الفهرس 
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Abstract
Imberi (Brycynus nurse) which belongs to family Alestedia, order of characiformes, is one of the African freshwater fish species and lives in Egypt and some other African Countries. The color of this species is silvery and often a blackish patch lies at the beginning of the lateral line. It feeds on algae, insects, planktonic crustaceans and less frequently small fishes mainly Haplochromis spp. and spawns in the summer season. B. nurse considers one of the marketable fishes of family Alestidae in Egypt and used in the salted fish industry.
To date, no information is available about the karyotype or sex-determination system in B. nurse or the majority species of the Alestidae family. So, the present study was carried out to:
1- Investigate the diploid chromosome number, chromosome feature and fundamental number (FN) in males and females of B. nurse.
2- Determine any possible morphologically differentiated sex chromosomes in B. nurse.
3- Investigate the molecular sex differences between males and females of B. nurse using RAPD, SCoT and ISSR markers.
To achieve that a total of 34 live adult specimens of B. nurse (17 males and 17 females) were collected from River Nile at Assiut city, Egypt, from March 2016 till June 2018. The collected specimens were transferred to the laboratory for analysis where 12 specimens (6 males and 6 females) were used for cytogenetic analysis and 22 specimens (11 males and 11 females) were used for molecular analysis.
1- Ctyogenetical analysis:
The collected specimens were injected intraperitoneal with 0.1% colchicine at a dose of 20 ul/g body weight for about three hours. After colchicine treatment, the specimens were sacrificed by decapitation and their sex was determined by histological analysis for their gonads. Then, intestine, gills and kidney tissues were removed immediately and sliced into 0.5 cm segments and immersed in a hypotonic solution of 0.075 M KCL for 30 min to swell the tissues. The swollen tissues were then fixed in cold freshly prepared Camoy’s fixative for 24 hours. Then, the slides were prepared according to the method of solid tissue technique, stained with Giemsa and examined under a total magnification of 1500 x under Olympus microscope. For karyotype analysis, ten well spread metaphases were captured and analyzed using Micromeasure software.
The results of the ctyogenetical analysis could be summarized as follow:
1. A total of 430 spread metaphases from six males and six females with at least 30 metaphases from each specimen were examined.
2. The count of chromosomes in the examined metaphase ranged from 41 (4.56%) to 45 (2.65 %) with the majority of cells with 44 chromosomes (79.77 %).
3. Karyotypic arrangement revealed three pairs of metacentric chromosomes, three pairs of submetacentric chromosomes, sixteen pairs of acrocentric chromosomes and FN was 56.
4. No morphologically differentiated heteromorphic sex chromosomes were detected where both males and females show the same diploid chromosome number (2n = 44)
5. The proposed karyotype formula for B. nurse is n = 3 M + 3 SM + 16 A, and FN=56.
2- Molecular analysis:
Genomic DNA was isolated from the caudal fins tissues from five males and five females of B. nurse. An equal amount of genomic DNA isolated from five specimens was mixed to prepare the bulked DNA sample for each gender. A total of 29 primers (9 RAPD primers and 10 primers for each SCoT and ISSR markers) were randomly selected and used in the present study to investigate the molecular sex differences between males and females of B. nurse. However, when some primers could successfully generate possible sex-specific bands after screening the bulked DNA samples, and to validate that, these primers were used further with individual DNA samples from a new group of specimens (6 males and 6 females) which allows increasing the number of tested individuals to increase the accuracy.
The results of the molecular analysis could be summarized as follow:
1. All 29 tested primers successfully generated a number of bands with DNA samples from males and females.
2. The total number of generated bands by RAPD primers was 75 bands, where primer OPE-04 produced the highest number of bands (13 bands) and primer OPA-11 produced the lowest number of bands (4 bands).
3. Among nine tested RAPD primers, only one primer OPI-18 successfully generated one possible female-specific band (660 pb) with the bulk DNA sample.
4. Interestingly, when this primer was used further with individual DNA samples from males and females, it generated the same specific band in all individual DNA samples from the females only suggesting that this band is female-specific.
5. The total number of generated bands by SCoT primers was 99 bands, where primer SCoT-18 produced the highest number of bands (18 bands) and primers SCoT-02 and SCoT-16 produced the lowest number of bands (3 bands). Out of ten SCoT primers tested, only two primers (SCoT-16 and SCoT-18) could generate possible female-specific bands (1150 bp and 720 bp, respectively) with the bulk DNA samples.
6. However, when these two primers were used further with the individual DNA samples from males and females only one primer (SCoT-18) produced female-specific band which was generated in all-female individuals tested and disappeared in all-male individuals tested.
7. Otherwise, the other primer (SCoT-16) which generated a polymorphic band in the bulked DNA samples from females when further used with the individual DNA samples from males and females could generate this polymorphic band in one male out of six tested males and two females out of six tested females. It appears that this band is not sex-specific and may result from the variation between the individuals.
8. The total number of generated bands by ISSR primers was 53 bands, where primer UBC-815 produced the highest number of bands (9 bands) and primers UBC-807, UBC-814, UBC-846, and UBC-873 produced the lowest number of bands (3 bands).
9. All of the ten tested ISSR primers failed to generate any sex-specific band.
10. According to molecular analysis results in this study, it seems that females of B. nurseare the heterogametic gender (ZW) and males are the homogametic gender (ZZ).
