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American Journal of Biology and Life Sciences 2015; 3(6): 223-227 Published online October 13, 2015 (http://www.openscienceonline.com/journal/ajbls)
A Comparative Study in Isolation of Saprolegniaceae Species from Infected Eggs, Larvae and Adults Common Carp Fish in Two Fish Hatcheries, in Middle of Iraq
Rana Hadi Al-Shammari1, *, Khalid Abdul Razak Habib2, Emaduldeen Al-Mukhtar2
1College of Science, Al-Mustansiriyah University, Baghdad, Iraq 2College of Science for Women, Baghdad University, Baghdad, Iraq
Email address
rana_ecology@yahoo.com (R. H. Al-Shammari)
To cite this article Rana Hadi Al-Shammari, Khalid Abdul Razak Habib, Emaduldeen Al-Mukhtar. A Comparative Study in Isolation of Saprolegniaceae Species
from Infected Eggs, Larvae and Adults Common Carp Fish in Two Fish Hatcheries, in Middle of Iraq. American Journal of Biology and Life
Sciences. Vol. 3, No. 6, 2015, pp. 223-227.
Abstract
This study was undertaken during the period from January 2010 till the end of July 2010, its aim was to find out if there are
differences in fungal infections in two main fish hatcheries (Al-Manahel and Al-Wahda ) in middle of Iraq. From 472 samples
147 samples from adult fish; 75 from larvae and 250 from eggs. The Saprolegniaceae genera (Aphanomyces, Achlya, Dictyuchus,
Saprolegniaparasitica and S. ferax). Saprolegniaparasitica was the most occurrence percentage in eggs and larvae (33.2% and
52%) respectively, Saprolegnia sp. the most frequent in larvae (46.6%) whereas Aphanomyces was the most frequent in eggs
(29.9%). The high percentage of infection in the early life stages of the fish because the lack of the defense system and depends
on the crowding and aggregations of eggs and larvae. Regarding natural Saprolegnia species infection in both hatcheries on adult
fish, there were no significant differences in fungal infections between the two hatcheries. Nevertheless, there were highly
significant differences (P<0.01) between months in percentage of fungal infection in fish body, which was 1% in both mouth and
eyes and 61% in the dorsal fin.
Keywords
Saprolegniaceae, Common Carp, Middle of Iraq
1. Introduction
Common carp proved to be the most successful cultivable
species because it presents throughout the year, acceptable by
consumer, tolerate adverse condition in captivity, accept
prepared foods and easy to manage (Moore, 2008).
Saprolegniaceae classified under the order Saprolegniales
Phylum Oomycota the term water molds used to designate
Saprolegniales recently, it has been moved to the new
kingdom Stramenopiles (Alexopouloset al., 1996; Dick,
2001). Most members of this order live in water, most
confined to fresh, clear water also some species are able to live
in brackish water of estuaries (Czeczuga, 1994).
The Saprolegniaceae genera particularly Achlya and
Saprolegnia are generally considered as opportunistic for fish
and their eggs (Bruno and Wood, 1999).The objectives of this
study to find if there are differences in fungal infection
between two fish hatcheries which are located in two
ecologically different locations, this study achieved for the
first time in Iraq as a comparative study of saprolegniases
between two fish hatcheries.
2. Materials and Methods
2.1. Fish Samples
Fish samples were collected from two fish hatcheries:
Manahel Hatchery (Mh) located on Musaiab sector –Babel
governorate (32°51'N, 44°19'E), water supply from Euphrates
and Wahda Hatchery (Wh) Located at Suwaera sector–Wasit
governorate (33°60'N, 44°47'E), water supply from Tigris,
middle part of Iraq during the period from January 2010 till
the end of July 2010.
224 Rana Hadi Al-Shammari et al.: A Comparative Study in Isolation of Saprolegniaceae Species from Infected Eggs, Larvae and Adults Common Carp Fish in Two Fish Hatcheries, in Middle of Iraq
Samples were collected with hand net in order to enumerate
the total count of fish, number of infected fish. They were
transferred to another pond after that another sample was
taken. The process repeated as a 10 samples for each pond (5-8
fish for each sample) then percentage of infected fish was
calculated proportionally to the stocking number of fish in
each pond (FAO, 1992).
Infected fish may exhibit either or both clinical symptoms
and Behavior signs (Carlander, 1977; FAO, 2002).
Clinical symptoms include excess mucous secretion;
change in normal coloration grayish or brownish areas over
the body, clinical signs of infection and morbidity mortality
rate. Erosion of scales, parts of fins and skin, paling of gills;
abdominal swelling; bulging of eyes; spots or patches over the
body and gills, fins, operculum, eyes; appearance of lesions,
hemorrhagic spots.
Behavioral signs like: slowing down or complete stoppage
of feeding; loss of equilibrium, swimming erratically or in
spirals; surfacing for gulping air and scraping against the floor
and sides of the pond.
2.2. Isolation of Saprolegniacea from Eggs,
Larvae and Adults Carp
Samples washed several time with sterile distilled water
then the eggs and larvae membrane with fungal mycelia were
separated and washed again and placed in sterile glass Petri
dishes with 9 ml of sterilized river water and 1 ml of
chloramphenicol with the concentration of 100 µg/ml added to
each sample.
