Gill filament differentiation and experimental colonization by symbiotic bacteria in aposymbiotic juveniles of Codakia orbicularis (Bivalvia: Lucinidae)

Summary

Codakia orbicularis is a tropical lucinid harboring gill endosymbionts which are environmentally transmitted from a free living-symbiont form to the new host generation after metamorphosis. Structural changes occurring in the cellular organization from incomplete gill filaments in young aposymbiotic juveniles to full differentiated gill filaments containing bacterial endosymbionts in reared symbiotic juveniles, were analyzed for juveniles from 250 μm to 2 μm shell-length. Aposymbiotic juveniles possess differentiated gill filaments with ciliated, intermediary, and lateral zones similar to those described in wild juveniles, except for the bacteriocytes which are lacking. Granule cells, which progressively differentiate during the morphogenesis of the gill filament, do not appear as a consequence of symbiosis. Experimental colonization of aposymbiotic juveniles by the free-living symbiont form has been obtained through the addition of unsterilized sand collected from the natural habitat of C. orbicularis. Two days after exposure to crude sand, symbiosis-competent bacteria enter by endocytosis at the apical pole of undifferentiated cells which progressively differentiate into classical bacteriocytes similar to those found in the adult gill filaments. Undifferentiated cells of aposymbiotic gill filaments remain receptive to bacteria several months after metamorphosis, and become bacteriocytes when aposymbiotic juveniles get contact with the symbiont free-living form. Therefore, the environmental transmission of symbionts does not appear to be restrained to a defined period of time during post-larval development in C. orbicularis.     

Gill-symbiosis in mytilidae associated with wood fall environments

Bivalves belonging to the genera Idas and Adipicola were collected from wood fall environments in the west Pacific (Vanuatu islands) between 300 and 890 m depths in 2004. Bacterial symbionts were checked by three complementary techniques: histological and DAPI staining, in situ hybridization (FISH), and TEM. No bacteria were detected inside the gills of the two species, rejecting the endosymbiosis hypothesis. However, results from our study demonstrated the existence of ectosymbionts colonizing microvilli differentiated at the apical surface of the cells constituting the lateral zone of gill filaments. These ectosymbionts are γ-Proteobacteria due to their strong hybridization with the specific probe GAM42; in contrast no hybridization was obtained from either gills or other host tissues by using the oligonucleotide probes specific to α- β- and δ-Proteobacteria. Based on TEM observations, these Gram-negative bacterial symbionts are not methanotrophic due to the lack of concentric stacking of intracellular membranes in their cytoplasm. Such ectosymbionts may represent thioautotrophic bacteria as already described in various Mytilidae from hydrothermal vents and cold seeps. Unfortunately, no phylogenetic analysis could be done in this study to compare their DNA sequence to that of other marine invertebrate symbionts described to date.     

Gill ultrastructure and symbiotic bacteria in Codakia orbicularis (Bivalvia,Lucinidae)

Summary The cellular organization of the gill, which harbors symbiotic bacteria, is described in juveniles and adults of Codakia orbicularis, a large tropical Lucinidae. The ciliary zone is similar in every species of Lucinidae described and includes the large clear cell which has been previously described as an intermediary cell. The intermediary zone is composed of a few narrow unciliated cells, which bind adjacent filaments together and constitute channels through which sea water circulates along the abfrontal part of the filaments. The lateral zone is more complex in C. orbicularis than in other Lucinidae, being composed of four cell types and differentiated into two distinct regions. The bacteriocytes and intercalary cells occupy the outermost bacteriocyte zone, while mucocytes and numerous cells crowded with proteinic, cystine-rich granules constitute the innermost secretory zone which has not been described in other species. The newly described granule cells are considered to be a key factor in the storage and metabolic conversion of sulfur compounds.     