11. In the future, SCoT marker can be a suitable and powerful marker to be used in genetic analysis studies in fish.
Imberi (Brycynus nurse) which belongs to family Alestedia, order of characiformes, is one of the African freshwater fish species and lives in Egypt and some other African Countries. The color of this species is silvery and often a blackish patch lies at the beginning of the lateral line. It feeds on algae, insects, planktonic crustaceans and less frequently small fishes mainly Haplochromis spp. and spawns in the summer season. B. nurse considers one of the marketable fishes of family Alestidae in Egypt and used in the salted fish industry.
To date, no information is available about the karyotype or sex-determination system in B. nurse or the majority species of the Alestidae family. So, the present study was carried out to:
1- Investigate the diploid chromosome number, chromosome feature and fundamental number (FN) in males and females of B. nurse.
2- Determine any possible morphologically differentiated sex chromosomes in B. nurse.
3- Investigate the molecular sex differences between males and females of B. nurse using RAPD, SCoT and ISSR markers.
To achieve that a total of 34 live adult specimens of B. nurse (17 males and 17 females) were collected from River Nile at Assiut city, Egypt, from March 2016 till June 2018. The collected specimens were transferred to the laboratory for analysis where 12 specimens (6 males and 6 females) were used for cytogenetic analysis and 22 specimens (11 males and 11 females) were used for molecular analysis.
1- Ctyogenetical analysis:
The collected specimens were injected intraperitoneal with 0.1% colchicine at a dose of 20 ul/g body weight for about three hours. After colchicine treatment, the specimens were sacrificed by decapitation and their sex was determined by histological analysis for their gonads. Then, intestine, gills and kidney tissues were removed immediately and sliced into 0.5 cm segments and immersed in a hypotonic solution of 0.075 M KCL for 30 min to swell the tissues. The swollen tissues were then fixed in cold freshly prepared Camoy’s fixative for 24 hours. Then, the slides were prepared according to the method of solid tissue technique, stained with Giemsa and examined under a total magnification of 1500 x under Olympus microscope. For karyotype analysis, ten well spread metaphases were captured and analyzed using Micromeasure software.
The results of the ctyogenetical analysis could be summarized as follow:
1. A total of 430 spread metaphases from six males and six females with at least 30 metaphases from each specimen were examined.
2. The count of chromosomes in the examined metaphase ranged from 41 (4.56%) to 45 (2.65 %) with the majority of cells with 44 chromosomes (79.77 %).
3. Karyotypic arrangement revealed three pairs of metacentric chromosomes, three pairs of submetacentric chromosomes, sixteen pairs of acrocentric chromosomes and FN was 56.
4. No morphologically differentiated heteromorphic sex chromosomes were detected where both males and females show the same diploid chromosome number (2n = 44)
5. The proposed karyotype formula for B. nurse is n = 3 M + 3 SM + 16 A, and FN=56.
2- Molecular analysis:
Genomic DNA was isolated from the caudal fins tissues from five males and five females of B. nurse. An equal amount of genomic DNA isolated from five specimens was mixed to prepare the bulked DNA sample for each gender. A total of 29 primers (9 RAPD primers and 10 primers for each SCoT and ISSR markers) were randomly selected and used in the present study to investigate the molecular sex differences between males and females of B. nurse. However, when some primers could successfully generate possible sex-specific bands after screening the bulked DNA samples, and to validate that, these primers were used further with individual DNA samples from a new group of specimens (6 males and 6 females) which allows increasing the number of tested individuals to increase the accuracy.
The results of the molecular analysis could be summarized as follow:
1. All 29 tested primers successfully generated a number of bands with DNA samples from males and females.
2. The total number of generated bands by RAPD primers was 75 bands, where primer OPE-04 produced the highest number of bands (13 bands) and primer OPA-11 produced the lowest number of bands (4 bands).
3. Among nine tested RAPD primers, only one primer OPI-18 successfully generated one possible female-specific band (660 pb) with the bulk DNA sample.
4. Interestingly, when this primer was used further with individual DNA samples from males and females, it generated the same specific band in all individual DNA samples from the females only suggesting that this band is female-specific.
5. The total number of generated bands by SCoT primers was 99 bands, where primer SCoT-18 produced the highest number of bands (18 bands) and primers SCoT-02 and SCoT-16 produced the lowest number of bands (3 bands). Out of ten SCoT primers tested, only two primers (SCoT-16 and SCoT-18) could generate possible female-specific bands (1150 bp and 720 bp, respectively) with the bulk DNA samples.
6. However, when these two primers were used further with the individual DNA samples from males and females only one primer (SCoT-18) produced female-specific band which was generated in all-female individuals tested and disappeared in all-male individuals tested.
7. Otherwise, the other primer (SCoT-16) which generated a polymorphic band in the bulked DNA samples from females when further used with the individual DNA samples from males and females could generate this polymorphic band in one male out of six tested males and two females out of six tested females. It appears that this band is not sex-specific and may result from the variation between the individuals.
8. The total number of generated bands by ISSR primers was 53 bands, where primer UBC-815 produced the highest number of bands (9 bands) and primers UBC-807, UBC-814, UBC-846, and UBC-873 produced the lowest number of bands (3 bands).
9. All of the ten tested ISSR primers failed to generate any sex-specific band.
10. According to molecular analysis results in this study, it seems that females of B. nurseare the heterogametic gender (ZW) and males are the homogametic gender (ZZ).
11. In the future, SCoT marker can be a suitable and powerful marker to be used in genetic analysis studies in fish.