scholarly journals Molecular Characterization of Sub-cuticular Bacteria in New Zealand Echinoderms

2021 ◽  
Author(s):  
◽  
Scott Anthony Lawrence
Keyword(s):  
Phylogenetic Analysis ◽  
New Zealand ◽  
16S Rrna ◽  
Culture Media ◽  
Molecular Techniques ◽  
The Body ◽  
Rrna Gene ◽  
Associated Bacteria ◽  
The Relationship

<p>Many echinoderms contain sub-cuticular bacteria (SCB), symbionts which reside in the lumen between the epidermal cells and the outer cuticle of the host. The relationship is very common, with ~60% of all echinoderms studied so far containing SCB. Currently, little is known about the function of the symbiosis, although it has been hypothesized that SCB may aid in host nutrition or antimicrobial defense. Whatever their function, the large numbers of SCB observed in many echinoderms (10 (to the power of 8) - 10 (to the power of 9) SCB g-1 AFDW host tissue) suggest that they may be important to the host. Factors contributing to the lack of knowledge about the echinoderm-SCB symbiosis include the difficulty associated with cultivating symbiotic bacteria, and the lack of studies identifying the SCB by molecular means. In this study, molecular techniques were employed to characterize the SCB of several common New Zealand echinoderms. The specific objectives of the study were to identify the SCB through sequencing of a region of the bacterial 16S rRNA gene, identify and locate SCB in situ through the use of fluorescence in situ hybridization (FISH), and cultivate SCB obtained from those echinoderms which were found to contain them. Phylogenetic analysis of 16S rRNA sequences obtained from echinoderm-associated bacteria resulted in the identification of four putative species of SCB. All four bacteria were isolated from samples of Stichopus mollis (class Holothuroidea), and two of the four were also found in samples of Patiriella sp (class Asteroidea). The first putative SCB belongs to the order Rhizobiales (a-proteobacteria), and is closely related to the SCB previously isolated from the brittle star Ophiactis balli. The second species belongs to the order Chromatiales (y-proteobacteria). Putative SCB species 3 falls within the Roseobacter clade (a-proteobacteria). The phylogenetic placement of the final putative SCB is more ambiguous, as this bacterium falls among members of the a- and y- subdivisions of the phylum Proteobacteria. The nearest relatives of this final bacterium are in the orders Rickettsiales and Thiotrichales. Results of FISH assays show that Patiriella sp. and S. mollis contain SCB, while a third species, Astrostole scabra (class Asteroidea) does not. The SCB community composition was found to vary between Patiriella sp. and S. mollis. In both species, the majority of the SCB present were found to belong to the a-subdivision of the phylum Proteobacteria (>80% in both species). However, in S. mollis, ~20% of the SCB community consists of bacteria belonging to the y-subdivision of the phylum Proteobacteria, whereas bacteria belonging to this subdivision were never observed in Patiriella sp. Cultivation experiments were carried out using a range of culture media, however results were inconclusive. Ten species of proteobacteria were successfully cultivated, three of which were obtained only from Patiriella sp. and S. mollis samples and were considered possible candidates for SCB. However, phylogenetic analysis of these three bacteria revealed that closely-related bacteria are predominantly free-living species. While the possibility remains that these three bacteria are in fact SCB, it seems more likely that they represent seawater or echinoderm surface-associated bacteria. This study contributes to the body of knowledge of the echinoderm-SCB symbiosis by identifying several potential SCB in Patiriella sp. and S. mollis, and is the first to identify SCB in situ through the use of FISH. An obvious goal in studies of the echinoderm-SCB symbiosis is to determine the function of the relationship. Potential functions of the symbiosis, based on the results obtained here, are discussed herein.</p>

scholarly journals Molecular Characterization of Sub-cuticular Bacteria in New Zealand Echinoderms

2021 ◽  
Author(s):  
◽  
Scott Anthony Lawrence
Keyword(s):  
Phylogenetic Analysis ◽  
New Zealand ◽  
16S Rrna ◽  
Culture Media ◽  
Molecular Techniques ◽  
The Body ◽  
Rrna Gene ◽  
Associated Bacteria ◽  
The Relationship

