Contribution of natural plankton to the diet of white leg shrimp Litopenaeus vannamei (Boone, 1931) post-larvae in fertilized pond conditions

Transcription

1 FACULTY OF BIOSCIENCE ENGINEERING Academic Year Contribution of natural plankton to the diet of white leg shrimp Litopenaeus vannamei (Boone, 1931) post-larvae in fertilized pond conditions By: Huynh Phuoc Vinh Promotors: Prof. Dr. Gilbert Van Stappen (Ghent University, Belgium) A dissertation submitted in partial fulfilment of the requirements for the degree of Master of Science in Aquaculture, Ghent University, Belgium.

2 COPYRIGHT The author and promoters give permission to put this thesis to disposal for consultation to copy parts of it for personal use. Any other use falls under the limitations of copyright, in particular, the obligation to explicitly mention the source when citing parts of this thesis. Ghent, September Prof. Dr. Gilbert Van Stappen Promoter (Ghent University, Belgium). Mr. Huynh Phuoc Vinh Author i

3 DEDICATION This is dedicated to my wife, my parents, my younger sister, and my professors who always trust in me. ii

4 ACKNOWLEDGEMENTS My sincere gratitude goes to the Flemish Interuniversity Council, Vlaamse Interuniversitaire Raad (VLIR) and the University Development Cooperation (UOS) for the financial support which helped me to undertake this master s program. Great thanks send to Prof. Dr. Gilbert Van Stappen, Assoc. Prof. Vu Ngoc Ut, Mr. Geert Van De Wiele, and staff members of the department of Applied Hydrobiology, College of Aquaculture and Fisheries, Can Tho University for their guidance and support throughout the course of this research. I would like to thanks to all staffs of the Laboratory of Aquaculture & Artemia Reference Center of Ghent University for supporting me during my master study period. Thanks also to my friends and classmates for making my time at Ghent University a great experience and a lot of fun. Finally, thanks to my mother and father for their encouragement and to my wife for her love. iii

5 LIST OF ABBREVIATIONS/ACRONYMS ANOVA Analysis of variances SD Standard deviation mg.l -1 Milligrams per litre, unit of measuring salinity inds.l -1 Individuals per liter FAO Food and Agriculture Organisation of the United Nations H Hour TL Total length L or ml Liter or milliliter o C N Z1 Z2 Z3 M1 M2 M3 PL Degree celcius Nauplius (plural = nauplii) First zoea stage of larvae Second zoea stage of larvae Third zoea stage of larvae First mysis stage of larvae Second Mysis stage of larvae Third mysis stage of larvae Post larvae T1,2, and 3 Treatment 1, 2, and 3 ARC Artemia Reference Center FAME Fatty acid methylated esters HUFA Highly unsaturated fatty acids EPA Eicosapentaenoic acid DHA Docosahexaenoic acid DPA Decosapentaenoic acid ARA Arachidonic acid n-3 Omega 3 fatty acids n-6 Omega 6 fatty acids iv

6 TABLE OF CONTENTS COPYRIGHT... i DEDICATION... ii ACKNOWLEDGEMENTS... iii LIST OF ABBREVIATIONS/ACRONYMS... iv TABLE OF CONTENTS... v LIST OF FIGURES... vii LIST OF TABLES... viii ABSTRACT... ix Chapter 1: INTRODUCTION... 1 Chapter 2: LITERATURE REVIEW Overview of white leg shrimp Litopenaeus vannamei Taxonomy Morphology Habitat and distribution Life cycle Nutrition requirements Culture status in Asia On growing techniques Apply fertilizers in shrimp pond Plankton in shrimp pond Phytoplankton Zooplankton Chapter 3: MATERIALS AND METHODS Study location and system Experimental design Sampling parameters and analytical methods Water parameters Plankton samples v

