Research Article

Total Bacterial Load and Enumeration of Pathogenic Bacteria vibrio spp., Aeromonas spp. and Pseudomonas spp. in the Production Line of Mud Crab (Scylla olivacea) Hatchery in Bangladesh

Md. Rashedul Islam1, Abul Farah Md. Hasanuzzaman1*, Md. Latiful Islam2, Ghausiatur Reza Banu1

1Khulna University, Bangladesh; 2Bangladesh Fisheries Research Institute (BFRI), Bangladesh.

Received | March 23, 2023; Accepted | April 20, 2023; Published | May 06, 2023

*Correspondence | Abul Farah Md Hasanuzzaman, Khulna University, Bangladesh; Email: [email protected]

Citation | Islam MR, M Hasanuzzaman AF, Islam ML, Banu GR (2023). Total bacterial load and enumeration of pathogenic bacteria vibrio spp., Aeromonas spp. and pseudomonas spp. In the production line of mud crab (scylla olivacea) hatchery in bangladesh. Adv. Anim. Vet. Sci. 11(6): 968-976.

DOI | http://dx.doi.org/10.17582/journal.aavs/2023/11.6.968.976

ISSN (Online) | 2307-8316

 

 

Copyright: 2023 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

The global demand for aquaculture products including mud crab causes rapid commercial exploitation of these species in recent years, and is projected to increase in the future. (Hungria et al., 2017). The increasing demand and high price of mud crab in the domestic and international market have motivated farmers to expand mud crab aquaculture utilizing locally available seeds (Azra and Ikhwanuddin, 2016). But, the sustainability of mud crab aquaculture is at risk because the crab farmers still rely mostly on the wild-caught crabs for seed stocks, fattening and soft-shelled crab production; thus rapid expansion and large-scale aquaculture of mud crabs is still inconvenient due to the small success rate of crablet production in the hatchery systems (Waiho et al., 2018). In Bangladesh, mud crab aquaculture is being rapidly intensified in the climatically vulnerable coastal region as an alternative livelihood for the coastal fisher folks (Hasanuzzaman et al., 2014). Due to high mortality of larvae in the hatchery, farmers depend on wild seeds and harvest mud crabs from the natural sources, which creates immense pressure on the wild stocks.

Disease is a key problem in aquaculture, regardless of the type of production and/or the species targeted. For instance, major obstacles to larval production in hatcheries include disease caused by opportunistic pathogens, such as vibrios (Sui et al., 2011; Zhang et al., 2014), a lack of knowledge of suitable diets (Basford et al., 2021) and cannibalistic behavior of some crab species (Romano and Zeng, 2017). Crustacean larvae are very much susceptible to disease during their initial developmental stages, where survivability, normal growth and development require specific biotic and abiotic conditions (Quinitio et al., 2001). Mud crab-producing countries across the world are working on various aspects of mud crab larviculture and larval rearing technology, which are being reported year round. Nevertheless, survival of mud carb larvae in commercial hatcheries is till now at unsatisfactory level; in most hatcheries, mass mortality occurs during transformation from zoea to crablet stage due to bacterial and fungal infection (Dan and Hamasaki, 2015). Molt death syndrome during the transition from zoea 5 to megalopa stage is also considered a major obstacle in the commercial production of crab larvae in hatchery systems (Hamasaki et al., 2002; Pates et al., 2017). A number of opportunistic pathogens includes, Vibrio spp., Aeromonas spp. and Pseudomonas spp. infect the zoeal stages of mud crabs and cause mass mortality during the production cycle (Gopal et al., 2005).

The disease problem is particularly severe in hatcheries, and in the past years many shrimp hatchery units were shut down due to invasion by pathogens where mostly by vibrios (Quinitio and Prado-Estepa, 2008). Frequent occurrence of disease has restricted the crab larval production in hatcheries, and this together with lack of suitable feeds are the prime hurdles in the sustainable development of the crab aquaculture sector (Coates and Rowley, 2022). In Bangladesh, two mud crab hatcheries (one government-owned and another private one) have been operated; the both hatcheries had too low survival percentage of larvae to run the hatcheries on a commercial scale. No specific research has been made on bacterial contamination in relation to such larval mortality in these hatcheries. Given with the scarcity of scientific information on the pathogenic bacterial population in crab hatcheries of Bangladesh, the current study is the first to attempt to determine the prevalence of Vibrio spp., Aeromonas spp., and Pseudomonas spp. in the mud crab hatchery production line. The documentation of scientific information on the prevalence of pathogenic bacteria and total bacterial load would be supportive for quality health management of the mud crab hatcheries.

MATERIALS AND METHODS

Sample collection

Desired experimental samples were collected from the crab hatchery of “Bangladesh Fisheries Research Institute (BFRI)”, Brackishwater Station, located at Paikgacha of the Khulna district. The samples were collected from four different cycles which were designated as batch 1, batch 2, batch 3 and batch 4 respectively. All the samples were collected from March 2020 to February 2022. From every single cycle berried crab, eggs, larvae, tank water, brood feed, larval feed and crablet samples were taken properly using sterile tools and apparatus. Laboratory analyses have been performed at “Fish and Shellfish Quality Control and Pathology Laboratory” under Fisheries Discipline of Khulna University, Bangladesh.

