4. Discussion
Initial assessment of the mass mortalities which have occurred
among spawner angelfish stocks and their egg progeny were
very complicated if multifactorial hypothesis was considered.
However, by progress of diagnostic investigations through
the entire event of mass kills, visual detection of hyphal masses
on both spawners and their egg progeny were presumptively
suggestive of a water mould infection. Ten pathogenic water
moulds were identified as Saprolegnia spp. based on morphological
characteristics and phylogenetic analysis. Our findings
confirm that Saprolegnia spp. are the major cause of Saprolegniasis
in ornamental fish production [43].
Identification of Saprolegnia spp. is complex and sometimes
confusing. However, several typical morphological features
involving asexual and sexual reproductive organs serve for
classical Saprolegnia identification [44]. In the current study,
the fact that 30% of the isolates were unable to develop sexual
stages even after prolonged incubation period cannot be neglected,
particularly after the addition of hemp seeds as biological
enhancer. The oogonia of these isolates are either
completely absent (30% of the retrieved isolates) or are formed
only after a prolonged period of time (70% of the retrieved isolates).
Hence, Saprolegnia which were pathogenic to angelfish
spawners/egg progeny have been identified as S. parasitica
according to criteria described by Hatai and Hoshiai [45].
Our data (unattainable sexual reproduction after 21 days incubation
for 30% of the isolates) coincide with Ristanovic´ and
Miller [46], Hatai and Hoshiai [45] and Hussein et al. [47]
who concluded that hemp seed could be of no identification
value if the oogonia or antheridia (sexual structures) were
not seen within 60 days of incubation at maximum (very long
incubation period compared to ours). Therefore, using molecular
tools such as PCR coupled with partial sequencing of inter-
transcribed spacer (ITS) gene was one of the most
important choices to distinguish S. parasitica from other water
moulds (Oomycetes) [39].
Non coding internal transcribed spacer regions (ITS1 and
ITS2) of the ribosomal DNA are considered to be the most accepted
genetic markers because of their relatively high sequence
variability [48] and the availability of primers that
would supply sequence data for Oomycetes [49]. These non
coding regions are located between two coding regions, the
18S and the 28S genes. Another coding region, the 5.8S gene,
is found between the ITS1 and ITS2. Thus, genetic sequential
analysis of these regions has been adopted to study the intragenic
as well as the inter-genic relationships among the 10 retrieved
Saprolegnia isolates [50]. Further, the phylogenetic
analysis based on the ITS rDNA region further confirmed
the taxonomic position of the 10 Saprolegnia isolates and con-
4. Discussion
Initial assessment of the mass mortalities which have occurred
among spawner angelfish stocks and their egg progeny were
very complicated if multifactorial hypothesis was considered.
However, by progress of diagnostic investigations through
the entire event of mass kills, visual detection of hyphal masses
on both spawners and their egg progeny were presumptively
suggestive of a water mould infection. Ten pathogenic water
moulds were identified as Saprolegnia spp. based on morphological
characteristics and phylogenetic analysis. Our findings
confirm that Saprolegnia spp. are the major cause of Saprolegniasis
in ornamental fish production [43].
Identification of Saprolegnia spp. is complex and sometimes
confusing. However, several typical morphological features
involving asexual and sexual reproductive organs serve for
classical Saprolegnia identification [44]. In the current study,
the fact that 30% of the isolates were unable to develop sexual
stages even after prolonged incubation period cannot be neglected,
particularly after the addition of hemp seeds as biological
enhancer. The oogonia of these isolates are either
completely absent (30% of the retrieved isolates) or are formed
only after a prolonged period of time (70% of the retrieved isolates).
Hence, Saprolegnia which were pathogenic to angelfish
spawners/egg progeny have been identified as S. parasitica
according to criteria described by Hatai and Hoshiai [45].
Our data (unattainable sexual reproduction after 21 days incubation
for 30% of the isolates) coincide with Ristanovic´ and
Miller [46], Hatai and Hoshiai [45] and Hussein et al. [47]
who concluded that hemp seed could be of no identification
value if the oogonia or antheridia (sexual structures) were
not seen within 60 days of incubation at maximum (very long
incubation period compared to ours). Therefore, using molecular
tools such as PCR coupled with partial sequencing of inter-
transcribed spacer (ITS) gene was one of the most
important choices to distinguish S. parasitica from other water
moulds (Oomycetes) [39].
Non coding internal transcribed spacer regions (ITS1 and
ITS2) of the ribosomal DNA are considered to be the most accepted
genetic markers because of their relatively high sequence
variability [48] and the availability of primers that
would supply sequence data for Oomycetes [49]. These non
coding regions are located between two coding regions, the
18S and the 28S genes. Another coding region, the 5.8S gene,
is found between the ITS1 and ITS2. Thus, genetic sequential
analysis of these regions has been adopted to study the intragenic
as well as the inter-genic relationships among the 10 retrieved
Saprolegnia isolates [50]. Further, the phylogenetic
analysis based on the ITS rDNA region further confirmed
the taxonomic position of the 10 Saprolegnia isolates and con-
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