of different cnidarian species.

Similarities and differences between endobacteria of nudibranchia and cnidaria

There are considerable morphological analogies between endobacteria observed in cnidarian tentacle and nudibranch cerata material (Table 2). This particularly applies to the aggregates containing the many coccoid bacteria with smooth surface found in several nudibranch species but also inside Sagartia elegans. Further similarities include rough coccoid forms observed in Berghia caerulescens and Tubularia indivisa. Additional indication is provided by 16S rDNA sequencing data, which showed perplexing similarities of endobacterial species found in tentacles and cerata (Doepke et al. 2011).

However, there are also certain differences concerning aggregate sizes, number of bacteria per aggregate and the many rod-shaped bacterial forms inside cnidarian tentacles. Rod-shaped bacteria are largely absent inside the cerata of nudibranchia.

Aggregate envelopes

Besides, the remarkable analogies concerning aggregates and bacterial shapes substantial questions remain. It is unclear whether the cnidarian and nudibranch hosts or else the endobacterial tenants do produce the envelopes. There is no investigation concerning the chemical composition and origin of aggregate envelopes.

Furthermore, the origin of dispersed bacteria has to be elucidated yet. They may occur only as an artefact after opening of tentacle and cerata tips (clipping procedure and Au resputtering of the opened surface), resulting in aggregate breakage and the release of bacteria.

Filaments

Filaments interconnecting bacteria were termed ‘nanowires’ by Gorby et al. (2005). The striking findings by El Naggar et al. (2008, 2010) and Nealson (2010) showed that nanowires formed by Shewanella oneidensis MR-1 are communication devices. In biofilm experiments, electron transport along bacterial nanowires could be measured and has been interpreted as cooperative respiration within the bacterial community. It is open whether this is a general mechanism.

Nevertheless, the development of interconnecting filaments between bacteria often occurs in nature. Duchow and Douglas (1949) reported this morphological feature for the photoheterotrophic Rhodomicrobium vaniellii. Funke et al. (1997) described a coryneform bacterium with filaments. Kuhner et al. (2000) and Matthies et al. (2001) studied anaerobic clostridia from acidic forest bog locations and suggested these appendages to serve as electron transport system.

Our current and previous SEM data (Schuett et al. 2007; Doepke et al. 2011) showed endobacterial filaments in cnidarian tentacles and nudibranch cerata. However, cnidarian microorganisms developing filaments were rod-shaped, in contrast to nudibranch endobacteria with coccoid forms.

Bacterial passage into the epidermis

An important question concerns the passage mechanism of bacteria to settle inside cerata or tentacles epidermis. Our previous examination showed that microbes displayed other pathogenic activities, notably necrotic traits which may allow for a microbial invasion (Schuett et al. 2007; Schuett and Doepke 2010; Doepke et al. 2011). Vibrio species with necrotic ability were detected in cnidarian species (Metridium senile, Tubularia indivisa) as well as in several nudibranch species (Berghia caerulescens, Coryphella lineata, Coryphella grancilis, Dendronotus frondosus, Polycera faeruensis, Polycera quadrilineata).

Do endobacteria of cerata originate from cnidarian tentacles?

The most exciting question concerns the origin of endobacteria inside nudibranchia, which predominantly prey on cnidarian species. Our observation favours the idea that bacteria inside cerata originate from cnidarian tentacles. Relevant indications like the absence of these microbes in the seawater (Schuett et al. 2007) support this assumption. The observed difference in endobacteria may be due to variations in diet, for example the spindle-like bacteria in Coryphella lineata and the two different oval forms in Berghia caerulescens and in Janolus cristatus. For the latter, nudibranch species (member of the family Zephyrinidae) Cimino and Ghiselin (1999) reported a possible diet switch from sponges to cnidaria. Further, major evidence supports the probability of bacterial transfer from cnidaria to nudibranchia: the uptake of the many unfired kleptocnides and their path through the complex nudibranch gut system to cnidosacks, their final destination in the tip region of cerata. In all likelihood, cnidarian microorganisms follow the same track. It can be assumed that bacteria reproducing inside cerata will find a milieu, which resembles that inside cnidarian tentacles.

Future research

The elucidation of the complex aspects of symbiosis is a great challenge to future research. To decode the black box, it requires a wide scope of experimental approaches, comprising intensified attempts in the fields of ecology, molecular biology, physiology, cell biology, structural chemistry, and physics, but also in the use of imaging systems. A major bottle neck in the analysis of physiological traits is due to the limited culturability of endobacteria. In order to bypass culture problems, Markert et al.(2007) and Gardebrecht et al. (2012) suggest the approach of physiological proteomics. However, this procedure is still in its infant state. It requires detailed preparatory proteomic information of endobacterial activities before it can become a powerful tool to enlighten complex black physiological and biochemical boxes.