<p>Many echinoderms contain sub-cuticular bacteria (SCB), symbionts which reside in the lumen between the epidermal cells and the outer cuticle of the host. The relationship is very common, with ~60% of all echinoderms studied so far containing SCB. Currently, little is known about the function of the symbiosis, although it has been hypothesized that SCB may aid in host nutrition or antimicrobial defense. Whatever their function, the large numbers of SCB observed in many echinoderms (10 (to the power of 8) - 10 (to the power of 9) SCB g-1 AFDW host tissue) suggest that they may be important to the host. Factors contributing to the lack of knowledge about the echinoderm-SCB symbiosis include the difficulty associated with cultivating symbiotic bacteria, and the lack of studies identifying the SCB by molecular means. In this study, molecular techniques were employed to characterize the SCB of several common New Zealand echinoderms. The specific objectives of the study were to identify the SCB through sequencing of a region of the bacterial 16S rRNA gene, identify and locate SCB in situ through the use of fluorescence in situ hybridization (FISH), and cultivate SCB obtained from those echinoderms which were found to contain them. Phylogenetic analysis of 16S rRNA sequences obtained from echinoderm-associated bacteria resulted in the identification of four putative species of SCB. All four bacteria were isolated from samples of Stichopus mollis (class Holothuroidea), and two of the four were also found in samples of Patiriella sp (class Asteroidea). The first putative SCB belongs to the order Rhizobiales (a-proteobacteria), and is closely related to the SCB previously isolated from the brittle star Ophiactis balli. The second species belongs to the order Chromatiales (y-proteobacteria). Putative SCB species 3 falls within the Roseobacter clade (a-proteobacteria). The phylogenetic placement of the final putative SCB is more ambiguous, as this bacterium falls among members of the a- and y- subdivisions of the phylum Proteobacteria. The nearest relatives of this final bacterium are in the orders Rickettsiales and Thiotrichales. Results of FISH assays show that Patiriella sp. and S. mollis contain SCB, while a third species, Astrostole scabra (class Asteroidea) does not. The SCB community composition was found to vary between Patiriella sp. and S. mollis. In both species, the majority of the SCB present were found to belong to the a-subdivision of the phylum Proteobacteria (>80% in both species). However, in S. mollis, ~20% of the SCB community consists of bacteria belonging to the y-subdivision of the phylum Proteobacteria, whereas bacteria belonging to this subdivision were never observed in Patiriella sp. Cultivation experiments were carried out using a range of culture media, however results were inconclusive. Ten species of proteobacteria were successfully cultivated, three of which were obtained only from Patiriella sp. and S. mollis samples and were considered possible candidates for SCB. However, phylogenetic analysis of these three bacteria revealed that closely-related bacteria are predominantly free-living species. While the possibility remains that these three bacteria are in fact SCB, it seems more likely that they represent seawater or echinoderm surface-associated bacteria. This study contributes to the body of knowledge of the echinoderm-SCB symbiosis by identifying several potential SCB in Patiriella sp. and S. mollis, and is the first to identify SCB in situ through the use of FISH. An obvious goal in studies of the echinoderm-SCB symbiosis is to determine the function of the relationship. Potential functions of the symbiosis, based on the results obtained here, are discussed herein.</p>

scholarly journals A universal subcuticular bacterial symbiont of a coral predator, the crown-of-thorns starfish, in the Indo-Pacific

2020 ◽  
Author(s):  
Naohisa Wada ◽  
Hideaki Yuasa ◽  
Rei Kajitani ◽  
Yasuhiro Gotoh ◽  
Yoshitoshi Ogura ◽  
...  
Keyword(s):  
Pacific Ocean ◽  
16S Rrna ◽  
Marine Invertebrates ◽  
Sequence Data ◽  
The Body ◽  
Rrna Gene ◽  
Ecological Knowledge ◽  
Reef Management ◽  
Genetic Traits ◽  
Associated Bacteria

