Introduction

Aquaculture has become an economic activity of great importance as a result of overfishing of wild populations and ecological degradation (Ariole and Eddo, 2015). Shrimp farming is an aquaculture business which occurs in either a marine or freshwater environment in order to produce shrimps or prawns to satisfy consumer demand which ordinary wild fishing alone could not meet. Many wild penaeid and non-penaeid shrimp species such as white shrimp (Litopenaeus vannamei), brown shrimp (Macrobrachium rosenbergii), fairy shrimp (Branchinella thailandensis), tiger shrimp (Penaeus monodon) and Streptocephalus sirindhornae have been cultivated in aquaculture (Farzanfar, 2006; Suantika et al., 2013). Shrimp farming can generate quick gains and meet loan conditions for farmers (Alvandi et al., 2004).

Vibriosis syndrome is one main problem of shrimp culture. Shrimp exposure to Vibrio sp. can be through different routes, such as injury, feed, gills, and mouth, where the bacteria cross the shrimp epithelium and colonize the host tissues (Martin et al., 2004). Vibrios cause several diseases which results in high mortality of cultured shrimp (Bondad-Reantaso et al., 2005). The application of effective probiotics in aquaculture is an excellent alternative for antibiotics in disease management due to antibiotic resistance and its epidemiological effects (Sahul Hameed et al., 2003). It is, therefore, imperative to develop microbial control strategies that will be eco-friendly and sustainable in aquaculture.

Information on aquatic indigenous probiotics in Nigeria is scarce. Therefore, the aim of the present work was to evaluate the effectiveness of antagonistic Pseudomonas aeruginosa previously isolated from healthy shrimp intestine (Ariole and Aungwa, 2013) as biocontrol agent against Vibrio infection in shrimp (Penaeus monodon) culture.

1 Results

1.1 Molecular identification

Molecular analysis conducted on Pseudomonas sp. Ps2 and Vibrio sp. V1 revealed that they are closely related to Pseudomonas aeruginosa strain XyC5 gi: 379070233 (Figure 1) and Vibrio mimicus strain XQ gi: 648833882 (Figure 2) respectively.

1.2 Pathogenicity tests

The antagonistic Pseudomonas aeruginosa was safe to Penaeus monodon postlarvae when compared with the control that showed mortality of 14% (Table 1). The shrimp became weak and sluggish after exposure to Vibrio mimicus and finally, mortality of the shrimp was observed. Other clinical signs of black disease such as lethargy, skin ulcers, swimming abnormality, and lack of appetite were very obvious. The thoracic appendage and exoskeleton showed the presence of black nodules. The LC₅₀ of Vibrio mimicus for Penaeus monodon postlarvae was estimated to be 3.2x10⁴ cfu/ml. Mortality of 6% was recorded among shrimp in control group (Table 2).

1.3 Histopathological test

There was no degeneration of the epithelial tissues and tubule lumen of the hepathopancreas (HP) of the shrimp from control group (Figure 3) as well as shrimp from the group inoculated with Pseudomonas aeruginosa (Figure 4). However, the shrimp infected with Vibrio mimicus showed an abnormal hepatopancreas. The epithelial tissues and the tubule lumen were degenerated. The nuclei were also disrupted (Figure 5).

1.4 Challenge test

No mortality was recorded in treatment group 1 (shrimp culture inoculated with Pseudomonas aeruginosa and challenged with Vibrio mimicus). The control group 2 with no addition of bacteria had 22% mortality while the control group 3 infected with Vibrio mimicus recorded the highest mortality of 54% (Table 3). Statistical analyses performed on the results of the water quality parameters measured during the culture period showed that the value of each parameter in each treatment group was not significantly different. Moreover, the water quality parameters were within the optimum ranges throughout the culture period (Table 4).

2 Discussion

In the present study, molecular analysis carried out on the antagonistic Pseudomonas sp. Ps2 and pathogenic Vibrio sp. V1 previously isolated from healthy and moribund shrimp (Penaeus monodon) intestines (Ariole and Aungwa, 2013) respectively, revealed that they are closely related to Pseudomonas aeruginosa (Figure 1) and Vibrio mimicus (Figure 2) respectively. Pseudomonas and Vibrio are common dwellers of aquatic habitat including ponds for shrimp culture and have been isolated by other workers (Vijayan et al., 2006; Chau et al., 2011). The inhibition of Vibrio mimicus by Pseudomonas aeruginosa may be as a result of its production of antagonistic substances such as pyocyanin and siderophores (Vijayan et al., 2006). Hadi et al. (2013) also reported strong antibacterial capabilities of four strains of Pseudomonas spp. against three shrimp pathogens V. cholerae, V. parahaemolyticus and V. alginolyticus.

