Biases during DNA extraction affect characterization of the microbiota associated with larvae of the Pacific white shrimp Litopenaeus vannamei

摘要

For in-depth characterization of the microbiota associated with shrimp larvae, careful selection of DNA isolation procedure is paramount for avoiding biases introduced in community profiling. Four E.Z.N.A.™ DNA extraction kits, i.e., Bacterial, Mollusc, Stool, and Tissue DNA Kits, abbreviated as Ba, Mo, St, and Ti, respectively, were initially evaluated with zoea 2 (Z2) larvae of the Pacific white shrimp (Litopenaeus vannamei) by 16S amplicon sequencing on a Illumina MiSeq platform. Further characterization of additional larval samples, specifically nauplii 5 (N5), mysis 1 (M1), and postlarvae 1 (P1), was performed with Ba and St kits to examine the changing microbiota profile during shrimp hatchery period. The results from the Z2 samples showed that DNA yields from the four kits varied significantly (P < 0.05), whereas no significant differences were detected in the α-diversity metrics of the microbiota. By contrast, the St kit, with the lowest DNA yield and quality, successfully recovered DNA from Gram-positive and gut-associated bacterial groups, whereas the Ba kit, which showed maximal microbiota similarity with the Mo kit, manifested the best reproducibility. Notably, significant differences were observed in relative abundances of most dominant taxa when comparing results from the Ba and St kits on Z2, M1, and P1 samples. In addition, the bacterial community identified shifted markedly with larval development regardless of the DNA extraction kits. The DNA recovery biases arising from the larval microbiota could be due to different protocols for cell lysis and purification. Therefore, combined application of different DNA extraction methods may facilitate identification of some biologically important groups owing to their complementary effects. This approach should receive adequate attention for a thorough understanding of the larvae-associated microbiota of the penaeid shrimp.