c-4. hatchery environment -...

Chapter 4

Microbial ecology of the hatchery environment of Macrobrachium rosenbergii

Chapter 4. Microbial ecology of the hatchery environment of Macrobrachium rosenbergii

Characterisation of the environmental microflora of M. rosenbergii and development of probiotic strains for its culture operations

85

4.1 Introduction

Production of healthy and quality seeds has been a major obstacle in the expansion of the

culture of M. rosenbergii. A complex mix of environmental factors, microbiological profiles and

management practices influence the success of the production cycle (Kennedy et al., 2006). As

per one of the projections made by Kurup (1994) 200 million PL of this fast growing prawn

species are required to extend their cultivation in India. To meet this demand several hatcheries

have been set up in India with the prime objective to supply good quality seed at the right time.

As per 2004-2005 survey, 77 hatcheries are under operation in the scampi industry for seed

production with 2247 million/ annum capacity and 36,990 ha of grow out ponds with a

production of 38,720 mt. However, the supply of PL remained at all times far below the

requirements mainly due to the low survival rate at different stages of the larval cycle.

Bacteriology is one of the most important areas determining the dynamics and health of

larvae and PL during the hatchery operations. With the recent development and innovations in

the culture techniques, the bacteriology of the cultured shrimp and prawn in the tropics is

receiving greater attention, since some species of bacteria associated with shrimp and prawn

cause disease under stress condition. There is a great microbial diversity in the environment and

a selection process usually starts between the environmental microflora and the larval gut once

the larvae start feeding. This selection process is generally beneficial to the host and the microbes

contribute in various ways such as helping the host digest difficult organic molecules by

providing nutritional factors and probably by competitively excluding the pathogenic ones from

the intestine.

It is important to understand the microflora associated with hatchery systems because

host microbe interactions have far reaching implications on larval health, development and

outbreaks of disease (Olafsen, 2001). Even in the natural estuarine and marine systems, different

types of bacteria colonize fish/ prawn eggs and larvae. In artificial hatchery systems, the flora

associated with prawn eggs and larvae may be different due to the various water treatment

measures, artificial feeds and higher density of population. Some of the bacteria may be

opportunistic pathogens causing diseases in stressed larval populations. On the other hand, some

of the microflora may be protective against pathogens (Hansen and Olafsen, 1999). Thus it

would be important to have data on the levels of bacteria and types of bacteria occurring in

hatchery systems at different intervals of operation.

The procedures used for microbial monitoring were effective and provide a basis for

routine monitoring efforts designed to detect changes in microbial numbers and composition and



86

allow informed decisions regarding microbial control measures. The study of bacterial

interaction with crustaceans, such as Artemia, prawn and shrimp are important, since it is

presumed that the bacteria provide, directly or indirectly, nutritional elements like vitamins,

essential amino acids, fatty acids, polyamines, and enzymes (Gorospe et al., 1996; Verschuere et

al., 2000b). Also, it is claimed that some bacteria in Artemia and shrimp cultures have the

capacity to control pathogenic or potentially pathogenic bacteria by means of competitive

exclusion, or by producing inhibitory effects (Verschuere et al., 2000a).

During the intensive hatching of eggs and rearing of larvae, various forms of interactions

between bacteria and the biological surfaces may occur. This may result in the formation of an

indigenous microflora or be the first step of infection. In the aquatic environment, bacteria travel

easily between habitats and hosts, and a better understanding of host–microbe interactions and

natural defences is imperative for the successful mass production of larvae. The continued

development of aquaculture relies on improved microbial control to prevent the proliferation and

spreading of pathogens. Interactions of microbes with larvae take place before the fish larvae

start drinking. Bacteria readily colonize on the surface of fertilized eggs during incubation and on

hatched larvae, especially under intensive conditions. Colonisation of bacteria has negative

effects on eggs and developing embryos (Bergh et al., 1997; Hansen et al., 1992a). The use of

antibiotics, water treatment, or disinfection reduces the growth of bacteria and inhibits the

proliferation of opportunistic bacteria.

The diverse flora that eventually develops on the egg surface reflects the bacterial

composition of the water; however, species-specific adhesion to surface receptors may also affect

the composition of the epiflora. Members of the adherent microflora may damage developing

eggs; however, we do not yet know whether a natural epiflora may bestow some protection in the

sense that a heterogenous epiflora may prevent microcolony formation or domination by

potentially harmful bacteria (Olafsen, 2001). Factors that may protect eggs from bacterial

invasion or infection are still poorly understood.

In aquaculture operations, feed is an important input and accounts for about 50–60% of

the recurring investment. The food for culture of M. rosenbergii larvae consists basically of

newly hatched Artemia nauplii (Lavens et al., 2000) supplemented with inert food (Valenti and

Daniels, 2000). Feed consumption by larvae is dependent upon numerous factors related to

culture conditions specific for site, and scientific knowledge is not enough to base an adequate



87

feeding schedule. Live feed organisms play an important role in the dietary regimen of cultivable

fish and shellfish, particularly in the larval stages.

Artemia is widely recognized as the best natural, storable live feed available, and is used

in marine finfish and crustacean hatcheries around the world because of its nutritional and

operational advantages (Sorgeloos et al., 1986). Artemia, however, are also considered a possible

vector for the introduction of viruses and bacteria into the rearing systems; live feeds are thought

to be responsible for the source of viral and bacterial infections (Tatani et al., 1985; Nicolas et

al., 1989; Mortensen et al., 1993; Skliris and Richards, 1998).

Bacteria can enter the hatchery system by various routes, most importantly through feed,

brood stock and rearing water. The qualitative and quantitative data on the microflora associated

with the system would help developing effective strategies for pathogen control (Skjermo and

Vadstein, 1999). Pathogenic bacteria can enter the hatchery systems from three principal routes:

rearing water, brood stock and feeds. Quantitative and qualitative aspects of bacterial flora

associated with these potential sources must be studied throughout the hatchery operations to

develop a disease management strategy. All the earlier studies on the bacteriology of M.

rosenbergii larval culture have been carried out on water and larval samples collected from

different hatcheries with different rearing methods and at different periods. Further, no

information on the bacterial flora of eggs, brooders and feed, except Artemia nauplii, is available

(Phatarpekar et al., 2002).

There is an increasing interest within the industry in the control or elimination of

antimicrobial use. Therefore, alternative methods need to be developed to maintain a healthy

microbial environment in the larval rearing tanks. One such method that is gaining acceptance

within the industry is the use of probiotic bacteria to control potential pathogen and maintain

good health to the larvae. The modern concept of probiotics was formulated 30 years ago

(Parker, 1974). Gatesoupe (1999) reported the first application of probiotics in aquaculture is

done by Kozasa (1986) by using Bacillus toyoi.

4.2 Review of literature

4.2.1 Bacteriology of hatchery environment

A significant factor affecting the outcome of hatchery fingerling production is the

microbiology of the rearing biotope. Successful aquaculture will rely on better insight into the

complex interactions between the cultured organisms and the bacterial communities that develop



88

in the rearing systems. Opportunistic bacterial pathogens, particularly Vibrio spp., are present as

part of the normal microbiota of marine fish and have been shown to be causative agents of

disease and mass mortality (Sedano et al., 1996; Pedersen et al., 1997). Larviculture tanks used

in intensive production can become high in organic matter, providing substrates for microbial

growth and leading to the proliferation of fast growing opportunistic bacteria. High microbial

numbers associated with live feed and/ or the fish’s rearing environment can result in poor

survival and performance of the larvae.

Miyamoto et al. (1983) identified 13 genera of bacteria from M. rosenbergii larvae,

larval cultural water and larval tissue from two Hawaiian hatcheries including Vibrio, Aeromonas

and Pseudomonas. A study of the microbiology of Macrobrachium larval culture water by

Fujioka and Greco (1984) recorded the isolation of many different species of Vibrio. The newly

hatched larval gut is reported to be sterile, it soon absorb microorganisms from the environment

by way of drinking and feeding from the surrounding environment (Olafsen, 1984; Hansen and

Olafsen, 1989). Colorni (1985) using selective media described the predominant flora in healthy

larvae as Aeromonas liquefaciens, Vibrio anguillarum and various other species of Vibrio,

Aeromonas and Pseudomonas spp. An estimation of aerobic heterotrophic bacterial flora

associated with tank water, tank sediment, tank surface and larval slurry in three M. rosenbergii

hatcheries in Malaysia was made by Anderson et al. (1989) and reported 16 genera of bacteria

from hatchery water and 15 genera from larval tissues. Alcaligenes, Bacillus, Plesiomonas and

Pasteurella was the frequently encountered genera in tank water and Alcaligenes, Vibrio and

Bacillus were the most abundant strains in larval tissues of M. rosenbergii.

