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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