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genetic study on the impacts of environmental factors on sexual maturation of some species of family mugilidae
1. Presented By
Hala Abd El-Samea Abd El-Hameed Abd El-Samea
ال على البيئية العوامل تأثير عن وراثية دراسة
نضج
البوري عائلة أنواع لبعض الجنسي
Laboratory of Genetics, Aquaculture
Division, National Institute of Oceanography
and Fisheries
Genetic Study on the Impacts of
Environmental Factors on Sexual Maturation
of Some Species of Family Mugilidae
2. Prof. Mohamed El-Said El-Mor
Prof. of Fish Biology, Department of Marine Science, Faculty of
Science, Suez Canal University
Dr. Mohamed Abd El-Haleem Hegazy El-kady
Associate Prof. of Molecular Genetics, National Institute of
Oceanography and Fisheries, Alexandria, Egypt
Dr. Amira Ahmed Ali
Lecturer of Fishery and Molecular Biology, Department of
Marine Science, Faculty of Science, Suez Canal University
3. Prof. Mohamed El-Said El-Mor
Prof. of Fish Biology, Department of Marine Science, Faculty
of Science, Suez Canal University
Prof. Dr. Mohamed Hamed Bahnasawy
Prof. of Fish biology, Zoology Department, Faculty of Science,
Damietta University.
Prof. Mohamed Kamel Hassan Farh
Prof. of Genetics and Molecular Genetics, Department of
Zoology, Faculty of Science, Port Said University
4. In the situation of looking for solutions to the
problem of mullet family fish, especially M.
cephalus and L. ramada fish, that cannot spawn
in captivity or industrial hatcheries, we
performed a genetic study to compare the
relative genetic expression of some genes that
controlling the reproductive process, which are
affected by environmental changes in the natural
and captive environments during the spawning
season.
Introduction
5.
6. The entire or almost all of the species in the family
Mugilidae are euryhaline. That are commonly known as
mullets or grey mullets, have ray-finned bodies.
Mugil cephalus and Liza ramada are two such individuals.
They are species inhabiting varied habitats, from shallow
brackish and marine areas close to lagoons, estuaries, and
river deltas, and surviving in high salinity levels as well as
abrupt changes of water quality. It is found in along the
shores of the Black, Mediterranean Sea, and East Atlantic.
7. Classification
Domain: Eukaryota
Kingdom: Animalia
Phylum: Chordata
Class: Actinopterygii
Order: Mugiliformes
Family: Mugilidae
Genus: Chelon
Species: L. ramada
C. ramada
Domain: Eukaryota
Kingdom: Animalia
Phylum: Chordata
Class: Actinopterygii
Order: Mugiliformes
Family: Mugilidae
Genus: Mugil
Species: M. cephalus
Mugil cephalus Liza ramada
8.
9.
10.
11. Liza ramada
Mugil cephalus
The head is short and flattened
A broad head and a thick
flattened above the eyes
Transparent adipose eyelid that
covers most of the eye
The mouth is broad with a narrow
upper lip
Lips are thin, with a bump at the tip
of the lower lip
It has an elongate body compressed
laterally.
The back is olive-green, sides are
silvery and shade to white.
Common names:
Chelon ramada, liza ramada
Trade name: Toubar
Common names: flathead mullet,
striped mullet, grey mullet
Trade name: Bouri
Its maximum length is 70 cm
Its length is typically 30 to 75 cm
It can reach a maximum weight of
three kilograms
It can reach a maximum weight of
eight kilograms
Spawning takes place at sea near the
coast from September and January
with one peak in December and
January
Spawning takes place at sea from
August to December with one peak in
September and October
12. They migrate to the sea to reproduce;
subsequently the fish fry approach the shore in
dense schools and enter lagoons, rivers, or even
lakes, where they continue to grow.
When grey mullets approach sexual maturity (1-2
years for L. ramada and 2-3 years for M. cephalus),
they move again towards the sea, where their
maturation is completed and their spawning takes
place.
Reproductive biology
13.