Since the larvae establish their bacterial flora partly in a non-selective way (Hansen and

Olafsen, 1990; Cahill, 1990), the initial bacterial environment is of utmost importance. In this

respect, the early colonisation of the gut by non-opportunistic bacteria may initiate a resident

microflora which could prevent the proliferation and colonisation of the gut of larvae by

opportunistic or pathogenic bacteria (Bergh, 1995; Skjermo et al., 1997). Huys et al. (2001)

reported large variation of survival in turbot larvae and concluded that there was no correlation

between the number of bacteria present in the gut of turbot larvae and the larval survival rate.

During their experiments, all replicates followed nearly the same rate of bacterial development in

the gut of turbot larvae going from 102 cfu larva-1 just before first feeding at day 3 to 105 cfu

larva-1 at day 9 after hatching.

Since the intestine of native fish larvae is virtually sterile, the bacteria present in the

environment and in the live feed are the initial colonisers. The composition of the intestinal



89

microflora in fish is influenced by, or directly derived from the microflora of the food (Muroga et

al., 1987). In juvenile and adult marine fish, the intestinal microflora is reported to be dominated

by vibrios (Newmann et al., 1972; Muroga et al., 1987). However, very little information is

available on the composition of the intestinal microflora and the colonisation of the intestine by

different bacterial species in very young fish larvae.

Diet is a fundamental aspect in larval culture of decapod crustaceans (Correia et al.,

2000; Valenti and Daniels, 2000). In general, species cultivated for commercial purposes are

small and have a small buccal opening and little vitellinic reserve. Therefore, exogenous food is

needed 1 or 2 days after hatching. The morphology, behavior and nutritional needs change

according to several developmental stages (Jones et al., 1997; Lavens et al., 2000). Generally,

live food supplies all the necessary nutrients for development and can contribute with exogenous

digestive enzymes that aid in digestion (Jones et al., 1993; Kamarudin et al., 1994). The

microflora of invertebrates and plankton may be dominated by opportunistic or potentially

pathogenic vibrios at certain times of the year, and marine invertebrates may harbour bacteria

that are pathogenic to other organisms. The defence factors of invertebrates are not fully

understood; however, bacteria may persist in their tissues and body fluids and, thus, marine

invertebrates or food organisms may serve as vectors for transfection of pathogens. A better

perception of these factors is essential to understand epidemiology in aquatic environments.

In general, decapod larvae do not specifically orientate towards a food resource. Rather,

they depend on chance encounter to capture food (Kurmaly et al., 1990). Little is known about

the importance of the microbial communities in live-feed production systems. The associated

microbiota is suspected to play a major role in the instability and variability of the live-feed

cultures themselves (Harzevili et al., 1997) and of the cultures of the marine predator-larvae

(Nicolas et al., 1989; Verdonck et al., 1997).

One of the most significant advances in aquaculture has been the introduction of Artemia

franciscana as food for fish and invertebrates larvae. Artemia nauplii have been reported as one

of the best foods for most organisms under culture (Sorgeloos et al., 1988). Artemia has been

used as a vector of growth promoter or drug carrier for therapeutic applications (Touraki et al.,

1996), as well as a biological control of toxic dinoflagellates in aquaculture systems (Oestman et

al., 1995). Most of the bacteria attached to Artemia cysts could be eliminated by chemical

treatment (Sorgeloos et al., 1977). Mortensen et al. (1993) showed the detection of infectious

pancreatic necrotic virus (IPNV) in Artemia pointed to the role of Artemia as a potential IPNV

reservoirs and vectors if they were subsequently eaten by fish.



90

According to the study of Lopez-Torres and Lizarraga-Partida (2001) Artemia nauplii are

vectors of Vibrio spp. in aquaculture activities, but these vibrios are introduced by the Artemia

hatching tanks operations and they are not associated with cysts. Dendrogramme group results

indicated that V. alginolyticus and Vibrio spp., isolated from Artemia hatching tanks, are

associated with isolates from mysis, zoea and post larvae shrimp stages tanks, indicating that

these vibrios remained associated with the different shrimp development stages. Verschuere et al.

(1997), who monitored the community level physiological profiles of the emerging microbial

communities in the culture water of Artemia juveniles in three identical culture series. Although

completely identical from the zootechnical point of view, the culture water of the three series

showed clearly distinct microbial communities developing in the first days of the experiment.

The same concept may be valid for the microbial communities developing in the culture water

and on the inner and outer surfaces of eggs and larval organisms. Obviously, due to the

heterogeneity of the microbial distribution in the air and water, in feeds, and on surfaces, the

stochastic factors are very important in the colonization of aquaculture environments. Austin and

Allen (1982), as well as Igarashi et al. (1989) have reported the presence of Vibrio spp. in

samples of Artemia cysts. Nevertheless, in a study (Lopez-Torres and Lizarraga-Partida, 2001),

where the authors have analysed 617 strains from 14 Artemia’s commercial brands, Vibrio spp.

was not detected, but rather a Gram-positive bacterial population capable to grow in the Vibrio-

selective media TCBS.

Artemia nauplii carry a large bacterial load that may be transferred from live preys into

the tanks of fish and shellfish larvae. Some bacteria have been reported to be the source of

diseases and high mortalities in fish larvae, and live feeds are thought to be responsible (Muroga

et al., 1987; Nicolas et al., 1989). Previous studies often recommend rinsing the nauplii in sterile

fresh or seawater (Austin and Allen, 1982), but some authors argue that rinsing has little effect

on the bacteria (Dehasque et al., 1991; Verdonck et al., 1991). The studies have also reported the

heavy bacterial load associated with Artemia nauplii (Verdonck et al., 1991). In this situation,

antibiotics have been tried to disinfect the live feed before introducing into the rearing systems

(Tanasomwang and Muroga, 1989, 1992; Gomez-Gil et al., 1994). Juvenile and adult brine

shrimp are used increasingly as suitable live diets for different aquaculture species (Sorgeloos et

al., 1998). The intensive culture of the brine shrimp Artemia has always suffered from

unpredictable results due to incidental crashes in individual production tanks (Verschuere et al.,

1997).



91

Several genera of bacteria such as Bacillus, Lactobacillus, Vibrio, Streptococcus,

Alteromonas, Aeromonas and Nitrosomonas have been used in the shrimps and molluscan

hatcheries (Burno et al., 2000). Normally these bacteria delivered directly into the water or via

live carriers such as Artemia nauplii or rotifers. As far as a good probiotic is concerned, it should

be a strain, which is capable of exerting a beneficial effect on the animal, should be non

pathogenic, should be present as viable cells, preferably in large numbers, should be capable of

surviving in the environment and gut and should be stable and capable of remaining viable for

long periods under storage and field conditions. The scientific information is rare on the use of

bacteria as probiotics in shrimp or prawn larviculture systems.

4.2.2 Physico-chemical parameters of the hatchery environment

Maintenance of right environmental parameters plays an important role in successful

larval rearing. The results of Law et al. (2002) indicated that M. rosenbergii egg hatchability is

extremely sensitive to hydrogen ion concentration in brackish water. In brackish water of 12 ppt

salinity, the highest hatching rate was detected at pH 7.0. While at pH of 5.0, 8.0, 9.0 and 10.0,

the hatching rates were zero. There is drastic reduction in hatching rates at pH 6.5 and 7.5.

Larvae are generally reared at 10-15 ppt salinity. The optimum water temperature range is 26-

31oC. Larvae must eat continuously to survive but do not actively search for food. Food density

is therefore critically important and must be maintained regardless of the density of prawn larvae.

Various live and inert feeds are used in different combinations. Newly hatched brine shrimp

nauplii (Artemia spp.), fish flesh and egg custard are frequently used as food and the feed is kept

suspended by vigorous aeration.

