Metabolomic profiling of wild rooibos (Aspalathus linearis) ecotypes and their antioxidant-derived phytopharmaceutical potential.

INTRODUCTION: Aspalathus linearis (commonly known as rooibos) is endemic to the Cape Floristic Region of South Africa and is a popular herbal drink and skin phytotherapeutic ingredient, with health benefits derived primarily from its unique phenolic content. Several, seemingly habitat-specific ecotypes from the Cederberg (Western Cape) and Northern Cape have morphological, ecological, genetic and biochemical differences. OBJECTIVES AND METHODS: Despite the commercial popularity of the cultivated variety, the uncultivated ecotypes are largely understudied. To address gaps in knowledge about the biochemical constituency, ultra-performance liquid chromatography-mass spectrometry analysis of fifteen populations was performed, enabling high-throughput metabolomic fingerprinting of 50% (v/v) methanolic extracts. Antioxidant screening of selected populations was performed via three assays and antimicrobial activity on two microbial species was assessed. The metabolomic results were corroborated with total phenolic and flavonoid screening of the extracts. RESULTS AND DISCUSSION: Site-specific chemical lineages of rooibos ecotypes were confirmed via multivariate data analyses. Important features identified via PLS-DA disclosed higher relative abundances of certain tentative metabolites (e.g., rutin, aspalathin and apiin) present in the Dobbelaarskop, Blomfontein, Welbedacht and Eselbank sites, in comparison to other locations. Several unknown novel metabolites (e.g., m/z 155.0369, 231.0513, 443.1197, 695.2883) are responsible for metabolomic separation of the populations, four of which showed higher amounts of key metabolites and were thus selected for bioactivity analysis. The Welbedacht and Eselbank site 2 populations consistently displayed higher antioxidant activities, with 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activities of 679.894 ± 3.427 µmol Trolox/g dry matter and 635.066 ± 5.140 µmol Trolox/g dry matter, respectively, in correlation with a high number of phenolic and flavonoid compounds. The contribution of the individual metabolites to the pharmacological effectiveness of rooibos remains unknown and as such, further structural elucidation and phytopharmacological testing is thus urgently needed.

Resprouters Versus Reseeders: Are Wild Rooibos Ecotypes Genetically Distinct?

Aspalathus linearis (Burm. F.) R. Dahlgren (Fabaceae) or rooibos, is a strict endemic species, limited to areas of the Cederberg (Western Cape) and the southern Bokkeveld plateau (Northern Cape) in the greater Cape Floristic Region (CFR) of South Africa. Wild rooibos, unlike the cultivated type, is variable in morphology, biochemistry, ecology and genetics, and these ecotypes are broadly distinguished into two main groups, namely, reseeders and resprouters, based on their fire-survival strategy. No previous assessment of genetic diversity or population structure using microsatellite markers has been conducted in A. linearis. This study aimed to test the hypothesis that wild rooibos ecotypes are distinct in genetic variability and that the ecotypes found in the Northern Cape are differentiated from those in the Cederberg that may be linked to a fire-survival strategy as well as distinct morphological and phytochemical differences. A phylogeographical and population genetic analyses of both chloroplast (trnLF intergenic region) and newly developed species-specific nuclear markers (microsatellites) was performed on six geographically representative wild rooibos populations. From the diversity indices, it was evident that the wild rooibos populations have low-to-moderate genetic diversity (He: 0.618-0.723; Ho: 0.528-0.704). The Jamaka population (Cederberg, Western Cape) had the lowest haplotype diversity (H = 0.286), and the lowest nucleotide diversity (π = 0.006) even though the data revealed large variations in haplotype diversity (h = 0.286-0.900) and nucleotide diversity (π = 0.006-0.025) between populations and amongst regions where wild rooibos populations are found. Our data suggests that populations of rooibos become less diverse from the Melkkraal population (Suid Bokkeveld, Northern Cape) down towards the Cederberg (Western Cape) populations, possibly indicative of clinal variation. The largest genetic differentiation was between Heuningvlei (Cederberg, Western Cape) and Jamaka (FST = 0.101) localities within the Cederberg mountainous region, and, Blomfontein (Northern Cape) and Jamaka (Cederberg) (FST = 0.101). There was also a significant isolation by distance (R2 = 0.296, p = 0.044). The presence of three main clusters is also clearly reflected in the discriminant analysis of principal components (DAPC) based on the microsatellite marker analyses. The correct and appropriate management of wild genetic resources of the species is urgently needed, considering that the wild Cederberg populations are genetically distinct from the wild Northern Cape plants and are delineated in accordance with ecological functional traits of reseeding or resprouting, respectively. The haplotype divergence of the ecotypes has also provided insights into the genetic history of these populations and highlighted the need for the establishment of appropriate conservation strategies for the protection of wild ecotypes.

Metagenomic assessment of body surface bacterial communities of the sea urchin, Tripneustes gratilla.

