Research Article: qPCR analysis of bivalve larvae feeding preferences when grazing on mixed microalgal diets

Date Published: June 29, 2017

Publisher: Public Library of Science

Author(s): Kai Liao, Wenbi Chen, Runtao Zhang, Haibo Zhou, Jilin Xu, Chengxu Zhou, Xiaojun Yan, Ruslan Kalendar.


Characterization of the feeding preferences of bivalve larvae would help improving the bivalve aquaculture and hatchery by providing appropriate microalgal diets. However, inaccurate and laborious identification and counting of microalgal species have challenged the selective feeding of bivalves. In the present study, we developed a highly specific and sensitive assay using quantitative polymerase chain reaction (qPCR) to assess the selective feeding of bivalve larvae based on species-specific primers targeting to microalgal 18S rDNA sequences. The assay exhibited good specificity. The detection limits of the qPCR assay were 769, 71, 781 and 21 18S rDNA copies for Chaetoceros calcitrans, Isochrysis galbana, Platymonas helgolandica and Nannochloropsis oculata, respectively. Using such assay, we found that C. calcitrans and I. galbana were preferentially ingested, whereas N. oculata was preferentially rejected in biodeposits of four bivalve species, Tegillarca gransa, Cyclina sinensis, Scapharca subcrenata and Sinonovacula constricta. Furthermore, our growth experiments revealed that C. calcitrans and I. galbana could significantly promote the shell growth, whereas feeding of N. oculata resulted in poorer growth of four bivalve species. These data indicated that qPCR might be useful in screening of efficient and reliable microalgal species for each bivalve species, leading to improved bivalve aquaculture and hatchery.

Partial Text

Live microalgae are the fundamental food sources for bivalves [1–3]. Previous study has revealed that some bivalves can distinguish their food from various types of particles, preferentially ingesting the high-quality ones, while rejecting those undesirable such as pseudofeces [4]. Therefore, it is important that the microalgae chosen as foods for bivalve aquaculture are preferentially ingested, and the characterization of feeding preferences of bivalves should help improving the bivalve aquaculture and hatchery by providing appropriate microalgal diets [5].

Investigation of the selective feeding of bivalves has been challenged by the inaccurate and laborious identification and counting of microalgal species. In the present study, qPCR was used to determine the removal rate of C. calcitrans, I. galbana, P. helgolandica and N. oculata from mixed algal suspensions of T. gransa, C. sinensis, S. subcrenata and S. constricta. In addition, we also used this method to quantify the proportion of each microalgal species in biodeposits of four bivalve species. The results indicated that this 18S rDNA-based method was highly specific for each microalgal species. A species-specific primer pair targeting to 18S rDNA exhibited no cross-reaction with other microalgal genomic DNA. Furthermore, the primers designed in our study were sensitive to microalgal genomic DNA extracted from mixed algal suspensions and biodeposits of bivalve larvae. The qPCR enabled the detection of 769 copies of C. calcitrans18S rDNA, 71 copies of I. galbana 18S rDNA, 781 copies of P. helgolandica 18S rDNA and 21 copies of N. oculata18S rDNA. Our data demonstrated that qPCR was an efficient way to evaluate feeding preferences of bivalve larvae, and such an approach and our findings might be broadly applicable.




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