4.3 Objectives of the present study

The review of literature indicates that microbial aspects of culture water, hatchery tank

environment, larvae and probiotics are important factors to the success of shrimp/ prawn

cultivation. It is envisaged in the present investigation to study the microbial ecology of a

commercial freshwater prawn hatchery, with and without the use of probiotics. The specific

objectives are as follows:

1. Monitoring of the physico-chemical parameters of hatchery environment of M.

rosenbergii.

2. Estimation of the load and characterisation of total viable bacteria associated with the

water and various larval stages of M. rosenbergii from tank treated with probiotic and

those without probiotic treatment.



92

3. Estimation of the load and characterization of total viable bacteria associated with

commercial probiotic consortium as well as feed items used in the hatchery operations of

M. rosenbergii.

4. Estimation of the load of health significant bacteria such as total coliforms and total

Vibrio like organisms in the water, various larval stages of M. rosenbergii, feed items

and commercial probiotics used in the hatchery.

4.4 Materials and methods

4.4.1 Description of the hatchery

The samples were collected from a private owned hatchery (Rosen Fisheries, Thrissur,

Kerala). The hatchery was one of the best M. rosenbergii seed production hatcheries in South

India. The hatchery was managed in scientific manner and consistently achieved good production

levels. The hatchery was maintained in clear water system with daily water exchange and has

also used commercial probiotics.

4.4.2 Collection of samples from the hatchery

The samples were collected during January and February 2005 from the hatchery.

Samples were collected from the hatchery during a full larval rearing cycle with 5-day interval in

between sampling. Samples were collected from 2 different systems, one set of tank with

incorporated probiotics and other without probiotics. All the samples except larvae and PL of M.

rosenbergii were kept in an ice-box and immediately brought to the laboratory for analysis.

Larvae and PL of M. rosenbergii were transported in live condition under oxygen packing.

4.4.2.1 Water samples from the hatchery system

Water samples from brooder tank, larval rearing tanks and Artemia hatching tank were

collected in sterile glass bottles.

4.4.2.2 M. rosenbergii larvae and PL

The larvae and PL were collected before feeding from the larval rearing tank using hand

net. The collected larvae were identified into different stages of growth by using the manual for

the culture of M. rosenbergii (New, 2002).

4.4.2.3 Feed items

Feed items (Live Artemia nauplii, Artemia cyst, Artemia flakes, egg custard and

commercial starter feed) used to feed various stages of larvae and PL of M. rosenbergii in the

hatchery was collected in sterile polythene bags.



93

4.4.2.4 Commercial probiotic consortium

Ready to add commercial probiotic consortium prepared by the workers of the hatchery

was collected in sterile plastic bottle.

4.4.3 Probiotic consortium application

Capacity of the tanks used for probiotic application was 5000 L. Four lakh larvae were

introduced into these tanks and commercial probiotics were applied (2.5 ml of probiotic solution

per tank) at 5 day intervals till PL stage.

4.4.4 Analysis of the physico-chemical parameters

The methods used for the analysis of the samples were described under the section 2.4.3.

4.4.5 Bacteriological analysis

Processing of the samples such as water, larvae and PL of M. rosenbergii samples for

bacteriological analysis were carried out as per the method mentioned in the section 2.4.4.1. The

feed samples were weighed and homogenized aseptically and serially diluted up to 105. For the

estimation of TVC, ½ ZMA for water and larval samples, TSA for egg custard, starter feed and

commercial probiotics, full strength ZMA for Artemia hatching water, Artemia cyst, Artemia

flake and Artemia nauplii were used. The methodology used was same as mentioned under

section 2.4.4.2. The media and methodology used for the estimation of TC and TVLO were same

as mentioned under section 2.4.4.3 to 2.4.4.4.

4.4.6 Isolation and identification of bacterial isolates

After recording the morphological characters and pigmentation representative types

constituting at least 20-40 numbers of colonies on plates were selected from each plate and re-

streaked onto TSA, ½ ZMA or ZMA plates to ensure purity. To isolate the Lactobacillus from

the probiotic consortium Lactobacillus MRS agar was also used. The method and different tests

used for the isolation and identification of the bacterial isolates are mentioned under the section

2.4.5.

4.4.7 Statistical analysis

Statistical analysis was done as per the method described under section 2.4.6.



94

4.5 Results

4.5.1 Physico-chemical parameters of the hatchery environment of M. rosenbergii

The physicochemical parameters of the water samples collected tanks without probiotics

and with probiotics are presented in Table 4.1. The temperature of water remained constant at

28.5 + 1oC, salinity ranged between zero to 15 ppt, pH varied between 7.2 to 7.6 and DO ranged

from 6.2 to 6.6 mg L-1 during the hatchery operations. There was no significant difference

between treatments. Result showed that all the physico-chemical parameters analysed were

within the optimal range for the larval rearing of M. rosenbergii in hatchery operations.

Table 4.1. Physico-chemical parameters of the water samples from the hatchery environment

Tanks WP Tanks WOP Sampling Period T

(oC) Salinity (ppt) pH DO

(mg L-1) T (oC) Salinity (ppt) pH DO

(mg L-1)

1st day (1)* 29.0 15.0 7.5 6.4 29.0 15.0 7.5 6.3

5th day (3) 29.0 14.0 7.5 6.4 29.0 14.0 7.6 6.4

10th day (5) 29.0 14.0 7.4 6.5 29.0 14.0 7.5 6.3

15th day (7) 29.0 14.0 7.4 6.4 29.5 14.0 7.5 6.5

20th day (10) 28.5 12.0 7.3 6.6 28.5 12.0 7.2 6.5

25th day (PL 2) 29.0 5.0 7.3 6.3 29.0 6.0 7.3 6.2

30th day (PL 7) 29.0 0.0 7.3 6.2 29.0 0.0 7.2 6.2 WOP and WP- samples from without probiotic and with probiotic treatment, *Figures in parenthesis indicate larval stage.

4.5.2 Bacteriology of water from hatchery environment of M. rosenbergii

4.5.2.1 Bacterial load of water

Variation in the total bacterial load in water samples from the hatchery tank with and

without probiotic application are presented in Figure 4.1 to 4.3. Result of TVC (Figure 4.1)

showed that there was no significant difference between the treatments, however there was

significant difference during the days of culture (P<0.001). In the tanks where probiotics were

not incorporateded, TVC of water ranged from 2.00 x 104 to 3.00 x 106 cfu ml-1. TVC of water

sample from probiotic treated water ranged between 2.00 x 104 to 4.70 x 106 cfu ml-1.



95

Figure 4.1. Total viable count (TVC) of water samples from the hatchery tanks without probiotic

and with probiotic treatment

Result of TC (Figure 4.2) showed that there was no significant difference between the

treatments, however there was significant difference during the days of culture (P<0.05). TC

count of water from without probiotic application ranged from 1.30 x 102 to 1.34 x 104 cfu ml-1

and that of probiotic treated water were 1.30 x 102 to 1.80 x 104 cfu ml-1. The TC load was found

to steadily increase from day 1 till 10th day, after which there was fluctuation in the pattern of

growth of TC.

Figure 4.2. Total coliform count (TC) of water samples from the hatchery tanks without probiotic

and with probiotic treatment



96

Result of TVLO (Figure 4.3) showed that there was no significant difference between the

treatments, however there was significant difference during the days of culture (P<0.05). TVLO

load highest during 10th day and lowest value was obtained on 1st day. TVLO count of water

from tanks without probiotic application ranged from 3.50 x 101 to 4.00 x 103 cfu ml-1 while that

of probiotic treated water ranged from 3.50 x 101 to 7.50 x 103 cfu ml-1.

Figure 4.3. Load of Vibrio like organisms (TVLO) of water samples from the hatchery tanks

without probiotic and with probiotic treatment

4.5.2.2 Bacteria of water from hatchery environment of M. rosenbergii

Ninety nine bacterial isolates of water samples from M. rosenbergii hatching tank

without probiotic application were characterised up to genera. The results are presented in Table

4.2. Out of 13 genera, Corynebacterium was the predominant genera followed by Bacillus and

Vibrio. Bacillus and Staphylococcus were isolated form all the samples.

