Effects of Microalgae Chlorella vulgaris Density on the Larval Performances of Fresh Water Prawn Macrobrachium rosenbergii (De Man, 1879)

The green water technique usually consists of adding microalgae cells to the rearing systems of molluscans, crustaceans or fish. The commercially available microalgae include Tetraselmis sp., Nannochloropsis sp., Chaetoceros sp., Sprirulina sp. and Chlorella sp. in addition to feed rotifers, moina, Artemia (live feeds) for fish larvae they are used to dose the larval tanks to promote “green water”. (Reitan et al., 1997; Muller-Feuga, 2000; Thépotet_al2016). The enrichment of live feed, generally with microalgae, is a common and necessary practice to boost the quality of the otherwise nutrient deficient feed (Fehéret_al2013). Addition of micoalgae has been shown to increase survival and developments in different aquatic organisms (Papandroulakis et al., 2002; Palmer et al., 2007; Hachéet_al2017). The better survival and growth in these controlled green-water systems are generally attributed by better direct and indirect nutrition of larvae, lower stress levels, enhance environmental conditions for feeding from increased turbidity, light scattering and attenuation and visual contrast enhancement, improve water quality due to stripping of nitrogenous substances and increase oxygenation rates, chemical and digestive stimulants and antibacterial properties of the microalgae (reviewed by Palmer et al., 2007). It has been also reported that active compounds in fresh and processed microalgae can inhibit pathogenic bacterial and fungal growth (Viso et al., 1987; Naviner et al., 1999; Marques et al., 2006; Sivakumar et al., 2018) and this in turn may reduce the risk of viral infection (Wang, 2003).
 
Giant freshwater prawn (Macrobrachium rosenbergii De Man, 1879), prevalently known as ‘scampi’ is widely accepted as one of the popular candidate species for the inland aquaculture of Southeast Asian and South Pacific countries (New, 2005). Due to its quick growth, high quality meat, stability, low protein diet requirements and high market demand (New, 2002) this species having great potential for farming in the Kingdom of Saudi Arabia. Although  grow out trials of M. rosenbergii has been conducted in Saudi Arabia (Siddiqui et al., 1996 and 1997) with varying success no  efforts have been taken so far for larval production of this species. Until now, the larvae of this species were imported from South Asian countries and this possibly the major constrains for successful expansion of this valued species in the locality. Larval stages of all crustaceans are highly sensitive and any sudden change may cause mass mortality (Anger, 2006). Larvae obtained from abroad not only reduce the possibility of survival but also increase the production cost. Based on huge potentialities of this species in the Kingdom, the Fish Resources Research Center of King Faisal University developed breeding protocols and established a live brood bank of M. rosenbergii successfully (Alsaqufi et al., 2018).

Green water system has been developed for larval rearing of several marine teleosts (Shields, 2001) while little information is available for crustacean species. There is one study until now conducted by Lober and Zeng (2009) who used green water enriched with Nannochloropsis spp for M. rosenbergii larvae and their results were encouraging. In a preliminary study, we compared the efficacy of various microalgae (Nannochloropsis, Isochrysis, Thalassiosira and Chlorella vulgaris) in green water system for the performances of M. rosenbergii larvae and confirmed that C. vulgaris performed better over other species (unpublished data). Chlorells vulgaris is a rich source of proteins, vitamins (B complex and ascorbic acid), minerals (potassium, sodium, magnesium, iron and calcium), ß-carotene, chlorophyll in addition, it has the ability to improve immunity, feed utilization, amelioration of stress, aquatic bioremediation, disease resistance in aquatic animals (Radhakrishnan et al., 2015; Sukri et al., 2016; Shi et al., 2017; Ahmad et al., 2020). Therefore, the present investigation aimed to explore the potential effects of C. vulgaris on the larval performance of M. rosenbergii. This study further optimized the concentration of C. vulgaris in larval rearing system.

Water quality parameters
 
Water used for the larval  rearing (5 and 12 ppt) and microalgae culture (12 ppt) were prepared in advance by diluting dechlorinated seawater (45 ppt) obtained from Aluqair beach, Saudi Arabia with treated hatchery tap water. Major water quality parameters were monitored in alternate days before the water exchange. Dissolved oxygen, pH, ammonia, nitrite, water temperature were measured with multiparameter tester (YSI professional plus, USA) and the salinity was observed using Atago (Japan) salinity refractometer. The others parameters like total hardness and alkalinity were determined by the standard methods (APHA).
 