Berried brood crabs weighing between 200 to 315 g with carapace length (CL) 7.4-8.0 cm and carapace width (CW) 10.2-10.8 cm were taken for analysis. Egg samples from spawned crabs were collected aseptically in small sterile falcon tubes. Tank water was taken direct inserting sterile falcon tubes inside the tank. To collect larval samples, harvested water was filtered through micro mesh sieves and the larvae were transferred in the sterile falcon tubes containing sterile saline water. Rotifer and Artemia used as live food for mud crab larvae were cultured inside the hatchery unit; rotifer and Artemia culture samples were taken in the falcon tubes. All the samples were immediately stored in an ice box and shipped to the pathology laboratory as earliest possible where the samples were stored at -20oC for further analysis.

Isolation and enumeration of bacteria

The samples were analyzed following the guideline of the Bacteriological Analytical Manual (Elliot et al., 1995) and Technical Specification (ISO, 2007). Brood crabs were dissected aseptically, then pooled samples using gill, gut, hepatopancreas and abdominal muscle samples were used. Brood, larvae, crablet and feed samples were pulverized with pestle and mortar vigilantly and mixed with sterile saline water to prepare the inoculum. A serially tenfold dilution (up to 10-6) of the stock solutions was prepared in alkaline saline peptone water (ASPW) to estimate the most probable number of the pathogenic Vibrio spp., Aeromonas spp. and Pseudomonas spp. Aliquots (0.1ml) from the stock solution; dilutions were plated on Thiosulfate Citrate Bile Salt Sucrose (TCBS) agar added with 2% sodium chloride for isolation of Vibrio spp. and the same aliquots were plated on Glutamate Starch Phenol-red (GSP) agar for the isolation of Aeromonas spp. and Pseudomonas spp. Aeromonas selective agar and Pseudomonas selective agar were further used to cross check outcome of Aeromonas spp. and Pseudomonas spp. respectively. TCBS plates were incubated at 35-37 oC for 24-48 h, while the GSP plates were incubated at 28-30 oC for 24-72 h. In case of total bacterial count, samples were cultured on nutrient agar plate and incubated at 25-30 oC for 18-24 h.

Identification of bacteria

External morphology of the colonies that appeared in TCBS agar and GSP agar were recorded for all the samples analyzed. For identification of species, the pure culture of isolated colonies were made on Trypticase Soy Agar (TSA) plates and Luria Bertani (LB) agar plates added with 1% NaCl. Several biochemical tests which included arginine dihydrolase (ADH), lysine decarboxylase (LDC), ornithine decarboxylase (ODC), motility, oxidase, indole production, fermentation of sugars (Klinger Iron Agar test and Triple Sugar-Iron Agar test), salt tolerant test and MR-VP tests were performed. To perform all the reactions, standardized techniques (Alsina and Blanch, 1994; Elliot et al., 1998) have been followed. Additionally, an identification kit (KB007 HiVibrio) was used for the identification of Vibrio at the species level.

Statistical analysis

Data were presented as means ± standard deviation. Data were analyzed using the Microsoft Excel and the SPSS version 26. Differences of data collected in different batches were analyzed by performing one-way ANOVA using Tukey’s honestly significant difference test. In both cases, the significance level was declared based on P< 0.05.

RESULTS

Prevalence of Vibrio spp. Aeromonas spp. and Pseudomonas spp. in the hatchery units

The samples from different units (brood, water, larvae and feeds) of the mud crab hatchery were found to be infected with chitinolytic bacteria pathogenic to mud crabs. Vibrio spp. were the most commonly identified bacteria in broods, larvae, larval tank water and in the feeds of both brood and larvae. Aeromonas spp. were isolated from all other units except for crablets, while Pseudomonas spp. were only detected in the brood crabs and larval feeds. Broods, brood’s tank water, larvae, larval rearing tank water and larval feed samples from all batches were infected with pathogenic Vibrio spp. 75% of broods, 65% of larvae and 100% of larval feed samples were contaminated with Vibrio spp. In the case of brood feeds, 60% sample from batch 1 and batch 2, and 40% sample from batch 3 were infected with Vibrio spp. 40% of the egg samples from batch 2 and batch 3 were found to be infected with Vibrio spp. Aeromonas contamination was found in half of the broods and brood’s tank water samples, 35% of the larvae and 40% of the larval tank water samples, and 60% of the larval feed samples. On the other hand, 60% of the broods, 40% of the brood tank water, and 60% of the larval feed samples were contaminated with Pseudomonas species (Figure 1). Both the crablet and its feed were found not to be contaminated with any of the pathogens.