Ninety two bacterial isolates of water samples from M. rosenbergii hatching tank with

probiotic application were characterised up to genera. The results are presented in Table 4.3. Out

of 14 genera, Vibrio and members of the family Enterobacteriaceae were the predominant Gram

negative genera. Enterobacteriaceae family was isolated form all the sampling period. Twelve

bacterial genera were found to be common in both the water samples. Aeromonas was isolated

from water samples from tank without probiotic and Flavobacterium and Lactobacillus from

probiotic treated tanks.



97

Table 4.2. Percentage occurrence of different genera of bacteria in the water samples from the

hatchery tank without probiotic application

Percentage occurrence at Genera 1st day

(18)10th day (30)*

20th day (28)*

30th day (23)*

Total (99)*

Gram Negative Acinetobacter -- -- 3.57 8.70 3.07 Aeromonas -- 6.67 -- -- 1.67 Alcaligenes -- -- -- 13.04 3.26 Cytophaga 11.11 -- 3.57 -- 3.67 Enterobacteriaceae 11.12 6.67 7.14 -- 6.23 Moraxella 16.66 -- 3.57 -- 5.06 Pseudomonas -- -- -- 8.70 2.18 Vibrio -- 50.00 3.57 -- 13.39 Gram Positive Bacillus 22.22 13.33 17.86 13.04 16.61 Corynebacterium 22.22 -- 25.00 26.09 18.33 Kurthia -- -- 3.57 4.35 1.98 Micrococcus 11.11 -- 10.72 4.35 6.54 Staphylococcus 5.56 13.33 3.57 17.38 9.96 Unidentified -- 10.00 17.86 4.35 8.05 Total 100.00 100.00 100.00 100.00 100.00

*Number of isolates Table 4.3. Percentage occurrence of different genera of bacteria in the water samples from the

hatchery tank with probiotic application


(18)10th day (25)*

20th day (25)*

30th day (24)*

Total (92)*

Gram Negative Acinetobacter -- -- -- 12.50 3.13 Alcaligenes -- -- 4.00 12.50 4.13 Cytophaga 11.11 -- 4.00 -- 3.78 Flavobacterium -- -- 4.00 -- 1.00 Enterobacteriaceae 11.12 32.00 16.00 8.33 16.86 Moraxella 16.66 -- 4.00 4.17 6.21 Pseudomonas -- -- 8.00 16.67 6.17 Vibrio -- 32.00 36.00 12.50 20.12 Gram Positive Bacillus 22.22 -- 4.00 -- 6.56 Corynebacterium 22.22 -- -- 12.50 8.68 Kurthia -- 8.00 -- 4.17 3.04 Lactobacillus -- -- 12.00 -- 3.00 Micrococcus 11.11 -- -- 8.33 4.86 Staphylococcus 5.56 16.00 -- -- 5.39 Unidentified -- 12.00 8.00 8.33 7.07 Total 100.00 100.00 100.00 100.00 100.00

*Number of isolates



98

4.5.3 Bacteriology of larvae from hatchery environment of M. rosenbergii

4.5.3.1 Bacterial load of larvae

Total bacterial load in larval samples from the hatchery tank without probiotic

application and probiotic treatment are presented in Figure 4.4 to 4.6. Result of TVC (Figure 4.4)

showed that there was no significant difference between the treatment, however there was

significant difference during the days of culture (P<0.001). The highest TVC was observed in

larval samples collected on 15th day and lowest count on those collected on 20th day. TVC count

of larvae from tanks without probiotic application ranged from 5.10 x 105 to 5.20 x 108 cfu g-1

and that from probiotic treated tanks ranged from 3.30 x 105 1.10 x 109 cfu g-1.

Figure 4.4. Total viable count (TVC) of larval and PL samples from the hatchery tanks without probiotic and with probiotic treatment

Result of TC (Figure 4.5) and TVLO (Figure 4.6) showed that there was no significant

difference between the treatments and during the days of culture. TC load of larvae from without

probiotic and probiotic treatment ranged from 2.50 x 102 to 2.78 x 105 cfu g-1 and 2.71 x 103 to

9.05 x 104 cfu g-1. TVLO load of larvae from without probiotic and probiotic treatment ranged

from 3.76 x 102 to 3.56 x 104 cfu g-1 and 3.44 x 103 to 2.00 x 104 cfu g-1.



99

Figure 4.5. Total coliform count (TC) of larval and PL samples from the hatchery tanks without probiotic and with probiotic treatment

Figure 4.6. Load of Vibrio like organisms (TVLO) of larval and PL samples from the hatchery

tanks without probiotic and with probiotic treatment 4.5.3.2 Bacteria of larvae from hatchery environment of M. rosenbergii

One hundred and five bacterial isolates of larval and PL samples from M. rosenbergii

hatching tank without probiotic application were characterised up to genera. The results are

presented in Table 4.4. Out of 10 genera, Corynebacterium, members of the family

Enterobacteriaceae and Alcaligenes were the frequently encountered genera.

One hundred and five bacterial isolates of larval and PL samples from M. rosenbergii

hatching tank with probiotic application were characterised up to genera. The results are

presented in Table 4.5. Out of 12 genera, Alcaligenes and Corynebacterium were predominant

Gram negative and Gram positive genera respectively. Members of the family

Enterobacteriaceae were the other frequently encountered genera.



100

Table 4.4. Percentage occurrence of different genera of bacteria in the larval and post larval

samples from the hatchery tank without probiotic application


(31) 10th day (24)*

20th day (25)*

30th day (25)*

Total (105)*

Gram Negative Acinetobacter 3.23 0.00 12.00 0.00 3.81 Alcaligenes 22.58 8.33 8.00 28.00 16.73 Cytophaga -- 8.33 -- -- 2.08 Enterobacteriaceae 16.13 8.34 16.00 32.00 18.12 Pseudomonas -- 4.17 4.00 8.00 4.04 Vibrio 3.23 8.33 -- -- 2.89

Gram Positive Bacillus -- 20.83 12.00 4.00 9.21 Corynebacterium 29.03 29.16 28.00 12.00 24.55 Kurthia 6.45 4.17 -- -- 2.66 Staphylococcus -- 4.17 -- 12.00 4.04 Unidentified 19.35 4.17 20.00 4.00 11.88 Total 100.00 100.00 100.00 100.00 100.00

*Number of isolates Table 4.5. Percentage occurrence of different genera of bacteria in the larval and post larval

samples from the hatchery tank with probiotic application


(31)10th day (27)*

20th day (24)*

30th day (23)*

Total (105)*

Gram Negative Acinetobacter 3.23 -- 4.17 -- 1.85 Aeromonas -- 3.70 -- -- 0.93 Alcaligenes 22.58 18.53 20.83 30.42 23.09 Enterobacteriaceae 16.13 18.51 12.50 17.39 16.14 Pseudomonas -- 3.70 8.33 -- 3.01 Vibrio 3.23 14.82 16.67 -- 8.68 Gram Positive Bacillus -- 11.11 8.33 17.39 9.21 Corynebacterium 29.03 18.52 12.50 8.70 17.19 Kurthia 6.45 3.70 4.17 4.35 4.67 Lactobacillus -- -- 4.17 8.70 3.22 Micrococcus -- -- -- 4.35 1.09 Staphylococcus -- -- -- 8.70 2.18 Unidentified 19.35 7.41 8.33 -- 8.77 Total 100.00 100.00 100.00 100.00 100.00

*Number of isolates



101

4.5.4 Bacteriology of feed items from hatchery environment of M. rosenbergii

4.5.4.1 Bacterial load of feed

Total bacterial load in Artemia hatching water and feed items such as Artemia cyst,

Artemia nauplii, Artemia flake, egg custard and starter feed used for feeding larvae and PL are

presented in Table 4.6. The result showed that there was significant difference between the

bacterial load (P<0.01) and also between different feed items (P<0.05). Among feed items higher

TVC was observed from Artemia nauplii while lowest TVC was obtained from starter feed

sample. TVC of feed items ranged from 3.50 x 105 to 3.20 x 107 cfu g-1. TC of feed items ranged

from zero to 1.10 x 105 cfu g-1 and TVLO ranged from zero to 8.68 x 105 cfu g-1. The highest load

of TVC, TC and TVLO were observed from Artemia nauplii and TC and TVLO were absent in

starter feed.