Sulphur-oxidizing extracellular bacteria in the gills of Mytilidae associated with wood falls

Six morphotypes of small mussels (Bivalvia: Mytilidae) were found attached to naturally sunken wood collected in the Bohol Sea (Philippines). These specimens are related to the large Bathymodiolus mussels that are found worldwide at cold seeps and hydrothermal vents. In these habitats, the mytilids harbour sulphur- and methane-oxidizing endosymbionts in their gills and depend on the energy and carbon provided by the symbionts. In this study, bacteria associated with the gills of wood-associated mussels are characterized using molecular and microscopic techniques. The existence of bacteria in the lateral zone of gill filaments in all specimens is demonstrated. Comparative analyses of 16S rRNA gene and adenosine 5'-phosphosulphate (APS) reductase gene sequences indicate that the bacteria are closely related to sulphur-oxidizing endosymbionts of Bathymodiolus. FISHs using specific probes confirm that sulphur oxidizers are by far the most abundant, if not the only bacteria present. Electron micrographs displayed mostly extracellular bacteria located between microvilli at the apical surface of host gill epithelial cells all along the lateral zone of each gill filament. In some specimens, occasional occurrence of intracellular bacteria with similar morphology was noted. This study provides the first molecular evidence for the presence of possible thiotrophic symbiosis in sunken wood ecosystems. With their epibiotic bacteria, wood-associated mussels display a less integrated type of interaction than described in their seep, vent and whale fall relatives.     

Electron microscopic study on the gill bars of amphioxus (Brachiostoma californiense) with special reference to Neurociliary control

Summary Both primary and secondary (tongue) bars of the pharyngeal gill basket are covered by epithelial cells that are continuous with the cells that line the atrium. Anterior and posterior faces of the gill bars are covered with lateral ciliated cells, which possess a single cilium, ringed by microvilli, and an elaborate basal mitochondria-rootlet apparatus. Pharyngeal faces of the gill bars are covered with ciliated pharyngeal cells, atrial faces by mucus secreting atrial cells. The surface epithelium rests on a stromal septum, a flattened tube of basal lamina which dilates to form the visceral blood vessel (along the pharyngeal face) and skeletal blood vessel (along the atrial face). This basal lamina surrounds paired skeletal rods which run through the longitudinal axis of the gill bars near the atrial face. Between the skeletal rods and atrial cells of primary gill bars is a coelomic channel lined by epithelioid coelomic cells. Neuronal processes, some with neurosecretory granules, are located among the bases of the atrial cells. Some axons may contact lateral ciliated cells where the latter meet atrial cells, but synaptoid endings have not been found here or elsewhere in the gill bars. Nervous tissue has not been identified among lateral ciliated cells even though ciliary activity of these cells is supposedly regulated by atrial nervous tissue.Supported by a Cottrell College Science Program Grant from Research Corporation. We thank Nancy Kelly and Gerhard Ott for excellent technical assistance and are grateful for the facilities provided by the Department of Zoology and Seaver Science Center, Pomona College.     

Ultrastructure, distribution, and transovarial transmission of symbiotic microorganisms in Nysius ericae and Nithecus jacobaeae (Heteroptera: Lygaeidae: Orsillinae)

The organization of the symbiotic system (i.e., distribution and ultrastructure of symbionts) and the mode of inheritance of symbionts in two species, Nysius ericae and Nithecus jacobaeae belonging to Heteroptera: Lygaeidae, are described. Like most hemipterans, Nysius ericae and Nithecus jacobaeae harbor obligate prokaryotic symbionts. The symbiotic bacteria are harbored in large, specialized cells termed bacteriocytes which are localized in the close vicinity of the ovaries as well as inside the ovaries. The ovaries are composed of seven ovarioles of the telotrophic type. Bacteriocytes occur in each ovariole in the basal part of tropharium termed the infection zone. The bacteriocytes form a ring surrounding the early previtellogenic oocytes. The cytoplasm of the bacteriocytes is tightly packed with large elongated bacteria. In the bacteriocytes of Nysius ericae, small, rod-shaped bacteria also occur. Both types of bacteria are transovarially transmitted from one generation to the next.     

