ABSTRACT
Missing values (NA) often occur in cancer research, which may be due to reasons such as data protection, data loss, or missing follow-up data. Such incomplete patient information can have an impact on prediction models and other data analyses. Imputation methods are a tool for dealing with NA. Cancer data is often presented in an ordered categorical form, such as tumour grading and staging, which requires special methods. This work compares mode imputation, k nearest neighbour (knn) imputation, and, in the context of Multiple Imputation by Chained Equations (MICE), logistic regression model with proportional odds (mice_polr) and random forest (mice_rf) on a real-world prostate cancer dataset provided by the Cancer Registry of Rhineland-Palatinate in Germany. Our dataset contains relevant information for the risk classification of patients and the time between date of diagnosis and date of death. For the imputation comparison, we use Rubin's (1974) Missing Completely At Random (MCAR) mechanism to remove 10%, 20%, 30%, and 50% observations. The results are evaluated and ranked based on the accuracy per patient. Mice_rf performs significantly best for each percentage of NA, followed by knn, and mice_polr performs significantly worst. Furthermore, our findings indicate that the accuracy of imputation methods increases with a lower number of categories, a relatively even proportion of patients in the categories, or a majority of patients in a particular category.
Subject(s)
Prostatic Neoplasms , Male , Humans , Germany , Registries , Data Interpretation, StatisticalABSTRACT
BACKGROUND: This double-blind, active-controlled, randomized, multinational study evaluated the efficacy, safety, pharmacokinetics (PK), and immunogenicity of PF-06438179/GP1111 (IxifiTM/Zessly®), an infliximab biosimilar, vs infliximab (Remicade®) reference product sourced from the European Union (infliximab-EU) in biologic-naïve patients with moderate to severe active rheumatoid arthritis (RA) despite methotrexate therapy. This paper reports results from the initial 30-week treatment period. METHODS: Patients (N = 650) were stratified by geographic region and randomized 1:1 to PF-06438179/GP1111 or infliximab-EU (3 mg/kg intravenous at weeks 0, 2, and 6, then every 8 weeks). Dose escalation to 5 mg/kg was allowed starting at week 14 for patients with inadequate RA response. The primary endpoint was American College of Rheumatology criteria for ≥ 20% clinical improvement (ACR20) response at week 14. Therapeutic equivalence was declared if the two-sided 95% CI for the treatment difference was within the symmetric equivalence margin of ± 13.5%. Statistical analysis was also performed with a two-sided 90% CI using an asymmetric equivalence margin (- 12.0%, 15.0%). RESULTS: Patients (80.3% female; 79.4% seropositive) had a mean RA duration of 6.9 years, and mean baseline Disease Activity Score in 28 joints, four components based on C-reactive protein was 6.0 in both arms. Week 14 ACR20 in the intention-to-treat population was 62.7% for PF-06438179/GP1111 and 64.1% for infliximab-EU. Week 14 ACR20 using nonresponder imputation was 61.1% for PF-06438179/GP1111 and 63.5% for infliximab-EU, and the 95% (- 9.92%, 5.11%) and 90% (- 8.75%, 4.02%) CIs for the treatment difference (- 2.39%) were entirely contained within the prespecified symmetric and asymmetric equivalence margins, respectively. No differences were observed between arms for secondary efficacy endpoints. Overall postdose antidrug antibody (ADA) rates through week 30 were 48.6% and 51.2% for PF-06438179/GP1111 and infliximab-EU, respectively. Efficacy and immunogenicity were similar between treatments for patients with dose escalation (at or after week 14), as well as between treatments for patients without dose escalation. Safety profiles of PF-06438179/GP1111 and infliximab-EU were similar, with no clinically meaningful differences observed between arms, including after ADA development. Serum drug concentrations were similar between arms at each time point during the initial 30-week treatment period. CONCLUSION: PF-06438179/GP1111 and infliximab-EU demonstrated similar efficacy, safety, immunogenicity, and PK with or without dose escalation in patients with moderate to severe active RA on background methotrexate. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02222493 . Registered on 21 August 2014. EudraCT, 2013-004148-49 . Registered on 14 July 2014.
