# 2003 Kluwer Academic Publishers. Printed in the Netherlands.

Influence of the antibacterial herbs, Solanumtrilobatum, Andrographis paniculata and Psoraleacorylifolia on the survival, growth and bacterialload of Penaeus monodon post larvae

T. CITARASU1,2,*, K. VENKATRAMALINGAM1, M. MICHEAL BABU1,

R. RAJA JEYA SEKAR1 and M. PETERMARIAN1

1Marine Biotechnology Laboratory, Centre for Marine Science and Technology, Manonmaniam

Sundaranar University, Rajakkamangalam 629502, Kanyakumari, India; 2Centre for Biotechnology,

Anna University, Chennai 600113, India; *Author for correspondence (e-mail: cittu_in@yahoo.com)

Received 27 March 2002; accepted in revised form 26 September 2003

Key words: Andrographis paniculata, Antimicrobial, Artemia franciscana, Bioencapsulation, Herbal

extracts, Larviculture, Penaeus monodon, Psoralea corylifolia, Solanum trilobatum

Abstract. The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to bio-

magnification and that in turn could lead to rejection of a whole consignment. The application of the

bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs

having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea

corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post

larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as

Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in

the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific

growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water

and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate

(8.42–9.1%) and increased bacterial load (2.86� 103 to 3.76� 105 cfu=g). The methanolic extracts of the

herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon

culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed

the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load

(1.12� 105 cfu=g).

Introduction

The application of antibiotics to cure bacterial diseases plays an important role in

shrimp larval culture. But it needs continuous and large volume of water exchange

and it also leads to the development of resistance strains of bacteria. Sparks (1981)

explained that the drug resistance may be due to a pre-existing factor in the mi-

croorganisms, or it may be due to some acquired factors. Resistance became a big

problem as the widespread use of antibiotics led to the elimination of sensitive

organisms from the population accompanying the resistance organisms (Pelczer

et al. 1993).

Increasingly, antibiotic resistance of bacterial pathogens is reported from all

areas of aquaculture, ranging from warm to cold water. Decreased efficacy has been

Aquaculture International 11: 583–595, 2003.

documented in antibiotics regardless of their mechanism of action. For example,

microbial resistance has been shown in different classes of antibiotics including

those affecting protein synthesis such as tetracycline and erythromycin, ami-

noglycosides like neomycin, antimetabolites such as the sulfa drugs and potentiated

salfonamides and quinolones like oxolinic acid (Dixon 1991).

Though most of the clinically used antibiotics are produced by soil micro-

organisms or fungi, higher plants have also been a source of antibiotics. Examples

for these are the bacteriostatic and antifungal properties of lichens, the antibiotic

action of allinine in Allium sativum and the antimicrobial action of berberines in

goldenseal (Hydrastis canadensis). They are effective in the treatment of infectious

diseases while simultaneously inhibiting many of the side effects that are often

associated with synthetic antimicrobials. In addition they are effective, without

deleterious side effects. Many plants have tropisms to specific organs or systems in

the body (Trease and Evans 1972).

The treatment of microbial diseases of fish by means of the currently applied

methods is rather difficult, non-effective, costly and involves environmental ha-

zards. A possible method to overcome these problems might be the oral adminis-

tration of antimicrobial compounds to the fish larvae through the food chain, using

the bioencapsulation technique as described by Verpraet et al. (1992). Live food

organisms especially Artemia and rotifers, are extensively used as the main food

sources for the larviculture of marine fish and shrimp. The two organisms are filter

feeders and are biological carriers for transferring essential nutrients to predator

larvae (Leger et al. 1986).

Alternative herb antibacterial methanolic extracts of S. trilobatum, A. paniculata

and P. corylifolia, which have proven to be very effective against bacterial patho-

gens, especially aquaculturaly important bacterial pathogens, were encapsulated in

Artemia (Citarasu 2000) and tested on Penaeus monodon cultures.

Materials and methods

The first stage P. monodon mysis were obtained from a commercial hatchery,

acclimated in the laboratory and reared till they reached the post larval stage.

Before starting the experiment, the post larvae were starved for 12 h.

After hatching, the nauplii were reared in culture tanks and fed with rice bran.

Four-day-old Artemia franciscana (San Francisco Bay brand) were used for en-

richment.

