Diet and Stomach Microbiota of Gulf Menhaden, a key forage filter feeding fish species

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Hanif, Ammar Wali
Jagus, Rosemary
Menhaden represent a family of important filter feeding forage fish that serves as a trophic link between plankton and piscivorous predators in the marine environment. Dietary analysis is difficult because diet items are small and >80 % of the stomach content is amorphous material. DNA metabarcoding combines mass-amplification of short DNA sequences (barcodes) with high-throughput sequencing. This application allows the simultaneous identification of many taxa within the same environmental sample, as well as the analysis of many samples simultaneously, providing a comprehensive assessment of diet items and gut microbiota. Here we present a methodological approach using DNA metabarcoding suitable for a small filter feeding fish to identify the stomach contents of juvenile Gulf menhaden (Brevoortia patronus), collected within Apalachicola Bay, Florida. I describe the optimization of DNA extraction, comparison of two primers and sequencing protocols, estimation of menhaden DNA contamination, quality filtering of sequences, post-sequence processing and taxonomic identification of recovered sequences. I characterized the prokaryotic community using 16S universal ribosomal RNA (rRNA) gene sequencing primers in the V3-V4 hypervariable regions. Using two different sequencing protocols employing different “universal” 16S rRNA gene sequencing primers. Although no difference in overall operational taxonomic units (OTUs) was found, the two sequencing protocols gave differences in the relative abundancies of several bacterial classes. The dominant OTUs resulting from 16S rRNA gene sequencing at the phylum level were assigned to Proteobacteria, Acidobacteria, Actinobacteria and Chloroflexi and included oil eating bacteria consistent with the Gulf of Mexico location. Stomach microbiota and diet were compared in juvenile Gulf menhaden, Brevoortia patronus, caught at two locations, Two Mile Channel and St. Vincent Sound, in Apalachicola Bay, FL in May and July of 2013. The stomach microbiota of samples from both locations showed a predominance of Proteobacteria, Chloroflexi, Bacteroidetes, Acidobacteria and Actinobacteria, although significant differences in composition at the class level were seen. The stomach microbiota from fish from Two-Mile Channel showed a higher level of taxonomic richness and there was a strong association between the microbiota and sampling location, correlating with differences in salinity. Approximately 1050 diet items were identified, although significant differences in the species represented were found in samples from the two locations. Members of the Stramenopile/ Alveolate/Rhizaria (SAR) clade accounted for 66 % representation in samples from Two Mile Channel, dominated by the diatoms Cyclotella and Skeletonema, as well as the ciliate Oligotrichia. In contrast, Metazoa (zooplankton) dominated in samples from St. Vincent Sound, accounting for over 80 % of the reads. These are mainly Acartia copepods. Since ciliates are considered to be microzooplankton, this means there is just over 60 % representation of phytoplankton in samples from Two Mile Channel and over 90 % representation of zooplankton in samples from St. Vincent Sound. Overall, I demonstrate the diversity of juvenile menhaden stomach contents that supports a characterization of menhaden as environmental samplers.