14. This fish species is considered a promising
candidate for aquaculture diversification in the
Mediterranean region and beyond due to its
robust resistance to captivity, rapid growth,
omnivorous feeding habits, and high market value
(Crosetti, 2015; Abou-Gabal, 2018).
It supports commercial and recreational fisheries
owing to the highly contained protein and vitamins
in the muscle tissue, the value of caviar from
females, and is a stable food source in many
countries (Whitfield et al. 2012).
Mullet aquaculture
15. The majority of commercial grey mullet
aquaculture production in Egypt revealed that
relies on wild-caught fry, which is less expensive
but not sustainable and time-consuming for
large-scale fish production (El-Dahhar, 2014;
Marimuthu, 2019; Ramos-Júdez et al. 2023).
16. Fish reproduction in captivity can be regulated by
modifying environmental parameters, such as
water temperature, salinity, photoperiod, and
social characteristics (Fiszbein et al. 2010;
Ramallo et al. 2014).
The internal regulating variables of fish can be
altered by injecting hormones or other
chemicals, or by placing the fish in a suitable
habitat, for controlling the reproductive cycle
(Das, 2012; Das et al. 2013).
17. Female vitellogenesis is not triggered and they
stay at previtellogenesis and, unavoidably,
aquaculture production is based mostly on the
acquisition of wild juveniles or broodstock, which
is not sustainable in the long run (Yousif et al.
2010).
Despite the closely related external appearance
between the two species, M. cephalus do not
undergo final oocyte maturation, ovulation, or
spawning in captivity but the thinlip grey mullet,
L. ramada can reach the final gonadal maturation
in fresh and brackish water but the natural
spawning was not observed (Mousa, 2010).
18. In fish and other vertebrates, the hypothalamic-
pituitary-gonadal axis regulates reproduction
through the coordinated communication of the
anterior pituitary, the gonads, and the brain.
The brain-pituitary-gonadal (BPG) axis, often referred
to as the hypothalamic-pituitary-gonadal (HPG) axis,
is essential for fish reproduction because it promotes
maturation in teleosts and other vertebrates through
hormonal and neurological feedback (Borella et al.
2020) as shown in figure (1).
Reproductive hormones
25. In the pars intermedia of fish, there is also another
type of secretory cell known as somatolactotropes,
which secretes somatolactin (Kaneko et al. 1993).
It has been established that somatolactin (Sl) is a
hormone that is a member of the growth
hormone/prolactin (GH/PRL) family (Zhu et al.
2004).
SLα is produced in the posterior pars intermedia by
somatolactotropes, while SLβ is produced by
somatolactotropes in the anterior pars intermedia of
the pituitary, (de Celis et al. 2003; Benedet, 2008).
Somatolactin (Slα)
26. Aim of the work
This study aims to using the gene expression technique to
identify the genes under ideal conditions in the wild and
captive habitats that regulate the proper gonadal
development of female M. cephalus and L. ramada during
the spawning season.
The relative expression levels of Kiss2, Gpr54, Drd2,
and Gnrh1 genes in the brain and Lhβ, Fshβ, and Slα in
the pituitary will be studied. Also the previously
mentioned genes will be under investigation in the
ovary.
Determining the relationships between the relative
mRNA expressions of the investigated genes in the
natural and captive habitats.
27. This is the first study to examine the genetic
composition of wild and captive populations of L.
ramada throughout the spawning season.
Looking for solutions to the problem of mullet family
fish, especially M. cephalus and L. ramada fish, that
cannot spawn in captivity or industrial hatcheries.
28. Materials and methods
Fish and Study area
1. The wild ripe females of M. cephalus and L.
ramada were collected during their spawning
seasons from the narrow outlets (Bogaz 2) that
connect Mediterranean Sea with El-Manzala
wetland from the side of El-Gamil and the
international coastal road, while the control or
immature samples were sampled from El-
Manzala wetland. The google map is shown in the
figure.
30. 2. The captive females of fish were captured from
outdoor earthen aquaculture farm at Ras El-Bar,
Damietta governorate. The location is shown in
figure.