Broodstock development
 
Post larvae (PL-23) of M. rosenbergii had an initial weight of 0.27± 0.02 g were obtained from Rosen Fisheries (PVT) Hatchery LTD, Thrissur, Kerala, India and transported to the fish rearing facilities of Fish Resources Research Center at King Faisal University, Kingdom of Saudi Arabia. After acclimatization, the PL were stocked in a 600-L fiber glass tanks with a density of 500 larvae/m³ for 30 days. The PL were then transferred into two 6000-L capacity fresh water recirculatory fiber glass tank system and stocked at a density of 200 larvae/m³. Tanks were provided with necessary hideouts in order to prevent cannibalism. The prawns were fed 35% protein contain artificial diet (ARASCO Company, KSA) twice in a day at the rate of 10% of their body weight for the first month, which had been reduced to 7% for second  month and subsequently to 5% for the remaining months. After six months of the rearing, healthy matured male and female prawns were segregated based on visual observation by their body color and anatomy. Selected matured prawns were moved to a 5000-L fiberglass recirculatory aquaculture system and stocked with a ratio of 1 male: 4 female to enable quick breeding process (New, 2002).
 
Hatching of larvae
 
Berried females with orange colored eggs were transferred to a 15-L hatching unit with 5 ppt for incubation. Embryo developmental stages were carefully monitored by a stereomicroscope (Leica, USA). Hatching occurred within ninth day after incubation. After hatching, spent female prawn was returned to brood rearing tanks and hatchlings were transferred to the hatching tank where salinity was gradually adjusted to 12 ppt.
 
Chlorella vulgaris culture
 
The microalgae Chlorella vulgaris were cultured in 5-liter conical flasks with commercially available f/2-Guillard medium. The continuous culture protocols were followed according to the methods developed by (Guillard, 1975).  The cell density was estimated using a hemocytometer in regular basis. The quantity of the microalgae required to adjust in volume and cell density in the treatment tanks were calculated by following formula (Lober and Zeng, 2009):
  Where
C1 denotes required microalgal density to the designated treatment; V1 indicates the amount of water required during water exchange; C2 represents cells concentration in algal tanks; V2 means the volume of the algae essential to formulate desired algal density for the respective experiment.
 
Experimental setup
 
Newly hatched larvae were stocked in 5-liter plastic aquaria at varying concentration of C. vulgaris such as 1) T1 (0×10⁵, control), 2) T2 (5×10⁵), 3) T3 (10×10⁵), 4) T4 (15×10⁵) and 5) T5 (20×10⁵) cells/ml and reared  until the conversion of first post larval stage at room temperature (30±1°C). Each treatment consisted of two replicates with 50 larvae. All the rearing tanks were maintained with salinity of 12 ppt, natural photoperiod (12L:12D) and continuous mild aeration. M. rosenbergii larvae were fed Artemia nauplii (OSI, Great Salt Lake, Utah, U.S.A) six times in a day at the concentration of 6 nauplii/ml. Artemia was produced by hatching brine shrimp cysts through decapsulation technique (Lavens and Sorgloos, 1996). Descapsulated Artemia cysts were incubated in sterile diluted seawater at 29°C and salinity of 30 ppt under constant aeration and 2000-lx illumination until hatching. Later, newly hatched nauplii were collected using a 100-µm mesh-size net and washed with fresh water for several times before feeding. Artemia culture was carried out in two times as one in the morning and other at evening in order to maintain the freshness of Artemia nauplii. Survival of M. rosenbergii larvae in each treatment was recorded every morning and larvae were transferred to new aquaria with respective conditions (e.g., microalgae concentration, ppt, food, temperature, etc.).
In this study, LSI (larval stage index) and LCI (larval condition index) were considered as larval developmental stages and larval quality, respectively. A total 75 larval samples (15 larvae × 5 treatments = 75) were randomly selected for LSI and LCI analyses. LSI was performed every alternative day while LCI was done every four day internals. Larval stage identification and condition were measured by a stereomicroscope (Leica model EZ4, USA) and photographs were taken by 5-Mega pixel HD camera (Switzerland) installed with stereomicroscope. Image analysis software (Leica LAS EZ, Version 20.0) was used for larval quality assessments. Each treatment was terminated when at least 25% larvae metamorphosed to first PL stage. LSI was assessed microscopically as per the method described by Manzi et al., (1977).
 
In this ni= represents number of larvae at stage Ei, n= denotes the number of larvae analyzed; E= larval developmental stage (zoea 1 to first PL, hereafter scored as 1 to 11).
 
On the other hand, LCI was determined in as per the method developed by Tayamen and Brown (1999):
   
In this P= total point assigned to each larvae examined, n=number of larvae assigned.
 
External phenotypic traits such as larval pigmentation, muscle gut ratio, gut lipid content, gut fullness, body coloration, setation, appearance of abdominal muscles, melanization, fouling organisms, swimming behavior and photo response were considered as condition factors. Tested larvae were given score ranging from 0.0 to 2.0, in this, 0.0 represents poor, 1.0 = indicates good and 2 denotes the excellent condition.
 