7 3.3.3 Shrimp digestive system content analysis Determination of fatty acid methyl esters (FAME) of shrimp larvae Data analysis Chapter 4: RESULTS Water parameters Plankton compositions in ponds Phytoplankton Zooplankton composition Contribution of plankton in shrimp growth Plankton composition in the digestive system Shrimps growth FAME content of shrimps in the three treatments Chapter 5: DISCUSSION Chapter 6: CONCLUSIONS AND RECOMMENDATIONS REFERENCES vi

8 LIST OF FIGURES Figure 2.1: The external morphology of Penaeid shrimp (after Motoh, 1981)... 4 Figure 2.2: The digestive system of L. vannamei (FAO, 2001)... 4 Figure 2.3: Penaeid shrimp larvae stages (after Motoh, 1979)... 6 Figure 2.4: Trends in shrimp (P. monodon and L vannamei) production in Asia-Pacific from 1990 to 2014 (Figure is collated from report of FAO, 2017) Figure 3.1 Study location citied from Google maps (2017) Figure 3.2: Samples collection on the farm Figure 3.3: Some pictures of shrimp dissection processes Figure 4.1: Variation of ph (bar) and temperature (line) at 6 A.M. over time during the sampling period Figure 4.2: Variation of ph (bar) and temperature (line) at 2 P.M. over time during the sampling period Figure 4.3: Some common phytoplankton species found in the white leg shrimp ponds Figure 4.4 Percentage compositions of phytoplankton divisions found in three treatments throughout the sampling period Figure 4.5: Daily percentage of phytoplankton groups in three treatments Figure 4.6: Phytoplankton density (cells/ml -1 ) in treatments during the study period Figure 4.7: Some common zooplankton found in the white leg shrimp ponds Figure 4.8: Percentage compositions of zooplankton groups found in three treatments throughout the sampling period Figure 4.9: Daily percentage of zooplankton groups in three treatments Figure 4.10: Zooplankton density (without protozoans) in treatments during the study period Figure 4.11: Gut fullness percentages of shrimps during the sampling period (n=30) Figure 4.12: Growth of shrimp in weight in treatments during the study period vii

9 LIST OF TABLES Table 4.1 Variation of TP, TN, and salinity in ponds during the sampling period Table 4.2: Sorensen similarity indexes (Cs) of phytoplankton composition in pairs of treatments and in all three treatments Table 4.3: Sorensen similarity indexes (Cs) of zooplankton composition in pairs of treatments and in all three treatments Table 4.4: Occurrences (%) of each plankton items in shrimp digestive system over the total number of sampled shrimps larvae (n=30) during the study in all treatments Table 4.5: Percentages (%) of each plankton items over the total plankton in the digestive system of shrimps during the study in all treatments (n=3) Table 4.6: Indices of electivity of shrimps during the study in all treatments Table 4.7: Averages of wet weight, dry weight, and total length of shrimps in three treatments 41 Table 4.8: Percentages (mean ± SD; n=3) of fatty acids over the total amount of fatty acid content in white leg shrimps (Litopenaeus vannamei) in this study viii