Subject(s)
Antirheumatic Agents/pharmacokinetics , Antirheumatic Agents/therapeutic use , Arthritis, Rheumatoid/drug therapy , Biosimilar Pharmaceuticals/pharmacokinetics , Biosimilar Pharmaceuticals/therapeutic use , Infliximab/pharmacokinetics , Infliximab/therapeutic use , Adult , Aged , Double-Blind Method , Female , Humans , Male , Methotrexate/therapeutic use , Middle Aged , Therapeutic EquivalencyABSTRACT
BACKGROUND: HX575 (biosimilar epoetin alfa) was approved in Europe in 2007 for the treatment of chronic kidney disease (CKD)-related anemia. This study assessed the clinical equivalence of HX575 with the US-licensed reference epoetin alfa (Epogen®/Procrit®, Amgen/Janssen) following subcutaneous (SC) administration in dialysis patients with CKD-related anemia. METHODS: This randomized, double-blind, parallel-group, multicenter study (NCT01693029) was conducted at 49 US clinical sites. Eligible patients were aged ≥18 years, had end-stage renal disease, were on hemodialysis or peritoneal dialysis for ≥6 months (or ≥12 months in the case of a failed kidney transplant), and were receiving treatment with stable SC doses of epoetin alfa. Eligible patients also had mean hemoglobin (Hb) concentration between 9.0 and 11.5 g/dL during the screening period. The primary endpoint was the mean absolute change in Hb concentration between the screening/baseline period (week-4 to week-1) and the evaluation period (weeks 21 to 28). RESULTS: Hb values at the end of the evaluation period and the Hb change from baseline to evaluation period were similar between treatment groups. The estimated difference between groups in mean absolute change in Hb concentration was -0.093 g/dL, with 90% CI (-0.23 to 0.04) entirely within the pre-specified equivalence limits (-0.5 to 0.5 g/dL). The safety profile of each medicine was similar and as expected in dialysis patients, and neither method of treatment led to the development of neutralizing, clinically relevant antibodies. CONCLUSIONS: SC HX575 in dialysis patients with renal anemia was therapeutically equivalent to the reference medicine in terms of maintaining stable Hb levels and safety.
Subject(s)
Anemia/drug therapy , Biosimilar Pharmaceuticals/therapeutic use , Epoetin Alfa/therapeutic use , Hematinics/therapeutic use , Kidney Failure, Chronic/drug therapy , Adult , Aged , Aged, 80 and over , Anemia/blood , Double-Blind Method , Female , Hemoglobins/analysis , Humans , Kidney Failure, Chronic/blood , Male , Middle Aged , Renal Dialysis , Therapeutic Equivalency , Treatment Outcome , United States , Young AdultABSTRACT
AIM: To assess the safety and immunogenicity of subcutaneous (SC) HX575 (epoetin-α) in dialysis- and nondialysis-dependent adult patients with chronic kidney disease (CKD). METHODS: Open-label, single-arm, multicenter study in patients (n = 416) from Germany, Italy, Poland, Romania, Russia, Turkey, and Ukraine. RESULTS: Mean (standard deviation (SD)) age was 52.3 (15.8) years, all patients were Caucasian, and similar proportions were male/female. 250 patients (60.1%) were erythropoiesis-stimulating agent (ESA)-naïve, and 166 (39.9%) were receiving ESA maintenance therapy at study start; mean (SD) on-study treatment duration with HX575 was 43.4 (15.8) weeks and 45.3 (13.7) weeks, respectively. Binding antierythropoietin (EPO) antibodies were detected by radioimmunoprecipitation (RIP) assay in 7 patients (1.7%; incidence 0.019); 5 of these were ESA-naïve at study entry. No patient developed neutralizing antibodies as determined in a cell-based epoetin neutralizing assay. Of the 7 patients with a positive binding anti-EPO RIP assay, 4 tested negative at later time points while continuing HX575 treatment. Three patients had low titers of anti-EPO antibodies at the last study assessment. There were no clinical signs of immunogenicity or hypersensitivity. CONCLUSIONS: SC HX575 was effective for correcting and maintaining correction of anemia, and the mean weekly dose remained stable over time.â©.