33. Sampling and sample elaboration procedure
Females of mature M. cephalus wild fishes were
collected from their natural habitat during the spawning
between October, 2021 and September 2022, while
females of ripe L. ramada were captured during
December, 2021 and December, 2022 from the same
location.
The study was conducted in the genetics and genetic
engineering laboratory at Al-Mataryiah Station for
Aquatic Resources (NIOF), in Egypt, where they were
brought alive. From the wild and captive samples, ten
ripe female specimens were accurately selected for
each group.
34.
35.
36.
37. All fish handling and procedures were approved by
the UPV/EHU Ethics Committee on Animal
Experimentation and by the regional authorities.
Dissections were immediately performed in situ.
Samples from the entirety of the brain or pituitary
and a section of the ovary were promptly
preserved in liquid nitrogen until further use.
38. Total RNA isolation (Trizol method)
Total RNA was extracted from the pituitary,
brain and ovary samples of M. cephalus and L.
ramada females using TRI reagent solution
(Transzol, China).
Almost 100 mg of the ovary was taken, whereas
in the case of the brain or the pituitary, the
entire tissue was used.
The used reagents
Trizol – Chloroform –Isopropanol – 75% diluted
ethanol – Elution buffer.
39.
40. The absorbance at A280 was also taken for each sample
to determine the purity of RNA. The following
formula was used to calculate the concentration of
RNA once the absorbance was determined:
O.D. Reading (A260) X (40 µg/ml l X dilution
factor = concentration in µg/l
Determination of total RNA concentration
The RNA quality is calculated by 260 absorbance /
280 absorbance to good quality the result would be
1.8 : 2.2
42. First-strand cDNA synthesis using SCRIPT RT-PCR two-step
Kit (RevertAid First Strand cDNA Synthesis Kit (Thermo
fisher)
43. Primer designing for target genes with β-actin as a
reference gene
The parameters that were considered for specific primer
designing
20-22 nucleotides in length
50- 60 GC content
Tm>60 °C; best result are obtained
PCR product from 100 to 250 base pairs
According to the NCBI, primers were specifically designed for
our investigation using partial or entire cDNA sequences were
specifically designed for our investigation using partial or entire
cDNA sequences.
Antisense primers for M. cephalus and L. ramada were created
and arranged in the following order: Lhβ, Fshβ, Slα, Gpr54,
Drd2, Gnrh1, and Kiss2 in the table.
44.
45. The examination of primer specificity
Checking primer specificity is crucial to ensure accurate
and specific amplification of the target DNA sequence by
the following:
By Utilize the Primer-BLAST tool provided by NCBI,
which integrates primer specificity checking with
the BLAST algorithm. It allows you to design and
check primers in one step against a specified target.
Perform a test PCR using your primers and the
intended template.
Verify the size of the amplified product matches
expectations.
Confirm specificity by sequencing the PCR product.
47. Preparation of the QRT-PCR assay
The reaction was established by a Real-Time PCR system
thermocycler (7300 Thermoscientific). Triplicate reactions
were performed for the positively transcribed cDNA and
their corresponding controls, respectively.
The 25 μl reaction volume consisted of 12.5 μL of SYBR
Green fluorescent dye master mix (Maxima, Thermo
Fisher), 0.6 pmol of the specific primer pair, 5 μL diluted
cDNA template and the remaining volume was RNase
water.
Cycling parameters were as follows: 50 °C for an initial
step for 2 min and 10 min at 95 °C, a denaturing step at
95°C for 15 s for 40 cycles, and 60 °C for an annealing step
at 30 s, and finally an extension step at 72 °C for 30 s.
48.
49. The cDNA from immature female mullet was used
as a calibrator. The data was normalized with a
single internal reference gene to simplify the
assessment of expression levels of the target
genes in various tissues. The investigation of RT-
QPCR assays was performed by the 2−ΔΔCt method
according to Livak and Schmittgen, (2001) and
the normalization according to the method of
Pfaffl (2001).
