A greener blue revolution - Part 2

I was already interested in herbivorous fish because of their low-trophic level and low/no requirement for wild-caught fish in their diets, but this finding highlighted the need to explore what seaweed could actually do to the immune system of herbivorous fish. Do they get less of an immune response since they eat seaweed on a daily basis?

Figure 1: mottled rabbitfish (Siganus fuscescens). Photo: Valentin Thepot

Selecting a promising fish species

Based on those insights, it was relatively easy to come up with the aims of the first feeding trial I conducted in my PhD: 1) to test as many seaweed species as possible (out of >11,000 seaweed species) to find the best performing species and 2) screen them for their immunostimulatory potential in a marine herbivorous fish: the mottled rabbitfish Siganus fuscescens.

I chose to work on this particular fish species because of its low-trophic level, but it also has tasty white flesh and, like other rabbitfish species, is capable of producing long chain highly unsaturated fatty acids (omega-3s) from short chain fatty acids. So, unlike its carnivorous counterparts (like salmon and sea bass) it does not require fishmeal and fish oil in its diet. S. fuscescens also has a wide geographical range in the Indo-West Pacific, as it is found from the north of Japan to the south of Australia, and is thus able to cope with temperature from 17°C to 31°C. The warming of the oceans due to climate change is already putting pressure on fish farmers growing temperate species with limited thermal ranges. The robustness of S. fuscescens in regards to some of the consequences of climate change is another reason why I wanted to work with this species.

In that trial, I investigated 11 seaweed species from the three groups (Rhodophyceae, Chlorophyceae and Phaeophyceae), which were collected around the Sunshine Coast or cultured by the USC Seaweed Research Group at the Bribie Island Research Centre, where I built my experimental setup and conducted all my feeding trials (Figure 1). In that trial, I also compared the different seaweed supplements to products, which I encountered in my literature search, and that are used either commercially or at the research level to promote fish immunity: β-glucan (Hilyses), sodium alginate, astaxanthin (Haematococcus pluvialis) and spirulina (Arthrospira platensis).

All supplements were added dry, at 3 percent by weight in commercially available fish pellets (Native Range, Ridley) and were fed to the rabbitfish at 3 percent body weight per day, twice a day. After two weeks of feeding, the fish were harvested, euthanised and blood was collected to assess their innate immune response. The innate immune system is the first line of defence in animals, and in ectotherm vertebrates like fish, the adaptive immune system is often sluggish, taking weeks to reach full potency. For this reason, the innate immune system is a key line of defence in fish when pathogens have been recognised by the fish immune cells.

Immune responses

In the blood of the fish I assessed the lysozyme activity (the enzyme liked with the destruction of gram positive bacteria), phagocytic activity (macrophages and other cells that are capable of engulfing and killing antigens), haemolytic activity (~35 proteins in the blood that form an attack membrane complex on the cell surface of antigens thus neutralising them) and the respiratory burst activity of leukocytes (white blood cells producing cytotoxic compounds to kill antigens).

Out of the four innate immune parameters of interest the most significant response was observed for the haemolytic activity (Figure 2), with the fish fed the red seaweed Asparagopsis taxiformis and the brown seaweed Dictyota intermedia. These showed four-fold increase and two-fold increases in their haemolytic activity respectively, compared to the control fish (Thépot et al., 2021b). This result with A. taxiformis was a clear indication of the potency of this species, but if a natural immunostimulant is to be used commercially then it should not have any detrimental effects on the growth and feed conversion ratio of the fish.

A follow-up trial

This is why in my next trial I used this seaweed and its methanolic extract to test whether 1) the dietary supplement had any growth or food efficiency effects and if 2) the methanolic extract with concentrated seaweed bioactives could outperform the dry seaweed. To do so I bred and reared S. fuscescens using adapted standard marine larval rearing protocols to have juvenile fish (2g; Figure 3), which would be fast growing and on which I could test the two above points. Because when we talk dietary supplements, the dose must also be considered, this is why in that second trial I decided to explore 1.5 percent (half dose) inclusion, 3 percent (same dose as the first trial) and 6 percent (double dose).