Diseased P. monodon (PL 24) were collected from semi-intensive cultured pond

system, Nellore, Andhra Pradesh, India. The larvae were ground well and were

serially diluted. The diluted samples of each dilution were plated in Zobell 2216 e

(Himedia) marine agar medium plates and were incubated at 28� 2 8C in a ther-

mostat incubator for 24 h. After incubation, colonies were isolated from this plate

and streaked three times on the same media in order to obtain pure isolates. They

were then stored in Zobell 2216 e marine agar slants for further identification.

Species level identification was carried out according to Bergey’s manual (Holt

584

et al. 1994). A detailed description is given in Table 1. Pseudomonas aeruginosa,

Staphylococcus aureus, Salmonella typhi and Vibrio sp. were isolated and these

were used to inoculate the P. monodon post larvae.

Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia were

selected based on their antibacterial characteristics (Nadkarni 1995) and extracted

in Soxhlet apparatus by following the method of Peach and Tracey (1956) using

methanol as a solvent.

The experimental set-up consisted of a series of small glass aquaria (2 l capacity).

The post larvae (PL 1) were introduced into the small glass aquaria at a stocking

rate of 10�l. Before introducing the post larvae, their length and weight were

measured. Three replicates and four trials were carried out for each treatment.

A monoculture of four fish pathogenic bacteria such as P. aeruginosa, Staph.

aureus, S. typhi and Vibrio sp. (previous isolates) were cultured in 100 ml Brain

Heart Infusion broth (BHI) (Himedia) in a 50 ml conical flask. For the culture of

Vibrio sp., 50% seawater (35%) was used for the medium preparation, replacing the

distilled water. This culture was kept in a shaker for 24 h and the bacterial cells

were later used for inoculating the post larval culture. The bacterial cells were

inoculated into the post larval culture water at a rate of more than 107 cfu=ml.

Eighty percent of the water was exchanged daily. After water exchange, 10 ml of

the cultured broth was taken, serially diluted and added to the post larval culture.

The extract obtained from the methanol extraction method was allowed to dry in

the shade. The dark green solid extracts obtained by this process were mixed with

ethanol (1 g ml�1). For enrichment at 400 mg l�1 (Citarasu 2000) the dissolved

herbal extract mixture was mixed with filtered sea water (30%) and heavy aeration

was performed to remove the effect of ethanol.

Table 1. Morphological and biochemical characteristics of the selected bacterial pathogens isolated

from the Penaeus monodon post larvae.

Tests Pathogens

P. aeruginosa Staph. aureus S. typhi Vibrio sp.

Colour of the colonies Pale yellow Black Black Yellow

Gram staining and

morphology

Green negative

rods

Gram positive

coccus

Gram negative

rods

Gram negative

rods

Motility Non motile Non motile Motile Motile

Baired parker agar � þ � �TSI agar slant � � þ �Catalase � þ � þOxidase þ � � þIndole red þ � � þMethyl red � þ � �Gelatin hydrolysis þ þ � þCoagulase test � þ � �Voges-pros kauer test � � � þ

(þ) Indicate positive response; (�) Indicate negative response.

585

Before enrichment, the 4-day-old Artemia was ‘gut defecated’ by keeping the

animals in a special defecater designed by Babu (1999). The defecated animals

were kept in the enrichment tank for 2 h to allow enrichment. The enrichment was

carried out at 30� 2 8C and 1000-lux illumination (Citarasu 2000). The enriched

Artemia and non-enriched Artemia were fed to the post larvae ad libitum. The food

density was adjusted four times per day. The debris, molted shell and dead post

larvae were removed during water exchange.

A detailed description of the treatments is given in Table 2.

Survival was assessed at the end of the experimental period of 25 days. The

weight (wet) gain was calculated by deducting the initial weight from final weight.

The specific growth rate was calculated by using the formula given below.

ðLnW2 � LnW1Þðt2 � t1Þ

� 100

where, Ln¼Logarithmic number, W2¼ Final weight at time t2, W1¼ Initial weight

at time t1.

After 25 days (termination of the experiment) 1 g of the P. monodon post larvae

were weighed aseptically and immersed in 50 ppm Formalin solution for 5 min (to

remove external bacteria present on the post larvae). Then they were washed

thoroughly under sterilized water for 30 sec to remove the remaining surface

bacteria and disinfectant. The washed samples were homogenized with 5 ml of 85%

sterile saline and diluted up to 10-fold. Sample of 0.5 ml was taken with the help of

sterilized pipette and poured into the appropriate specific agars such as Cetrimide,

Table 2. Detailed description of different treatments.