Subject(s)
Anemia/drug therapy , Epoetin Alfa/adverse effects , Hematinics/adverse effects , Recombinant Proteins/adverse effects , Renal Dialysis/adverse effects , Renal Insufficiency, Chronic/complications , Adult , Aged , Anemia/etiology , Epoetin Alfa/therapeutic use , Erythropoietin , Europe , Female , Hematinics/therapeutic use , Humans , Male , Middle Aged , Recombinant Proteins/therapeutic use , Renal Insufficiency, Chronic/therapyABSTRACT
Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.
Subject(s)
Deltaproteobacteria/classification , Deltaproteobacteria/metabolism , Drug Discovery , Animals , Biosynthetic Pathways/genetics , Deltaproteobacteria/genetics , Deltaproteobacteria/physiology , Environmental Microbiology , Genes, Bacterial/genetics , Genome, Bacterial/genetics , Metagenomics , Molecular Sequence Data , Multigene Family/genetics , Peptides/metabolism , Polyketides/metabolism , Porifera/metabolism , Porifera/microbiology , Single-Cell Analysis , SymbiosisABSTRACT
The oviparous sponge Ectyoplasia ferox is commonly found in Florida and the Bahamas. Every year in August and/or September about 6 days after a full moon, E. ferox will shed embryo-containing spawning material into the seawater from which hundreds to thousands of larvae will hatch per host individual. In order to investigate vertical microbial transmission in E. ferox, 16S rRNA gene library construction and denaturing gradient gel electrophoresis was employed. Microbial symbionts from six phyla and the unknown lineage SAUL were shown to be vertically transmitted. The identification of 21 VT clusters, of which 19 were situated within sponge-specific or sponge-coral-specific clusters, indicated that a large fraction of the symbiotic microbial consortium was present in the sexual reproductive stages. Spawning led to a 50 % reduction of microbial numbers in the adult sponge mesohyl. We furthermore provide the first evidence that the symbiotic microbial consortia of E. ferox were generally metabolically active within the reproductive stages. Finally, we propose E. ferox as a model system for vertical transmission owing to the ease of experimental access to all sexual reproductive stages, and to experimental tractability in the laboratory including the possibility of rearing symbiont-free juvenile sponges.
Subject(s)
Bacteria/isolation & purification , Porifera/microbiology , Symbiosis , Animals , Bacteria/classification , Bacteria/genetics , DNA, Bacterial/genetics , Denaturing Gradient Gel Electrophoresis , Gene Library , Larva/microbiology , Larva/physiology , Phylogeny , Porifera/physiology , RNA, Ribosomal, 16S/genetics , Reproduction , Sequence Analysis, DNAABSTRACT
Numerous studies have reported the existence of sponge-specific 16S ribosomal RNA (rRNA) gene sequence clusters, representing bacteria found in sponges but not detected in other environments, such as seawater. The advent of deep-sequencing technologies allows us to examine the rare microbial biosphere in order to establish whether these bacteria are truly sponge specific, or are more widely distributed but only at abundances below the detection limit of conventional molecular approaches. We screened >12 million publicly available 16S rRNA gene pyrotags derived from 649 seawater, sediment, hydrothermal vent and coral samples from temperate, tropical and polar regions. We detected 77 of the 173 previously described sponge-specific clusters in seawater or other non-sponge samples, albeit generally at extremely low abundances. Sequences representing the candidate phylum 'Poribacteria', previously thought to be largely restricted to sponges, were recovered from 46 (out of 411) seawater and 41 (out of 129) sediment samples. While the presence of an organism does not imply that it is active in situ, our results do suggest that many 'sponge-specific' bacteria occur more widely outside of sponge hosts than previously thought.
Subject(s)
Bacteria/classification , Biodiversity , Porifera/microbiology , Seawater/microbiology , Animals , Bacteria/genetics , Bacteria/isolation & purification , DNA, Bacterial/genetics , Geologic Sediments/microbiology , High-Throughput Nucleotide Sequencing , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Species SpecificityABSTRACT
It has long been recognized that sponges differ in the abundance of associated microorganisms, and they are therefore termed either 'low microbial abundance' (LMA) or 'high microbial abundance' (HMA) sponges. Many previous studies concentrated on the dense microbial communities in HMA sponges, whereas little is known about microorganisms in LMA sponges. Here, two LMA sponges from the Red Sea, two from the Caribbean and one from the South Pacific were investigated. With up to only five bacterial phyla per sponge, all LMA sponges showed lower phylum-level diversity than typical HMA sponges. Interestingly, each LMA sponge was dominated by a large clade within either Cyanobacteria or different classes of Proteobacteria. The overall similarity of bacterial communities among LMA sponges determined by operational taxonomic unit and UniFrac analysis was low. Also the number of sponge-specific clusters, which indicate bacteria specifically associated with sponges and which are numerous in HMA sponges, was low. A biogeographical or host-dependent distribution pattern was not observed. In conclusion, bacterial community profiles of LMA sponges are clearly different from profiles of HMA sponges and, remarkably, each LMA sponge seems to harbour its own unique bacterial community.