4.5.4.2 Bacteria of feed items from hatchery environment of M. rosenbergii

One hundred and fifty one bacterial isolates from Artemia hatching water and feed items

used in M. rosenbergii hatchery were characterised up to genera. The results are presented in

Table 4.7. The results revealed the dominance of Vibrio in Artemia hatching water and on

Artemia nauplii. Other frequently encountered genera included Aeromonas and Bacillus from

Artemia hatching water, Vibrio, Acinetobacter and Micrococcus from Artemia nauplii. Bacillus

was the predominant genera in Artemia cyst, Artemia flake, egg custard and starter feed.

Table 4.6. Total bacterial load of Artemia hatching environment and feed items used for feeding

larvae and PL in hatchery

Total bacterial load (cfu ml-1 or g-1) Samples

TVC TC TVLO

Artemia hatching water 2.35 x 105 4.67 x 103 2.32 x 104 Artemia cyst 1.00 x 106 4.50 x 104 5.00 x 102 Artemia nauplii 3.20 x 107 5.60 x 104 8.68 x 105 Artemia flake 4.50 x 105 6.00 x 102 3.40 x 102 Egg custard 2.50 x 107 1.10 x 105 4.00 x 102 Starter feed 3.50 x 105 ND# ND

#Not detected, TVC-Total viable count, TC-Total coliform, TVLO-Total Vibrio like organism



102

Table 4.7. Percentage occurrence of different genera of bacteria in Artemia hatching environment and feed items used for feeding larvae and PL in hatchery

Percentage occurrence at

Genera Artemia hatching water (23)*

Artemia cyst (20)

Artemia nauplii (28)

Artemia flake (27)

Egg custard (28)

Starter feed (25)

Gram Negative Acinetobacter -- -- 10.71 -- -- -- Aeromonas 21.74 -- 7.14 -- -- -- Alcaligenes -- 5.00 3.58 -- -- -- Cytophaga 8.70 -- -- -- -- -- Enterobacteriaceae 8.70 10.00 7.14 14.81 14.29 16.00 Moraxella -- 5.00 -- -- -- -- Vibrio 26.09 15.00 46.43 11.11 -- -- Gram Positive Bacillus 13.04 20.00 7.14 22.22 42.86 32.00 Corynebacterium -- 15.00 -- 7.41 21.43 20.00 Micrococcus 8.70 15.00 10.72 18.52 7.14 24.00 Staphylococcus -- -- -- 11.11 -- -- Unidentified 13.03 15.00 7.14 14.82 14.28 8.00 Total 100.00 100.00 100.00 100.00 100.00 100.00

*Number of isolates

4.5.5 Bacteriology of commercial probiotic consortium used in the hatchery of M. rosenbergii

Load of TVC, TC and TVLO of commercial probiotics were estimated. While TVC load of 2.20 x 106 cfu ml-1 were observed, TC and TVLO were absent in the commercial probiotics. Forty two bacterial isolates from commercial probiotics used in the M. rosenbergii hatchery were characterised up to genera. The results are presented in Table 4.8. Lactobacillus was the predominant genera followed by Corynebacterium.

Table 4.8. Percentage occurrence of different genera of bacteria from probiotic consortium used in the hatchery of M. rosenbergii.

Genera % occurrence (42)* Gram NegativeAcinetobacter 2.38 Alcaligenes 7.14 Enterobacteriaceae 11.90 Gram Positive Bacillus 7.14 Corynebacterium 19.05 Lactobacillus 33.33 Micrococcus 2.38 Staphylococcus 2.38 Unidentified 14.29 Total 100.00

*Number of isolates



103

4.5.6 Comparison of bacteriology of various samples from hatchery environment of M. rosenbergii

4.5.6.1 Bacterial load of various samples from hatchery environment

The load of TVC, TC and TVLO (Figure 4.7) of various samples from the hatchery

environment showed that there was significant difference among different organism such as

TVC, TC and TVLO (P<0.001) as well as the load of these organism among various samples

(P<0.01). TVC load was generally high followed by TC and TVLO, however, the TVLO was

found to be higher than TC from Artemia nauplii samples. TC and TVLO were not detected from

the starter feed and probiotic consortium samples. The highest TVC was observed from Artemia

nauplii followed by larval samples from probiotic treated tanks and egg custard samples.

Figure 4.7. Total bacterial load (TVC, TC and TVLO) of various samples from hatchery environment of M. rosenbergii

4.5.6.2 Bacteria of various samples from hatchery environment

The result of Gram negative (Figure 4.8 a – h) and Gram positive (Figure 4.9 a – f)

bacterial genera from various samples from hatchery environment of M. rosenbergii showed

significant difference among genera (P<0.001). While Bacillus was the predominant genera,

followed by Vibrio, Corynebacterium and members of the family Enterobacteriaceae were the

other frequently encountered genera. Flavobacterium was encountered only in water samples

treated with probiotics (Figure not included). Members of the family Enterobacteriaceae and

Bacillus were identified from all the samples from M. rosenbergii’s hatchery environment. The

percentage occurrence of bacterial genera such as Vibrio, Bacillus and Lactobacillus among

various samples was found to vary considerably.



104

Figure 4.8 a – h. Percentage occurrence of Gram negative bacterial genera isolated from various samples of M. rosenbergii’s hatchery environment

a. Acinetobacter b. Aeromonas

c. Alcaligenes d. Cytophaga

e. Enterobacteriaceae family f. Moraxella

g. Pseudomonas h. Vibrio



105

Figure 4.9 a - f. Percentage occurrence of Gram positive bacterial genera isolated from various samples of M. rosenbergii’s hatchery environment

4.6 Discussion

4.6.1 Physico-chemical parameters

The physico-chemical parameters of the hatchery environment agreed with that

suggested by New (1990; 2002). The temperature of water remained constant at 28.5 + 1oC and

salinity ranged between zero to 15 ppt from the hatchery water in the present study. The rearing

of M. rosenbergii larvae was generally done at 10-15 ppt, optimum temperature of 26-31oC with

a pH in between 7.0-8.3. However, many changes in the chemical water quality of larval rearing

a. Bacillus b. Corynebacterium

c. Kurthia d. Lactobacillus

e. Micrococcus f. Staphylococcus



106

water can occur. Hydrogen ion concentration is one of the important water quality parameters for

aquaculture. The optimum pH value for freshwater fish is normally between 6.5 and 8.5, while

the optimum value recommended for marine aquaculture is between 7.5 and 8.5 (Law, 1988).

In present study, pH varied between 7.2 to 7.6 and DO ranged from 6.2 to 6.6 mg L-1

during the hatchery operations. Low pH of water has been reported to cause retarded growth in

P. monodon, disturbed ion regulation in crayfish and tiger shrimp and acid-base imbalance in

crayfish and M. rosenbergii (Allan and Maguire, 1992; Chen and Lee, 1997a). Chen and Chen

(2003) indicated that the molting frequency of M. rosenbergii placed in pH 6.8, 6.2 and 5.6 was

significantly lower than those maintained in pH 7.4 and 8.2. The change of pH in water may

enhance the production of toxic compounds, such as the production of toxic unionized ammonia

(NH3) at higher pH and hydrogen sulfide (H2S) in lower pH. This may cause some physiological

changes in the organisms, which will lead to harmful effects.

Guisande et al. (1998) reported that the quality of eggs could be affected by biotic

factors such as egg and larval abundance and abiotic factors such as temperature and salinity. It

was reported that at a temperature below 24-26oC the larvae will not grow well and the time to

attain metamorphosis will be longer and temperature over 33oC are normally lethal. It is also

reported that environmental parameters such as temperature and DO affect the immune system of

crustaceans (Truscott and White, 1990; Le Moullac et al., 1998). DO is one of the water quality

parameter that is more closely related to the survival. Avault (1986) reported that M. rosenbergii

will be under stress if DO falls below 2 mg L-1. DO lower than 0.5 mg L-1 found to be lethal level

for the prawns. Cheng et al. (2003b) noticed that all prawns kept at 2.75 and 1.75 mg L-1 DO

showed sluggish behaviour and ceased feeding for the initial 2 days and considered it as a

hypoxic for M. rosenbergii.