Isolation of Saprolegniacea from adults was carried out
from naturally infected fish, samples were taken from fish
showing skin lesions, eye, fins, gills, mouth and inoculated
onto CMA medium plates and incubated at 20 ± 2ºC for 3-4
days, subculture on the same media was done for purification.
2.3. Statistical Analysis
All the statistical analyses were performed using SPSS
Statistics 17.0 software 1997. A comparative approach
employed for comparing data sets, which included application
of ANOVA and Correlations Coefficient with probability level
0.05.
Occurrence and frequency of Saprolegniacaea species
calculated according to (Pitt and Hocking 1997) as follow:
Occurrence % = no. of samples in which genus occur *100 / no. of total samples
Frequency % = no. of isolates of each genus *100 / no. of isolates of all genera
3. Results
The infected Carp fish eggs covered with some White or grey
threads of cotton -like mycelia. 67 isolates were obtained from
eggs and identified as: Aphanomyces sp. (20 isolates),
Saprolegnia parasitica (17 isolates), Saprolegnia sp. (12
isolates), Achlya sp. (12 isolates), S. ferax (3 isolates) and
Dictyuchus sp. (3 isolates). Were as 15 isolates were obtained
from larvae and identified as: Aphanomyces sp. (1 isolate),
Saprolegnia parasitica (4 isolates), S. ferax (3 isolates). In
addition to 15 isolates were obtained from larvae and identified
as: Aphanomyces sp. (1 isolate), Saprolegnia parasitica (4
isolates), 18 isolates were obtained from larvae and identified as:
Saprolegniaparasitica (3 isolates) and Saprolegnia sp. (15
isolates). S. parasitica recorded the highest occurrence
percentage in both eggs and larvae; Aphanomyces sp. recorded
the highest frequency percentage in eggs while Saprolegnia sp.
was the highest frequency percentage in larvae and adults (table
1), although different genera like Saprolegnia, Aphanomyces,
Achlya and Dictyuchus but Saprolegnia is the most abounded
this result agreed with(Klinger and Floyd,1996). First record of
Saprolegnia infection in khashnei fish (Mugil abu) in Iraq by
Herzog (1969), after that infection with water mold recorded in
16 species of fishes (Mhaisen, 2015).One of the difficulties of
studying saprolegniasis is that many Saprolegniaceae isolates
obtained from infected fish and fish eggs and river water do not
develop sexual structure in the laboratory conditions so they
cannot be identified to the species level using classical
taxonomic criteria (Fregeneda – Grandes et al., 2000).
Table 1. Occurrence and frequency of Saprolegniaceac genera isolated from eggs, larvae and adults Common Carp fish in two hatcheries (Manahel and Wahda).
Source Genera No. of isolates No. of samples Frequency% Occurrence%
Wh Mh total Wh Mh total Wh Mh total Wh Mh total
Eggs
Aphanomyces sp. 12 6 20 32 8 40 17.9 12 29.9 12.8 3.4 16
S. parasitica 9 8 17 67 16 83 13.4 11.9 25.3 26.8 6.4 33.2
Achlya sp. 3 9 12 34 9 43 4.4 13.5 17.9 13.6 3.6 17.2
Saprolegnia sp. 5 7 12 41 23 64 7.5 10.4 17.9 16.6 9.2 25.6
Dictyuchus sp. 0 3 3 0 11 11 0 4.5 4.5 0 4.4 4.4
S. ferax 0 3 3 0 9 9 0 4.5 4.5 0 3.6 3.6
Total 4 29 38 67 174 76 250 43.2 56.8 100 69.4 30.6 100
Larvae
Saprolegnia sp. 6 1 7 9 8 17 40 6.6 46.6 12 10.7 22.7
S.parasitica 2 2 4 18 21 39 13.3 13.3 26.6 24 28 52
S. ferax 0 3 3 5 9 14 0 20.2 20.2 6.6 12 18.6
Aphanomyces sp. 0 1 1 2 3 5 0 6.6 6.6 2.7 4 6.7
Total 2 8 7 15 34 41 75 53.3 46.7 100 45.3 54.7 100
Adults S. parasitica 2 1 3 3 4 7 2.1 2.7 4.8 2 2.8 4.8
Saprolegnia sp. 5 10 15 60 80 140 40.7 54.5 95.2 38.1 57.1 95.2
Total 1 7 11 18 63 84 147 42.8 57.2 100 40.1 59.9 100
American Journal of Biology and Life Sciences 2015; 3(6): 223-227 225
According to Saprolegniasis in adult fish 461 Carp fish
were examined during January and February (fungal infection
occurred only in these two months), as shown in (Table 2),
percentage of Saprolegniasis in January was 31% in Mh and
34% in Wh, the percentage increased to 38% in Mh and Wh.
