Introduction
Buffeljags Abalone is a commercial abalone farm in South Africa that practices integrated multi-trophic aquaculture (IMTA) by growing the green seaweed Ulva lacinulata in D-shaped paddle-raceways receiving effluent water from adjacent Haliotis midae raceways. This practice allows for bioremediation of farm effluent , partial recirculation of water and the cultivated seaweed is often used as supplementary feed. Seaweeds cultivated in IMTA have also been reported to have a modulatory effect on the microbiome.
Microorganisms play vital roles in aquaculture systems, including DOM and POM decomposition, fermentation, nitrification, nutrient cycling and protection against pathogenic microorganisms. Host associated bacteria can synthesise essential amino acids, enhance digestion efficiency by supplying enzymes , produce essential micronutrients and metabolites (e.g., SCFAs), and enhance growth, health and development/morphology of the host. The microbiome of an IMTA and the associated species, is likely influenced by feeds and environmental factors but also by changing conditions during grow-out. Therefore, t he aim of this study was to characterise the bacterial microbiome of hatchery-produced juvenile abalone (3 - 10 mm shell length (SL)) and the sources of bacterial introductions (feeds and seawater) , and to compare this with the microbiome of adult abalone (± 70 mm SL), and their rearing environment , cultivated grown in an integrated abalone-Ulva IMTA system.
Materials and Methods
Hatchery-representative samples were collected from three tanks (L x W x D: 0.68 m x 0.50 m x 0.12 m) in the Buffeljags Abalone commercial hatchery, each stocked with 25 000 juvenile abalone . The juvenile abalone were fed a mixed diet of wild diatoms, formulated feed, Ulva lacinulata and Gracilaria . Samples (n = 36) for next-generation sequencing (NGS) included abalone intestines, abalone faeces , each feed , and bacterial cells in incoming seawater (500 mL) collected on 0.22 µm filters. Samples were collected in triplicate on the day that animals were moved from settlement plates to the rearing tanks and then once per month for two months. G row-out representative samples (n = 60 ) were collected from three separate abalone-Ulva IMTA systems at Buffeljags over the course of a year. S amples included the abalone effluent water entering the Ulva paddle raceway, bioremediated water returning to abalone raceways after being mixed with 50% fresh seawater, and Ulva grown in the raceways. Simultaneously, the Ulva raceway that supplies the abalone hatchery with seawater was also sampled. Ulva in this system is not grown in abalone effluent and served as a non-IMTA control. Intestinal samples (n = 30) from adult abalone fed diets supplemented with or without IMTA grown Ulva (or components of Ulva ) were also included in this study to compare to that of juvenile abalone.
A 16S rDNA fragment was amplified to characterise bacterial communities and raw sequence data was processed using QIIME2. Bacterial 16S reads were mapped against the SILVA 16S rRNA reference database for taxonomic identification of amplicon sequence variants (ASVs) . MicrobiomeAnalyst was used to assess within- and between sample bacterial diversity, as well as to quantify and visualise taxonomic abundance , perform differential abundance analyses and to identify putative functional capabilities of the taxa . Data from the respective sample sets (hatchery, grow-out and adult abalone) were treated in the same way to allow for comparisons between the datasets.
Results & Discussion
J uvenile abalone digestive tract bacteria were dominated by the genera Formosa (36 %), Psychrilyobacter (11 %), Vibrio (11 %) and Mycoplasma (5%), all of which are known colonisers of adult abalone digestive systems and had high abundances in adult abalone digestive systems in the current study. Over time, abalone digestive tracts and their associated microbiome became more specialised, with a lower overall diversity in adult abalone guts when compared to juveniles.
Faeces collected from juvenile abalone largely reflected their gut bacterial communities with the exception of Tenacibaculum, which increased in abundance from 3% in the intestines to 17% in the faeces. This could be the result of nutrient availability causing bacterial proliferation or as a result of the a balone digestive systems being a niche environment capable of selecting for/against specific bacteria. Similar observations were made for the genera Rubidimonas and Lewinella, which were present across all environmental and feed samples, but absent from juvenile and adult abalone gut samples. These results are further supported by the lower bacterial diversity observed for juvenile abalone digestive tracts (Chao1; ANOVA F-value 22.23, P < 0.05) and adult abalone digestive tracts when compared to water and feed samples.
The gut bacterial communities of juvenile abalone were introduced by the incoming seawater, as well as the respective feeds, as a high abundance of Vibrio (19%), Psychrilyobacter (18%), Tenacibaculum (8%), Formosa (5%) and Psychromonas (4%) were observed in water and digestive tract samples. T he Ulva -, Gracilaria - and diatom-associated microbiome also contributed to enteric bacteria of juvenile abalone, with th e bacterial microbiome of Gracilaria and the diatoms showing a greater extent of similarity to that of juvenile abalone gut and faec al samples . Conversely , Ulva samples showed a distinct microbial profile, with a high abundance of Granulosicoccus (15%), Rhodobacteraceae (11%) and Saprospiraceae (9%), as well as other bacteria that are known for their involvement in Ulva morphogenesis and development. These results are similar to those obtained for samples from the grow-out IMTA system, where Ulva was also colonised by bacteria contributing to development of Ulva and nutrient cycling. In the abalone-Ulva IMTA , Ulva modulates its surface microbiome and that of the abalone effluent, reducing the abundance of certain genera, including k nown opportunistic pathogens, without causing a collapse in bacterial diversity of the bioremediated seawater, acting as a positive indicator for system health.
Conclusion
Incoming sea water and diet shapes the bacterial microbiome of the juvenile abalone gut , whereas inclusion (integration) of Ulva positively modulates the microbiome and contributes towards the functioning of IMTA on a commercial abalone farm . The complex interactions between microbial diversity, animal health and productivity has been observed in various aquaculture systems .
This study contributes towards the understanding of the bacterial dynamics, their sources of introduction and their roles at different abalone production stages in an integrated abalone-Ulva system.
This study received funding from the EU Horizon 2020 Research & Innovation Programme ASTRAL Project under Grant Agreement No. 863034