10 ABSTRACT This study was conducted to determine the contribution of plankton to growth and live feed selection of white leg shrimp (Litopenaeus vannamei) in the intensive culture ponds under stimulating condition by applying a commercial nutrient product (as a kind of fertilizer) within the first 20 days of the culture period. In this study, the most suitable period of application of this fertilizer to promote the best growth of plankton as initial food for the newly stocked postlarvae (PL) was determined. Fertilizer was applied daily at a dose of 1 kg per 1000 m 3 during the first 10 days. Three treatments with 3 replicates each were randomly set up in 9 earthen ponds with an area of 2,000-2,500 m 2 in which commercial fertilizer was applied at different periods: (i) 4 days before stocking; (ii) 2 days before stocking and (iii) the control, in which no fertilizer was applied but PL was fed with fine pellets of commercial diet 4-5 times per day right after stocking. A total of 47 species of zooplankton belonging to 5 main groups (Protozoans, Rotifers, Cladocerans, Copepods, and a group of other taxa) and a total of 126 species of phytoplankton belonging to 5 divisions ((Bacillariophyta, Dinophyta, Chlorophyta, Cyanophyta, and Euglenophyta) were recorded in which Protozoans and Bacillariophyta were most abundant. Protozoans were also the main group which mostly contributed to the densities of zooplankton with the highest density of 46 thousand inds.l -1 whereas the highest density of phytoplankton was obtained at the level of 296 cells.ml -1. In the digestive system of shrimp, the most encountered plankton groups were nauplii of copepods, other stages of copepods, rotifers (Brachionus plicatilis), and Bacillariophyta. Analysis of digestive system contents indicated that shrimps without commercial feed had been actively selecting those zooplankton groups for their diets within the first 10 days, but not phytoplankton. Shrimps with commercial feed did not seriously choose plankton for their diets. Shrimps in treatment 1 and 2 were significantly (P<0.05) growing faster than shrimps in treatment 3 in both length and weight from day 10 onwards. The fatty acid methylated esters content of shrimp in the three treatments was not significantly different (P>0.05). In conclusion, in the ponds which were induced by commercial fertilizer, plankton species composition and abundance were higher than in the conventional ones. Applying different periods of fertilizer gave similar results. Copepods and rotifer B. plicatilis are important as initial feed for white leg shrimp in the pond during the first 10 days of stocking that improved shrimp growth afterwards. Key words: Plankton, fertilizer, white-leg shrimp, species composition, density ix

11 Chapter 1: INTRODUCTION The production of the white-leg shrimp, Litopenaeus vannamei has exceeded black tiger shrimp production since 2003 by 309,172 tons (59% of world production - FAO 2011). That was the result of the widespread of the diseases in combination with the lack of a good improvement program of the black tiger culture industry. This motivated many Asian countries such as China, Thailand, Vietnam, Bangladesh, and Indonesia to begin culturing L. vannamei. In Asia, the total farmed production of L. vannamei has increased from 1.27 million tons in 2008 to 2.30 million tons in 2014, the highest production in the region in history. The introduced species L. vannamei is now completely overtaking regional shrimp production that was dominated before by the native shrimp, P. monodon (FAO, 2017). During the first months of culture in earthen ponds, the feeding base of most shrimp larvae is composed partially of the natural food, i.e. - plankton for instance produced in the pond (Rothlisberg, 1998). Many authors have emphasized the importance of natural food items in the diet of penaeid shrimps (Tacon et al., 2002; Focken et al., 1996; Moorthy & Altaff, 2002). Natural food or live food plays a very important role in aquaculture, especially in rearing and improved extensive culture systems. Live food includes algae, zooplankton (rotifers, water flea, copepods, ) and benthos (oligochaetes, polychaetes, molluscs, small crustaceans, ). For the early stages of shrimp, zooplankton organisms are important and irreplaceable food as their size is small (size of rotifers ranges µm, water flea are µm ) and suitable for the larval to postlarval stages of shrimp. Moreover, they have high nutritional value, especially in terms of HUFAs (EPA, DHA, ARA) and essential enzymes for growth that are not met by artificial feed provision. Zooplankton organisms are thus commonly used as initial food for larval stages of many crustacean species in aquaculture (Lavens and Sorgeloos, 1996). Many studies have confirmed that zooplankton has higher nutritional value than artificial feed to meet the nutritional requirements, particularly proteins for the larval stages which require high protein contents (42% for omnivorous and 52% for carnivorous) to sustain (Tacon, 1990). Spolaore et al. (2006) suggested that in aquaculture, microalgae not only provide food for zooplanktons, stabilize and improve the quality of the culture medium, but also stimulate immunity of their consumers. The value of microalgae to the nutrition of white shrimp in aquaculture has also been documented to be substantial, and thus it is critical to determine more specifically the contribution of various essential nutrients from natural productivity to optimize diet for shrimp 1