4.6.2 Bacterial load of various samples from M. rosenbergii’s hatchery environment

4.6.2.1 Bacterial load of water from hatchery

TVC load of water from tanks without probiotic and probiotic treatment were 2.00 x 104

to 3.00 x 106 and 2.00 x 104 to 4.70 x 106 cfu ml-1 respectively. TVC load was similar to that of

reported by Phatarpekar et al. (2002) who reported 1.1 x 104 to 2.4 x 106 ml-1 from brooder tank

and spawning tank waters of M. rosenbergii. Anderson et al. (1989) also reported similar load of

bacteria in water from M. rosenbergii’s hatchery. Sahul Hameed et al. (2003) reported slightly

lower TVC load in water from larval rearing tank of M. rosenbergii hatchery suggested that the

use of antibiotics for controlling bacterial pathogens in the system may be the reason for the low



107

THB load. Aquacop (1977) and Miyamoto et al. (1983) also reported the similar range of

bacterial count. The fluctuation in the range of THB load depend on the method used in the

hatchery system and quality of feed, removal of unconsumed feed, dead larvae and other

suspended solids (Aquacop, 1977; Anderson et al., 1989). Otta et al. (2001) studied bacteriology

from several shrimp hatcheries located along the east and west coast of India and reported the

total plate counts of raw sea water on tryptic soya agar ranged from 102 to 104 cfu ml-1, whereas

it ranged from 104 to 106 cfu ml-1 in larval tanks.

4.6.2.2 Bacterial load of larvae and PL of M. rosenbergii

The TVC load of bacteria associated with larvae and PL from tanks without probiotics

and probiotic treated tank was 1.67 x 107 to 1.10 x 109 and 1.67 x 107 to 5.20 x 108 cfu g-1. The

result showed fluctuations in number during different larval stages from both the tanks. The

findings are in agreement with the observation of Sahul Hameed et al. (2003) who studied the

bacteriology of larvae of M. rosenbergii from hatchery. Sahul Hameed et al. (2003) also reported

the numbers of aerobic heterotrophic bacteria from eggs, larvae and PL, which were found to

vary from 7.9 x 104 to 8.2 x 106, 0.8 x 105 to 81.1 x 105 and 38.3 x 105 to 10.9 x 106 cfu g-1

respectively. Karunasagar et al. (1994) encountered a total bacterial count of 1.7 to 2.5 x 107 cfu

g-1 in larvae and 1.2 x 103 to 2.0 x 107 cfu g-1 in PL of P. monodon. The observation differs from

those of Miyamoto et al. (1983) and Anderson et al. (1989) on larvae of M. rosenbergii but

agreed with Yasuda and Kitao (1980) and Singh (1986) on larvae and PL of P. japonicus and P.

indicus. Miyamoto et al. (1983) and Anderson et al. (1989) noted a general trend of increasing

bacterial cell count per gram of larval tissue with advancing age of larvae. Huys et al. (2001)

observed an increase in TVC of larvae from stages just before first feeding up to 9th day.

However, the results of the study of Munro et al. (1994) and Huys et al. (2001) clearly

demonstrated that there was no correlation between the number of bacteria present in the larval

gut and larval survival rates.

The TVC of larvae was found to decrease drastically on 20th day sampling. The reason

for sudden decrease of TVC at 20th day sampling may be due to the changes in the feeding habit.

It was reported by Barros and Valenti (2003) that the larval stages between II and VI, live food

intake were significantly higher compared to common aquaculture dry feed and egg custard. A

significant intake of dry feed and egg custard was observed from larval stage VI onwards. The

development of the digestive tract and increase enzyme activity from stage VI onwards

(Kamarudin et al., 1994; Kumlu and Jones, 1995) may also help to explain the increasing

acceptance of inert diets. The preference of larvae from stages VI, VII and VIII to ingest wet



108

rather than dry diets might indicate a preference for clear and soft particles during this transition

period. Meyers and Hagood (1984) observed that M. rosenbergii larvae from stage VI onwards

preferred particles of diets with lighter colors. New and Shingholka (1985) recommend egg

custard from stage III onwards (about the fourth day of culture). Daniels et al. (1992)

recommended diet supplementation from stages V to VI (between the 8th and 10th day). The

sudden decrease in the TVC load towards 20th day of sampling is quite likely due to change in

the feeding habit of M. rosenbergii larvae, as suggested by researchers mentioned above.

TVC of rearing water of the study environment were lower than that of larvae. Miyamoto

et al. (1983) and Sahul Hameed et al. (2003) also reported higher bacterial count in M.

rosenbergii larvae than in rearing water. Larval surface provide a suitable microenvironment for

bacterial growth, which may be responsible for association of greater number of bacteria with

eggs and larvae. The composition of the microflora is believed to be of great importance for

development and health of the host. Colorni (1985) also observed rich intestinal flora in less than

24 hour old larvae. Sahul Hameed (1993) suggested that interaction between the larval surface

and bacterial adhesion was responsible for the association of greater number of bacteria.

4.6.2.3 Bacterial load of feed items used in M. rosenbergii hatchery

Diet is a fundamental aspect in larval culture of crustaceans. During the developmental

stages, their morphology, behaviour and nutritional needs change. A wide variety of feeds are

employed by different hatcheries, including the nauplii of brine shrimp (Artemia spp.),

freshwater cladoceran (Moina spp.), fish eggs, squid flesh, frozen adult Artemia, flaked adult

Artemia, fish flesh, egg custard, worms and commercial feeds (New, 2002). One of the most

significant advances in aquaculture has been the introduction of Artemia as food for fish and

invertebrates larvae. Artemia nauplii have been reported as one of the best foods for most

organisms under culture. Artemia has been used as vectors of growth promoter or drug carrier for

therapeutic applications (Ozkizilcik and Chu, 1994; Touraki et al., 1996). Mass mortality of

larvae in the hatcheries is often attributed to opportunistic bacteria. Feeds are one of the principal

routes of pathogenic bacteria to enter the hatchery systems and feed composition affects the

commensal microflora and results in the proliferation of bacteria that normally have restricted

opportunities to grow. Study of the bacterial flora of larval turbot (Nicolas et al., 1989; Munro et

al., 1994) has shown that within 3 to 4 days after feeding, the number of gut microflora of larvae

reached 104 to 105 cfu larvae-1 for various food sources and larval rearing conditions.



109

The bacterial load of feeds studied in the present investigation agreed to some extend

with those of Muroga et al. (1987) who reported the total bacterial count of 2.1 x 107 cfu g-1 for

live feeds (rotifer and Artemia) and 1.2 x 104 cfu g-1 for artificial feeds. Trust and Wood (1973)

reported 103-104 aerobic bacteria per gram of fish diet. The present findings are also in agreement

with the observations of Raghavan (2003) that the THB in the dry and exposed feeds ranged

from 3.3 x 104 to 1.1 x 106 cfu g-1 and 9.4 x 106 to 4.8 x 107 cfu g-1 respectively. The results of the

present study indicated that the bacterial counts were within the recommended limits. In the

study of Sahul Hameed and Balasubramanian (2000), the total number of aerobic heterotrophic

bacterial flora ranged from 3.8 x 103 to 8.1 x 103 cfu nauplius-1 on seawater nutrient agar. Kerry

et al. (1997) reported a TVC load of 1.3 x 104 cfu g-1 in 12 samples of the commercial feed

pellets.

4.6.2.4 Bacterial load of commercial probiotics used in the hatchery of M. rosenbergii

TVC of probiotic solution used in the hatchery under present study had a TVC of 2.20 x

106 cfu ml-1. The use of probiotic microorganisms has proven advantageous in domestic animal

production (Vanbelle et al., 1990). In aquatic ecosystems, the intimate relationships between

microorganisms and other biota and the constant flow of water through the digestive tract of fish

and invertebrates will also affect their indigenous microflora. Against this background, we may

assume that the natural microbiota on eggs and larvae may help to protect against colonisation by

a harmful microflora. The microflora of intensive larval rearing systems, however, differs

dramatically from that in the natural system, and is influenced by rearing techniques, nutrients,

disinfection techniques, and use of antibiotics. It is not yet known to what extent the natural

microflora of fish may be protective towards pathogen colonisation (Hansen and Olafsen, 1999).