 

Determination the load of total bacteria and some chitinolytic bacteria in the hatchery units

Broods: The bacteriological analysis showed that total bacteria count (TBC), total Vibrio count (TVC), total Aeromonas count (TAC) and total Pseudomonas count (TPC) in the brood samples ranged between log10 5.78 and log10 6.83 cfu g-1, log10 2.66 and log10 4.86 cfu g-1, log10 2.74 and log10 3.88 cfu g-1, log10 2.78 and log10 3.66 cfu g-1 respectively. The highest TBC and TVC were recorded in the batch 3, while the highest TAC and TPC were recorded in the batch 1 and batch 4 respectively (Table 1). In the study period, the average concentration of total bacteria, Vibrio spp., Aeromonas spp. and Pseudomonas spp. were log10 6.21±0.43 cfu g-1, log10 3.86±0.61 cfu g-1, log10 3.40±0.31 cfu g-1 and log10 2.71±0.31 cfu g-1 respectively. Data analysis showed that, the concentration of Vibrio spp. was significantly higher (p<0.05) in the batch 1 and batch 3 compared to the batch 2 and batch 4. However, the data showed no significant differences (p>0.05) for Aeromonas spp. and Pseudomonas spp. count among different batches.

Brood’s tank water

Tank water from the brood rearing unit was sampled during the collection of the broods. TBC, TVC, TAC and TPC varied from log10 5.60 to log10 7.58 cfu mL-1, log10

 

Table 1: Total bacteria and pathogenic bacterial load in broods from mud crab hatchery

Sample source	Bacterial count (log10 cfu g-1)
	Total bacterial count	Vibrio spp. count	Aeromonas spp. count	Pseudomonas spp. count
Batch 1	6.40±0.35	4.18±0.38 a	3.62±0.24	2.72±0.15
Batch 2	5.94±0.19	3.40±0.29 b	3.26±0.33	2.57±0.34
Batch 3	6.69±0.12	4.44±0.43 a	3.36±0.28	2.59±0.34
Batch 4	5.82±0.27	3.41±0.53 b	3.35±0.32	2.95±0.22

* Different superscript letters in the same column indicates significant differences (p<0.05)

 

Table 2: Total bacteria and pathogenic bacterial load in brood’s tank water of mud crab hatchery

Sample source	Bacterial count (log10 cfu mL-1)
	Total bacterial count	Vibrio spp. count	Aeromonas spp. count	Pseudomonas spp. count
Batch 1	7.13±0.48 a	4.14±0.50 a	3.29±0.37	ND
Batch 2	6.06±0.45 b	3.32±0.36 b	2.84±0.15	ND
Batch 3	6.94±0.58 a	3.74±0.65 a, b	3.08±0.33	2.51±0.26
Batch 4	5.95±0.40 b	3.33±0.36 b	2.88±0.17	2.58±0.38

*ND- not detected

* Different superscript letters in the same column indicates significant differences (p<0.05)

2.62 to log10 4.78 cfu mL-1, log10 2.54 to log10 3.81 cfu mL-1 and log10 2.15 to log10 3.08 cfu mL-1 respectively. The highest TBC and TVC were calculated in the batch 3, while the highest TAC and TPC were calculated in the batch 1 and batch 4 respectively (Table 2). Though Vibrio spp. and Aeromonas spp. were detected in the samples collected from all batches, Pseudomonas spp. was not detected in the tank water samples collected from the batch 1 and batch 2. In the entire study period, the average count of total bacteria, Vibrio spp., Aeromonas spp. and Pseudomonas spp. were log10 6.52±0.70 cfu mL-1, log10 3.63±0.57 cfu mL-1, log10 3.02±0.32 cfu mL-1 and log10 2.54±0.32 cfu mL-1 respectively. The mean Vibrio concentration of the batch 2 and batch 4 were significantly (p<0.05) lower compared to the mean Vibrio count of the batch 1. However, the data showed no significant difference (p>0.05) of Aeromonas ssp. and Pseudomonas spp. count among the batches.

Brood’s feeds: Tilapia fish collected from local sources were degutted, chopped into small pieces, then was thoroughly to remove dirt and pathogens to use as feed for the broods. Feed collected from different batches were found to be infected with Vibrio spp. but free of Aeromonas spp. and Pseudomonas spp. The TVC in feed sample ranged from log10 2.75 cfu g-1 to log10 3.08 cfu g-1; the highest count was recorded in the batch 3 and the lowest was in the batch 2, while the average concentration was recorded as log10 2.88±0.25 cfu g-1. The differences of Vibrio count among different batches were non-significant (p>0.05). On the other hand, the TBC of brood’s feed ranged from log10 5.68 to log10 6.65 cfu g-1. The highest load was recorded in the batch 1 and the lowest was recorded in the batch 2, and the average count was log10 6.25±0.32 cfu g-1. The data showed no significant difference (p>0.05) in total count among the batches.

Eggs from spawned crab: The egg samples collected from different batches were analyzed. The TBC ranged from log10 4.64 to log10 5.56 cfu g-1. The highest concentration was detected in the batch 3 and the lowest count was found in the batch 4. The average concentration of total bacteria was log10 4.93±0.39 cfu g-1. All the samples from four different batches were free of Aeromonas spp. and Pseudomonas spp., and no pathogenic Vibrio spp. were detected in the samples from the batch 1 and batch 3. However, one sample (20%) of the batch 2 and two samples (40%) from the batch 3 were found to be infected with Vibrio spp. The TVC in egg samples varied from log10 2.45 to log10 2.92 cfu g-1. The highest concentration of Vibrio spp. was recorded in the batch 3 and it was lowest in the batch 2. The average concentration of Vibrio spp. was log10 2.73±0.13 cfu g-1. The data showed no significant difference (p>0.05) in TBC and TVC among different batches.