Treatments Inoculation Enrichment

1 No No

2 P. aeruginosa No

3 Staph. aureus No

4 S. typhi No

5 Vibrio sp. No

6 P. aeruginosa S. trilobatum

7 Staph. aureus S. trilobatum

8 S. typhi S. trilobatum

9 Vibrio sp. S. trilobatum

10 P. aeruginosa A. paniculata

11 Staph. aureus A. paniculata

12 S. typhi A. paniculata

13 Vibrio sp. A. paniculata

14 P. aeruginosa P. corylifolia

15 Staph. aureus P. corylifolia

16 S. typhi P. corylifolia

17 Vibrio sp. P. corylifolia

586

Manitol salt, Bismuth sulphide and TCBS agar. Triplicates were maintained in each

sample and incubated at 37 8C for 24 h.

The total bacterial count (cfu=g) was calculated as follows:

¼ Number of colonies � dilution factor

Weight of the sample ðmgÞ

The data obtained were analyzed statistically through the Students Newman Keuls

(SNK) test following Zar (1974).

Results

P. monodon post larvae reared in the non-inoculated seawater successfully com-

pleted the PL 25 stage with 90� 8.52% survival. When the bacterial inoculations of

P. aeruginosa, Staph. aureus, S.typhi and Vibrio sp. were given individually in the

culture water, survival decreased to 30� 3.7, 20� 3.5, 10� 1.85 and 25� 1.93%,

respectively. When three methanolic extracts of herbs were given through Artemia

to the post larvae, survival was increased for all types of bacterial inoculation. P.

corylifolia helped to increase maximum larval survival to of 72� 5.15, 60� 7.25

and 55� 3.78% in P. aerugionosa, Staph. aureus and S. typhi inoculations, re-

spectively. S. trilobatum helped to increase the survival to 52.5� 2.46% in the

Vibrio sp. inoculation (Tables 3–6).

A specific growth rate of 12.40% was observed when the post larvae were reared

in the non-inoculated and non-enriched treatment. The specific growth rate was

decreased to 9.14, 9.10, 8.86 and 7.54% in the P. aeruginosa, Vibrio sp., Staph.

aureus and S. typhi inoculated post larval cultures, respectively. The three anti-

bacterial herb extracts gave a significantly higher specific growth rate (P< 0.05) in

the non-enriched Artemia fed group compared to other treatments. The maximum

weight gain was of 41.44 mg when no bacterial inoculation was given. When

different pathogenic inoculations were given in the larviculture, the weight de-

creased to 18.25, 16.86, 15.51 and 11.75 mg for the P. aerugionosa, Vibrio, Staph.

aureus and S. typhi inoculations, respectively. When the different herb extract

enriched Artemia were fed the shrimp gained more weight. Among the four in-

oculations the A. paniculata enriched Artemia fed group achieved the highest gain.

In Vibrio inoculation, the A. paniculata fed groups had maximum of 201% weight

gain (Tables 3–6).

The effects of the three antibacterial herb extracts on the four pathogenic bac-

terial loads on P. monodon post larvae (PL 1 – 25) are given Table 7. The lowest

level (2.8� 103� 1.8� 102 cfu) of P. aeruginosa were isolated from the P. aeru-

ginosa inoculated post larval culture. For the other three pathogens inoculated from

the post larval culture, the load was 3.27� 105� 1.78� 104, 1.76� 105� 7.2� 103

and 3.76� 105� 6.73� 103 cfu=g, respectively. Methanolic extract of the herb

enriched Artemia fed groups were found to have reduced bacterial load in cultures

in which the post larvae were reared. In the P. aeruginosa inoculated post larval

587

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592

culture, the load was 1.3� 103� 1� 101, 6.0� 102� 1.34� 102 and 1.14� 103�1.34� 102 cfu=g post larvae in S. trilobatum, A. paniculata and P. corylifolia,

respectively in enriched Artemia fed group. Similarly the post larvae reared in the