Subject(s)
Biodiversity , Cyanobacteria/classification , Microbial Consortia , Porifera/microbiology , Proteobacteria/classification , Animals , Caribbean Region , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , Indian Ocean , Microscopy, Electron, Transmission , Pacific Ocean , Phylogeny , RNA, Ribosomal, 16S/geneticsABSTRACT
Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world's oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.
Subject(s)
Bacteria/classification , Biodiversity , Metagenome , Porifera/microbiology , Animals , Bacteria/genetics , DNA, Bacterial/genetics , Genes, rRNA , Geography , Oceans and Seas , Phylogeny , Porifera/genetics , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Species Specificity , Tropical ClimateABSTRACT
Some marine sponges harbor dense and phylogenetically complex microbial communities [high microbial abundance (HMA) sponges] whereas others contain only few and less diverse microorganisms [low microbial abundance (LMA) sponges]. We focused on the phylum Chloroflexi that frequently occurs in sponges to investigate the different associations with three HMA and three LMA sponges from New Zealand. By applying a range of microscopical and molecular techniques a clear dichotomy between HMA and LMA sponges was observed: Chloroflexi bacteria were more abundant and diverse in HMA than in LMA sponges. Moreover, different HMA sponges contain similar Chloroflexi communities whereas LMA sponges harbor different and more variable communities which partly resemble Chloroflexi seawater communities. A comprehensive phylogenetic analysis of our own and publicly available sponge-derived Chloroflexi 16S rRNA gene sequences (> 780 sequences) revealed the enormous diversity of this phylum within sponges including 29 sponge-specific and sponge-coral clusters (SSC/SCC) as well as a 'supercluster' consisting of > 250 sponge-derived and a single nonsponge-derived 16S rRNA gene sequence. Interestingly, the majority of sequences obtained from HMA sponges, but only a few from LMA sponges, fell into SSC/SCC clusters. This indicates a much more specific association of Chloroflexi bacteria with HMA sponges and suggests an ecologically important role for these prominent bacteria.
Subject(s)
Biodiversity , Chloroflexi/genetics , Phylogeny , Porifera/microbiology , Animals , Bacterial Typing Techniques , Chloroflexi/classification , Chloroflexi/growth & development , DNA, Bacterial/genetics , Denaturing Gradient Gel Electrophoresis , Gene Library , In Situ Hybridization, Fluorescence , New Zealand , RNA, Ribosomal, 16S/genetics , Seawater/microbiologyABSTRACT
The phylogenetic diversity of microorganisms in marine sponges is becoming increasingly well described, yet relatively little is known about the activities of these symbionts. Given the seemingly favourable environment provided to microbes by their sponge hosts, as indicated by the extraordinarily high abundance of sponge symbionts, we hypothesized that the majority of sponge-associated bacteria are active in situ. To test this hypothesis we compared, for the first time in sponges, 16S rRNA gene- vs 16S rRNA-derived bacterial community profiles to gain insights into symbiont composition and activity, respectively. Clone libraries revealed a highly diverse bacterial community in Ancorina alata, and a much lower diversity in Polymastia sp., which were identified by electron microscopy as a high- and a low-microbial abundance sponge, respectively. Substantial overlap between DNA and RNA libraries was evident at both phylum and phylotype levels, indicating in situ activity for a large fraction of sponge-associated bacteria. This active fraction included uncultivated, sponge-specific lineages within, for example, Actinobacteria, Chloroflexi and Gemmatimonadetes. This study shows the potential of RNA vs DNA comparisons based on the 16S rRNA gene to provide insights into the activity of sponge-associated microorganisms.
