Abstract: This study was conducted for 4 weeks to determine the effects of different concentrations probiotic Bacillus sp., on angelfish growth, larvae survival rate and tolerance against environmental stressor (high temperature). This experiment was carried out in a completely random design. Artemia urmiana naupli was used as a vector to carry probiotic bacillus to digestive tract of Pterophyllum scalare larvae. Nauplii with three concentrations of bacteria, 1x10⁷, 2x10⁷ and 3x10⁷ bacteria mL^-1 in suspension of broth for 10 h were bioencapsulated and angelfish larvae were fed by them. Angelfish larvae were fed 4 times a day. The control treatment was fed by unbioencapsulated Artemia nauplii. At the end of experiment, half of larvae in each treatment were challenged by high temperature (36°C) and their survival was calculated. The results indicated that the probiotic bacillus could influence on growth, survival rate and viability against high temperature stress in angelfish larvae. The final body length in experimental treatments had significant difference in comparison to control (p<0.05). In experimental treatments the survival rate did not show the significant difference (p>0.05) in comparison with control. Nevertheless, viability against high temperature stress in T1 (bioencapsulated Artemia with 1x10⁷ cfu mL^-1) was increased significantly (p<0.05) in comparison with control. The result indicate that different concentrations of bacteria could influence on growth, larvae survival rate and tolerance against environmental stressor (high temperature) in angelfish larvae and the findings can be useful in the performance of larviculture of this species.

INTRODUCTION

The word probiotic is constructed from the Latin word pro (for) and the Greek word bios (life). The definition of a probiotic differs greatly depending on the source but the 1st generally, accepted definition was proposed by Fuller (1989) as a live microbial feed supplement which beneficially affects the host animal by improving its microbial balance. Animal gut microflora consists of hundreds of different bacterial strains (Walker and Duffy, 1998) able to promote digestion and absorption of nutrients to increase body resistance to infectious diseases (Tannock, 1988) to yield positive affects on growth and to improve general animal welfare (FAO/WHO, 2001). FAO has now designated the use of probiotics as a major means for the improvement of aquatic environmental quality (Subasinghe et al., 2003). In the last decade, the scientific community carefully examined roles and effects of probiotics in aquaculture as an alternative to antimicrobial drugs, demonstrating positive effects on fish survival (Villamil et al., 2002), growth (Burr et al., 2005), stress resistance (Smith and Davey, 1993; Rollo et al., 2006), immunosystem enhancement (Erickson and Hubbard, 2000; Picchietti et al., 2007) and finally general welfare (Silvi et al., 2008). Most studies on the effects of probiotics on cultured aquatic animals have emphasized a reduction in mortality or the improved resistance against putative pathogens (Irianto and Austin, 2002). However, the beneficial effects are sometimes temporal, depending on the time of exposure (Verschuere et al., 2000). As most fish contain a specific intestinal microbiota established at the juvenile stage (Olafsen, 2001), the colonization of probiotics to fish intestines requires adequate probiotics presented in ambient Microbial Community (MC) and their interaction with MC should not be neglected. In aquaculture, captive rearing conditions generally can be sources of stress, triggering high mortality, mainly during larval rearing. The use of natural prophylactic supplements in place of chemotherapeutics in aquaculture has received a great deal of attention in the past decade such preventive products include probiotics. These biotics can be applied through external bathing or dietary supplementation and have been demonstrated to improve growth performance, feed utilization, digestibility of dietary ingredients, disease resistance and stimulate the immune response of aquatic animals (Gatesoupe, 2008; Kesarcodi-Watson et al., 2008; Wang et al., 2008; Merrifield et al., 2010). Aquatic probiotics have been defined as live microbial supplements that can modulate microbial communities and improve microbial balance, thus providing benefits to the host (Gram et al., 1999). Furthermore, probiotic Bacillus species have been shown to improve digestive enzyme activities, growth and survival of crustaceans (Liu et al., 2009; Rengpipat et al., 1998, 2003; Wang, 2007; Zhou et al., 2009). The beneficial effects of these probiotics include higher growth and feed efficiency, prevention of intestinal disorders and pre-digestion of anti-nutritional factors present in the ingredients. Also, existing literature on probiotics usually focused on resistance to some aquatic pathogens such as Vibrio sp. (Villamil et al., 2003; Planas et al., 2006), Amyloodinium ocellatum (Li et al., 2005) and Carnobacterium sp. These studies mainly focused on effects of probiotics on enhancement of survival and nutritional parameters such as feed efficiency and feed conversion ratio. Bacillus can act positively on cultured organisms by enhancing survival and growth (Gomez-Gil et al., 2000) by stimulating the digestive (Ziaei-Nejad et al., 2006) and immune systems (Gatesoupe, 1999) and by improving water quality in terms of bioremediation (Kennedy et al., 1998; Moriarty, 1998). Several studies demonstrated these positive effects using a single or two probiotic strains and just few studies described the effects of a mixture of probiotics in fish and shrimp aquaculture (Balcazar, 2003; Lara-Flores et al., 2003; Ziaei-Nejad et al., 2006). Concurrently, Bacillus species can be found in marine environment and are part of the microflora of several marine species (Kennedy et al., 1998; Hovda et al., 2007). little studies had been carried out to incorporate probiotics into a freahwater species common carp, C. carpio (Yanbo and Zirong, 2006) and crustaceans, Indian white shrimp Fenneropenaeus indicus (Ziaei-Nejad et al., 2006) and shrimp Penaeus vannamei (Wang, 2007) based on growth performances and digestive enzyme activities. As stated above, the application of probiotics in aquaculture as the environment friendly treatments has been also increasing rapidly (Gatesoupe, 1999, 2002, 2007) and some papers were associated with the effect of probiotics in fish and other marine organisms (Mohanty et al., 1993; Sharma and Bhukhar, 2000). Probiotics is usually defined as live microbial feed supplements that are administered in such a way as to enter the gastrointestinal tract and to be kept alive; this beneficially affects the host animal by improving its intestinal microbial balance and in turn its health (Gatesoupe, 1999, 2007). As described by several researchers, probiotics in aquaculture have been demonstrated to have several modes of action: competitive exclusion of pathogenic bacteria through the production of inhibitory compounds; improvement of water quality; enhancement of immune response of host species and enhancement of nutrition of host species through the production of supplemental digestive enzymes (Thompson et al., 1999; Verschuere et al., 2000). The brine shrimp Artemia sp. are common live food organisms used for the rearing of marine fish larvae. These have been considered as possible vectors for the delivery of different substances such as nutrients and probiotics (Gatesoupe, 1991). This positive effect of probiotics may be attributed to their ability to outcompete other bacteria (Austin et al., 1995) or to produce micronutrients important for the development of fish larvae (Ringo et al., 1992). Several bacteria have been used as probiotics in the larval culture of aquatic organisms and they can be either delivered directly into the water or via live carrier such as Artemia nauplii and rotifers or else added to pelleted dry food (Gomez-Gil et al., 2000). The aim of this study was to evaluate the effects of probiotic bacillus on the growth, larvae survival rate and tolerance against environmental stressor (high temperature) in angelfish larvae.