Gill anatomy and the evolution of symbiosis in the bivalve family Thyasiridae

Among families of bivalves with chemoautotrophic symbionts, the Thyasiridae may vary the most in their anatomical characters and in the extent of their nutritional reliance upon symbionts. Since only a fraction of thyasirid species are symbiotic, and the symbionts are mostly observed to be extracellular, this group may be representative of early stages in the evolution of bacterium- bivalve symbioses. To better understand the distribution of symbiosis among thyasirid genera, and the relationships between gill structure and symbiont occurrence, the gills of 26 thyasirid species were studied by light and electron microscopy. Observations revealed three gill types, which are generally constrained within genera or subgenera. Symbionts were found in two gill types: the most simple, homorhabdic filibranch morphotype, and the most derived and thickened morphotype, which resembles the gill structure of other chemosymbiotic bivalves. In all observable cases, the symbionts were located extracellularly among the microvilli of the bacteriocytes. Among individuals of the species Thyasira (Parathyasira) equalis, the quantity of symbionts varied. The results suggest an evolutionary sequence: a homorhabdic filibranch gill structure with few symbionts among the epithelial cell microvilli eventually thickened abfrontally, and thereby offered a larger surface for colonization by symbionts. Eventually, the symbionts persisted and grew in vacuoles within epithelial cells.     

Environmental acquisition of thiotrophic endosymbionts by deep-sea mussels of the genus bathymodiolus

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally "leaky" system of vertical transmission could not be excluded.     

Patterns of host cell inheritance in the bacterial symbiosis of whiteflies

Whiteflies possess bacterial symbionts Candidatus Portiera aleyrodidium that are housed in specialized cells called bacteriocytes and are faithfully transmitted via the ovary to insect offspring. In one whitefly species studied previously, Bemisia tabaci MEAM1, transmission is mediated by somatic inheritance of bacteriocytes, with a single bacteriocyte transferred to each oocyte and persisting through embryogenesis to the next generation. Here, we investigate the mode of bacteriocyte transmission in two whitefly species, B. tabaci MED, the sister species of MEAM1, and the phylogenetically distant species Trialeurodes vaporariorum. Microsatellite analysis supported by microscopical studies demonstrates that B. tabaci MED bacteriocytes are genetically different from other somatic cells and persist through embryogenesis, as for MEAM1, but T. vaporariorum bacteriocytes are genetically identical to other somatic cells of the insect, likely mediated by the degradation of maternal bacteriocytes in the embryo. These two alternative modes of transmission provide a first demonstration among insect symbioses that the cellular processes underlying vertical transmission of bacterial symbionts can diversify among related host species associated with a single lineage of symbiotic bacteria.     

Coexistence of multiple proteobacterial endosymbionts in the gills of the wood-boring Bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae)

Wood-boring bivalves of the family Teredinidae (commonly called shipworms) are known to harbor dense populations of gram-negative bacteria within specialized cells (bacteriocytes) in their gills. These symbionts are thought to provide enzymes, e.g., cellulase and dinitrogenase, which assist the host in utilizing wood as a primary food source. A cellulolytic, dinitrogen-fixing bacterium, Teredinibacter turnerae, has been isolated from the gill tissues of numerous teredinid bivalves and has been proposed to constitute the sole or predominant symbiont of this bivalve family. Here we demonstrate that one teredinid species, Lyrodus pedicellatus, contains at least four distinct bacterial 16S rRNA types within its gill bacteriocytes, one of which is identical to that of T. turnerae. Phylogenetic analyses indicate that the three newly detected ribotypes are derived from gamma proteobacteria that are related to but distinct (>6.5% sequence divergence) from T. turnerae. In situ hybridizations with 16S rRNA-directed probes demonstrated that the pattern of occurrence of symbiont ribotypes within bacteriocytes was predictable and specific, with some bacteriocytes containing two symbiont ribotypes. However, only two of the six possible pairwise combinations of the four ribotypes were observed to cooccur within the same host cells. The results presented here are consistent with the existence of a complex multiple symbiosis in this shipworm species.     