Normalization to QRT- PCR reaction
53. Total length and total weight of female M. cephalus in wild
and captive habitats
Female M. cephalus Wild Captive
Immature (Control)
Length
Weight
23.03±4.88
235.57±39.33
19.4±0.91
169.28±55.19
Mature
Length 44.29±1.79 47.22±2.43
Weight 954.06±109.18 1143.75±190.9
56. The transcription pattern of Kiss2 in the brain and ovary
of M. cephalus
1.14
1.15
4.27
1.03
6.77
1.07
10.47
2.87
57. The transcription pattern of Gpr54 (Kissr2) in the brain and
ovary of M. cephalus
1.07
4.07
1.03
5.35
1
5
1
7.83
58. The transcription pattern of Gnrh1 in the brain and ovary
of M. cephalus
1.04
1.78
1.07
5.64
1.02
6.64
1.12
13.37
59. The transcription pattern of Drd2 in the brain and
ovary of M. cephalus
1.06
1.86
1
1.35 1.16
7.94
1.03
4
60. Correlation between Kiss2, Gpr54, Gnrh1 and Drd2 at wild
and captive environments
The various patterns of correlation between the analyzed
gene expression profiles in the brain and ovary of wild
females and captive ones. The investigated genes showed
a direct Pearson association with significant values in the
brains of wild females in relation to the values of captives
throughout the spawning period.
Moreover a direct correlation was observed in the ovaries
of wild females for Kiss2, Gnrh1 and Drd2 with their
counterparts in the captive ones, while Gpr54 revealed an
inverse correlation in the ovaries within the two habitats.
65. The expression patterns of three genes (Fshβ, Lhβ, and
Slα) in the pituitaries and ovaries of wild females seem
to be significantly correlated with those of their
counterparts in captive females.
The direct relationship between Fshβ and Lhβ in the
pituitaries and ovaries was observed.
Furthermore, the direct association of Slα in the ovary
was displayed as well, while, the negative correlation
for Slα in the pituitary was observed.
Correlation between Fshβ, Lhβ, and Slα in the wild and
captive habitats of M. cephalus
66. Correlation between the different genes of M. cephalus in
the wild habitat
Wild habitat Gnrh1 Fshβ Drd2 Kiss2 Gpr54 Lhβ Slα
Kiss2 0.95 0.66* 0.77
Gpr54 -0.69 -0.01 0.42 -0.59
Gnrh1 -0.73* -0.69 0.93
Drd2 0.29 -0.91 0.44
Fshβ 0.86* -0.96 0.79 0.99
Lhβ 0.99* -0.82* 0.98*
Slα 0.99 -0.9 0.88
67. Captive habitat Gnrh1 Fshβ Drd2 Kiss2 Gpr54 Lhβ Slα
Kiss2 0.25 0.99 0.97 0.92
Gpr54 0.024 0.93 0.97 0.94
Gnrh1 0.33* 0.25 -0.14*
Drd2 0.99 0.95 0.78
Fshβ 0.4* 0.86 0.74
Lhβ 0.86 0.89 0.99
Slα -0.32* 0.83 0.98
Correlation between the different genes of M. cephalus in
the captive habitats
68.
69. Total length and total weight of female L. ramada in the
wild and captive habitats
Female
L.ramada
Wild Captivity
Immature
(Control)
Length
Weight
21.4±2.63
187.97±48.61
25.9±2.76
166.53±47.49
Mature
Length 44±3.63 33±1.9
Weight 639.29±164.9 352.55±66.79
72. The transcription pattern of Kiss2 in the brain and
ovary of L. ramada
1.08
1.05
5.76
1.07
5.33
1.02
6.67
2.37
73. The transcription pattern of Gpr54 (Kissr2) in the brain
and ovary of L. ramada
1.19
1.8
1.05
2.6
1
3.9
1.04
4.25
1.08
74. The transcription pattern of Grnh1 in the brain and ovary
of L. ramada
1.08
1.5
1.02
2.1
1.19
3.33
1.07
6.97
75. The transcription pattern of Drd2 in the brain and ovary
of L. ramada
1.04
3.07
1.07
4.22
1.01
1.46
1
1.97
76. The varying patterns of correlation between the analyzed
gene expressions profiles in the brain and ovary of wild
and captive females.
