Download Expert meeting: David Clayton

Expert meeting: David Clayton
Location: RBINS (Brussels)
November 17, 2015
Authored by: Sofie Derycke, Loic Kéver and Maarten Van Steenberge
Expert meeting: David Clayton
17/11/2015
Present : Loic Kéver, Koen Herten, Jos Snoeks, Erik Verheyen, Sofie Derycke, Maarten Van Steenberge, Jonas, Pascal
Poncin, Jeroen Van Houdt
Excused : Eric Parmentier
10.00h – 11.15h: Seminar by David Clayton: “Acute isolation leads to a shift in
neurogenomic state across the songbird forebrain” (open to all interested scientists)
This talk explained the relevance of songbirds as a study system for vocal communication
and learning behaviour and presented unpublished data on gene expression data in the
forebrain of zebrafinches in isolation and when exposed to con- and heterospecific songs.
11.30h-12.15h: Presentation GENBAS project by Sofie Derycke (GENBAS partners only)
Overview of the aims of the project
Expert meeting: David Clayton | 11/17/2015
12.30h-13.15h: Lunch at l’ Oriento
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13.30h-17.15h: Presentations on progress of each work package and planning of future
actions
1/ Behavioural and acoustic experiments (presented by Loic Kéver):
Reproductions have been recorded for O. ventralis (4) and O. nasuta (3).
Important to keep in mind that the set-up for both species is somewhat
different: for O. ventralis, one male is present in the tank, for O. nasuta 3
males are present. Based on the literature on fish behavior, some
behaviours will be redefined (“quivering” will be replaced by “swimming on
the spot, tail wagging”; “spasm” will be named “inviting”)
Other behaviors will be added to the ethogram
“inviting’ behaviour can be accompanied by a very simple sound, most
likely for close range communication
Ethograms will be adjusted, and statistics recalculated based on reanalyses
of the movies
Nest shape experiments did not show a preference of O. nasuta for a
particular bower; test will be redone with gravid female because she may
show more interest in a nest than non-gravid females and because nongravid females might also try to avoid nests of conspecifics.
Heterospecific setting: 1 male O. nasuta with 3 females O. ventralis: they
interact but females become dominant.
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Follow-up tanks of O. ventralis and O. nasuta in which reproduction has
taken place. When a females lays the first egg, she will be killed and brain
will be disected and stored in RNA later.
Another experiment will be initiated to investigate the gene expression
when a female is confronted with a con-or heterospecific male:
o 1F On + 1M On
o 1F On + 1 M Ov
o 1F On + 1 M Ob
o 1F On + 1 F On
o 1F On
 5 treatments * 3 replica’s * 6 brainparts = 90 RNA samples
 15 O. nasuta females will be sacrificied; they all need to be mature;
3 females On and 3 males of On, Ov and Ob are also needed
 after the whole procedure has been done, we can consider to
repeat the experiment with O. ventralis
 fish will be acclimatised for 1 day, and than allowed to see each
other for 30 min; this time frame will be tested by Loic to ensure
behavioral responses are present during this period. A grid will be
used so that they can see and smell each other, but agression
should be reduced by the fact that they cannot physically interact
with each other
2/ Gene expression part (presented by Koen Herten and Sofie Derycke):
- We have performed RNAseq analysis (Quantseq 3’ from Lexogen) on 60 samples
from control females of O. nasuta and O. ventralis.
- reads: 50-55% of the reads map to the tilapia genome, only 12-17% maps in gene
regions => for each sample, we retained roughly 200 000 reads, which is
substantially lower than normal RNA seq data. Koen will remap the reads to the O.
ventralis transcriptome that has been published by the group of Walter Salzburger
to explore whether we can retrieve more reads per sample
-reads that did map against genes of Oreochromis indicated that 7189 genes were
expressed, of which 64% were expressed in all brainparts. The number of genes
uniquely expressed in a particular brain region ranged between 69-238. All major
pathway groups and organism systems were expressed in all brain regions.
-Differential expression (DE) was investigated between the two species: only 93
genes were DE, indicating that under control conditions the females of both species
are experiencing the same cues in the same way
-Of the DE genes, 64% are downregulated in O. ventralis => need for validation of
this result, can it be because of differential mapping of the reads to Oreochromis?
-The most pronounced difference (10-12 times downregulated in O. ventralis) was
found in the TERC gene, a component of telomerase
Expert meeting: David Clayton | 11/17/2015
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3/ Genotype-by-sequencing (by Sofie Derycke)
- testrun has been analysed, ca 15 000 SNPs are found shared between all specimens.
These SNPs were distributed across the scaffolds of the nile tilapia genome, and
were present in both coding and non coding regions.
- Library preparation of the full dataset is at full speed now. Some questions with the
protocol needed to be answered:
o Can we clean-up only a part of the PCR product (to safe costs on Ampure
beads)? Yes
o Transfer of beads during clean-up is not a problem (magnetic block also used
in Genomics core)
o It is normal that not all primer dimers are removed, as long as the fraction is
much less than that of the PCR product, it is ok.
o If a good correlation is observed between picogreen and qPCR, than we can
choose the picogreen method to quantify (will be much quicker and cheaper)
o If we do need to use qPCR: standards are run in duplicates, individual
samples should be run individually
o Standard size to convert pmol to ng/microliter: doesn’t matter, as long as you
take the same size for all samples.
o Prepare a pool of 20 nM. No need to concentrate, just take out 30 microliter
for the Blue Pippin. We assume everything is homogeneously distributed in
the pool, so taking a fraction will still contain all fragments of all specimens
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Final library preparation steps will be performed in the next weeks.
Expert meeting: David Clayton | 11/17/2015
4/ Alternative classifications of Ophthalmotilapia and Cyathopharynx (presented by
Maarten Van Steenberge)
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overview of the different varieties of the Ophthalmotilapia as found in the literature from
aquariophiles. Distribution of varieties throughout the lake is reflected in the haplotype
network. Haplotypes are grouped somewhat according to varieties, and in many instances
better than according to species. However, most of these varieties cannot be verified
anymore, because colours disappear when specimens are fixed in ethanol. So the grouping
can equally well represent geographic clustering. Specimens need to be checked to see
whether varieties can still be found (nasuta OB eg should still be visible). For
Cyathopharynx, measurements have been performed in ca 200 specimens. Until now, we
had difficulties to define a priori groups to separate the morphometric data into distinct
species.
5/ Year report has to be submitted by the 30st of November. Contributions from all
partners are required.
Meeting ends at 17.15h, after which a dinner was offered to David Clayton.