Mechanical sensitivity and cell coupling in the ciliated epithelial cells ofMytilus edulis gill

Summary Transmission electron microscopy has not provided strong evidence for gap junctions inMytilus edulis gill tissue, in spite of extensive physiological evidence for coupled ciliary arrest in lateral cells and coupled activation in abfrontal cells. To investigate the kinds and relative distribution of cell junctions and also to determine whether ciliary membrane particle differences exist in these two types of oppositely mechanically sensitive cells, we analyzed the structure of these and two other ciliated cell types (frontal and laterofrontal) by freeze-fracture replication. Gap junctions occur in all four ciliated cell types, but they are relatively small and of variable morphology, often consisting of elongate, winding complexes of membrane particles. Statistically, such structures rarely would be recognized as gap junctions in thin sections. Gap junctions appear to be most abundant between the highly coupled abfrontal cells, minimal between laterofrontal cells, and not evident in the epithelial cells that separate coupled ciliated cell types. The ciliary necklaces of the mechanically activated abfrontal cilia are typically 4- or 5-stranded while those of the remaining three cell types are mainly 3-stranded. In developing gill tips, ciliated cells have abundant gap junctions and newly formed cilia have a full complement of necklace particles. Nascent lateral cilia are not mechanically sensitive, indicating that the acquisition of mechanosensitivity does not correlate with the presence of ciliary necklace or other membrane particles. Lateral and laterofrontal cells become sensitive to neurotransmitters soon after the appearance of the latter during development, but mechanosensitivity of both lateral and abfrontal cells arises substantially later.     

Sea-land transitions in isopods: pattern of symbiont distribution in two species of intertidal isopods <Emphasis Type="Italic">Ligia pallasii</Emphasis> and <Emphasis Type="Italic">Ligia occidentalis</Emphasis> in the Eastern Pacific

Studies of microbial associations of intertidal isopods in the primitive genus Ligia (Oniscidea, Isopoda) can help our understanding of the formation of symbioses during sea-land transitions, as terrestrial Oniscidean isopods have previously been found to house symbionts in their hepatopancreas. Ligia pallasii and Ligia occidentalis co-occur in the high intertidal zone along the Eastern Pacific with a large zone of range overlap and both species showing patchy distributions. In 16S rRNA clone libraries mycoplasma-like bacteria (Firmicutes), related to symbionts described from terrestrial isopods, were the most common bacteria present in both host species. There was greater overall microbial diversity in Ligia pallasii compared with L. occidentalis. Populations of both Ligia species along an extensive area of the eastern Pacific coastline were screened for the presence of mycoplasma-like symbionts with symbiont-specific primers. Symbionts were present in all host populations from both species but not in all individuals. Phylogenetically, symbionts of intertidal isopods cluster together. Host habitat, in addition to host phylogeny appears to influence the phylogenetic relation of symbionts.     

Symbiotic microorganisms in <Emphasis Type="Italic">Puto superbus</Emphasis> (Leonardi, 1907) (Insecta,Hemiptera, Coccomorpha: Putoidae)

The scale insect Puto superbus (Putoidae) lives in mutualistic symbiotic association with bacteria. Molecular phylogenetic analyses have revealed that symbionts of P. superbus belong to the gammaproteobacterial genus Sodalis. In the adult females, symbionts occur both in the bacteriocytes constituting compact bacteriomes and in individual bacteriocytes, which are dispersed among ovarioles. The bacteriocytes also house a few small, rod-shaped Wolbachia bacteria in addition to the numerous large, elongated Sodalis-allied bacteria. The symbiotic microorganisms are transovarially transmitted from generation to generation. In adult females which have choriogenic oocytes in the ovarioles, the bacteriocytes gather around the basal part of the tropharium. Next, the entire bacteriocytes pass through the follicular epithelium surrounding the neck region of the ovariole and enter the space between oocyte and follicular epithelium (perivitelline space). In the perivitelline space, the bacteriocytes assemble extracellularly in the deep depression of the oolemma at the anterior pole of the oocyte, forming a “symbiont ball”.     