Sea urchins, including Tripneustes gratilla, are susceptible to a disease known as bald sea urchin disease, which has the potential to lead to economic losses in this emerging aquaculture industry in South Africa. This disease is characterized by lesions that form on sea urchin exoskeletal surfaces. This study aimed to characterize the body surface bacterial communities associated with T. gratilla, using a 16S rDNA gene metagenomics approach, to provide insight into the bacterial agents associated with this aquaculture species, as well as with this balding disease. Bacterial samples were collected from non-lesioned healthy animals obtained from natural locations along the eastern coast of South Africa, as well as from different cultured cohorts: non-lesioned healthy-, lesioned diseased- and non-lesioned stressed animals. A total of 1,067,515 individual bacterial operational taxonomic units (OTUs) were identified, belonging to 133 family-, 123 genus- and 113 species level OTU groups. Alpha diversity analyses, based on Chao1, Shannon and Simpson indices, showed that there were no statistically significant differences (ANOVA; P > 0.05) between the respective cohorts, as all cohorts displayed a high degree of bacterial diversity. Similarly, beta diversity analyses (Non-metric multidimensional scaling) showed a large degree of overlapping OTUs across the four cohorts. Within each cohort, various OTUs commonly associated with marine environments were found, predominantly belonging to the families Vibrionaceae, Saprospiraceae, Flavobacteriaceae and Sphingomonadaceae. Differential abundance analysis (DESeq2) revealed that OTUs that are differentially abundant across cohorts were likely not responsible for this balding disease, suggesting that complex bacterial agents, rather than a specific pathogenic agent, are likely causing this disease. Furthermore, the putative metabolic functions assigned to the bacterial communities showed that heterotrophic bacteria appear to be responsible for tissue lysis of degrading animal matter. The results from this study, obtained through univariate and multivariate-based approaches, contributes to future management strategies of this emerging aquaculture species by providing insight into the bacterial communities associated with both natural and cultured environments.

Testing and validating a modified CTAB DNA extraction method to enable molecular parentage analysis of fertilized eggs and larvae of an emerging South African aquaculture species, the dusky kob Argyrosomus japonicus.

This study describes the successful implementation of a modified cetyltrimethyl ammonium bromide (CTAB) protocol to isolate genomic DNA and amplify 14 microsatellite markers from fertilized eggs and larvae of an emerging South African farmed marine fish species, the dusky kob Argyrosomus japonicus. To test and validate the efficiency of this method, genetic data were utilized to resolve parentage and kinship of first-generation (F1) offspring produced in mass-spawning events of wild broodstock fish in a commercial hatchery.

Identification of naturally occurring hybrids between two overexploited sciaenid species along the South African coast.

Hybridisation between fish species can play a significant role in evolutionary processes and can influence management and conservation planning, however, this phenomenon has been widely understudied, especially in marine organisms. The distribution limits of two sciaenid species (silver kob, Argyrosomus inodorus, and dusky kob, A. japonicus) partly overlap along the South African coast, where both species have undergone severe depletion due to overfishing. Following the identification of a number of possible cases of species misidentification or hybridisation (21 out of 422 individuals), nuclear and mitochondrial DNA data (12microsatellite loci and 562bp of the COI gene) were analysed to investigate the genetic composition of these individuals. Results indicated a field-based species misidentification rate of approximately 2.8% and a rate of natural hybridisation of 0.7%. Interestingly, all hybrid fish resulted from first-generation (F1) hybridisation events, which occurred exclusively between silver kob females and dusky kob males. Whether hybridisation is the result of natural events (such as secondary contact following a shift in distribution range), or anthropogenic activities (size-selective pressure due to overfishing), these findings have important implications for critical recovery and future management of these species in the wild.

New microsatellite markers for the abalone Haliotis midae developed by 454 pyrosequencing and in silico analyses.

Farming of Haliotis midae is the most lucrative aquaculture venture in South Africa. The genome of this species needs to be studied to assist in selective breeding programs aimed at increasing overall yield, and molecular markers will be required to attain this goal. We identified and characterized 82 polymorphic microsatellite loci by using repeat-enriched genomic libraries and high-throughput pyrosequencing technology. The observed number of alleles ranged from 2 to 21, expected heterozygosity from 0.063 to 0.968, observed heterozygosity from 0.000 to 1.000, and polymorphic information content from 0.059 to 0.934. Three loci gave significant hits to other haliotid genes and/or microsatellite loci; hits to genes were always located in the 5'/3'-UTR or intron region. Many of these newly designed markers would be useful for parentage, population and linkage studies.

Preliminary investigation to determine the cytotoxicity of various cryoprotectants on southern African abalone (Haliotis midae) embryos.

Cryopreservation could provide stock quantities of embryos for transgenic research. This study aimed to determine the least toxic cryoprotective agent for Haliotis midae embryos. They were exposed for 30min to concentrations varying from 5% to 20% of the following cryopreservatives: methanol (MET), polyethylene glycol (PEG), dimethyl sulfoxide (ME(2)SO) and glycerol (GLY). In contrast to cryopreservation studies done in other molluscs, PEG showed the least toxicity to H. midae embryos in concentrations ranging from 5% to 15%. MET was also less toxic than ME(2)SO and GLY at correlating concentrations. GLY showed the most toxic effects with most embryos dead or abnormal at concentrations above 15%.

Discovery and evaluation of single nucleotide polymorphisms (SNPs) for Haliotis midae: a targeted EST approach.

In this study, we describe the first set of SNP markers for the South African abalone, Haliotis midae. A cDNA library was constructed from which ESTs were selected for the screening of SNPs. The observed frequency of SNPs in this species was estimated at one every 185 bp. When characterized in wild-caught abalone, the minor allele frequencies and F(ST) estimates for every SNP indicated that these markers may potentially be useful for population analysis, parentage assignment and linkage mapping in Haliotis midae. No linkage disequilibrium was observed between SNPs originating from different EST sequences. These SNPs, together with additional SNPs currently being developed, will provide a useful complementary set of markers to the currently available genetic markers in abalone.