Ten newly converted PLs from each experimental tank were pre-weighed individually and dried in hot air oven at temperature of 60°C for 48 hours for dry mass analysis. A high precision microbalance (Sartorius, Germany) was used to measure the dry mass samples.
 
Statistical analyses
 
All the data were statistically tested by one-way ANOVA. Tukey’s tests were performed to evaluate any significant differences among treatments at 0.05 significance level. The analyses were performed using the software SPSS Version 16.0 for windows 8.

Water quality parameters during the study period were ranged from 28.9 to 29.2°C, 5.61 to 6.11 ppm, 7.9 to 8.5, 133 to 145 ppm, 220 to 232 ppm, 0.02 to 0.089 ppm and 0.13 to 0.30 ppm for temperature, DO, pH, alkalinity, hardiness, nitrite, ammonia, respectively. There were no significant variations observed in the water quality parameters among the treatments during the experimental period.
 
Larvae exposed to moderate concentrations of C. vulgaris performed better than high and low concentrations. Fig 1 shows the survival rates upon time and the appearance of the first PL under various concentrations of C. vulgaris. Survival rates of larvae treated with or without C. vulgaris decreased gradually with time and remained constant after metamorphosis of first PL stage. At the end of the experiment, survival rates were parallel for T2, T3, T4 and T5 but varied significantly (P<0.05) from T1. Highest larval survival was observed in T3 (60.83%) followed by T4 (56.91%), T2 (48.39%), T5 (40.93%) and T1 (30.65%), respectively. Larval metamorphosis occurred significantly earlier (P<0.05) in T3 than other treatments, while prolong time required to metamorphosis for T1 (clear water). The PL appearance was first witnessed in T3 that took an average duration of 25 days followed by T4, with 28 days; while T2, T5 and T1 recorded 31, 32 and 39 days, respectively (Table 1). A similar developmental pattern was observed for larval weight at first PL stage, larvae treated with moderate concentrations of C. vulgaris exhibited higher weight gain than high and low concentrations of C. vulgaris. Weight gain among C. vulgaris treated larvae were comparable but plummeted significantly (P<0.05) from untreated control (T1).
 

 

 
The mean values of the larval stage index (LSI) attained in the experimental tanks are presented in Table 2. Larval development was significantly (P<0.05) faster for T3 than those obtained for T4, T2, T5, T1, in this sequence. Larvae converted from zoea to first PL stage within 24 days when they were treated with C. vulgaris at the concentration of 10×10⁵ cells/ml (T3). Larvae treated with T2 (5×10⁵ cells/ml), T4 (15×10⁵ cells/ml) and T5 (20×10⁵) showed a similar pattern of larval development (28 to 32 days), while T1
(control, no microalgae) required prolonged period (40 days).
 

 
The larval condition index (LCI) based on the microscopic observation is shown in Table 3. Overall, the larvae appeared healthier regardless of C. vulgaris concentrations and experimental periods but LCI scores gradually decreased with time except T3 and T4. Although the LCI values between the treatments during stocking was insignificant (1.90 to 1.96) significant variations (P>0.05) occurred at the end of the study. The highest LCI values at the end of 25 days rearing were recorded in T3 (1.8) followed by T4 (1.7), T2 (1.47), T5 (1.4) and T1 (1.1) respectively.
 

 
Successful rearing of crustacean larvae not only relies on food quality but also depends on the water quality. Any inclusion of fouling organisms or undesirable particles in the water system alters the water parameters that ultimately hamper sustainable production. The present investigation reveals the inclusion of microalgae C. vulgaris at a suitable concentration in the larval rearing system improved the larval growth and development greatly. Larval survival, larval development (LSI) and larval quality (LCI) was found significantly higher in all C. vulgaris treated tanks in contract to untreated control. Besides, the improved larval performances especially exposed to C. vulgaris at varying concentrations resulted in higher dry weight gain of the newly settled post larvae. The incorporation of C. vulgaris in larval rearing tanks did show any negative impact on water quality parameters throughout the study periods.

Water quality and dietary requirements considered as the important elements influencing the metamorphosis and survival of crustacean larvae (Anger, 2006).  In present study, the observed water quality parameters in treated and untreated tanks were found within the optimal range as suggested by New (2002). Application of microalgae in larval rearing tank not only improves water quality but also removes nitrogenous substances in the system (Cohen et al., 1976; Mallasen and Valentini, 2006). It has been reported that microalgae are able to produce several bioactive compounds that can obstruct the entry of various harmful pathogens (Lober and Zeng, 2009). This study clearly observed the better survival in the presence of moderate of microalgae. Addition of microalgae contributes beneficial bacteria in to the larval rearing systems that may influence intestinal and water microbiota (Salvesen et al., 2000; Muller-Fegua, 2000; Jakhar et al., 2016; Maliwat et al., 2017; Pakravan et al., 2017;). Likewise, Gatesoupe (1999) documented that the gut microbiota of fish larvae clearly influenced by the media they survive.
 