Symbionts in the oocytes of Blattella germanica (L.) (Dictyoptera : Blattellidae): Their mode of transmission

In Blattodea, the transmission of symbionts from one generation to the next is of transovarial type. The symbionts in Blattella germanica (L.) (Dictyoptera : Blattellidae) reach the ovary inside bacteriocytes in the 5-day-old nymphal instar, and are located among the ovarioles. Between the 11th and 17th day, the symbionts leave the bacteriocytes, cross the ovariole sheath, the tunica propria, and the follicular epithelium, and reach the space between the latter and the oocyte. In 17-day-old nymphs, the symbionts are in contact with the microvilli of the oocyte, remaining in this position until the end of the vitellogenic phase, when the microvillar border is progressively reduced. At this stage, the symbionts are actively phagocytosed by the oocyte before chorion formation.     

Ultrastructural reinvestigation of the trophosome in adults of Riftia pachyptila (Annelida, Siboglinidae)

Abstract. The trophosome of adults of Riftia pachyptila (Vestimentifera) was reinvestigated using 3-dimensional ultrastructural reconstruction and quantitative morphological analysis. The symbionts make up 24.1%, the symbiont-containing cells (bacteriocytes) are 70.5% of the trophosome's volume. The trophosome is composed of lobules that have a central axial blood vessel surrounded by a myoepithelium containing bacteriocytes, in turn surrounded by an apolar tissue of bacteriocytes. Part of the splanchnic peritoneum lining the trunk coelom encases the bacteriocytes and forms a ramifying network of peripheral blood vessels. Based on the morphology and ultrastructure of the adult, we hypothesize a mesodermal rather than endodermal origin of trophosome and its constitute bacteriocytes. Some of the central bacteriocytes are part of the myoepithelium surrounding the axial blood vessel and act as stem cells for a proliferating tissue produced in the center and ultimately degraded at the periphery of each lobule. Similarly, the rod-shaped symbionts in the center act as stem cells and exhibit a simple cell cycle. Differentiation into cocci takes place in the median and peripheral zone. Lysis of cocci occurs in the degenerative zone.     

Embryonic development and endosymbiont transmission mode in the symbiotic clam Lucinoma aequizonata (Bivalvia: Lucinidae)

Summary

Lucinoma aequizonata is a large lucinid clam which lives in reducing mud around 500 m deep. Adults harbor intracellular chemoautotrophic sulfur-oxidizing bacteria in specialized gill cells called bacteriocytes. The embryonic and early larval development of L. aequizonata is described by using light and scanning electron microscopy. Gametes were obtained by injection of 0.2ml of 4 mM serotonin solution in seawater into the posterior adductor muscle. The oocytes, 200 μm in diameter, are surrounded by a glycoprotein capsule which gives to the egg a total diameter of 500μm. The development which occurs at 10°C is slow. The first polar body is detected 2.5 h after contact between sperm and oocytes (T_o+2.5 h), and the first cleavage begins 10 h later (T_o+12.5 h). The following successive cleavages produce a nonciliated morula, then a ciliated gastrula which begins to rotate within the egg-capsule at T_o+4.5 days. At this stage, the first shell pellicle appears on the dorsal side of the embryo. At T₀+8 days, the trochophore larvae develop discrete ciliary bands which constitute the prototroch. Typical straight-hinge veligers, D-shaped larvae, hatch from the egg-capsule 12 days after fertilization. The newly hatched larvae are 240 μm in length and 200μm in height, and the straight hinge 150μm long. To elucidate the symbiont transmission mode, two symbiont-specific primers were designed and used in amplifications by PCR. This primer set was unsuccessful in amplifying symbiont DNA targets from mature gonads, spawned oocytes, eggs, and veligers whereas successful amplifications were obtained from symbiont-containing gill tissues. These data rule out the vertical transmission mode and strongly suggest that the symbionts are environmentally transmitted to the new host generation in L. aequizonata as for all tropical lucinids examined to date.     