AbstractBackgroundPopulation outbreaks of the crown-of-thorns starfish (Acanthaster planci sensu lato; COTS), a primary predator of reef-building corals in the Indo-Pacific Ocean, are major concerns in coral reef management. While biological and ecological knowledge of COTS has been accumulating since the 1960s, little is known about its associated bacteria. The aim of this study was to provide fundamental information on dominant COTS-associated bacteria through a multifaceted molecular approach.MethodsA total of 205 COTS individuals from 17 locations throughout the Indo-Pacific Ocean were examined for the presence of COTS-associated bacteria. We conducted 16S rRNA metabarcoding of COTS to determine the bacterial profiles of different parts of the body, and generated a full-length 16S rRNA gene sequence from a single dominant bacterium, which we designated COTS27. We performed phylogenetic analysis to determine the taxonomy, screening of COTS27 across the Indo-Pacific, FISH to visualize it within the COTS tissues, and reconstruction of the chromosome from the hologenome sequence data.ResultsWe discovered that a single bacterium exists at high densities in the subcuticular space in COTS forming a biofilm-like structure between the cuticle and the epidermis. COTS27 belongs to a clade that presumably represents a distinct order (so-called marine spirochetes) in the phylum Spirochaetes and is universally present in COTS throughout the Indo-Pacific Ocean. The reconstructed genome of COTS27 includes some genetic traits that are probably linked to adaptation to marine environments and evolution as an extracellular endosymbiont in subcuticular spaces.ConclusionsCOTS27 can be found in three allopatrically speciated COTS species, ranging from northern Red Sea to the Pacific, implying that symbiotic relationship arose before the speciation (approximately 2 million years ago). The universal association of COTS27 with COTS and nearly mono-specific association at least with the Indo-Pacific COTS potentially provides a useful model system for studying symbiont-host interactions in marine invertebrates.

Ultrastructural and molecular characterization of endosymbionts of the reed beetle genusMacroplea(Chrysomelidae, Donaciinae), and proposal of “CandidatusMacropleicola appendiculatae” and “CandidatusMacropleicola muticae”

2009 ◽  
Vol 55 (11) ◽  
pp. 1250-1260 ◽  
Author(s):  
Gregor Kölsch ◽  
Corinna Matz-Grund ◽  
Bo V. Pedersen
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
Fluorescent In Situ Hybridization ◽  
Malpighian Tubules ◽  
Rrna Gene ◽  
Beetle Species ◽  
Bacterial Symbionts ◽  
The 16S Rrna Gene

Intracellular bacterial symbionts are known from various insect groups, particularly from those feeding on unbalanced diets, where the bacteria provide essential nutrients to the host. In the case of reed beetles (Coleoptera: Chrysomelidae, Donaciinae), however, the endosymbionts appear to be associated with specialized “glands” that secrete a material used for the beetles’ unusual water-tight cocoon. These glands were discovered over a century ago, but the bacteria they contain have yet to be characterized and placed in a phylogenetic context. Here, we describe the ultrastructure of two endosymbiotic species (“ Candidatus Macropleicola appendiculatae” and “ Candidatus Macropleicola muticae”) that reside in cells of the Malpighian tubules of the reed beetle species Macroplea appendiculata and Macroplea mutica , respectively. Fluorescent in situ hybridization using oligonucleotides targeting the 16S rRNA gene specific to Macroplea symbionts verified the localization of the symbionts in these organs. Phylogenetic analysis of 16S rRNA placed “Candidatus Macropleicola” in a clade of typically endosymbiotic Enterobacteriaceae (γ-proteobacteria). Finally, we discuss the evidence available for the hypothesis that the beetle larvae use a secretion produced by the bacteria for the formation of an underwater cocoon.

scholarly journals Coral-Associated Bacteria and Their Role in the Biogeochemical Cycling of Sulfur

2009 ◽  
Vol 75 (11) ◽  
pp. 3492-3501 ◽  
Author(s):  
Jean-Baptiste Raina ◽  
Dianne Tapiolas ◽  
Bette L. Willis ◽  
David G. Bourne
Keyword(s):  
16S Rrna ◽  
Acrylic Acid ◽  
16S Rrna Gene ◽  
Bacterial Communities ◽  
Sulfur Compounds ◽  
Molecular Techniques ◽  
Rrna Gene ◽  
Coral Mucus ◽  
Associated Bacteria ◽  
Cooling Effects

