Work package 1: Pairing experiments
Task 1.1: Installation of the tanks, experimental set up. Tanks for the proposed experiments will be set up and tested using live fish.
Task 1.2: Recording and characterisation of the behaviour during con- and heterospecific encounters. In an experimental set up females of each of the four Ophthalmotilapia species will be confronted with males from the same or from a different species. Using visual and video recorded monitoring, the pairing behaviour of each of the four species in both conditions will be described in detail.
Task 1.3: Recording and characterisation of acoustic communication during con- and heterospecific encounters. Acoustic communication during con- and heterospecific pairing interactions will be recorded using hydrophones. We will characterise these sounds for temporal (number of pulse, pulse period, pulse and call lengths, etc.) and spectral characters (main frequency, harmonics, etc.) for the four species.
Task 1.4: Data analysis behaviour and acoustics. The data obtained during the behavioural and acoustic experiments will be analysed for differences between species and between experimental conditions (con- versus heterospecific). Depending on the nature of the data appropriate statistical test will be applied (parametric, non-parametric, anova,…).
Work package 2: Specimen and tissue collection
Task 2.1: Purchase, import and determination of live fishes. Live fish will be purchased from a reliable Zambian trader in tropical fishes (African Fish Aquaculture, Kitwe, Zambia). Detailed morphological and molecular identification of specimens will be conducted to ascertain correct species identification.
Task 2.1: DNA extraction samples genotyping by sequencing. DNA from approximately 300 samples present in frozen tissue collections will be extracted. DNA quality and quantity will be analysed using high resolution platform (Agilent Bioanalyzer).
Task 2.2: Brain dissection and RNA extraction samples. Brains of fish from the behavioural and acoustic experiments will be dissected and preserved for RNA extraction. RNA will be extracted for approximately 120 samples. RNA quality and quantity will be analysed using high resolution platform (Agilent Bioanalyzer)..
Work package 3: Next Generation Sequencing
Task 3.1: Genotyping by sequencing. Genomic libraries for genotyping by sequencing will be constructed for 300 samples from four Ophthalmotilapia species and outgroups from the Ectodini. These libraries will be sequenced on a next generation sequencing platform to generate a genome wide screening of the variation within and among species.
Task 3.2: RNA sequencing. RNA extracted from brain tissue will be reversely transcribed to cDNA libraries for 120 samples taken during pairing experiments. The libraries will be sequenced on a next generation sequencing platform to generate transcriptome sequences of the genes expressed in the brain.
Work package 4: Genomics data analysis
Task 4.1: Preparatory data analysis for genotyping by sequencing. Available cichlid reference genomes will be analysed in silico to determine the correct protocol (cutting enzyme) for the preparation of the libraries for genotyping by sequencing.
Task 4.2: Data analysis genotyping by sequencing. Sequences generated using genotyping by sequencing will be mapped to a reference genome and analysed for genetic variation. The phylogeny of the four Ophthalmotilapia species and the outgroups will be investigated to ascertain the sister species status. Introgression among species will be determined and the genome wide genetic variation will be characterised for position in the genome and source (coding or non-coding regions). Genes in gene regions with higher or lower variation compared to the average will be analysed for functionality and physiological process.
Task 4.3: Data analysis RNA sequences. Sequences generated by RNA sequencing will be assembled and mapped to a reference genome. The gene assemblies will be analysed for differential expression among different experimental conditions. The retrieved genes will be annotated using GO-blast.
Work package 5: Data Integration
Task 5.1: Integration of the behaviour and acoustics with the gene expression. Results from the behavioural and acoustics observations will be integrated with the results from the RNA sequencing analysis. We aim at linking specific gene expression profiles with behavioural patterns and acoustic profiles.
Task 5.2: Integration of behaviour and acoustics with introgression patterns. The data obtained by the genotyping by sequencing will reveal detailed introgression patterns among species. We aim at relating these patterns with differential patterns for mate pairing and acoustics to understand the role of behaviour and acoustics in prezygotic isolation.
Task 5.3: Integration of the gene expression analysis with the genotyping by sequencing. We want to examine whether the part of the genome that is expressed during mate recognition in the brains of the female cichlids corresponds with one or more of the genomic areas with increased or decreased differentiation within or among the Ophthalmotilapia species. This will shed light on the mechanisms of selection acting upon these genes (diversifying vs. balancing selection).
Work package 6: Project coordination
Task 6.1: Coordination of the project. Regular meetings will be organised to ascertain a fluent coordination among the network partners. These meetings will consist of informal and formal meetings to discuss progress, study approach and novel developments relating to the study subject. All or a selection of the partners will attend these meetings according to the necessity.
Task 6.2: Reporting and administration. All mandatory administrative and scientific reports will be coordinated by RBINS. Each partner will contribute for the designated work packages.
Task 6.3 Communication with the follow up committee. For the annual network meetings a selection of the members of the FUC will be invited to participate. This selection will depend on the needs of the project at the different stages of the project.
Task 6.4 Dissemination of the results. The obtained results will be disseminated through scientific contributions on conferences or through the publication of papers in peer reviewed journals. These results will also be translated into publications that are accessible for a broad audience. This can be done through displays in the RBINS, publications in written popular media (newspapers, scientific oriented magazines), oral interviews on radio, etc. Each partner will contribute to the dissemination of results according to the designated work packages.