However, there is increasing evidence that microflora manipulation, or addition of probiotic

microorganisms (Gatesoupe, 1999; Gomez-Gil et al., 2000) in aquaculture.

4.6.3 Health significant bacteria from hatchery environment of M. rosenbergii

Water samples from hatchery tank without probiotic and with probiotic treatment had TC

and TVLO within a range of 1.30 x 102 to 1.80 x 104 and 3.50 x 101 to 7.50 x 103 cfu g-1

respectively. TC and TVLO of larval and PL samples ranged from 2.50 x 102 to 9.05 x 104 and

3.76 x 102 to 3.56 x 104 cfu g-1 respectively. Phatarpekar et al. (2002) reported almost similar

range of TC from hatchery water samples. However the TVLO load was one log higher than

those encountered in the present study. Eddy and Jones (2002) reported Vibrio count of 1.8 x 103

to 2.5 x 106 cfu ml-1 from summer flounder hatchery waters. The high load of Vibrio may be due



110

to the fully marine habitat of the hatchery in case of flounder. Sahul Hameed et al. (2003) also

noted high TVLO count than the present study. They observed 1.00 x 103 to 3.54 x 105 cfu ml-1,

2.00 x 104 to 1.32 x 106 and 1.7 x 105 to 5.36 x 106 cfu g-1 from water, larvae and PL respectively

from the hatchery system of M. rosenbergii. However the result of TVLO encountered in present

study is in agreement with Kennedy et al. (2006) who reported 101 to 103 cfu ml-1 from water and

102 to 104 cfu per 10 larvae from the larval rearing set up of M. rosenbergii.

In the present study, TC and TVLO were not encountered from probiotics preparations

and Starter feed. TC and TVLO results of feed items and its environment (Artemia hatching

water, Artemia cyst, Artemia nauplii, Artemia flake and egg custard) ranged from 6.00 x 102 to

1.10 x 105 and 3.40 x 102 to 6.85 x 105 cfu g-1 respectively. Lizarraga-Partida et al. (1997)

reported TVLO count as high as 1 x 106 to 9 x 106 cfu g-1 bacteria from Artemia nauplii, one log

increase result than present study. The study of Sahul Hameed and Balasubramanian (2000)

reported TVLO count of Artemia nauplii ranged from 9.4 x 102 to 4.3 x 103 cfu nauplius-1.

Vibrios are known to be pathogenic to prawns (Anderson et al., 1989) and systemic

infection and necrotic appendages caused by Vibrio have been reported in hatcheries (New,

1995). Fujioka and Greco (1984) isolated V. fluvialis, V. alginolyticus and V. cholerae from M.

rosenbergii larvae. Singh (1990) observed relationship between the abundance of the members of

family Vibrionaceae and mortality of M. rosenbergii larvae during the midlarval cycle.

4.6.4 Bacteria of various samples from M. rosenbergii’s hatchery environment

4.6.4.1 Bacteria of water samples

A significant factor affecting the outcome of hatchery production is the microbiology of

the rearing biotope. The intensive rearing of various species in aquaculture has revealed intimate

relationships between the organism and bacteria that eventually may affect establishment of a

normal mucosal microflora or result in disease epizootics. Interactions between bacteria and

mucosal surfaces play important roles both at the egg and larval stages. Bacterial adhesion and

colonization of the egg surface occur within hours after fertilization. The diverse flora which

eventually develops on the egg appears to reflect the bacterial composition and load of the

ambient water, but species-specific adhesion at the egg surface may also play a role in

development of the egg epiflora (Hansen and Olafsen, 1999). Ingestion of bacteria at the yolk sac

stage results in establishment of a primary intestinal microflora which seems to persist beyond

first feeding. Establishment of a gut microflora is likely to undergo several stages, resulting in an

adult microflora weeks to months after first feeding. Ingested bacteria may serve as an exogenous



111

supply of nutrients or essential factors at an early life stage. Early exposure to high bacterial

densities is probably important for immune tolerance, and thus for the establishment of a

protective intestinal microflora (Hansen and Olafsen, 1999). Successful rearing of early life

stages of several aquatic fish and prawn species depends on knowledge of the complex

interactions among the cultured organisms and the bacterial communities which develop at the

mucosal surfaces and in the ambient water and rearing systems.

Altogether 15 genera were identified from water samples from tank without probiotic

and with probiotics treatment. The microbial diversity was similar to those observed by

Anderson et al. (1989) who isolated 16 genera of bacteria from hatchery water. Eight of the

genera were also common to those observed by Anderson et al. (1989). Phatarpekar et al. (2002)

and Kennedy et al. (2006) also reported 11 and 14 genera of bacteria from M. rosenbergii’s

hatchery water during larval rearing operation respectively. Many of the genera encountered by

these researchers were also encountered in the present study. The results of the present study give

an idea about the autochthonous bacterial genera that are present in the hatchery set up of M.

rosenbergii.

In the present study Vibrio was found to be one among the most predominant genera

which agreed with findings of Otta et al. (2001) who reported the proportion of Vibrio species

ranged from 50% to 73%. A mixed bacterial flora was observed in hatchery water; however, in

the larval tanks, the flora in the larvae was predominantly made up of Vibrio species. Kennedy et

al. (2006) also reported the predominance of Vibrio in the water samples analysed. Opportunistic

bacterial pathogens, particularly Vibrio spp., are present as part of the normal microbiota of

marine fish and have been shown to be causative agents of disease and mass mortality (Sedano et

al., 1996; Pedersen et al., 1997).

Other genera that are frequently encountered in water during the present study were

Corynebacterium, Bacillus and members of the family Enterobacteriaceae. Twelve bacterial

genera were found to be common in water samples from tanks without probiotic and those

treated with probiotics. Aeromonas was isolated from water samples from tank without probiotics

while Flavobacterium and Lactobacillus from tanks treated with probiotics. The result of the

present study are in agreement with that of Anderson et al. (1989) and Kennedy et al. (2006) who

reported Bacillus as one of the predominant genera from M. rosenbergii’s larval rearing water.

Incidence of Lactobacillus was noted only in the probiotic treated water and this may be due to

the addition of probiotics that contained 33% Lactobacillus. However, Kennedy et al. (2006)



112

reported the presence of Lactobacillus from the larval rearing water of M. rosenbergii. It was

reported that supply water can be a major source of the microbes associated with an aquaculture

facility (Douillet and Pickering, 1999).

4.6.4.2 Bacteria of larvae and PL of M. rosenbergii

In present study, 13 bacterial genera were isolated from the larvae and PL of M.

rosenbergii. Corynebacterium, Alcaligenes and members of the family Enterobacteriaceae were

the predominant genera. An understanding of the characteristics or features and role of the

indigenous microflora of larvae may help to improve feed and conditions for the intensive mass

rearing of healthy individual. The information concerning the bacterial flora of crustaceans,

particularly freshwater prawn, appears to be limited. The bacteria associated with larvae and

post-larvae of M. rosenbergii showed fluctuations in number during different larval stages. Sahul

Hameed et al. (2003) and Kennedy et al. (2006) also observed a similar trend. Munro et al.

(1995) postulated that control on the bacterial diversity present, rather than the total bacterial

density in the gut might be important in ensuring high larval survival rates.

Anderson et al. (1989) and Phatarpekar et al. (2002) observed predominance of

Alcaligenes from the larval sample of M. rosenbergii. Kennedy et al. (2006) reported the

predominance of Pseudomonas and Aeromonas. Phatarpekar et al. (2002) mentioned that the

absence of major mortalities and abnormalities in larvae may be attributed to the absence of

Aeromonas and Vibrio. Yasuda and Kitao (1980) noted an association between poor larval

growth and dominance of Aeromonas in the gut of P. japonicus. Aeromonas spp. and Vibrio spp.

isolated from diseased prawns were also reported to be pathogenic (Sahul Hameed, 1989).

Strom and Olafsen (1990) demonstrated that composition of the intestinal microflora of

wild captured juvenile cod changed after subsequent feeding on a commercial diet. With the

onset of first feeding, the intestinal microbiota becomes increasingly dominated by the bacteria

associated with the live feed (Munro et al., 1994; Grisez et al., 1997). However, it was reported,

during larval development, no dominant and persistent colonisation of the intestine by any given

bacterial species was observed from the intestinal samples of sea bream and sea bass larvae and

fluctuations in the composition of the dominant microflora appeared to reflect the bacterial

composition of the ingested live feed (Grisez et al., 1997). The authors also could not observe

any selection towards the genus Vibrio until the larvae reached the end of larval life stage.



