Biomarker responses in Nile tilapia (Oreochromis niloticus) larvae as a tool to assess aquatic pollution in Egypt

Abstract

With a global farmed production of around 5.5 million tonnes annually, the tropical cichlid Nile tilapia (Oreochromis niloticus) is one of the most important farmed fish species worldwide, with Egypt being the leading producer on the African continent. In arid countries including Egypt, water resources are scarce and agricultural drainage as well as treated sewage are the primary water suppliers for aquaculture. Accordingly, fish are potentially exposed to a wide range of pollutants, which can build up to levels exceeding maximally acceptable concentrations in tilapia fillets. Despite a clear need to assess water quality in Egypt, comprehensive tools to measure water suitability for aquaculture and predict potential environmental impacts are currently lacking. This thesis systematically assessed the potential of Nile tilapia (Oreochromis niloticus) larvae at age of seven days post-fertilization (PF) to express biomarkers following exposure to diverse classes of environmental pollutants. This was accomplished through an integrated approach combining genome searches with transcriptomic and biochemical analyses. The study started with a characterization of the key gene superfamilies involved in the biochemical defensome of Nile tilapia (‎Chapter 2) through performing BLAST homology searches using the homologous gene sequences from different model species followed by validation of the identity of isolated sequences by reverse BLAST searches and phylogenetic analyses. The analysis identified 76 cytochrome P450 (CYP) genes in Nile tilapia, which were classified into 10 clans and 17 families, with a notable expansion observed for genes of clan 2. The findings also revealed 54 genes belonging to the ATP-binding cassette (ABC) transporter superfamily, distributed across eight subfamilies (10 ABCAs, 11 ABCBs, 15 ABCCs, 4 ABCDs, 1 ABCE, 3 ABCFs, 8 ABCGs, and 2 ABCHs). Moreover, 21 glutathione S-transferase (GST), 4 heat shock protein 90 (HSP90), 19 HSP70 genes as well as 34 UDP-glucuronosyltransferase (UGT) genes were characterized in the Nile tilapia genome. These findings underscore the adaptive genetic complexity of Nile tilapia in responding to environmental stressors and pollutants. To explore their utility for biomarker studies, larvae of O. niloticus at 7d PF were exposed to six waterborne toxic chemicals provided as single compound treatments, namely gemfibrozil (GEM), malathion, azoxystrobin, bisphenol A (BPA), Benzo[a]pyrene (BaP), and Cd, as well as three mixture treatments including BaP + BPA, Cd + BaP, and Cd + BPA at two different sublethal concentrations for 24 hours. Then, the transcript expression of target biomarker genes including cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), glutathione S-transferase alpha 1 (GSTA1), superoxide dismutase (SOD), glutathione peroxidase (GPX), Bcl-2 associated X protein (BAX), caspase-3 (Casp3), B-cell lymphoma (BCL-2), and metallothionein (MT) was examined using RT-qPCR (‎Chapter 3). The results showed that SOD, GPX, CYP1A1, GSTA1, and BCL-2 genes were transcriptionally upregulated in larvae following exposure to most of the chemicals, compared to controls (p < 0.05), while no statistically significant effects were observed for the genes MT, BAX, and Casp3. These results confirm the sensitivity and specificity of SOD, GPX, CYP1A1, GSTA1, and BCL-2 biomarker genes in Nile tilapia larvae. ‎Chapter 4 examined the comprehensive transcriptional responses of tilapia larvae, employing RNA-seq following exposure to the above chemicals in single compound exposures. As a result of quantification of the expression abundance and differentially expressed gene (DEG) analysis, 347, 294, 238, 131, 54, and 37 DEGs were identified in BPA, azoxystrobin, malathion, GEM, BaP, and Cd, respectively. Gene ontology (GO) function, ingenuity pathway analysis (IPA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed an enrichment of detoxification and oxidative stress pathways following larval exposure to all pollutants. Enrichment of immunoinflammatory response, endocrine disruption, lipid metabolism, and neurological pathways was observed in response to exposure to most of the chemicals. In addition, GEM, BPA, and BaP caused an alteration of the MAP kinase cascade, whereas exposure of larvae to malathion, BaP, and Cd induced DNA damage responses. These results suggested the potential of Nile tilapia larvae at 7d PF to express novel molecular biomarkers that can provide deeper mechanistic insights into stress responses triggered by these pollutants. Finally, ‎Chapter 5 investigated the response of the oxidative stress biomarkers oxylipins in tilapia larvae following 24h exposure to two concentrations of GEM and Cd. Following isoprostane extraction and quantification from tilapia larvae tissues, a significant higher level of 13-HOTrE, 9-HODE, PGA2 isomer, 14-HDHA, along with lower 5-HEPE, 12 12-HEPE were displayed in tilapia larvae following exposure to both concentrations of GEM as compared to control treatment. Moreover, there was a significant increase in levels of 12- HETE, PGF2α, TxB2, 14-HDHA, and 17-HDHA in tilapia larvae following Cd exposure. From these results, we can conclude that Cd targeted inflammatory oxidative burst through regulated enzymatic signalling via activation of both COX and LOX signalling pathways, whereas GEM appears to trigger a non-enzymatic oxidative stress profile. Taken together, this thesis concluded that Nile tilapia larvae at age of 7d PF can be a promising model for studying stress responses triggered by various water pollutants. By examining these biomarker responses and applying the findings to water management in Egypt, we can gain mechanistic insights into the actions of these pollutants, which can support the development of an environmental monitoring system tailored to tilapia aquaculture and sustainable water resource management.