MATERIALS AND METHODS

Preparing of probiotic bacillus: The probiotic bacillus was prepared from Protexin Co. (Iran-Nikotak). The five species of probiotic bacillus as bacterial blend under the commercial title of Protexin aquatic were used for bioencapsulation of A. urmiana. The blends of probiotic bacillii (Bacillus licheniformis, Bacillus subtilis, Bacillus polymixa, Bacillus laterosporus and Bacillus circulans) from suspension of spores with special media were provided. Three concentrations of bacterial suspension, 1x10⁷, 2x10⁷ and 3x10⁷ bacteries mL^-1 (cfu mL^-1) were provided by Protexin Co. and the Colony Forming Unit (CFU) of probiotic bacillii were tested by microbial culture in Tryptic Soy Agar (TSA) (Rengpipat et al., 1998).

Artemia cyst hatching and bioencapsulation: The cysts of A. urmiana from the center of Artemia and Aquatic Animals in Urmia (Iran) were used for this study. The corions of the cysts were removed chemically by using the methodology that proposed by Sorgeloos et al. (1977). This process is known as decapsulation. Hatching of the decapsulated cysts was performed in glass cone with 1 L of seawater (3.0% salinity) at a density of 5.0 g L^-1 and incubated at 30°C with constant illumination and aeration through setting air pump (Gomez-Gil et al., 1998). The bioencapsulation of Artemia nauplii were accomplished with density of 2 g live nauplii L^-1 (Makridis et al., 2001) for 10 h and with three concentration of 1x10⁷ (T1), 2x10⁷ (T2) and 3x10⁷ (T3) bacteries mL^-1 in suspension of broth.

Experimental design: The experiments were conducted from June to August 2010 in Institute of Ornamental Fish Hatchery in Babol, Iran. After yolk sac absorption, larvae were divided to twelve glass aquaria (80x30x40 cm) with three replicates for experimental and control treatments. This experiment was conducted in a completely randomized design with four treatments (treatment 1-3 and control).

The density of fish larvae in per tank were 10 fish L^-1. Angelfish larvae in control and experimental treatments were fed 4 times a day (8.00, 12.00, 16.00 and 20.00). The control treatment was fed unbioencapsulated Artemia nauplii. Gentle aeration was provided by air stones. During the experiment, the water quality parameters were monitored during the trial and average value for temperature, dissolved oxygen, hydrogen ion concentration (pH) and salinity were 26±2°C, 5.7-7.7 mg L^-1, 6.9-7.8 units and 0.1 mg L^-1, respectively. Dark cycle of 12:12 h was maintained during the experiment. Angelfish larvae were reared for 30 days. Fish from each aquarium were counted and their length was measured to monitor growth and mortalities were recorded.

High temperature challenge: At the end of experiment for evaluation of the larval quality, half of the larvae (in each replicate) were challenged by high temperature. In this propose, larvae were transferred to other aquariums and temperature was increased to 36°C (10°C higher than cultivation temperature) and survival duration was calculated.