Drd2 and Kiss2 genes showed a reverse correlation in the
ovaries of wild and captive females while the direct
association in the brains during the spawning period was
observed, whereas Gnrh1 and Gpr54 showed a direct
correlation with significant values in the brains and
ovaries of wild females compared to the values of
captives at the same time.
Correlation between Kiss2, Gpr54, Gnrh1 and Drd2 in
the wild and captive female of L. ramada
78. The transcription pattern of Fshβ in the pituitary and ovary
of L. ramada
1.1
4.5
1.12
7.16
1.08
9.3
3.62
1.08
79. The transcription pattern of Lhβ in the pituitary and ovary of
L. ramada
1.03
6.26
5.93
1
5.63
1.13
1
3.04
80. The transcription pattern of Slα in the pituitary and ovary
of L. ramada
1.1
1.25
2.85
1.21
2.18
1.17
3.5
1.71
81. The expression patterns of Fshβ, Lhβ, and Slα genes in
the pituitary and ovary of wild females seem to be
significantly correlated with those of their counterparts
in captive ones.
Fshβ and Slα in the pituitaries and ovaries displayed a
direct association between the wild and captive, while
Lhβ displayed an inverse correlation in the pituitaries
and ovaries
Correlation between Fshβ, Lhβ, and Slα in the wild and
captive habitats of L. Ramada
82. Wild habitat Gnrh1 Fshβ Drd2 Kiss2 Gpr54 Lhβ Slα
Kiss2 0.71* 0.012 0.74 -0.68
Gpr54 -0.89* -0.02 -0.02
Gnrh1 0.71* 1*
Drd2 0.92 0.37* -0.46
Fshβ 0.93 0.68
Lhβ -0.07* -0.75 0.66
Slα 0.03 0.72 0.43 -1
Correlation between the different genes of L. ramada in
the wild habitat
83. Captive habitat Gnrh1 Fshβ Drd2 Kiss2 Gpr54 Lhβ Slα
Kiss2 0.98 0.93 -0.59*
Gpr54 0.88* 0.49 0.78 -0.97*
Gnrh1 0.93 0.98 0.51
Drd2 0.84* -0.25
Fshβ 0.98 0.65
Lhβ -0.73* -0.44* 0.22
Slα 0.78 0.89 0.66* 0.04
Correlation between the different genes of L. ramada in
the captive habitat
84.
85. In conclusion, it was shown that fish kept in captivity
expressed the least amounts of Kiss2, Gpr54, Grnh1,
Fshβ and Lhβ. When compared to the wild female M.
cephalus and L. ramada.
Gnrh1 expressions in the brains was higher and
tended to correlate with the expression of Fshβ, Lhβ,
and Slα in the pituitary of wild females, while the
expression of Lhβ and Fshβ in the pituitaries of captive
fishes may be inversely correlated with Gnrh1
transcription levels in the brains.
Whereas the expression levels of Drd2 are more likely
to be stumpy in the wild fishes than in the captive
ones.
86.
87. We suggest that this study clarify the function of the
kisspeptin system (Kiss2 and Gpr54) and how it
contributes to spawning because of the observed
variations in its gene expression in the two habitats
and kiss2 relation to Lhβ secretion.
Consequently, it is important to investigate it in a lab
setting by injecting it into captive mullet fish (M.
cephalus and L. ramada) and determining how much
of their impacts on the spawning process.
88. According to the study, Gnrh1 and Drd2 have a
significant impact on reproduction, and their
inverse relationship in the wild helps the
spawning process to be completed, whereas direct
correlation in captivity prevents it from happening.
The research provides additional context for the
involvement of Slα, since it was found to have
statistically significant relationships with Gnrh1 and
to have a noticeable less increase in gene
expression in the captive.