Larvae: The analysis of larval samples showed that all the zoea stages of all batches were free of Pseudomonas contamination and the zoea stages of the batch 1 were free of Aeromonas contamination. The TBC, TVC and TAC of the larval samples raged between log10 4.52 and log10 5.68 cfu g-1, log10 2.28 and log10 3.64 cfu g-1, log10 2.56 and log10 3.28 cfu g-1. The maximum average of TBC and TVC was detected in zoea 3 of the batch 1, and for TAC, the maximum average was noticed in zoea 4 of the batch 4 (Table 3). The data showed no significant (p>0.05) difference for TBC, TVC and TAC among the different batches. However, Vibrio spp. count of zoea stage 3 and 4 was found significantly higher (p<0.05) than other zoea stages of the batch 1 and batch 3.

 

Table 3: Total bacteria and pathogenic bacterial load at the zoea stages of mud crab larvae

Sample Source	Bacterial Types	Bacterial load ( log10 cfu g-1) at larval stages
		Zoea 1	Zoea 2	Zoea 3	Zoea 4	Zoea 5
	TBC	4.68±0.15	4.85±0.24	5.56±0.21	5.18±0.12	4.84±0.15
Batch 1	TVC		2.93±0.16b	3.49±0.18a	3.04±0.15a	2.88±0.12b
	TAC
	TPC
Batch 2	TBC	4.64±0.15	4.84±0.24	5.08±0.31	4.86±0.35	4.66±0.28
	TVC		2.56±0.16	2.64±0.10	2.52±0.15	2.38±0.10
	TAC				2.88±0.25	2.76±0.15
	TPC
Batch 3	TBC	4.72±0.28	4.94±0.32	5.52±0.18	5.11±0.22	4.78±0.16
	TVC	2.66±0.11b	2.72±0.12b	3.21±0.21a	2.92±0.15a	2.68±0.16b
	TAC				2.75±0.14	2.88±0.10
	TPC
Batch 4	TBC	4.66±0.35	4.72±0.36	4.95±0.26	4.76±0.28	4.65±0.24
	TVC		2.72±0.15	2.93±0.22	2.88±0.16	2.52±0.12
	TAC			2.64±0.12	3.08±0.16	2.91±0.15
	TPC

*Empty fields indicates- ‘not detected’

* Different superscript letters in the same row indicates significant differences (p<0.05)

 

Table 4: Total bacteria and pathogenic bacterial load in the water samples from larval rearing tank

Study periods	Bacterial count (log10 cfu mL-1)
	Total bacterial count	Vibrio spp. count	Aeromonas spp. count	Pseudomonas spp. count
Batch 1	5.34±0.30	3.53±0.10 a	2.79±0.12	ND
Batch 2	5.16±0.29	3.02±0.19 b	2.65±0.06	ND
Batch 3	4.91±0.13	3.21±0.0.23a, b	2.88±0.07	ND
Batch 4	4.98±0.15	2.90±0.14 b	2.75±0.06	2.67±0.10

*ND- not detected

* Different superscript letters in the same column indicates significant differences (p<0.05)

Tank (larval rearing) water: The larval tank water had a TBC from log10 4.74 to log10 5.65 cfu mL-1. Though Vibrio spp. and Aeromonas spp. was identified in the samples collected from all batches, Pseudomonas spp. was only detected in the batch 4. The highest average TBC and TVC was found in the batch 1, while the highest mean TAC was found in the batch 3 (Table 4). The data of TBC and TAC showed no significant (p= 0.431 and p= 0.122 respectively) differences among the batches, but the data of TVC, showed a significant difference (p= 0.03) among the batches.

Larval feed: Artemia and rotifers are widely used as live feed for larvae of many crustacean species in the hatchery units. In the larval feed, TBC ranged from log10 4.86 to log10 6.60 cfu mL-1 in Artemia culture and log10 4.92 to log10 6.18 cfu mL-1 in rotifer culture. In both cases of Artemia and rotifer cultures, TBC was recorded higher in the batch 1 and batch 3 compared to the batch 2 and batch 4 (Figure 2). In case of pathogenic bacterial infection, Vibrio spp. were frequently detected in all batches of Artemia and rotifer cultures. The TVC in Artemia and rotifer culture ranged from log10 2.74 to log10 3.56 cfu mL-1 and log10 2.83 to log10 3.62 cfu mL-1 respectively. The Vibrio count in the batch 2 for both Artemia and rotifer was significantly lower (p<0.05) than in other batches. In Artemia culture, Aeromonas spp. was identified in the batch 1, 2 and 3, while Pseudomonas spp. was identified in the batch 3 and 4. On the other hand in rotifer culture, both Aeromonas spp. and Pseudomonas spp. were identified in the batch 2, 3 and 4, but not detected in the batch 1. The TAC in Artemia culture ranged from log10 2.81 to log10 3.04 cfu mL-1 and the TAC was found from log10 2.93 to log10 3.18 cfu mL-1in rotifer culture. The TPC ranged from log10 2.68 to log10 2.98 cfu mL-1 in Artemia culture and from log10 2.93 to log10 3.08 cfu mL-1 in rotifer culture. The data analysis showed that both TAC and TPC did not significantly differ (p>0.05) among different batches of the Artemia and rotifer cultures.