There were no significant differences in fungal infections
between the two hatcheries. Nevertheless, there were highly
significant differences (P<0.01) between the months
according to the fungal infection, it’s clear from results when
temperature decrease fungal infection increase (Paxton and
Willoughby, 2000). Regarding water temperature, fish are cold
blooded animals primarily dependent upon water as a medium
in which to live. Fish can tolerate wide range of water
temperature they can distinguish a rise in temperature from
a fall but the physiological mechanism for such recognizing is
not known (Hatai and Hoshia, 1994; Grandes et al., 2001).
Temperature stress, particularly cold temperatures can
completely halt the activity of immune system eliminatingthis
defense against invading disease organisms (Knightsand Lasee,
1996).
These results agreed with Al-Kazzaz et al., (2002) who
record egg-invaded with fungi during autumn and winter
artificial propagation of the common carp (Cyprinus carpio
L.)in Baghdad. Poor water quality (water with low circulation,
low dissolved oxygen, or high ammonia and high organic
loads including the presence of dead eggs, are often associated
with infections, (Klinger and Floyd, 1996)
Table 2. Natural fungal infection on Common Carp fish.
hatcheries
months of infection
Manahel Hatchery Wahada Hatchery
a b c d E A b c d e
Jan.-2010 165 51 31 10 19 122 34 28 9 26
Feb.-2010 86 33 38 14 42 88 29 33 12 41
Mar.-2010 0 0 0 0 0 0 0 0 0 0
Apr.-2010 0 0 0 0 0 0 0 0 0 0
May-2010 0 0 0 0 0 0 0 0 0 0
Jun-2010 0 0 0 0 0 0 0 0 0 0
July-2010 0 0 0 0 0 0 0 0 0 0
Total 251 84 69 24 61 210 63 61 21 67
Mh: Manahel hatchery Wh: wahda hatchery a = No. of examined Carp fish.
b = No. of infected Carp fish. c= % infected Carp fish. d = No. of dead Carp fish.
e = % of dead Carp fish.
Clinical signs of Saprolegniasis
Figure 1. Location of cutaneous lesions on Common Carp fish with
Saprolegnia.A: infection in the base of pelvic fin, B: infection in the anal fin
and in the base of caudal fin, C: infection in the dorsal fin and in the
operculum.
Infected Carp fish showed anorexia and abnormal
swimming movements. The natural infected Carp fish
revealed focal grayish –White patches on the head regions as
well as skin, fins and occasionally gills. In advanced stages of
infection, Saprolegniasis spread out to cover the Whole body
(Figure1). Signs of saprolegniasis on Carp fish resembled
the recorded sings and lesions which were pathogenomonic
caused by S. parasitica (Fregeneda-Grandes et al., 2001;
Osman et al., 2008). Saprolegniasis among fishes in
aquaculture or in an aquarium may be confined to one fish, a
few fishes or the entire population depending on the reason for
the fungal invasion (Post, 1987).
The cutaneous lesions were more frequent, particularly on
dorsal, followed by the caudal, anal and pictorial fins (Figure
2).The results of fungal infection show fins and bases of fins
were recorded the highest infection percentages
Lesions on the rest of the body appeared mainly on the
caudal and dorsal regions around the fins and on the opercula,
nostrils, eyes and gills (Figure3).
The percentage of body surface invaded by Saprolegnia in
the 461 Common Carp fish, which died, or still alive varied
between 1% in eyes and nostrils and 61 % in dorsal fin Figure
(3).InJapan, Hatai and Hoshiai (1994) indicate that in Miyagi
Prefecture, there is an annual mortality rate of 50% in coho
salmon (Oncorhynchuskisutch) due to S. parasitica. Losses
have also been reported in elver (Anguilla anguilla) culture in
Japan, and in southeastern United States, major loses occur in
channel catfish aquadulture due to a condition called "winter
kill" a condition occurs during winter months when the colder
weather suppresses the catfish immune system rendering them
susceptible to saprolegniasis. Some catfish farmers have
reported losses of up to 50%, an economic loss of $ 40 million
(Bruno and Wood, 1994).
226 Rana Hadi Al-Shammari et al.: A Comparative Study in Isolation of Saprolegniaceae Species from Infected Eggs, Larvae and Adults Common Carp Fish in Two Fish Hatcheries, in Middle of Iraq
Figure 2. Location of cutaneous lesions on Common Carp fish infected with Saprolegnia.
Figure 3. Schematic shape shows Location of cutaneous lesions of the current study on Common Carp fish infected with Saprolegnia species.
4. Conclusion
1. There were no significant differences in fungal infections
between the two hatcheries.
2. Fungal infection depends on the quality of eggs. Eggs of
poor quality have mortality rate as high as that of good quality,
when the fungi occurred over the dead eggs, they spread
around healthy eggs, causing the loss of oxygen for breathing
and thus die.
3. Fungal infection in adult fish depend on many factors
which are: a limited space with no in or outflow of water, the
density of fish population and inhabitance of fish surrounded
by their own metabolic wastes, Injuries from handling the
environmental factors especially water temperature when it
decrease fungal infection increase, physical conditions,
genetic resistance, host age and sex, also play a part in
determining the susceptibility of the fishes to diseases.
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