ABSTRACT Marine bacteria play a central role in the degradation of dimethylsulfoniopropionate (DMSP) to dimethyl sulfide (DMS) and acrylic acid, DMS being critical to cloud formation and thereby cooling effects on the climate. High concentrations of DMSP and DMS have been reported in scleractinian coral tissues although, to date, there have been no investigations into the influence of these organic sulfur compounds on coral-associated bacteria. Two coral species, Montipora aequituberculata and Acropora millepora, were sampled and their bacterial communities were characterized by both culture-dependent and molecular techniques. Four genera, Roseobacter, Spongiobacter, Vibrio, and Alteromonas, which were isolated on media with either DMSP or DMS as the sole carbon source, comprised the majority of clones retrieved from coral mucus and tissue 16S rRNA gene clone libraries. Clones affiliated with Roseobacter sp. constituted 28% of the M. aequituberculata tissue libraries, while 59% of the clones from the A. millepora libraries were affiliated with sequences related to the Spongiobacter genus. Vibrio spp. were commonly isolated from DMS and acrylic acid enrichments and were also present in 16S rRNA gene libraries from coral mucus, suggesting that under “normal” environmental conditions, they are a natural component of coral-associated communities. Genes homologous to dddD, and dddL, previously implicated in DMSP degradation, were also characterized from isolated strains, confirming that bacteria associated with corals have the potential to metabolize this sulfur compound when present in coral tissues. Our results demonstrate that DMSP, DMS, and acrylic acid potentially act as nutrient sources for coral-associated bacteria and that these sulfur compounds are likely to play a role in structuring bacterial communities in corals, with important consequences for the health of both corals and coral reef ecosystems.

scholarly journals Layered Structure of Bacterial and Archaeal Communities and Their In Situ Activities in Anaerobic Granules

2007 ◽  
Vol 73 (22) ◽  
pp. 7300-7307 ◽  
Author(s):  
Hisashi Satoh ◽  
Yuki Miura ◽  
Ikuo Tsushima ◽  
Satoshi Okabe
Keyword(s):  
Spatial Distribution ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Clone Library ◽  
Layered Structure ◽  
Effective Diffusion ◽  
Middle Layer ◽  
Molecular Techniques ◽  
Rrna Gene

ABSTRACT The microbial community structure and spatial distribution of microorganisms and their in situ activities in anaerobic granules were investigated by 16S rRNA gene-based molecular techniques and microsensors for CH4, H2, pH, and the oxidation-reduction potential (ORP). The 16S rRNA gene-cloning analysis revealed that the clones related to the phyla Alphaproteobacteria (detection frequency, 51%), Firmicutes (20%), Chloroflexi (9%), and Betaproteobacteria (8%) dominated the bacterial clone library, and the predominant clones in the archaeal clone library were affiliated with Methanosaeta (73%). In situ hybridization with oligonucleotide probes at the phylum level revealed that these microorganisms were numerically abundant in the granule. A layered structure of microorganisms was found in the granule, where Chloroflexi and Betaproteobacteria were present in the outer shell of the granule, Firmicutes were found in the middle layer, and aceticlastic Archaea were restricted to the inner layer. Microsensor measurements for CH4, H2, pH, and ORP revealed that acid and H2 production occurred in the upper part of the granule, below which H2 consumption and CH4 production were detected. Direct comparison of the in situ activity distribution with the spatial distribution of the microorganisms implied that Chloroflexi contributed to the degradation of complex organic compounds in the outermost layer, H2 was produced mainly by Firmicutes in the middle layer, and Methanosaeta produced CH4 in the inner layer. We determined the effective diffusion coefficient for H2 in the anaerobic granules to be 2.66 × 10−5 cm2 s−1, which was 57% in water.

