Microbial and chemical patterns during a disease outbreak in the sponge, Aplysina aerophoba, from Slovenia

Samples of Aplysina aerophoba were collected by scuba at 3-7 m depth at Pacug, Slovenia for microbiological and chemical comparison between sponge lesions and unaffected tissue on 10 diseased sponges and 10 healthy controls. To estimate disease incidence, a survey was performed along a randomly selected 15m line transect: all healthy and diseased specimens found within 0.5 m to either side were recorded: 53 Aplysina aerophoba colonies were recorded of which, 22 presented visible disease symptoms (black patches, white necrotic tissue or exposed skeletal fibres).DGGE was used to explore the similarity in microbial communities between healthy and diseased samples. A total of 65 unique bands were used to compare community similarity between samples, of these, 24 were sequenced to obtain phylogenetic detail. 16S rRNA gene sequencing disclosed bacterial phyla with representatives of the Alpha-, Gamma-, Delta- and Epsilon-proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chloroflexi, Firmicutes and Cyanobacteria. The Bacteroidetes (13, 19 and 21) and Epsilonproteobacteria (20 and 24) were only detected in diseased sponges.A more comprehensive phylogenetic comparison between the diseased and healthy samples using clone sequence data showed considerable differences proportions of the microbial community composition. Rarefaction analysis was performed to determine the number of unique bacterial clones as a proportion of the estimated total diversity.To determine if any eukaryotic organisms were specifically associated with the lesions on Aplysina aerophoba, DGGE was conducted with universal eukaryotic primers. A large number of bands were detected, but a single band was present in all diseased samples (with the exception of D2) and absent from the unaffected tissue of diseased sponges and healthy controls. 18S rRNA gene sequencing of this band revealed 96% sequence homology to the Harpactic copepod, Tisbe furcata.A maximum-likelihood phylogenetic tree was constructed from analysis of all 16S rRNA gene sequences retrieved from DGGE analysis. HPLC-UV-MS was used to acquire chemical profiles of healthy, diseased and unaffected portions of diseased Aplysina aerophoba tissue.
The study aimed to understand the microbiology associated with 'Aplysina Black Patch Syndrome' and to determine if shifts in sponge metabolite production occur with disease.

研究人员于斯洛文尼亚帕库格（Pacug）海域3-7米水深处，通过水肺潜水采集气生阿普萨海绵（Aplysina aerophoba）样本，用于开展10条患病海绵与10条健康对照海绵的病灶组织与健康组织的微生物学及化学对比分析。
为估算病害发生率，研究人员沿随机布设的15米样线开展调查：记录样线两侧0.5米范围内的所有健康与患病海绵个体。本次调查共记录到53个气生阿普萨海绵（Aplysina aerophoba）群落，其中22个表现出可见病害症状（黑色斑块、白色坏死组织或裸露的骨骼纤维）。
采用变性梯度凝胶电泳（DGGE）分析健康与患病样本的微生物群落相似性，共选取65个特异性条带用于样本间群落相似性对比，其中24个条带被测序以获取系统发育信息。
通过16S核糖体RNA（16S rRNA）基因测序，共鉴定出涵盖α-、γ-、δ-及ε-变形菌纲、酸杆菌门（Acidobacteria）、拟杆菌门（Bacteroidetes）、放线菌门（Actinobacteria）、绿弯菌门（Chloroflexi）、厚壁菌门（Firmicutes）以及蓝细菌门（Cyanobacteria）的细菌类群。
拟杆菌门（条带13、19与21）和ε-变形菌纲（条带20与24）仅在患病海绵中被检测到。
利用克隆测序数据对患病与健康样本开展更全面的系统发育对比分析，结果显示二者的微生物群落组成占比存在显著差异。
采用稀化曲线分析（Rarefaction analysis）估算独特细菌克隆子数量占预估总多样性的比例。
为明确是否存在与气生阿普萨海绵病灶特异性结合的真核生物，研究人员使用通用真核引物开展变性梯度凝胶电泳（DGGE）分析。
本次检测到大量条带，但存在一条仅在所有患病样本（D2除外）中出现的特异性条带，且该条带未在患病海绵的健康组织与健康对照样本中检出。
对该条带开展18S核糖体RNA（18S rRNA）基因测序，结果显示其与猛水蚤目（Harpactic）的叉胸猛水蚤（Tisbe furcata）的序列同源性达96%。
基于从DGGE分析中获取的全部16S rRNA基因序列，构建了最大似然系统发育树（maximum-likelihood phylogenetic tree）。
采用高效液相色谱-紫外-质谱联用技术（HPLC-UV-MS）获取健康海绵组织、患病海绵的病灶组织以及患病海绵自身的非病灶健康组织的化学图谱。
本研究旨在明确与‘气生阿普萨海绵黑斑病综合征（Aplysina Black Patch Syndrome）’相关的微生物学特征，并探究海绵代谢产物的合成是否随病害发生出现变化。