 

Crablet rearing unit: Just after molting, megalopa stage of mud crab larvae turned into crablet which were immediately transferred into crablet rearing unit from larval rearing unit. The TBC of crablet samples was between log10 4.93 and log10 2.91 cfu g-1. The crablet feed and tank water had the TBC from log10 4.20 to log10 4.34 cfu g-1 and log10 4.65 to log10 5.68 cfu g-1 respectively. There was no Vibrio spp., Aeromonas spp. and Pseudomonas spp. infection in the crablet, crablet feed and crablet rearing tank water samples collected from the hatchery unit.

Species identified and their proportion

Selective agar media, several biochemical tests and identification kits were used for identifying the genus Vibrio, Aeromonas and Pseudomonas at the species level. The Vibrio species identified from the samples were V. alginolyticus (8%), V. parahaemolyticus (12%), V. mimicus (16%), V. harveyi (12%), V. fluvialis (6%), V. vulnificus (8%), V. cholerae (6 %) and V. spendidus (6%), while about 26 percent of the Vibrio colonies grown in TCBS plates were unspecified. A. hydrophila was accounted for 80% of the isolates of Aeromonas spp., and the remainder identified as A. caviae and A. veronii. On the other hand, all the Pseudomonas species were identified as P. aeruginosa.

Water quality parameters of larval rearing tank

In captive condition, maintaining water quality parameter within optimum range are very much crucial for the survival of mud crab larvae. All the water quality parameters of the larval rearing tanks were found within the acceptable limit. The water temperature ranged from 29.1 to 31 ℃, water salinity ranged from 27 to 30 ppt, pH ranged from 7.7 to 8.8, dissolve oxygen ranged from 5.5 to 6.7 mg L-1 and NH4 ranged from 0.00 to 0.22 mg L-1. The differences of the mean value of the water quality parameters among different batches were statistically non-significant (p>0.05).

DISCUSSION

The present study aimed to identify the prevalence rate and load of pathogenic Vibrio spp. Aeromonas spp. and Pseudomonas spp. in the mud crab hatchery units, as the presence and excess load of such pathogenic bacteria can be fatal for the larval production. In mud crab hatchery, vibriosis is a major concern causing high mortality and inconsistent survival of mud crab larvae (Mann et al., 1998). Among Vibrio spp., V. hraveyi, V. parahaemolyticus, and V. alginolyticus are highly pathogenic to mud crab zoea (Boer et al., 1993). Opportunistic marine bacteria may infect any stage of the production cycle but the possibility of vertical transmission could not be ignored. Transmission of pathogenic Vibrio harveyi through feces of adult female was responsible for heavy mortality during larval rearing of blue swimming crab P. pelagicus (Talpur et al., 2011).

Among the Vibrio spp. isolated in this study, V. alginolyticus, V. fluvialis, V. harveyi, V. parahaemolyticus and V. vulnificus were predominant, and these Vibrio spp. are considered as pathogenic to adult and larval crustacean (Hisbi et al., 2000; Gopal et al., 2005, Liu et al., 2016). Gunasekaran, et al. (2019) reported V. alginolyticus involvement in mortality of S. serrata in hatchery unit and in culture farms. V. harveyi is often an opportunistic bacteria and pathogenic at 102-103 cfu mL-1 to zoeal stages of mud crab (Jithendran et al., 2010). The present study also identified Aeromonas spp. and Pseudomonas spp. infection in the hatchery units. Aeromonas spp. are common micro flora of wild and reared crustacean and are often considered as opportunistic pathogen (Lightner, 1993). Aeromonas spp. has been associated with the soft shell syndrome in P. monodon (Baticados et al., 1986; Uddin et al., 2008). The genus Pseudomonas was found to cause red spot syndrome in Litopenaeus vannamei, which lead to mass mortality of larvae and post larvae in the hatchery (Soltani et al., 2010).

