n-3 PUFA biosynthesis by the copepod Apocyclops royi determined by fatty acid profile and gene expression analysis

Abstract

The cyclopoid copepod Apocyclops royi (Lindberg 1940) is one of two dominant mesozooplankton species in brackish Taiwanese aquaculture ponds. Periodically low n-3 polyunsaturated fatty acid (PUFA) content in seston could potentially be a limiting factor for zooplankton diversity. Apocyclops royi ’s potential ability to biosynthesize n-3 PUFA was investigated through a short-term feeding experiment on four species of microalgae . Furthermore, we analyzed the expression of genes encoding putative fatty acid elongase ( ELO ) and desaturase ( FAD ) enzymes in A. royi on long-term diets of the PUFA-poor Dunaliella tertiolecta and the PUFA-rich Isochrysis galbana . The copepods exhibited high contents of docosahexaenoic acid (DHA, C22:6n-3) (>20% of total fatty acid) even when DHA-starved for two generations, and no significant differences were found in absolute DHA content between treatments. Transcripts correlating to the four enzymes Elovl4, Elovl5, Fad Δ5 and Fad Δ6 in the n-3 PUFA biosynthetic pathway were identified. Gene expression analysis revealed a significantly higher expression of two desaturases similar to Fad Δ6 in copepods fed PUFA-lacking algae compared to copepods fed algae with high PUFA content. These findings suggest a highly active n-3 PUFA biosynthesis and capability of DHA production in A. royi when fed low-PUFA diets.

The whole genome sequence and mRNA transcriptome of the tropical cyclopoid copepod Apocyclops royi

“…An overview of the experimental and bioinformatical workflow can be seen in Fig 1C. Animal husbandry, sampling, RNA purification, RNA sequencing strategy and initial RNA data processing is also described in a recent paper which used the mRNA dataset presented here to analyze the fatty acid metabolism genes and differential expression based on feeding regime [9] (in review). Briefly, an Apocyclops royi animal culture obtained from Tungkang Biotechnology Research Center in Taiwan was split in two which were kept in 100 L tanks on separate microalga feeding regimes.…”

“…Animals from each feeding regime were sampled as described in [9]. Briefly, animals were starved for 2 h to empty their guts and a 53 µm filter was used to separate all the life stages (nauplii, copepodites and adults) from the sea water.…”

“…as described in [9], RNA was extracted with RNeasy (Qiagen, Venlo, Nederlands) according to protocol. Before extraction, all RNAlater was removed and the animal tissue was disrupted with a 1.5 mL RNase-Free Pellet Pestle (Kimble Chase, Vineland, New Jersey, USA) mounted on a Kontes Pellet Pestle motor (Kimble Chase, Vineland, New Jersey, USA) for 1 min on ice in 20 µl buffer RTL, before adding the remaining 320 µl Buffer RTL.…”

“…The RNA sequencing library strategy is described in [9]. Briefly, an mRNA sequencing library was produced for each of the eight replicates with the Truseq stranded mRNA kit (Illumina, San Diego, California, United States) and SuperscriptII reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…It has a life cycle of 7-8 days [5], and can tolerate temperatures of 15-35 °C [6], and salinities of 0-35 psu [7]. In a recent publication, we report its ability to biosynthesize the polyunsaturated fatty acid Docosahexaenoic acid (DHA) from alpha -Linolenic acid [8, 9] which makes Apocyclops royi an interesting organism for copepod physiological studies.…”