Subject(s)
Bacteria/classification , Biodiversity , DNA, Bacterial/genetics , Genes, rRNA/genetics , Porifera/microbiology , RNA, Bacterial/genetics , RNA, Ribosomal, 16S/genetics , Animals , Bacteria/genetics , Bacteria/isolation & purification , DNA, Bacterial/chemistry , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
Palytoxin (PTX), one of the most potent and chemically complex marine toxins, is predominantly found in zoanthid corals and sporadically in dinoflagellates. Its biosynthesis and metabolic pathways are largely unknown. However, the widespread occurrence of the toxin in phylogenetically distinct marine organisms is consistent with its production by microorganisms and subsequent accumulation in the food chain. To investigate a possible microbial origin, bacteria from two zoanthid corals (Palythoa caribaeorum, Zoanthus pulchellus) and one sponge (Neofibularia nolitangere) were isolated. More than 250 bacteria were screened for hemolysis using a newly developed PTX-screening assay of which 7% showed PTX-like hemolytic activity. 16S rRNA gene sequencing revealed that these bacterial isolates belonged to strains of Bacillus cereus group (n = 11) as well as the genera Brevibacterium (n = 4) and Acinetobacter (n = 2). The results indicate the presence of Na+/K+-ATPase toxins and possibly PTX in hemolytic bacteria from P. caribaeorum.
Subject(s)
Acrylamides/metabolism , Anthozoa/microbiology , Bacteria/classification , Bacteria/isolation & purification , Hemolysin Proteins/biosynthesis , Animals , Cluster Analysis , Cnidarian Venoms , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNAABSTRACT
Many marine sponges, hereafter termed high-microbial-abundance (HMA) sponges, harbor large and complex microbial consortia, including bacteria and archaea, within their mesohyl matrices. To investigate vertical microbial transmission as a strategy to maintain these complex associations, an extensive phylogenetic analysis was carried out with the 16S rRNA gene sequences of reproductive (n = 136) and adult (n = 88) material from five different Caribbean species, as well as all published 16S rRNA gene sequences from sponge offspring (n = 116). The overall microbial diversity, including members of at least 13 bacterial phyla and one archaeal phylum, in sponge reproductive stages is high. In total, 28 vertical-transmission clusters, defined as clusters of phylotypes that are found both in adult sponges and their offspring, were identified. They are distributed among at least 10 bacterial phyla and one archaeal phylum, demonstrating that the complex adult microbial community is collectively transmitted through reproductive stages. Indications of host-species specificity and cospeciation were not observed. Mechanistic insights were provided using a combined electron microscopy and fluorescence in situ hybridization analysis, and an indirect mechanism of vertical transmission via nurse cells is proposed for the oviparous sponge Ectyoplasia ferox. Based on these phylogenetic and mechanistic results, we suggest the following symbiont transmission model: entire microbial consortia are vertically transmitted in sponges. While vertical transmission is clearly present, additional environmental transfer between adult individuals of the same and even different species might obscure possible signals of cospeciation. We propose that associations of HMA sponges with highly sponge-specific microbial communities are maintained by this combination of vertical and horizontal symbiont transmission.
Subject(s)
Archaea/classification , Bacteria/classification , Biodiversity , Porifera/microbiology , Porifera/physiology , Animals , Archaea/isolation & purification , Archaea/physiology , Bacteria/isolation & purification , Bacterial Physiological Phenomena , DNA, Archaeal/chemistry , DNA, Archaeal/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Genes, rRNA , In Situ Hybridization, Fluorescence , Microscopy, Electron, Transmission , Molecular Sequence Data , Phylogeny , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Sequence Homology, Nucleic Acid , SymbiosisABSTRACT
The potential for nitrification in the Mediterranean sponge Aplysina aerophoba was assessed using a combined physiological and molecular approach. Nitrate excretion rates in whole sponges reached values of up to 344 nmol g(-1) dry weight (wt) h(-1) (unstimulated) and 1325 nmol g(-1) dry wt h(-1) (stimulated). Addition of nitrapyrin, a nitrification-specific inhibitor, effectively inhibited nitrate excretion. Ammonium was taken up by sponges in spring and excreted in fall, the sponges thus serving as either an ammonium sink or ammonium source. Nitrosospira cluster 1 and Crenarchaeota group I.1A 16S rRNA and amoA genes were recovered from A. aerophoba and other sponges from different world's oceans. The archaeal 16S rRNA genes formed a sponge-specific subcluster, indicating that their representatives are members of the stable microbial community of sponges. On the other hand, clustering was not evident for Nitrosospira rRNA genes which is consistent with their presence in sediment and seawater samples. The presence of both Nitrosospira cluster 1 and crenarchaeal group 1 phylotypes in sponge tissue was confirmed using fluorescently labelled 16S rRNA gene probes. This study contributes to an ongoing effort to link microbial diversity with metabolic functions in the phylogenetically diverse, elusive and so far uncultivated microbial communities of marine sponges.