The safety of Pseudomonas aeruginosa (Table 1) and the virulence of Vibrio mimicus (Table 2) to Penaeus monodon postlarvae as well as the probiotic potential of Pseudomonas aeruginosa (Table 3) were established in this study. This affirms that Pseudomonas aeruginosa strain XyC5 is a beneficial bacterium and has no pathogenic effects on shrimp. The safety of Pseudomonas sp. to fish and shellfish has been reported (Vaschuere et al., 2000; Chau et al., 2011; Nour and El-Ghiet, 2011; Hadi et al., 2013). The high mortality of the test shrimp during the four-day pathogenicity test of Vibrio mimicus strain XQ could be as a result of the establishment of vibriosis, a popular disease that affects aquatic animals. This is indicative of high virulence of Vibrio species as has been reported by several authors (Chen et al., 2000; Alday-Sanz et al., 2002; Martin et al., 2004; Lavilla-Pitogo, 2013; Hadi et al., 2013;). The LC₅₀ which was the concentration able to kill fifty percent of the test organisms was found to be 3.2x10⁴ cfu/ml revealing Vibrio mimicus as a shrimp pathogen. Vijayan et al. (2006) also reported similar findings on the pathogenicity of V. parahaemolyticus a common shrimp pathogen of penaeid and non-penaeid rearing systems. Furthermore, Alday-Sanz et al. (2002) reported how P. monodon larvae suffered mortalities within 48 hr of immersion challenge with strains of V. harveyi and V. splendidus.

Mortalities due to vibriosis occur when shrimps are stressed by factors such as: poor water quality, crowding, high water temperature, low DO (dissolved oxygen) and poor water exchange (Martin et al., 2004). However, the water quality parameters were within the optimum ranges throughout the culture period (Table 4). The stability of the shrimp culture water quality may be attributed to the buffering capacity of the bicarbonate incorporated at the bottom of each tank as culture substrate. A similar result was reported by Suantika et al. (2013).

Information on the health status and pathogenicity of cultural fish and shellfish are usually provided through histopathological analysis which helps to provide details on their internal organs. The use of hepatopancreas as the main tissue of target during any infection and an index to check the stress induced conditions and health status of experimental shrimp is well documented (Pazir et al., 2012). The histopathological result of the experimental group treated with Pseudomonas aeruginosa revealed normal vacuoles and tubule epithelial cells (Figure 4). This result showed that the shrimp internal organs were not affected by the probiotic candidate. Contrary to this, the destruction of the hepatopancreas tissue (Figure 5) of the group challenged with Vibrio mimicus may be as a result of the established disease which affected the shrimp and led to their mortalities. This is related to a work done by Pazir et al. (2012) on both P. monodon and P.vannamei of which they found out Early Mortality Syndrome (EMS) caused by Vibrio parahaemolyticus. Another histopathological result comparable to this was that reported by Prachumwat et al. (2012). They found out that epithelial cells of the HP tubules were disfunctioned. This destructive pathology strongly implies a lethal/toxic etiology and also suggests that the pattern of disease spread was related to the infectious agent. These results indicate that the indigenous non-pathogenic and antagonistic Pseudomonas aeruginosa strain XyC5 could be beneficial as biological control agent for management of vibriosis in shrimp aquaculture.

3 Materials and Methods

3.1 Collection and acclimatization of shrimp (Penaeus monodon) postlarvae

Healthy live shrimp (Penaeus monodon) were collected from Sombriero River in Tombia, Rivers State of Nigeria, with the assistance of local fishermen. They were carefully transported to the laboratory in aerated tanks containing habitat water. The shrimp were acclimatized at room temperature for two days in aerated tanks containing habitat water. The average length and weight of the shrimp used were 5.1 ± 0.1 cm and 1.5 ± 0.1 g respectively.