Calculations and statistical analysis: The following variable was calculated (Ai et al., 2006):

N_t and N₀ were final and initial numbers of larvae in each replicate, respectively and t is the experimental period in days. Results are presented as means±SD. Significant differences among treatments were determined by Analysis of Variance (ANOVA) and the differences between means were tested with Duncan’s multiple-range test using SPSS 16.0 programme. Differences were considered significant at p<0.05.

RESULTS AND DISCUSSION

Effect of probiotic bacillus on growth and viability: Among the three different concentration of probiotic bacillus in bioencapsulation of Artemia nauplii which was fed by angelfish larvae, the highest results obtained in T1 (bioencapsulated Artemia with 1x10⁷ cfu mL^-1). As shown in the Fig. 1, growth of angelfish larvae was significantly (p<0.05) affected by probiotic bacillus. In the experimental treatments, growth was significantly (p<0.05) higher than control treatment. The highest average of length was obtained in the experimental treatment of T1 however, there was no significant difference among experimental treatments (p>0.05). The survival rate of angelfish larvae at the end of experiment is showed in Fig. 2. The significant difference was not observed in experimental treatments in comparison with control (p>0.05).

Effect of probiotic bacillus on larval tolerance: As shown in the Fig. 3, differences in larval tolerance against high temperature stress (36°C) were observed among experimental groups and they were significant among them. The highest and lowest times of survival in 36°C were observed in T1 and T3, respectively.

Fig. 1:	Growth rate in experimental groups after 10 and 20 days

Fig. 2:	Survival rate (%) of larvae in day 20th

Fig. 3:	Larval viability against high temperature stress

The current study demonstrates different concentrations of bacteria for enrichment Artemia with commercial Bacillus sp. was significantly difference in growth between experimental treatments in comparison with control. Similar findings have previously been documented in preliminary trials on larval H. gammarus (Daniels et al., 2006). Also, this result indicates that different concentrations of bacteria not differed significantly in survival. Since, the 1st use of probiotics in aquaculture, a growing number of studies have demonstrated their ability to control potential pathogens and to increase the growth rates and welfare of farmed aquatic animals (Gatesoupe, 1991; Lara-Flores et al., 2003; Carnevali et al., 2004; Macey and Coyne, 2005; Wang et al., 2005; Yanbo and Zirong, 2006). Here, we report for the 1st time, an enhancement of the growth rate of the Pterophyllum scalare, one of the most important ornamental species in the Iran. The positive effect of Bacillus were observed by Gatesoupe (1991) in using Bacillus toyoi on turbot (Scophthalmus maximus Linnaeus, 1758), Swain et al. (1996) in Indian carps that improved the growth factors and feeding performance and Ghosh et al. (2003) on the Rohu. Noh et al. (1994) and Bogut et al. (1998) showed that a commercial probiotic preparation of Streptococcus faecium improved the growth and feed efficiency of carp (Cyprinus carpio).

Bagheri et al. (2008) found that supplementation of trout starter diet with the proper density of commercial Bacillus probiotic could be beneficial for growth and survival of rainbow trout fry. Ghosh et al. (2002) indicated that the B. circulans, B. subtilis and Bacillus pamilus, isolated from the gut of Rohu have extracellular protease, amylase and cellulose and play an important role in the nutrition of Rohu fingerlings. The photosynthetic bacteria and Bacillus sp. (isolated from the pond of common carp) was used in diet of common carp (Cyprinus carpio Linnaeus, 1758) by Yanbo and Zirong (2006). It is important to consider the possibility of using different species as suggested by Noh et al. (1994) and Bogut et al. (1998). Here, we studied the effects of a combination of Bacillus sp. (Bacillus licheniformis, Bacillus subtilis, Bacillus polymixa, Bacillus laterosporus and Bacillus circulans) at different concentrations of enrichment on growth and survival in angelfish larvae. The results indicated that always growth and survival rate do not increase with increasing concentrations of probiotics, similar results were reported by Jafaryan et al. (2010).

Cavalli et al. (2003) evaluated the effect of dietary supplementation of vitamins C and E on maternal performance and larval quality of the prawn Macrobrachium rosenbergii. They tested the tolerance of newly hatched and 8 days old larvae of M. rosenbergii to ammonia exposure. Their results shown newly hatched and 8 days old larvae tolerance tended to increase with increasing levels of AA and higher dietary levels of α-tocopherol acetate did not affect the tolerance to ammonia of newly hatched larvae but it positively augmented the ammonia tolerance of 8 days old larvae.

The study documented that larval survival duration against high temperature stress was promoted in T1 and T2 but resistance against stress in T3 was similar to control group. As a result larvae tolerance tended to decrease with increasing levels of probiotic.

CONCLUSION

In this study, this experiment indicated that the probiotic bacillus have the highest ability to promote the growth in Pterophyllum scalare larvae. Different concentrations of probiotic bacilluse had the same effects on the growth in angelfish larvae but the best survival duration against environmental steressor (high temperature) was obtained in T1 (bioencapsulated Artemia with 1x10⁷ cfu mL^-1). In general, the findings can be useful in the performance of larviculture of this species.