Periodic Reporting for period 1 - HALOSPATH (Halogen metabolism of brown macroalgae and its biological significance in the context of pathologies - An omics approach)
Reporting period: 2020-11-23 to 2022-11-22
Macroalgal pathogens are a threat to the sustainable development of macroalgal mariculture and natural populations in marine ecosystems, causing disease outbreaks and significant losses. The lack of proper posteriori containment and disease management measures has increased the urgency to study macroalgal pathologies. The nature and epidemiology of macroalgal pathogens is dramatically understudied, limited to their phylotaxonomy. Recent studies have provided some genomic and proteomic information in brown macroalgae but nothing is known about the nature of metabolites, involved in macroalgal host-pathogen interactions. HALOSPATH intended to elucidate the dynamic changes in the metabolic profiles in defense mechanisms of two brown macroalgae Ectocarpus and Laminaria against pathologies, focussing on identifying the roles of any halometabolites involved in the process, using an integrated approach of time-series comparative metabolomics, and RNA-seq transcriptomics. The metabolomic datasets were generated for Ectocarpus siliculosus infected with oomycetes Eurychasma dicksonii 4018/1 and 4018/4, identifying 21 differentially expressed metabolites in the infection. Lipidomic analysis identified 419 lipid metabolites of which 231 lipids were significantly involved in lipid remodelling during host-pathogen interaction in Ectocarpus-Eurychasma pathosystem showing the upregulation of fatty acyls and sphingolipids concomitant with the downregulation of phospholipids with increasing infection. The metabolomic analysis of Laminaria digitata from two different geographic locations co-cultivated with Laminariocolax tomentosoides displayed distinct DEM profiles. 115 genes were differentially expressed in L. digitata co-cultivated with L. tomentosoides mostly involved in photosynthesis, ROS metabolism, cell wall and post-translational modification and autophagy-induced defense responses. Thus, HALOSPATH provided in-depth insights into the metabolic and transcriptomic changes occurring during the infective success of these pathogens in Ectoacrpus and Laminaria that could be exploited for better understanding of host-pathogen interaction in seaweeds.
The two pathosystems Ectocarpus siliculosus - Eurychasma dicksonii (CCAP 4018/1 and 4018/4) (EEP) and Laminaria digitata - Laminariocolax tomentosoides H (LLP) were established under laboratory conditions, making them amenable for metabolomic and molecular studies (WP1). The metabolomic analysis was conducted for all the established pathosystems (EEP and LLP) and RNA-seq transcriptomics for LLP, revealing differentially expressed metabolites (DEMs) and genes (DEGs) involved in the host-pathogen interaction of 2 brown algae. The metabolomic analysis of EEP showed similar patterns of DEMs between E. siliculosus infected with Eu. dicksonii 4018/1 and 4018/4, although the relative level DEMs were significantly different between the two pathosystems. Further, 419 lipid metabolites belonging to 4 lipid categories (fatty acyls, glycerolipids, glycerophospholipids and sphingolipids), 15 main lipid classes and 35 lipid sub-classes were identified in E. siliculosus – Eu. dicksonii 408/1 pathosystem. 231 lipid molecules were significantly differentially expressed during host-pathogen interaction suggesting the upregulation of fatty acyls and sphingolipids concomitant with downregulation of phospholipids with the increase in oomycetes infection. Further, metabolite profiles of organic and aqueous extracts were obtained for L. digitata sporophytes of 2 different geographic origins co-cultivated with L. tomentosoides H. 69 metabolites were identified in the organic and 40 in the aqueous extract of LLP including 2 halogenated metabolites. The DEM profiles of co-cultivated L. digitata samples were significantly different from control algae. More than 90% of DEMs in the organic extract were downregulated in co-incubated L. digitata in both the pathosystems though their relative contents were different. On contrary, DEMs identified in the aqueous extract had significantly different expression patterns between the two LLP pathosystems. RNA-seq transcriptomic analysis was performed with L. digitata co-incubated with L. tomentosoides H for 3 and 5 days. The data was processed with Seq2Fun (www.Seq2Fun.ca) differentially expressed genes were identified using the DESeq pipeline of ExpressAnalyst (https://www.expressanalyst.ca). 115 DEGs were identified in L. digitata co-incubated with L. tomentosoides H for 5 days as compared with 21 DEGs at day3 of co-cultivation. The co-incubation of L. digitata with L. tomentosoides put both the host alga and the endophyte alga under selection pressure to survive. The endophytic algae attempt to get attached to the host surface while the host algae initiate ROS-mediated defence response and down-regulates its metabolic photosynthetic and oxidative phosphorylation thereby switching to a defensive low energy state. There was a considerable evidence of cell wall modification, post-translational modification and autophagy induced systemic defence responses in co-cultivated L. digitata Additionally, L. digitata collected from Aberdeenshire coast were divided in 3 groups, healthy, grazed and diseased based on the morphological symptoms. The metabolites were exhaustively extracted from these algae using methanol-dichloromethane solvent system for 5 days. The obtained were successively fractionated on silica multiple times to obtain a total of 112 fractions. All the 112 fractions were tested at MEDINA, Spain during secondment for antimicrobial and anticancer activities. None of the extracts showed any promising biological activity against the tested microbial or cancer cell lines.
The results obtained in this project will be mainly disseminated as publications. We envisage to publish 4 articles based on the results obtained above. After publication, the results will be shared on the University’s PURE website, ResearchGate and social media platforms. One podcast has been made online (24 September, 2022), Seaweed the Marine Commodity highlighting the importance of seaweed industry, current challenges and seaweed disease problem. Also, the concept and objectives of the HALOSPATH project were shared on the School of Biological Sciences (University of Aberdeen) Twitter webpage (7 March, 2022) for International Women’s Day.
The results obtained in this project will be mainly disseminated as publications. We envisage to publish 4 articles based on the results obtained above. After publication, the results will be shared on the University’s PURE website, ResearchGate and social media platforms. One podcast has been made online (24 September, 2022), Seaweed the Marine Commodity highlighting the importance of seaweed industry, current challenges and seaweed disease problem. Also, the concept and objectives of the HALOSPATH project were shared on the School of Biological Sciences (University of Aberdeen) Twitter webpage (7 March, 2022) for International Women’s Day.
The project deliverables were delayed due to COVID restrictions during the start of the period. It is envisaged that the ongoing de novo RNA-seq assembly with Trinity, and functional annotation as well as whole genome sequencing of Laminaria in future, will provide further in-depth insights of the genetic metabolic regulation in LLP. The obtained data sets will be submitted to EMBL/ NCBI in the public domain that will help other researchers and seaweed practitioners to design further experiments for understanding host-pathogen interactions in brown seaweeds.