113

In the present study Vibrio spp. were isolated from the larvae. Tanasomvang and Muroga

(1989) demonstrated that increased colonization of marine larvae with Vibrio spp. resulted in

delayed development of digestive system and increased mortality. This result agrees with earlier

reports of Yasuda and Kitao (1980), Colorni (1985) and Anderson et al. (1989). However,

Phatarpekar et al. (2002) did not report Vibrio from the larvae of M. rosenbergii. In probiotics

treated larval sample high prevalence of Vibrio spp. was observed compared to the larval samples

from rearing tanks without application of probiotics. The absence of Aeromonas may influence

the proliferation of Vibrio in the system. This was agreed with the study of Austin et al. (1995)

who reported that probiont Vibrio applied to Salmon could reduce the number of Aeromonas.

Anderson et al. (1989) observed high prevalence of Vibrio in washed larval slurries of M.

rosenbergii. Miyamoto et al. (1983) and Colorni (1985) also recorded Vibrio from the larvae.

Sahul Hammed (1993) and Singh (1986) found Vibrio as the predominant genera in larvae of P.

indicus. Out of 15 genera isolated from water 13 were common in larvae. The results were

similar to Anderson et al. (1989) who reported 14 out of 16 genera from larval tissue slurries

common to that of water. Yasuda and Kitao (1980) also reported similar results from the intestine

and that of water.

4.6.4.3 Bacteria of feed items used in M. rosenbergii hatchery

The present findings of generic diversity in Artemia hatching water and hatchery feed

items agreed with David and Dixon (1993) who reported that live brine shrimp contain variety of

bacterial flora within their systems, and it appears that this flora fluctuated in diversity with

changes in salinity. Vibrio and Aeromonas in Artemia hatching water and Vibrio, Acinetobacter

and Micrococcus in Artemia nauplii were the predominant genera. The result agreed with that of

Kennedy et al. (2006) who reported the predominance of Vibrio in all the 7 samples examined.

Kennedy et al. (2006) also noted Pseudomonas, Aeromonas and Bacillus as the common species

and isolated Acinetobacter and Microccous from 2 samples.

Austin and Allen (1982) studied the aerobic heterotrophic bacteria associated with

commercial cysts of Artemia and reported Aeromonas, Bacillus, Micrococcus and

Staphylococcus from cyst-hatching water. Sorgeloos et al. (1986) noted the dominance of Vibrio

spp. during hatching of Artemia. Artemia cysts are broken during hatching and a reserve organic

substance, glycerol, is excreted to hatching water. Glycerol is an organic substrate that is utilized

efficiently by Vibrio spp. A very low inoculum of this population could become dominant,

utilizing glycerol rather than the Gram-positive population. A study by Straub and Dixon (1993)

on adult Artemia from hypersaline ponds in California showed the presence of V. alginolyticus,



114

V. fluvialis, V. metschnikovii, V. mimicus and others at high salinity levels. Artemia nauplii carry

a heavy bacterial load and some bacterial species especially Vibrio spp. have been reported to be

the source of infections and high mortalities in fish larvae, and live feed organisms are thought to

be responsible (Muroga et al., 1987; Nicolas et al., 1989; Muroga et al., 1994; Verdonck et al.,

1994).

The present study revealed the predominance of Bacillus, Corynebacterium and

Micrococcus from Artemia cyst, Artemia flake, egg custard and starter feed. Microbiological

studies have demonstrated that Artemia cysts carry bacteria in the shell (Lopez-Torres and

Lizarraga-Partida, 2001). Bacillus could survive the freezing and sealing process used to package

of brine shrimp and that may be the reason for their predominance. Austin and Allen (1982)

studied the aerobic heterotrophic bacteria associated with commercial cysts of Artemia and

reported presence of Bacillus, Erwinia herbicola, Micrococcus, Staphylococcus and V.

parahaemolyticus from dehydrated cysts. Igarashi et al. (1989) reported the presence of Gram

positive rods identified as Corynebacterium spp. and Bacillus from Artemia cyst.

The bacterial population changes observed in the comparative study (Lopez-Torres and

Lizarraga-Partida, 2001) performed under laboratory and hatchery conditions show that the

Vibrio introduced with Artemia nauplii as a vector came from hatchery operations, not from

cysts. The air supply, hatching water, or hatching tanks could be the sources of Vibrio spp. Duan

et al. (1995) indicate that the bacteria produce organic substances that develop films on surfaces

exposed to seawater. Those films are composed of polysaccharides, mainly of glucose and

galactose (Rodriguez and Bhosle, 1991) which could protect bacteria against washing and

chlorination of walls of the tanks. These findings together with deficient cleaning of hatching

tanks could explain how Vibrio spp. is seeded during the hatching operation. Trust and Wood

(1973) had suggested that the ingredients used in feed formulation partially determine the nature

of its microflora. Farm-made feeds used in the traditional shrimp culture systems are generally

made from marine animal ingredients such as prawn meal, fishmeal and clam meat. Studies

(Trust, 1971; Trust and Money, 1972) have shown that commercial fish feeds contain mixed

microflora including bacterial species potentially pathogenic to man and aquatic animals.

4.6.4.4 Bacteria of commercial probiotics used in M. rosenbergii hatchery

Bacteria belonging to 8 genera were isolated from the commercial probiotic preparation

that is used in the hatchery under study. Out of 8 genera, Lactobacillus was found to be the

dominant genera followed by Corynebacterium and members of the family Enterobacteriaceae



115

from probiotic sample. The amount of the probiotic bacteria added into the system was

insufficient as it contains only 2.0 x 106 cfu ml-1. Lactic acid bacteria produced growth inhibiting

factors that could inhibit various Vibrio species especially V. anguillarum (Olafsen, 2001).

Gatesoupe (1991) reported that commercial preparation of live L. plantarum decreased the

proportion of A. salmonicida like bacteria associated with rotifers. It is demonstrated that L.

lactis (AR 21) exhibited an inhibitory effect against V. anguillarum in rotifer culture at sub

tropical conditions. The ability of Lactobacillus to produce organic acids, H2O2 and other

compounds which depress the growth of other microorganisms is partially responsible for their

positive effect on host and the host’s microflora. Queiroz and Boyd (1998) confirmed that a

commercial inoculums of Bacillus species increased survival and production of channel cat fish.

4.7 Conclusion

Bacteriology of the hatchery environment of M. rosenbergii was studied with the help of

various samples such as water used in the rearing tanks, larvae and PL, live Artemia nauplii,

Artemia cyst, Artemia flakes, egg custard and commercial starter feed used to feed the larvae,

and probiotics that are used in a commercial M. rosenbergii hatchery. Samples were collected

from tanks with probiotics application as well as those without probiotic treatment. Load of

different groups of bacteria such as TVC, TC and TVLO showed an increasing trend at the

beginning of larval rearing tank followed by a decrease after 10-15 days of rearing. At the end of

30 days rearing period, reduction in TVC and TC in water were more pronounced in the tanks

with probiotic application. While the TVC load was 2-3 logs higher than the TC and TVLO,

there was no significant variation between the TC and TVLO load of samples from hatchery.

Starter feed and commercial probiotics that are used in the hatchery did not show any TC and

TVLO.

Characterisation of heterotrophic bacterial genera showed good diversity in the water

samples used in the hatchery. Unlike in the culture environment samples Corynebacterium was

found to be the predominant Gram positive genera in the hatchery water samples. Lactobacillus

was encountered in the water samples from the rearing tanks where commercial probiotics were

applied. Gram positive genera were more diverse in the hatchery samples when compared to the

samples from natural and culture environment of M. rosenbergii. Among the Gram negative

genera Vibrio was found to be predominant in the hatchery water samples indicating the

significance of this organism as a cause of mortality of prawn larvae in hatchery environment.

There was considerable percentage of Vibrio in all Artemia samples (Artemia hatching water,

Artemia cyst and Artemia nauplii) and their percentage of incidence was found to be increasing

till 20th day of larval rearing.

c-4. hatchery environment -...

Documents