Vibrio spp. Aeromonas spp. and Pseudomonas spp. were dominant in the broods and brood rearing tanks; The TBC, TVC, TAC and TPC in brood crabs calculated in the present study were found similar as described by Lavilla-Pitogo et al. (2001). Warmer temperature might be the reason for higher concentration of Vibrio spp. in the batch 1 and 3 compared to other two batches. There are very few studies available on pathogenic bacterial association with mud crab eggs and larvae. The Vibrio load in zoea in the present study was found almost similar to the Vibrio load in shrimp larvae mentioned by several authors (Soto-Rodriguez et al., 2006; Hassan et al., 2017). Spawned crab may be treated with suitable antibiotics or medical plant extract to reduce the bacterial load in eggs and lessen the chance of vertical transmission. The infection rate and load of Vibrio were found higher at the middle stage of larviculture, such as at zoea 3. As time progress during larviculture bacterial load increased and this might be due to intervention of live feeds which often carry pathogenic bacteria. On the other hand decreasing Vibrio load at later stages (zoea 4, zoea 5) might be the effect of probiotics which ensure adequate growth of beneficial bacteria that inhibit the proliferation of pathogenic Vibrio and other chitinolytic bacteria. Studies demonstrated that both rotifer and Artemia are vector for introducing potentially harmful bacteria such as Vibrio spp. In spite of special efforts are being made to keep the cultures as clean as possible, high-density cultures create high organic load hence rapidly colonized by bacteria which results in poor growth and survival of fish and crustacean larvae (Dhert et al., 2001). Furthermore, the lack of a strong biosecurity in the Artemia and rotifer culture units may increase the risk of contamination by various pathogens, as such culture units provide a suitable environment with adequate nutrients from the feeds (type of algae) given for Artemia and rotifer enrichment for the growth of various pathogenic bacteria. Any breach of the biosecurity line could result in tank water contamination, which could explain variations in both the total count and the pathogenic bacterial load in larval rearing tank water from different batches. The larval feeds from all batches were infected with Vibrio spp., mostly by V. alginolyticus. Though concentration was low, prevalence of both Aeromonas spp. and Pseudomonas spp. were common in larval feeds. The TVC, TAC and TPC were found almost similar to the findings of Interminense et al. (2014). Overall, in the present study, the load of pathogenic Vibrio, Aeromonas, and Pseudomonas identified in the different components of the hatchery was similar to the findings of many authors (Nakamura et al., 1995; Roza and Hatai, 1999; Lavilla-pitogo and de la Pena, 2004; Quinitio and Parado-Estepa, 2008; Dan and Hamasaki, 2015) who conducted such experiments in the production line, particularly with S. serrata and S. paramamosain.

The total load of Vibrio and Aeromonas was much higher in the hatchery systems particularly in the warmer months while maximum load of Pseudomonas was observed in the wet season and in the winter months. Similar result was reported (Gopal et al., 2005; Liu et al., 2016) in farms and wild condition but inside the controlled environment of hatchery unit such result indicates improper handling either of physical or biological components or breakdown of bio-security line by any means. Water quality parameters, particularly salinity, temperature and pH are considered to be the most important ones for larval rearing and culture of fish and crustacean (Ojwala et al., 2018). Suitable water quality variables ensure proper utilization of nutrients by plankton and better growth and survival of larvae and juveniles. Having all of the water quality variables in the larval rearing tanks within their optimum range indicated that these variables had no adverse effect on larval survivability. The notable finding from the hatchery unit was a pathogen-free crablet.

CONCLUSION

Successful production and survival of mud crab larvae in hatcheries is the prime need to ensure the sustainability of this sector as wild stock is the only source of crabs being harvested and stocked in the mud crab farms of Bangladesh. Through this study, scientific knowledge regarding the association of total bacteria and some pathogenic bacteria in the mud crab hatchery production units has been documented. The prevalence rate and load of pathogenic bacteria detected in broods, eggs and larval feeds might be matters of great concern because vertical and horizontal transmissions of such pathogens in larval production units are quite possible. Therefore, more experimental efforts are needed to diminish the chances of pathogen transmission from brood to offspring, and contamination from external sources during hatchery operation. The scientific use of prebiotics, probiotics and antibiotics to produce viable percentages of crablets, which are indeed inevitable to flourish the aquaculture of mud crab (Scylla olivacea) in Bangladesh.

ACKNOWLEDGEMENTs

This work was supported by the Bangladesh Agricultural Research Council (BARC) under the National Agricultural Technology Program-Phase II Project (Project ID: 029, NATP-2, BARC).

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest.

novelty statement

This study is the first one that has been carried out to determine the bacterial load and identification of pathogenic bacteria in the larval production line of mud crab (Scylla olivacea) hatchery in Bangladesh. Therefore, all the findings of this research are novel.

authors contribution

Md. Rashedul Islam: Collection and analysis of samples; data mining; interpretation and analysis of data; write up of the manuscript draft.

Abul Farah Md. Hasanuzzaman: Conceptualization and design of the research work; data analysis; comprehensive review of the manuscript.

Md. Latiful Islam: Concept of sampling design and collection of water quality data. Ghausiatur Reza Banu: Conceptualization of the research work; supervision of the experiments; revision of the manuscript draft.

ETHICAL APPROVAL

The Research Ethics Committee of Khulna University Research Cell, Bangladesh, had given ethical approval (Ref No: KU AEC-2021/08/15; Date: August 17, 2021). All procedures carried out involving crab during the present study followed the institutional guideline, following the international and national guidelines.

REFERENCES

Alsina M, Blanch AR (1994). A set of keys for biochemical identification of Vibrio species. J. Appl. Bacteriol., 76: 79-85.