The direct beneficial effects of microalgae in larvae rearing systems are not clear but it is predicted that several factors responsible for better growth and development. The primary reason probably related to the nutritional improvement in the early larval stages by the presence of microalgal cells. While working on M. rosenbergii larvae, Lober and Zeng (2009) found the presence of micoalgal cells in the larval gut although, they were uncertain whether those cells were actively consumed or accidentally ingested. Similarly, algal residues were witnessed in the larval gut in several instances during present investigation. Occurrence of microalgae or its constituents, even at small quantity in the gut can activate the production of digestive enzymes and enhance growth performances (Reitan et al., 1993; Brito et al., 2004). Brown et al., (1997) stated the presence of several growth promoting pigments such as chlorophyll, carotenoids and phycobiliproteins in microalgae. Radhakrishnan et al., (2015); Sukri et al., (2016) and Maliwat et al., (2017) described the beneficial effects of C. vulgaris on digestive enzyme activities in M. rosenbergii. Furthermore, Xu et al., (2014) and Shi et al., (2017) witnessed certain improvements of amylase tryptsin, lipase and protease activities in aquatic animals when treated with Chlorella species.
 
In addition, larvae may be benefited from the indirect nutrition by the supplementation of live Artemia that depends microalgae as their food source. Even though Artemia is considered as the best live feed for the larvae of many crustacean species lacks several essential nutrient components (like HUFA, DHA and EPA) in it may limit the survival and growth of crustacean and fish larvae (Légeret_al1986; Felix et al., 2020). C. vulgaris contains valuable source of LC-PUFAs with presence of ARA, DHA and EPA (Pakravan et al., 2017; Maliwat et al., 2017) that possibly enhanced the nutritional profile of Artemia.
 
High microalgae rations affect the survivorship of cultures, because those cells that are not eaten by larvae suffer microbial decomposition processes, exposing cultures to noxious bacteria and fungi (Loosanoff and Davis, 1963; Liu et al., 2006). This study confirmed that higher algal concentration caused a marked decline in growth, survivability, larval developments and LCI score particularly in T5 treatment with microalgal concentration of 20×10⁵ cells/ml. This agrees with findings of Hurley et al., (1997), who found that excessive microalgal addition are less productive and an optimal concentration is required for the better larval survival. Furthermore, decline in the larval performance observed in the higher algal density tanks could be associated with restriction of larval free movements due to the thick concentration of algae that possibly obstruct its foraging behavior. 
 
Tank color and light influence the feeding efficiency, embryo developmental stage, survival and growth of fish species (Downing et al., 2001; Puvanendran and Brown, 2002). The clear water tank reflects more light and attracts larvae towards the tank walls due to their positive phototaxis response which may results in increased agonistic interactions and competition for food and space (Maciel and Valenti, 2014). Yasharian et al., (2005) found significantly improved survival of M. rosenbergii larval in red and green tanks than those raised in blue and white tanks. In the present study, addition of chlorella may possibly controlled overall light distribution in the rearing tank that helped in the uniform dispersal of Artemia nauplii and prawns larvae in the water column, consequently contributed proper predation activity and non-stressful environment.
M. rosenbergii larvae rearing with C. vulgaris improved the growth performances as measured by final dry weight, survival, LSI and LCI. Similar context, Lober and Zeng (2009) observed the improved growth performances for M. rosenbergii larvae reared in combination of Artemia and Nannochloropsis sp. (as green water) than without green water. Under the best experimental conditions, this study proved better growth parameters in most cases (dry weight: 0.908 mg, LSI: 24 days and LCI: 1.8) than reported by Lober and Zeng (2009) (dry weight: 0.852 mg, survival: 70.8% and LSI: 30.06 days) except survival (61 vs 71%). The lowering survival in this study could be stocking density, high stocking densities may increase competition for resources as food and space, generating, cannibalism and mortality (David et al., 2016). Density-dependent stocking of M rosenberii larvae under C vulgaries should be explored in future studies.

The present study optimized the best rearing condition of M. rosenbergii larvae under green water system. Based on the results obtained in this study ascertained that the incorporation of C. vulgaris at a concentration of 10×10⁵ cells/ml could be advantageous for the overall development of newly hatched M. rosenberii larvae. These promising results could be recommended in commercial prawn hatcheries towards sustainable eco-friendly larval production of this valued species.

The authors extend their appreciation to the Deputyship for Research and Innovation, Ministry of Education in Saudi Arabia for funding this research work through the project number IFT20134.