After four weeks on the different diets offered at 3 percent body weight per day, the juvenile S. fuscescens receiving the double dose A. taxiformis extract diet outperformed the other fish with regards to both growth and feed efficiency (Thépot et al., 2021c; Figure 3).

Figure 2: Haemolytic activity (left) of the fish fed diets supplemented with different seaweed and immunostimulants (the asterisks indicate statistical significance).

Figure 3: Juvenile S. fuscescens (1 month old; left) and specific growth rates (a) and food conversion ratio (b) of juvenile S. fuscescens fed dry A. taxiformis (‘whole’: W), A. taxiformis methanolic extract (‘Extract’: E) and the residual biomass from the extraction (‘Residue”: R) at 1.5 percent, 3 percent and 6 percent dietary inclusion.

From those two trials I knew that A. taxiformis supplements could promote the growth and improve feed efficiencies and the immune response of the mottled rabbitfish. By that stage I was wondering if those results were rabbitfish-specific. Would I get similar responses in a completely different fish, maybe a temperate freshwater carnivore?

Although scientific publications are the usual currency in academia this project also produced two international patents, one on the immune boosting properties of A. taxiformis in fish and one on its growth promoting effects

A shift to salmon

More questions = more trials! So, with that in mind the last feeding trial in my PhD investigated the dietary effects of A. taxiformis supplements (dry and extract) in Atlantic salmon (Salmo salar) parr.

I found that, after four weeks of feeding the salmon ad libitum with the three seaweed supplemented diets, the fish were significantly heavier than the control fish (Thépot et al., 2022) and this was especially so for the fish fed the double dose seaweed extract (Figure 4). Albeit lower than for the control fish, the seaweed supplements had no statistically significant effects on the FCR of the fish.

Figure 4: Relative weight gain, feed intake and FCR (top) of Atlantic salmon parr fed the unsupplemented control diet (‘Control’), lipopolysaccharide from E. coli (‘LPS’ at 0.01 percent), A. taxiformis as whole seaweed (‘Whole’ at 3 percent inclusion) as extract (‘Extract’ at 3 percent) and double dose extract (‘Extract×2’). Haemolytic activity of Atlantic salmon fed the different diets for 2 and 4 weeks (middle).

Juvenile mottled rabbitfish. Photo: Valentin Thepot

Although scientific publications are the usual currency in academia this project also produced two international patents, one on the immune boosting properties of A. taxiformis in fish and one on its growth promoting effects. These facilitated the establishment of a collaborative partnership with one of Australia’s most important aquaculture business. The patents were also filed because, regardless of the clear positive effects of A. taxiformis in fish during this project, there must also be a clear economic incentive for an uptake of this technology at scale.

Conclusions

This PhD journey has been extremely rewarding, with multiple publications (scientific and patents) but the most rewarding element was to be able to shine the light on two potential solutions – low trophic level marine fish and seaweed functional ingredients – to two important issues in aquaculture: 1) reliance on wild sourced ingredients for aquafeeds and 2) disease outbreaks.

We only have one planet, with limited and connected resources, so I hope to see more research and commercial interest in the use of seaweeds as functional ingredients for farmed fish and in the development of novel low trophic aquaculture candidate fish.

For my part, I am still working with Siganus spp. and helping colleagues overseas, including in Indonesia, under the Australian Centre for International Agricultural Research project ‘Accelerating the development of finfish mariculture in Cambodia through south-south research cooperation with Indonesia’.

I am in the process of transitioning from academia to join the Anindilyakwa Land Council on the remote Groote Eylandt in the Northern Territory. In my position of aquaculture coordinator, I will help develop a sustainable aquaculture industry to facilitate the transition from mining operations towards a more long-term solution for this remote community. I will keep on working on different marine species, including seaweed, to continue on my path of improving the productivity and environmental footprint of this industry. I’d love to ultimately make this blue revolution a greener one.