Stap. aureus inoculation reflected the same degree of bacterial load reduction. The

load was 1.05� 105� 8.82� 103, 9.48� 104� 6.65� 103 and 8.76� 104� 6.9�102 cfu=g of post larvae for the above extracts, respectively. But for the S. typhi

inoculation the load was 1.15� 105� 7.82� 103, 9.2� 104� 5� 102 and 8.52�104� 3.63� 103 cfu=g of post larvae in S. trilobatum, A. paniculata and P. cor-

ylifolia, respectively. P. monodon could tolerate the Vibrio sp. population to the tune

of 1.46� 105� 6.5� 103 cfu=g. A further increase in the inoculation reduced

survival to 25%. If the herbal extract helped to increase the survival more than 45%,

the bacterial load also decreased to 1.27� 105� 8.5� 103–1.12� 105� 5.9�103 cfu=g. Among the three antibacterial methanolic extracts, A. paniculata helped

to reduce the bacterial load to a minimum level.

Discussion

Some bacterial species have been reported to be a source of infections and cause

high mortalities in fish larvae (Muroga et al. 1987; Nicolas et al. 1989). Improved

disease control may be obtained by oral delivery of the drug to the predator larva. In

this respect it has recently been demonstrated that an excellent tool to incorporate

drugs as well as vaccines is by bioencapsulation via Brachionus and Artemia to the

infected larvae (Verpraet et al. 1992). High doses of more than 290 mg=g dry

weights of Trimethoprim and Sulfamethoxazole can be bioencapsulated in Artemia

and incorporated into sea bass fry. Upon administration of these enriched Artemia

nauplii high therapeutic levels in the fish tissue of 22.6 mg=g dry weights were

reached within 3 h of feeding (Chair et al. 1991). In the present study, P. monodon

post larvae (PL 1–25) reared in the bacteria inoculated water (P. aeruginosa,

Staph. aureus, S. typhi and Virbio sp.) showed a poor survival (10–30%), a low

specific growth rate (7.54–9.14%) and an increased bacterial load. The methanolic

herbal extracts (S. trilobatum, A. paniculata and P. corylifolia) helped to increase

the survival and growth and reduced bacterial load. Among the three treatments,

post larvae fed with P. corylifolia enriched Artemia showed better growth perfor-

mance and a lower bacterial load. The overall performance of A. paniculata was

found to be more effective when compared to the others. Among the four cultures

inoculated with pathogen, the P. aeruginosa load was reduced to the lowest level

(6.0� 102 cfu=g in the A. paniculata enriched Artemia fed group). Also A. pani-

culata extract reduced the S. typhi load to 8.52� 104 cfu=g. The lowest level of

Vibrio sp. load was found (1.12� 105 cfu=g) in the P. corylifolia enriched Artemia

fed group.

Similar findings were reported in the Atlantic salmon (Salmo salar L.) fed with

the immunostimulant algibind wet feed and challenged with Aeromonas salmo-

nonicida. The Algibind wet feed fed group showed improved survival and growth

(Nordmo and Holth 1995). The results obtained from the present study showed that

593

the larvae fed with the enriched herbal medicine had higher survival, growth and a

lower bacterial load. The herbal extract fed to the post larvae might have improved

the immunostimulant effects in their body by enhancing the microbicidal activity of

hemolymph and pagocytosis of cells. The dietary incorporation of beta-1, 3-glucan

from Schizophyllum communae enhanced the resistance of post larvae, juvenile and

adult P. monodon to white spot syndrome virus (Chang et al. 1999). Also Song and

Sung (1993) found out that wheat germ agglutinin (WGA), a lectin administrated as

feed additive promoted bacterial resistance of Penaeus orientalis.

Conclusions

1. Post larvae reared in the non-inoculated water had the highest survival (90%),

weight gain, specific growth and a lower body bacterial load.

2. When different bacterial inoculations were given in the culture without the

application of herbal products, the survival, weight gain and specific growth

were poor and the bacterial load increased nearly three times.

3. When antibacterial herbal extracts were fed to the P. monodon post larvae

(reared in the different bacterial inoculation) through Artemia, the survival and

weight gain were increased more than one-fold and the extracts also helped to

reduce the bacterial load by a factor of two.

4. Among the three herbal extracts, A. paniculata performed well in the control of

bacterial load in the various bacterial pathogen inoculated P. monodon post

larval culture media.

Acknowledgement

One of the authors Dr. M. Michael Babu gratefully acknowledges the International

foundation for Sciences (IFS, Sweden), for the research funding, under the research

grant No-F=3291-1.

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