Phylogenetic and phylogeographic diversity of tilapia, and their gut-bacterial microbiota

概要

Doctoral Thesis

Phylogenetic and phylogeographic diversity of
tilapia, and their gut-bacterial microbiota

(Summary)

Patrick Senam Kofi, Fatsi
Department of Bioresource Science
Graduate School of Biosphere Science
Hiroshima University

September 2020

Chapter 1: Background information
The introduction and distribution of tilapia to the Asian-Pacific region begun the wider
distribution events for the well-adapted reproductive populations of tilapia across the region.
The introduction of tilapia to Asia was celebrated as the solution to the short supply of animal
protein in the region. The year 1954 marked the initial introduction of the first tilapia species
to Japan from Thailand, which subsequently grew up to a total of nine species, from different
parts of the world, by the year 1984. However, since tilapia was introduced to Japan, very little
or nothing is known about the existing tilapia fauna in the freshwater systems of Japan. This
study was conducted to establish the degree of phylogenetic and phylogeographic diversity of
tilapia species found in Japan, and also to understand the quantitative diversity of the gut
bacterial microflora.

Chapter 2: Population genetics and taxonomic signatures of wild tilapia in japan, based
on mitochondrial DNA control region analysis.
Non-native tilapia species were repeatedly introduced to Asian and South American countries
because of their adaptiveness to new environments and new environmental conditions. Such
founder populations rapidly adapted by evolving outstanding ecological and physiological
features, making them highly successful invaders throughout the tropical and subtropical
regions. In Japan, nine tilapia species were introduced. In recent years, several established
populations were introgressed by the accidental release of aquaculture strains of Oreochromis
niloticus, causing reductions in population sizes, genetic diversity, and genetic perturbation
upon introgressive hybridization. This study aimed to assess the levels of genetic integrity,
genetic population structure, and biogeographic relationships of wild tilapia species found in
Japan. Phylogenetic and phylogeographic analysis based on mtDNA control region sequences
yielded 52 distinct haplotypes, indicating significant differences in genetic variability among

the analyzed populations. Analysis of molecular variance (AMOVA) showed high levels of
variation within populations, except for tilapia species representing reproductively
incompatible taxonomic units to O. niloticus aquaculture stains. Overall, a significant genetic
structure emerged among the 11 populations analyzed. Our results indicated that the
accidental release of O. niloticus via aquaculture contributed significantly to the current
genetic differentiation among populations within a few generations, showing clear signals of
mtDNA introgression across species boundaries.

Chapter 3: Morphological disparity between the largest genetically monophyletic taxa
of tilapia in the Kyushu and Okinawa ecogeographical regions of Japan.
Post-introduction of non-native tilapia into Japan about 6.5 decades ago, saw the distribution
and adaptation leading to the subsequent increase in the freshwater systems across the
country. In this study, we used two multivariate statistical methods to investigate the
morphological disparities relative to phenotypic plasticity and/or adaptive evolution for the
most prominent genetically monophyletic taxon of tilapia species retrieved from five
populations in Kyushu and Okinawa ecogeographical regions.

Even though conductivity

and salinity levels greatly vary for Kyushu than Okinawa populations, the PCA and LDA
largely indicated significant morphological differentiation between all Okinawa than Kyushu
populations, whereas showing a higher degree of convergence between the Kyushu and
Ishigakijima populations contrary to the other populations of Miyakojima and Naha.
Additionally, allometric growth varies significantly in Okinawa than Kyushu, attesting
morphological variations in Okinawa to adaptive or phenotypic plasticity than Kyushu. The
statistical relationship between the ANOSIM and PERMANOVA also indicated a similar
degree of differentiation between all populations. However, these observed variations are
either due to genetic and/or environmental events influencing the morphology of tilapia

populations in the wild, we also strongly believed the equal opposite whereby morphological
variations are imported with different aquaculture strains of tilapia.

Chapter 4: Characterization of environmental and GIT bacteria microbiome of tilapia.
The study of GI microbiome is the ultimate scientific viewpoint for constructive development,
prediction, and manipulation schemes aimed at successful prescription and adoption of
prominent core microbiome as prebiotics/probiotics candidates. This investigation focused on
the impact of niche bacterial microbiome on GIT colonization of resident teleost fauna by
characterizing the environmental and gut microbiota of resident tilapia fauna. The
microbiome of the environment and gut of tilapia was profiled using a culture-dependent
method and identified by 16S rRNA sequencing. The 16S rRNA sequences generated OTUs
dominated by Bacillus and Pseudomonas respectively, in terms of relative abundance.
Comparatively, Proteobacteria dominated other representative phyla. Nevertheless,
pathogenic bacteria such as Vibrio, Enterobacter, Plesiomonas, and Klebsiella were only
recorded from the gut, but Aeromonas and Pseudomonas were found common between the
environment and gut microbiota. The coexistence and/or cohabitation of opportunistic and/or
pathogenic bacteria amidst fermentative/biodegradative/bioconversion, bioremediation, and
antimicrobial synthesizing bacteria (example, Rhodococcus and Acinetobacter), suggested a
complex relationship that could competitively, sequentially, or symbiotically reduce disease
incidences. This study indicated that the gut microbiome contained selected bacterial flora
some of which could be adopted as functional prebiotic/probiotics in tilapia aquaculture.

Chapter 5: General Discussion
Phylogenetic and phylogeographic analysis based on mtDNA control region sequences

indicated that the introduction of O. niloticus via aquaculture contributed significantly to the
current genetic differentiation among populations within a few generations, showing clear
signals of mtDNA introgression across species boundaries. Successively, the two multivariate
statistical methods used to investigate morphological disparities relative to phenotypic
plasticity and/or adaptive evolution for the most prominent genetically monophyletic taxon
of tilapia species retrieved from five populations in Kyushu and Okinawa ecogeographical
regions, largely indicated significant morphological differentiation between all Okinawa than
Kyushu populations, whereas the degree of convergence between the Kyushu and
Ishigakijima populations contrary to Miyakojima and Naha suggested that allometric growth
varies significantly in Okinawa than Kyushu. Additionally, the statistical relationship
between the ANOSIM and PERMANOVA also indicated a similar degree of differentiation
between all populations. Finally, the total cultivable bacteria associated with the gut
microbiota yielded 30 predominant pure culture isolates differentiated by colony
morphologies, and taxonomically composed of Actinobacteria, Bacteroidetes, Firmicutes,
and Proteobacteria. Overall, our result indicated that the tilapia gut microbiome seemed
respectively dominated by Proteobacteria and Firmicutes totaling about 90 % of the gut
bacteria microflora, whereas Actinobacteria and Bacteroidetes represented a lesser
percentage. The findings from our study suggested that the gut of tilapia and other fishes
such as carp, salmon, zebrafish, and rainbow trout are selective of the bacteria phyla of
Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria with similar marginal
abundance for Proteobacteria and Firmicutes as the predominant phyla respective of rearing
or growth environmental conditions.