Subject(s)
Ammonia/metabolism , Crenarchaeota/classification , Crenarchaeota/metabolism , Nitrites/metabolism , Nitrosomonadaceae/classification , Nitrosomonadaceae/metabolism , Porifera/microbiology , Animals , Cluster Analysis , Crenarchaeota/genetics , DNA, Archaeal/chemistry , DNA, Archaeal/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Enzyme Inhibitors/pharmacology , Genes, Archaeal , Genes, Bacterial , Molecular Sequence Data , Nitrates/metabolism , Nitrosomonadaceae/genetics , Phylogeny , Picolines/pharmacology , RNA, Ribosomal, 16S/genetics , Seasons , Sequence Analysis, DNAABSTRACT
Many marine demosponges contain large amounts of phylogenetically complex yet highly sponge-specific microbial consortia within the mesohyl matrix, but little is known about how these microorganisms are acquired by their hosts. Settlement experiments were performed with the viviparous Caribbean demosponge Ircinia felix to investigate the role of larvae in the vertical transmission of the sponge-associated microbial community. Inspections by electron microscopy revealed large amounts of morphologically diverse microorganisms in the center of I. felix larvae, while the outer rim appeared to be devoid of microorganisms. In juveniles, microorganisms were found between densely packed sponge cells. Denaturing gradient gel electrophoresis (DGGE) was performed to compare the bacterial community profiles of adults, larvae, and juvenile sponges. Adults and larvae were highly similar in DGGE band numbers and banding patterns. Larvae released by the same adult individual contained highly similar DGGE banding patterns, whereas larvae released by different adult individuals showed slightly different DGGE banding patterns. Over 200 bands were excised, sequenced, and phylogenetically analyzed. The bacterial diversity of adult I. felix and its larvae was comparably high, while juveniles showed reduced diversity. In total, 13 vertically transmitted sequence clusters, hereafter termed "IF clusters," that contained sequences from both the adult sponge and offspring (larvae and/or juveniles) were found. The IF clusters belonged to at least four different eubacterial phyla and one possibly novel eubacterial lineage. In summary, it could be shown that in I. felix, vertical transmission of microorganisms through the larvae is an important mechanism for the establishment of the sponge-microbe association.
Subject(s)
Bacteria/classification , Porifera/microbiology , Water Microbiology , Animals , Bacteria/genetics , Electrophoresis , Larva/microbiology , Microscopy, Electron, Scanning , Phylogeny , Porifera/ultrastructureABSTRACT
18S ribosomal DNA and internal transcribed spacer 2 (ITS-2) full-length sequences, each of which was sequenced three times, were used to construct phylogenetic trees with alignments based on secondary structures, in order to elucidate genealogical relationships within the Aplysinidae (Verongida). The first poriferan ITS-2 secondary structures are reported. Altogether 11 Aplysina sponges and 3 additional sponges (Verongula gigantea, Aiolochroia crassa, Smenospongia aurea) from tropical and subtropical oceans were analyzed. Based on these molecular studies, S. aurea, which is currently affiliated with the Dictyoceratida, should be reclassified to the Verongida. Aplysina appears as monophyletic. A soft form of Aplysina lacunosa was separated from other Aplysina and stands at a basal position in both 18S and ITS-2 trees. Based on ITS-2 sequence information, the Aplysina sponges could be distinguished into a single Caribbean-Eastern Pacific cluster and a Mediterranean cluster. The species concept for Aplysina sponges as well as a phylogenetic history with a possibly Tethyan origin is discussed.