scholarly journals Molecular Evidence for Association of Chlamydiales Bacteria with Epitheliocystis in Leafy Seadragon (Phycodurus eques), Silver Perch (Bidyanus bidyanus), and Barramundi (Lates calcarifer)

2006 ◽  
Vol 72 (1) ◽  
pp. 284-290 ◽  
Author(s):  
Adam Meijer ◽  
Paul J. M. Roholl ◽  
Jacobus M. Ossewaarde ◽  
Brian Jones ◽  
Barbara F. Nowak
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Molecular Evidence ◽  
Rrna Gene ◽  
Lates Calcarifer ◽  
The Family ◽  
First Time ◽  
Specific Sequences

ABSTRACT Epitheliocystis in leafy seadragon (Phycodurus eques), silver perch (Bidyanus bidyanus), and barramundi (Lates calcarifer), previously associated with chlamydial bacterial infection using ultrastructural analysis, was further investigated by using molecular and immunocytochemical methods. Morphologically, all three species showed epitheliocystis cysts in the gills, and barramundi also showed lymphocystis cysts in the skin. From gill cysts of all three species and from skin cysts of barramundi 16S rRNA gene fragments were amplified by PCR and sequenced, which clustered by phylogenetic analysis together with other chlamydia-like organisms in the order Chlamydiales in a lineage separate from the family Chlamydiaceae. By using in situ RNA hybridization, 16S rRNA Chlamydiales-specific sequences were detected in gill cysts of silver perch and in gill and skin cysts of barramundi. By applying immunocytochemistry, chlamydial antigens (lipopolysaccharide and/or membrane protein) were detected in gill cysts of leafy seadragon and in gill and skin cysts of barramundi, but not in gill cysts of silver perch. In conclusion, this is the first time epitheliocystis agents of leafy seadragon, silver perch and barramundi have been undoubtedly identified as belonging to bacteria of the order Chlamydiales by molecular methods. In addition, the results suggested that lymphocystis cysts, known to be caused by iridovirus infection, could be coinfected with the epitheliocystis agent.

scholarly journals Molecular Characterization of Copy’s Frog Hydrophylax Leptoglossa Based on 16s rRNA Gene

2021 ◽  
Vol 49 (1) ◽  
pp. 105-115
Author(s):  
Mehrab Chowdhury ◽  
Abu Sinha ◽  
Md Salim Mahabub Rony ◽  
Hawa Jahan ◽  
Ashfaqul Muid Khandaker ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Genomic Dna ◽  
Molecular Techniques ◽  
The Body ◽  
Monophyletic Group ◽  
Morphological Identification ◽  
Rrna Gene ◽  
Anuran Species

Copy’s frog, commonly observed in Bangladesh, is ecologically important for evergreen forest. Sample was collected from Satchari National park, Hobigonj, Sylhet. Attempts were made to combine morphological and molecular techniques for identification and analysis of evolutionary relation of Copy’s frog with anuran species. Morphological identification of Hydrophylax sp. was conducted based on distinguishing characteristics of the body and different body measurement parameter. The species was identified morphologically as Hydrophylax leptoglossa based on the taxonomic key of finger toes and formula was 3>1>4>2. Simultaneously genomic DNA was isolated using CTAB extraction method. The 16S rRNA gene was amplified from the genomic DNA by polymerase chain reaction method using the pair of universal primers. Amplified product was a 308 bp fragment that generated AT/GC ratio of 1.08% once sequenced. In the present study comparative analysis of intraspecific and interspecific variation was conducted based on nucleotide sequence of 16S rRNA gene collected from GenBank. The intraspecific variation compares between Southeast Asia and Bangladesh was 1.29% for 16S rRNA gene. The interspecific genetic divergence was studied between H. leptoglossa and H. tytleri was observed 14.29%. The nucleotide sequence of the present study will be used as DNA barcode for that particular individual collected from Sylhet, Bangladesh. Furthermore, a molecular phylogenetic tree was constructed, where all the anuran species from a monophyletic group. Four species of Hydrophylax form monophyletic group and is consistent with relation based on morphology. Bangladesh J. Zool. 49(1): 105-115, 2021

scholarly journals Molecular detection of Campylobacter species from human and cattle faecal samples in Kilosa District, Tanzania