Azra MN, Ikhwanuddin M (2016). A review of maturation diets for mud crab genus Scylla broodstock: present research, problems and future perspective. Saudi J. Biol. Sci., 23 (2):257–267. https://doi.org/10.1016/j.sjbs.2015.03.011

Basford AJ, Makings N, Mos B, White CA, Dworjanyn S (2021). Greenwater, but not live feed enrichment, promotes development, survival, and growth of larval Portunus armatus. Aquaculture, 534:736331. https://doi.org/10.1016/j.aquaculture.2020.736331

Baticados CL,Coleso RM, Duremdez RS (1986). Studies on the chronic soft shell syndrome in the tiger prawn, Penaeus monodon Fabricius from brackish water ponds. Aquaculture, 56(3-4): 271–285. https://doi.org/10.1016/0044-8486(86)90342-X

Boer DR, Zafran, Parenrengi A, Abmad T (1993). Preliminary study on luminescent vibrio infection on mangrove crab, Scylla serrata larvae. Res. J. Coastal Aquac., 9(3): 119-123.

Coates CJ, Rowley A (2022). Emerging diseases and epizootics in crabs under cultivation. Front. Mar. Sci., 8: 809759. https://doi.org/10.3389/fmars.2021.809759

Dan S,Hamasaki K (2015). Evaluation of the effects of probiotics in controlling bacterial necrosis symptoms in larvae of the mud crab Scylla serrata during mass seed production. Aquac. Int. 23(1): 277–296. https://doi.org/10.1007/s10499-014-9815-1

Dhert P, Rombaut G, Suantika G, Sorgeloos P (2001). Advancement of rotifer culture and manipulation techniques in Europe. Aquaculture, 200(1-2): 129-146. https://doi.org/10.1016/S0044-8486(01)00697-4

Elliot EL, Kaysner CA, Jackson L, Tamplin ML (1995). Vibrio cholerae, V. parahaemolyticus, V. vulnificus and other Vibrio spp., p. 901-927. In FDA Bacteriological Analytical Manual, 8th ed. AOAC International, Gaithersburg, MD.

Elliot EL, Kaysner CA, Jackson L, Tamplin ML (1998). Vibrio cholerae, V. parahaemolyticus, V. vulnificus and other Vibrio species. In: Food and Drug Administration Bacteriological Manual, 8th edn. (Revision A), AOAC International, Gaitheisbarg, MD.

Gopal S, Otta SK, Kumar S, Karunasagar I, Nishibuchi M, Karunasagar I (2005). The occurrence of Vibrio species in tropical shrimp culture environments; implications for food safety. Int. J. food microbiology, 102(2): 151-159. https://doi.org/10.1016/j.ijfoodmicro.2004.12.011

Gunasekaran T, Gopalakrishnan A, Deivasigamani B, Muhilvannan S, Kathirkaman P (2019). Vibrio alginolytigus causing shell disease in the mud crab Scylla serrata (Forskal 1775). Ind. J. Geo Mar. Sci., 48 (9): 1359-1363.

Hamasaki K, Suprayudi MA, Takeuchi T (2002). Mass mortality during metamorphosis to megalops in the seed production of mud crab Scylla serrata (Crustacea, Decapoda, Portunida). Fish. Sci., 68(6): 1226–1232. https://doi.org/10.1046/j.1444-2906.2002.00559.x

Hasan MAR, Haque MIM, Hossain A, Hasan M,Rahman MS (2017). Bacterial Loads in Shrimp and Fish Hatchery Environments of Bangladesh. J. Asiatic Society of Bangladesh, Science, 43(1): 37-47.

Hasanuzzaman AFM, Arafat ST, Huq KA (2014). Mud Crab (Scylla spp.) Aquaculture in the south-west Sundarbans region of Bangladesh. Iraqi J. Aquac., 11(1): 57-83.

Hisbi D, Vandenberghe J, Robles R, Verdonck L, Swings J, Sorgeloos P (2000). Characterization of Vibrio and related bacteria associated with shrimp Penaeus monodon larvae in Indonesia. Asian Fish. Sci., 13: 57-64. https://doi.org/10.33997/j.afs.2000.13.1.007

Hungria DB, Tavares CP, Pereira LA, da Silva UDAT, Ostrensky A (2017). Global status of production and commercialization of soft-shell crabs. Aquacult. Int., 25(6): 2213–2226. https://doi.org/10.1007/s10499-017-0183-5

Interaminense JA, Ferreira Calazans N, do Valle BC, Lyra Vogeley J, Peixoto S, Soares R, Lima Filho JV (2014). Vibrio spp. control at brine shrimp, Artemia, hatching and enrichment. J. World Aquac. Society, 45(1): 65-74. https://doi.org/10.1111/jwas.12096

ISO (2007). Microbiology of food and animal feeding stuffs- Horizontal method for the detection of potentially entero-pathogenic Vibrio spp. ISO/TS 21872-1:2007(E), Switzerland.

Jithendran KP, Poornima M, Balasubbramanian CP, Kulasekarandian S (2010). Diseases of mud crabs (Scylla spp.): an overview. Ind. J. Fish. 57: 55–63.

Lavilla-Pitogo CR, Marcial HS, Pedrajas SAG, Quinitio ET, Millamena OM (2001). Problems associated with tank-held mud crab (Scylla spp.) broodstock. Asian Fish. Sci., 14(2): 217-224.

Lavilla-Pitogo CR, de la Peña LD (2004). Disease in farmed mud crab Scylla spp. Diagnosis, prevention and control. Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo, Philippines, pp.89.

