Molecular-genetic approaches to species identif ication of platyhelminthes of the genus Ligophorus (Monogenea) parasitising f lathead mullet
Mugil cephalus L., 1758 (flathead mullet) is a valuable commercial fish and a promising object of artificial breeding in the Black Sea and the Sea of Azov, and the study of its parasite fauna is important for fishery and mariculture. Monogeneans of the genus Ligophorus are common ectoparasites dwell...
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Published in | Vavilovskiĭ zhurnal genetiki i selekt͡s︡ii Vol. 26; no. 3; pp. 290 - 297 |
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Main Authors | , , , , |
Format | Journal Article |
Language | English |
Published |
Russia
The Federal Research Center Institute of Cytology and Genetics of Siberian Branch of the Russian Academy of Sciences
01.05.2022
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Abstract | Mugil cephalus L., 1758 (flathead mullet) is a valuable commercial fish and a promising object of artificial breeding in the Black Sea and the Sea of Azov, and the study of its parasite fauna is important for fishery and mariculture. Monogeneans of the genus Ligophorus are common ectoparasites dwelling on the gills of mullets. Two representatives of this genus parasitise flathead mullet in the Azov-Black Sea region, namely Ligophorus mediterraneus Sarabeev, Balbuena et Euzet, 2005 and Ligophorus cephali Rubtsova, Balbuena, Sarabeev, Blasco-
Costa et Euzet, 2006. Morphological identification of these species requires spending much time and a high level
of experience in monogenean taxonomy. For quick and correct species identification of these parasites, we have
developed a genotyping approach based on the polymerase chain reaction of allele-specific gene sites for various
Monogenea species. A fragment of the 28S ribosomal gene, which includes conserved and variable sites, was
chosen as a genetic marker. Three approaches were used as follows: amplified fragment length analysis, allelespecific
PCR with endpoint detection and allele-specific real-time PCR using SYBR Green intercalating dye. The
first approach was by obtaining PCR products of different lengths that were specific either to L. mediterraneus or
to L. cephali. This approach was implemented due to the presence of several variable sites located at a distance
from each other. The PCR mixture contained three primers: one forward and two reverse. The forward primer
was complementary to the conserved site, which did not differ between species. Reverse primers were speciesspecific
and, for each species, they were complementary to different DNA regions located 100 bp apart. As a result,
L. mediterraneus was characterized by shorter amplicons than L. cephali. For the second and third approaches,
a pair of primers was designed according to the following principle: the forward primer was complementary to
both species, since it was selected for the conserved gene region. Reverse primers were species-specific and were
designed for the 28S variable region. The two parasite species were distinguished by three-point mutations. Thus,
one pair of primers was complementary to L. mediterraneus, the other, to L. cephali. The amplified fragment length
analysis and the allele-specific real-time PCR demonstrated 100 % coincidence of genotyping results compared
with Sanger sequencing. The developed genotyping protocols can be used not only to distinguish two species of
Ligophorus from flathead mullet in ecological studies and veterinary practice but also for further development of
similar approaches for other monogeneans, among which there are many pathogenic species. |
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AbstractList | Mugil cephalus L., 1758 (flathead mullet) is a valuable commercial fish and a promising object of artificial breeding in the Black Sea and the Sea of Azov, and the study of its parasite fauna is important for fishery and mariculture. Monogeneans of the genus Ligophorus are common ectoparasites dwelling on the gills of mullets. Two representatives of this genus parasitise flathead mullet in the Azov-Black Sea region, namely Ligophorus mediterraneus Sarabeev, Balbuena et Euzet, 2005 and Ligophorus cephali Rubtsova, Balbuena, Sarabeev, Blasco-
Costa et Euzet, 2006. Morphological identification of these species requires spending much time and a high level
of experience in monogenean taxonomy. For quick and correct species identification of these parasites, we have
developed a genotyping approach based on the polymerase chain reaction of allele-specific gene sites for various
Monogenea species. A fragment of the 28S ribosomal gene, which includes conserved and variable sites, was
chosen as a genetic marker. Three approaches were used as follows: amplified fragment length analysis, allelespecific
PCR with endpoint detection and allele-specific real-time PCR using SYBR Green intercalating dye. The
first approach was by obtaining PCR products of different lengths that were specific either to L. mediterraneus or
to L. cephali. This approach was implemented due to the presence of several variable sites located at a distance
from each other. The PCR mixture contained three primers: one forward and two reverse. The forward primer
was complementary to the conserved site, which did not differ between species. Reverse primers were speciesspecific
and, for each species, they were complementary to different DNA regions located 100 bp apart. As a result,
L. mediterraneus was characterized by shorter amplicons than L. cephali. For the second and third approaches,
a pair of primers was designed according to the following principle: the forward primer was complementary to
both species, since it was selected for the conserved gene region. Reverse primers were species-specific and were
designed for the 28S variable region. The two parasite species were distinguished by three-point mutations. Thus,
one pair of primers was complementary to L. mediterraneus, the other, to L. cephali. The amplified fragment length
analysis and the allele-specific real-time PCR demonstrated 100 % coincidence of genotyping results compared
with Sanger sequencing. The developed genotyping protocols can be used not only to distinguish two species of
Ligophorus from flathead mullet in ecological studies and veterinary practice but also for further development of
similar approaches for other monogeneans, among which there are many pathogenic species. |
Author | Vodiasova, E A Atopkin, D M Shikhat, O V Dmitrieva, E V Chelebieva, E S |
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Title | Molecular-genetic approaches to species identif ication of platyhelminthes of the genus Ligophorus (Monogenea) parasitising f lathead mullet |
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