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Noel Gahamanyi ◽  
Leonard E.G. Mboera ◽  
Mecky I. Matee ◽  
Dieudonné Mutangana ◽  
Raghavendra G. Amachawadi ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
Molecular Techniques ◽  
16S Rrna Sequencing ◽  
Rrna Gene ◽  
Faecal Samples ◽  
Animal Pathogens ◽  
Rrna Sequencing ◽  
Campylobacter Species ◽  
Reference Strains

A growing number of Campylobacter species other than C. jejuni and C. coli have been considered as emerging human and animal pathogens but their contribution to human gastroenteritis is poorly documented. This study aimed at detecting Campylobacter species from human and cattle faecal samples in Kilosa District, Tanzania using molecular techniques without culture. Seventy (70) faecal samples were collected from five diarrheic and 65 non-diarrheic human patients attending Kilosa District Hospital in Tanzania from July to October 2019. During the same period, 30 faecal samples were also collected from healthy cattle in the same district. Genus and species identification of Campylobacter was conducted on the samples using molecular techniques [the polymerase chain reaction (PCR) and 16S rRNA sequencing]. Phylogenetic analysis was carried out by comparison of the 16S rRNA gene sequences to reference strains by the Neighbor-Joining method in MEGA X. Campylobacter species detection rate by PCR was 65.7% (46/70) and 20% (6/30) in humans and cattle, respectively.  There were five human diarrheic cases, four of which were positive for Campylobacter and of these, two were children ≤15 years of age. In humans, 16S rRNA sequencing revealed that C. concisus was the most predominant species occurring at a frequency of 37.8% (14/37), followed by uncultured Campylobacter spp. 24.3% (9/37) and C. hominis 21.6% (8/37). The least represented species were C. jejuni and C. lanienae, all occurring at 2.7% (1/37). In cattle, five (100%) sequenced PCR products matched with C. lanienae. Phylogenetic analysis revealed that with the exception of C. lanienae, 16S rRNA sequences of Campylobacter species were closely related to the reference strains used (Percent identity: 90.51-96.56%). Based on our findings, we recommend that molecular techniques, mainly PCR be adopted for the direct detection of Campylobacter species during laboratory screening and surveillance studies.

scholarly journals Mycoplasma ovis comb. nov. (formerly Eperythrozoon ovis), an epierythrocytic agent of haemolytic anaemia in sheep and goats

2004 ◽  
Vol 54 (2) ◽  
pp. 365-371 ◽  
Author(s):  
Harold Neimark ◽  
Brent Hoff ◽  
Martin Ganter
Keyword(s):  
Phylogenetic Analysis ◽  
16S Rrna ◽  
Haemolytic Anaemia ◽  
Blood Feeding ◽  
Rrna Gene ◽  
New Approach ◽  
Sheep And Goats ◽  
Animal Hosts ◽  
The Relationship ◽  
The 16S Rrna Gene

Eperythrozoon ovis, an erythrocytic agent that causes haemolytic anaemia in sheep and goats, occurs worldwide and is currently thought to be a rickettsia. To determine the relationship between this agent and other haemotrophic bacterial parasites, the 16S rRNA gene of this organism was sequenced. Phylogenetic analysis revealed that this wall-less bacterium is not a rickettsia, but a mycoplasma. This mycoplasma is related closely to several other uncultivated, epierythrocytic mycoplasmas that comprise a recently identified group, the haemotrophic mycoplasmas (haemoplasmas). The haemoplasma group is composed of former Eperythrozoon and Haemobartonella species, as well as newly identified epierythrocytic mycoplasmas. Haemoplasmas parasitize the surface of erythrocytes of a wide variety of vertebrate animal hosts and are transmitted mainly by blood-feeding arthropod vectors. Recognition that E. ovis is a mycoplasma provides a new approach to dealing with this bacterium. It is proposed that E. ovis should be reclassified as Mycoplasma ovis comb. nov.

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