3.2 Molecular characterization of Pseudomonas sp. Ps2 and Vibrio sp. V1

Chromosomal DNA extraction was performed with Zymo Research Bacterial DNA MiniPrep Kit. The DNA samples were quantified using NanoDrop ND-2000 Spectrophotometer. The 16S rRNA region of the rRNA genes of the isolates was amplified using the 27F:5‟AGAGTTTGATCMTGGCTCAG3‟and1492R:5‟TACGGYTACCTTGTTACGACTT 3‟ primers on an ABI 9700 Applied Biosystems thermal cycler at a final volume of 50 microliters for 35 cycles. The PCR mix included: the X2 Dream taq Master mix supplied by Inqaba, South Africa (taq polymerase, DNTPs, MgCl), the primers at a concentration of 0.4 M and the extracted DNA as template. The PCR conditions were as follows: Initial denaturation of DNA template strands at 95ºC for 5 minutes; subsequent denaturation at 95ºC for 30 seconds; annealing of primers at 52ºC for 30 seconds; elongation at 72ºC for 30 seconds for 35 cycles and final elongation at 72ºC for 5 minutes. The product was resolved on a 1% Agarose gel at 120V for 15 minutes and visualized on a UV Trans illuminator. The amplified 16S products were sequenced on a 3 500 genetic analyser using the Bigdye-Termination technique by Inqaba South Africa. The sequences were edited using the bioinformatics algorithm Bioedit. Similar sequences were downloaded from the National Biotechnology Information Center (NCBI) data base using Blast-N, and these sequences were aligned using ClustalX. Neighbor-Joining mechanism was used to infer the history of evolution in MEGA 6.0 and Jukes-Cantor method was used to compute the distances in evolution.

3.3 Pathogenicity tests of Pseudomonas aeruginosa and Vibrio mimicus to shrimp culture

A stock concentration of Pseudomonas aeruginosa suspension grown on cetrimide agar for 24 hours and that of Vibrio mimicus suspension grown on TCBS agar for 24 hours were prepared by diluting each bacterial suspension and adjusting to McFarland No.1 standard turbidity (equivalent to 3 x 10⁸ cfu/ml). Ten-fold serial dilutions with normal saline were carried out to obtain 3x10⁷, 3x10⁶, 3x10⁵, 3x10⁴ and 3x10³ cfu/ml. Different tanks of 4 L capacity, each containing 2 L habitat water buffered using sterile bicarbonate powder and constantly aerated were set up. Fifty (50) healthy shrimp were stocked into each of the tanks.  Then 0.1 ml of each suspension was introduced into each tank. Shrimp grown in habitat water alone served as control. The experiment was carried out in triplicate. The shrimp mortalities, if any, were monitored and recorded daily for 4 days. The mortality rate of the animal was calculated for each treatment. Probit Analysis Package (SPSS version 20) was used to determine LC₅₀ of Vibrio mimicus.

3.4 Histopathological examination of the hepatopancreas of experimental shrimp

The five-step procedure (fixation, dehydration and clearing, embedding, sectioning, and staining and mounting) for histological study of the test shrimp after pathogenicity test was carried out according to the method described by Wiss et al. (2010). The sections were viewed and the micrographs of the tissues of the test animal were compared with the control.

3.5 Probiotic application and infection of shrimp culture by immersion method

Three experimental groups were set up using 4 L tanks each containing 2 L of habitat water. The tanks were buffered using sterile bicarbonate powder and constantly aerated. Fifty (50) healthy shrimp were stocked into each of the tanks. To shrimp culture in treatment group 1, 0.1 ml 3x10⁵ cfu/ml fresh culture of Pseudomonas aeruginosa was added and challenged with 0.1 ml 3x10⁵ cfu/ml fresh cultures of pathogenic Vibrio mimicus on the third day. There was no addition of bacteria in treatment group 2 shrimp culture (control). Treatment group 3 was infected with 0.1 ml 3x10⁵ cfu/ml fresh culture of Vibrio mimicus. Mortalities were recorded daily for 12 days. The experiment was conducted in three replicates. Water quality parameters (pH, temperature, ammonia, nitrite, nitrate, salinity and dissolved oxygen) were monitored during the culture period.

3.6 Determination of water quality

Water quality parameters such as pH, temperature, dissolved oxygen (DO) and salinity were measured daily, while the concentrations of nitrate, nitrite and ammonia were determined at two days interval. Water samples were collected in plastic bottles and examined according to the methods described in APHA (2005).

3.7 Statistical analysis

One way ANOVA was used to analyze the differences in shrimp mortality rates and Duncan’s multiple range test was used to test significant differences among means (p ˂ 0.05). Two Sample t-test was used to determine differences (p ˂ 0.05) between water quality results, while LC₅₀ was calculated using Probit Analysis. All statistics were performed with SPSS for Windows, version 20.

Authors’ contributions

CNA conceived, designed and supervised the research work. NGA contributed during design, sample collection, laboratory experiments and data analysis. NGA also wrote the first draft while CNA wrote the final manuscript. All the authors read and approved the final manuscript.

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