Unveiling protist diversity associated with the Pacific oyster Crassostrea gigas using blocking and excluding primers

Abstract

Background: Microbiome of macroorganisms might directly or indirectly influence host development and homeostasis. Many studies focused on the diversity and distribution of prokaryotes within these assemblages, but the eukaryotic microbial compartment remains underexplored so far. Results: To tackle this issue, we compared blocking and excluding primers to analyze microeukaryotic communities associated with Crassostrea gigas oysters. High-throughput sequencing of 18S rRNA genes variable loops revealed that excluding primers performed better by not amplifying oyster DNA, whereas the blocking primer did not totally prevent host contaminations. However, blocking and excluding primers showed similar pattern of alpha and beta diversities when protist communities were sequenced using metabarcoding. Alveolata, Stramenopiles and Archaeplastida were the main protist phyla associated with oysters. In particular, Codonellopsis, Cyclotella, Gymnodinium, Polarella, Trichodina, and Woloszynskia were the dominant genera. The potential pathogen Alexandrium was also found in high abundances within some samples. Conclusions: Our study revealed the main protist taxa within oysters as well as the occurrence of potential oyster pathogens. These new primer sets are promising tools to better understand oyster homeostasis and disease development, such as the Pacific Oyster Mortality Syndrome (POMS) targeting juveniles.

Revealing the composition of the eukaryotic microbiome of oyster spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

“…3a-b ). Nevertheless, given that there are differences in primer design among the three methods, as well as differences in PCR conditions (Table S3) [ 54 , 57 , 63 , 70 , 102 ], it is not surprising that there were differences among the taxa detected (Fig. 3b ).…”

“…Such “blocking primers” typically use a short blocking-oligonucleotide with a modified 3′ end that binds to the 18S rRNA gene of the host, and prevents “universal” 18S primers from amplifying host sequences [ 60 ]. Such an approach has been successfully applied to krill [ 60 ], fish [ 61 , 62 ], coral [ 63 ], primates [ 64 ], shrimp [ 65 , 66 ], flying squid [ 67 ], mosquitos [ 68 , 69 ] and Pacific oysters [ 57 ], although a large proportion of the sequences can still be host-derived (e.g., up to 92% in coral, 42% in krill, and 45% in fish) [ 57 , 63 , 71 ]. This approach also requires designing and optimizing the blocking primers for each animal host, which remains a challenge [ 70 , 71 ].…”

Revealing the Composition of the Eukaryotic Microbiome of Oyster Spat by CRISPR-Cas Selective Amplicon Sequencing (CCSAS)

“…This 30-nt oyster-blocking primer was modified at the 3' end with theSpacer C3 CPG (3 hydrocarbons) and contained a 10bp overlap with the reverse primer 18SV4-R-Nxt, which prevents the amplification of the 18S rRNA gene from Pacific oysters, and thus enriches the proportion of amplicons from microeukaryotes [57] . In the first PCR, the [57], which included an initial incubation of 15 min at 96°C followed by 35 cycles of denaturation at 96°C for 30 s, annealing at 52°C for 30 s and elongation at 72°C for 60 s, and a final elongation for 10 min at 72°C. The first PCR product was purified using magnetic Agencourt SPRI beads (Beckman Coulter) at a ratio of 1:1 (vol:vol) for beads:DNA to remove fragments less < 200bp (e.g.…”

“…Preparation of the sequencing library for the 18S amplicons obtained using blocking primers was similar to that described above, except that the first PCR used the primer set 18SV4-F-Nxt / 18SV4-R-Nxt and the oysterblocking primer 18SV4-Block-oyster (Table S3), which was adapted from Clerissi et al [57] , to amplify a ~377 bp fragment of 18S rRNA gene that is specific to microeukaryotes but not Pacific oysters. This 30-nt oyster-blocking primer was modified at the 3' end with theSpacer C3 CPG (3 hydrocarbons) and contained a 10bp overlap with the reverse primer 18SV4-R-Nxt, which prevents the amplification of the 18S rRNA gene from Pacific oysters, and thus enriches the proportion of amplicons from microeukaryotes [57] [57], which included an initial incubation of 15 min at 96°C followed by 35 cycles of denaturation at 96°C for 30 s, annealing at 52°C for 30 s and elongation at 72°C for 60 s, and a final elongation for 10 min at 72°C. The first PCR product was purified using magnetic Agencourt SPRI beads (Beckman Coulter) at a ratio of 1:1 (vol:vol) for beads:DNA to remove fragments less < 200bp (e.g.…”

“…Such "blocking primers" typically use a short blocking-oligonucleotide with a modified 3' end that binds to the 18S rRNA gene of the host, and prevents "universal" 18S primers from amplifying host sequences [60]. Such an approach has been successfully applied to krill [60], fish [61][62], coral [63], primates [64], shrimp [65][66], flying squid [67], mosquitos [68][69] and Pacific oysters [57], although a large proportion of the sequences can still be host-derived (e.g. up to 92% in coral, 42% in krill, and 45% in fish) [57,72,63].…”