Lightner DV (1993). Diseases of cultured penaeid shrimps.In: Mc Vey, J.P. (Ed.), CRC Handbook of Mariculture, 2nd ed.,CRC Press, Boca Raton, pp. 393– 486.

Liu R, Chen H, Zhang R, Zhou Z, Hou Z, Gao D, et al. (2016). Comparative transcriptome analysis of Vibrio splendidus JZ6 reveals the mechanism of its pathogenicity at low temperatures. Appl. Environ. Microbiol., 82(7): 2050–2061.

https://doi.org/10.1128/AEM.03486-15

Mann D, Akasawa T, Pizzuto M (1998). Development of a hatchery system for larvae of the mud crab Scylla serrata at the Bribie Island Aquaculture Research Centre. In: Mud Crab Aquaculture and Biology: Proceedings of an International Scientific Forum held in Darwin, Australia. 21-24 April 1997. pp. 153-158.

Nakamura K, Nakamura M, Hatai K (1995). Lagenidium infection in eggs and larvae of mangrove crab (Scylla serrata) produced in Indonesia. Mycoscience, 36(4): 399-404. https://doi.org/10.1007/BF02268623

Ojwala RA, Otachi EO, Kitaka NK(2018). Effect of water quality on the parasite assemblages infecting Nile tilapia in selected fish farms in Nakuru County, Kenya. Parasitol. Res., 117(11): 3459-3471. https://doi.org/10.1007/s10499-017-0183-510.1007/s00436-018-6042-0

Pates GS, Quinitio ET, Quinitio GF, Parado-Estepa FD (2017). Morphological deformities in mud crab Scylla serrata juveniles exposed to antibiotics during the larval stage. Aquac. Res. 48 (5): 2102–2112. https://doi.org/10.1007/s10499-017-0183-510.1111/are.13046

Quinitio ET, Parado-Estepa FD, Millamena OM, Rodriguez E, Borlongan E (2001). Seed production of mud crab Scylla serrata juveniles. Asian Fish. Sci., 14(2): 161–174.

Quinitio ET, Parado-Estepa FD (2008). Biology and Hatchery of Mud Crab Scylla spp. (2nd Edition). 44 pp. Aquaculture Extension Manual No. 34.. SEAFDEC/AQD, Iloilo, Philippines.

Romano N, Zeng C (2017). Cannibalism of decapod crustaceans and implications for their aquaculture: A review of its prevalence, influencing factors, and mitigating methods. Rev. Fish. Sci. Aquac., 25(1): 42–69. https://doi.org/10.1080/23308249.2016.1221379

Roza D, Hatai K (1999). Pathogenicity of fungi isolated from the larvae of the mangrove crab, Scylla serrata, in Indonesia. Mycoscience, 40(5): 427-431. https://doi.org/10.1007/BF02464397

Soltani M, Ahmadi MR, Yavari H, Mirzargar S (2010). Red-pink colony-producing Pseudomonas sp. is the causative agent of mass mortality in larvae and post-larvae of Litopenaeus vannamei raised in hatcheries in south Iran. Int. J. Vet. Res., 4(2): 89-94.

Soto-Rodríguez S, Armenta M, Gomez-Gil B (2006). Effects of enrofloxacin and florfenicol on survival and bacterial population in an experimental infection with luminescent Vibrio campbellii in shrimp larvae of Litopenaeus vannamei. Aquaculture, 255(1-4): 48-54. https://doi.org/10.1016/j.aquaculture.2005.11.035

Sui L, Wille M, Cheng Y, Wu X, Sorgeloos P (2011). Larviculture techniques for Chinese mitten crab Eriocheir sinensis. Aquaculture, 315 (1-2): 16–19. https://doi.org/10.1016/j.aquaculture.2010.06.021

Talpur AD, Memon AJ, Khan MI, Ikhwanuddin M, Daniel MD, Abol-Munafi AB (2011). A novel of gut pathogenic bacteria of blue swimming crab Portunus pelagicus (Linneaus, 1758) and pathogenicity of Vibrio harveyi a transmission agent in larval culture under hatchery conditions. Res. J. Appl. Sci., 6(2): 116-127.

Uddin SA, ZafarM, Noman ASM, Sharmin A(2008). Isolation and Identification of Vibrio spp. and Aeromonas spp. in wild and hatchery reared Penaeus monodon postlarvae from Cox’s Bazar, Bangladesh. Pakistan J. Mar. Sci., 17:161–167.

Waiho K, Fazhan H, Quinitio ET, Baylon JC, Fujaya Y, Azmie G, Wu Q, Shi X, Ikhwanuddin M, Ma H (2018). Larval rearing of mud crab (Scylla): what lies ahead. Aquaculture, 493: 37-50. https://doi.org/10.1016/j.aquaculture.2018.04.047

Zhang XJ, Bai XS, Yan BL, Bi KR, Qin L (2014). Vibrio harveyi as a causative agent of mass mortalities of megalopa in the seed production of swimming crab Portunus trituberculatus. Aquacult. Int. 22: 661–672. https://doi.org/10.1007/s10499-013-9695-9