Environmental Acquisition of Thiotrophic Endosymbionts by Deep-Sea Mussels of the Genus Bathymodiolus

Deep-sea Bathymodiolus mussels, depending on species and location, have the capacity to host sulfur-oxidizing (thiotrophic) and methanotrophic eubacteria in gill bacteriocytes, although little is known about the mussels' mode of symbiont acquisition. Previous studies of Bathymodiolus host and symbiont relationships have been based on collections of nonoverlapping species across wide-ranging geographic settings, creating an apparent model for vertical transmission. We present genetic and cytological evidence for the environmental acquisition of thiotrophic endosymbionts by vent mussels from the Mid-Atlantic Ridge. Open pit structures in cell membranes of the gill surface revealed likely sites for endocytosis of free-living bacteria. A population genetic analysis of the thiotrophic symbionts exploited a hybrid zone where two Bathymodiolus species intergrade. Northern Bathymodiolus azoricus and southern Bathymodiolus puteoserpentis possess species-specific DNA sequences that identify both their symbiont strains (internal transcribed spacer regions) and their mitochondria (ND4). However, the northern and southern symbiont-mitochondrial pairs were decoupled in the hybrid zone. Such decoupling of symbiont-mitochondrial pairs would not occur if the two elements were transmitted strictly vertically through the germ line. Taken together, these findings are consistent with an environmental source of thiotrophic symbionts in Bathymodiolus mussels, although an environmentally “leaky” system of vertical transmission could not be excluded.     

Unusual features of intercellular junctions in the larvacean Oikopleura dioica

Summary In the pelagic larvacean Oikopleura dioica, the epithelium lining the alimentary tract consists of ciliated and unciliated cell types. The ciliated cells also exhibit an apical border of long microvilli. Between the microvilli, the cellular membrane often projects deeply down into the cytoplasm; the membranes of these invaginations and those of apicolateral interdigitations may be associated with one another by tight junctions. Some of these junctions may be autocellular. The tight junctions are seen by freeze-fracture to be very simple in construction, composed of a single row of intramembranous particles, which may be fused into a P-face ridge. There is a dense cytoplasmic fuzz associated with these tight junctions which may extend into adjoining zonula adhaerens-like regions. The invaginations of the apical membranes are, in addition, associated by gap junctions which may also be autocellular. More conventional homocellular and heterocellular tight and gap junctions occur along the lateral borders of ciliated cells and between ciliated and unciliated cells. These gap junctions possess a reduced intercellular cleft and typical P-face connexons arranged in macular plaques, with complementary E-face pits. Both cell types exhibit extensive stacks of basal and lateral interdigitations. The tight junctions found here are unusual in that they are associated with a dense cytoplasmic fuzz which is normally more characteristic of zonulae adhaerentes.     

Scanning electron microscopy of the oviduct of Salamandra salamandra (L.) (Amphibia, Urodela)

The oviduct of non-pregnant females of the ovoviviparous salamander, Salamandra salamandra, was examined using SEM-techniques. In the luminal epithelium polygonal ciliated cells were found along the entire surface of the oviduct, except the uterus, and non-ciliated cells with a varying number of short or long microvilli. The ciliated cells occur in the most anterior portion of the oviduct, the pars recta; they are sparsely distributed in the p. convoluta I, but abundant in the p. convoluta II and III. Non-ciliated cells comprise several small gland cells, restricted to the p. convoluta I, II, III, and undifferentiated cells both provided with microvilli, but difficult to be discerned from their surface appearance. The p. convoluta I, II, III is characterized by three types of secretory cells forming tubular glands, each type confined to a given zone. The secretory cells have slender microvilli at their surfaces. In freeze-cracked glands details of their secretory products can be visualized. The findings are compared to previously published TEM-investigations and discussed with regard to some functions of the oviduct during reproduction.