The role of tissue type, sampling and nucleic acid purification methodology on the inferred composition of Pacific oyster ( Crassostrea gigas ) microbiome

Abstract

Aims This study evaluated methods to sample and extract nucleic acids from Pacific oysters to accurately determine the microbiome associated with different tissues. Methods and Results Samples were collected from haemolymph, gill, gut and adductor muscle, using swabs and homogenates of solid tissues. Nucleic acids were extracted from fresh and frozen samples using three different commercial kits. The bacterial DNA yield varied between methods (P < 0·05) and each tissue harboured a unique microbiota, except for gill and muscle. Higher bacterial DNA yields were obtained by swabbing compared to tissue homogenates and from fresh tissues compared to frozen tissues, without impacting the bacterial community composition estimated by 16S rRNA gene (V1–V3 region) sequencing. Despite the higher bacterial DNA yields with QIAamp® DNA Microbiome Kit, the E.Z.N.A.® Mollusc DNA Kit identified twice as many operational taxonomic units (OTUs) and eliminated PCR inhibition from gut tissues. Conclusions Sampling and nucleic acid purification substantially affected the quantity and diversity of bacteria identified in Pacific oyster microbiome studies and a fit‐for‐purpose strategy is recommended. Significance and Impact of the Study Accurate identification of Pacific oyster microbial diversity is instrumental for understanding the polymicrobial aetiology of Pacific oyster mortality diseases which greatly impact oyster production.

Pearl Oyster Bacterial Community Structure Is Governed by Location and Tissue-Type, but Vibrio Species Are Shared Among Oyster Tissues

“…This study revealed that, as has been observed in other oyster species ( King et al, 2020 ), discrete bacterial communities are associated with different pearl oyster tissue types. This finding supports the hypothesis that each tissue type represents a unique microenvironment for bacterial colonization and offers differences in niche space available for bacterial colonization ( Dubé et al, 2019 ; Pathirana et al, 2019 ; King et al, 2020 ). The lack of association with water at each farm also supports the unique nature of the microbial communities in oyster tissues.…”

DNA Enrichment Methods for Microbial Symbionts in Marine Bivalves

“…Given many symbiotic microorganisms that cannot be cultivated in vitro, the current research on bivalve symbionts mainly relies on culture-independent technologies including nano-scale secondary ion mass spectrometry (NanoSIMS) [20], high-throughput sequencing [17], matrix-assisted laser desorption/ionization MSI (MALDI-MSI) [21], and synchrotron-radiation based microcomputed tomography (SRµCT) [22]. The widely used high-throughput sequencing technology allows us to investigate the diversity of microorganisms and the mechanisms of symbiosis in mussels without the need for prior cultivation [17,23]. Though highthroughput sequencing is powerful for symbiosis research, minimizing the contamination of host DNA is still a challenge [24].…”

“…These methods might result in the quantity of host DNA deeply exceeding microbial DNA, limiting the sensitivity of nucleic acidbased microbial diagnostic systems [33][34][35]. Another approache is that using differential centrifugation [36,37] and filtration [23,38] pre-treatment methods or host DNA depletion kit [23] to enrich microbial cells in consideration of the contamination of host DNA. Differential centrifugation and filtration are easy-handling methods that can be achieved at the sampling site.…”

“…While the NEBNext Microbiome DNA Enrichment kit is not fit for invertebrate samples due to the low levels of invertebrate genome methylation [40]. Employing QIAamp DNA Microbiome kit acquired higher bacterial DNA yields from oyster tissue, while the microbial diversity was reduced [23]. Though the HostZERO Microbial DNA kit could effectively deplete host DNA in clinical samples [41], food [42], and coral [43], it has not been applied in host DNA depletion for bivalve samples.…”

“…In addition, Ahannach et al, 2021 found that different microbial DNA enrichment methods can introduce some biases into the microbial diversity profile and cause potential taxonomic composition differences in skin and saliva samples [41]. To date, the efficiency of these different methods for depleting host DNA from bivalve tissues and the bias introduced by these methods to the composition of these samples have rarely been evaluated [23].…”