12 (Tacon et al., 2002). Inducing live food in the shrimp rearing ponds will contribute to increase the survival rate and quality of shrimp larvae and ultimately increase the production efficiency. In shrimp culture pond conditions, the production of natural food depends chiefly on the availability of nutrients and primary productivity and it varies depending upon the hydro biological parameters and the primary producers present. Some studies on the occurrence of plankton and their succession in culture ponds for freshwater fish, prawn, and black tiger shrimp P. monodon have been done; however, such information on L. vannamei culture ponds is limited. The present study was therefore carried out to quantify the variation in the abundance and composition of plankton organisms in intensive white leg shrimp L. vannamei aquaculture ponds, with and without fertilization, and to understand the role of those plankton items in the growth of L. vannamei during the first 20 days after stocking. The general objective of the study is to evaluate the contribution of plankton in the diet of white leg shrimp in intensive grow-out ponds within the first 20 days of the stocking. We focus on the following specific objectives: I. To evaluate the effect of the period during which fertilizer is applied on the abundance and species composition of the plankton II. To determine composition and abundance of natural plankton organisms in the digestive system of shrimp in conditions with and without supplement feed during the first 10 days of the stocking III. To evaluate the growth of shrimp in two conditions (1) with commercial feed and no enhancement of natural productivity, and (2) without commercial feed in the first 10 days and enhancement of natural productivity 2

13 Chapter 2: LITERATURE REVIEW 2.1 Overview of white leg shrimp L. vannamei Taxonomy Penaeid shrimps are a group of crustaceans belonging to the largest phylum the Arthropoda. These animals have a structure called exoskeleton covering the whole body to protect all muscle and organs. White leg shrimp L. vannamei is one of over 42,000 species of the subphylum Crustacea (Hickman et al., 2006). According to Fransen and Grave (2015), the detailed taxonomic classification of white leg shrimp is shown below. Kingdom: Animalia Phylum: Arthropoda Subphylum: Crustacea Superclass: Multicrustacea Class: Malacostraca Subclass: Eumalacostraca Order: Decapoda Family: Penaeidae Genus: Litopenaeus Species: Litopenaeus vannamei (Boone, 1931) Accepted name: Penaeus vannamei (Boone, 1931) Common names: Pacific white leg shrimp; White leg shrimp; White shrimp Morphology Shrimps in the Penaeus family have a well-structured body that supports for their swimming. Their body is laterally compressed, elongate decapods, and a well-developed abdomen. Each segment (somite) is enclosed by a dorsal tergum and ventral sternum. The whole body of these species can be clearly distinguished by two parts, the head part called cephalothorax, and the tail part called abdomen. The cephalothorax is original including the head (five somites) and thorax (eight somites) fused together and completely covered by the carapace. Gills are covered by the branchiostegite that is the pleura of the cephalothorax. The appendages of the cephalothorax are modified into different forms. The prominent rostrum is a high median blade with 7-10 dorsal and 2-4 ventral teeth. The compound eyes with the sensory functions are stalked and laterally 3

14 mobile. The five somites of the head bear pairs of antennules, antennae, mandibles, maxillae 1, and maxillae 2. The thorax has three pairs of maxillipeds and five pairs of pereiopods (legs), the first three functioned in feeding, and the last two used for walking. The abdomen consists of six somites, the first five with paired pleopods (swimming legs), and the last somite connects to the telson. The anus is located on the ventral surface of the telson (Dall et al., 1990). Figure 2.1: The external morphology of Penaeid shrimp (after Motoh, 1981) Stomach Hepatopancreas Eye stalk Heart Hindgut Abdominal segment Esophagus Antenna Anus Pereopods Pleopods Figure 2.2: The digestive system of L. vannamei (FAO, 2001) 4

15 Sex determination of this species at the mature stage is quite easy due to the ventral external structures of their genital system in both male and female. The male has two pairs of modified abdominal appendages called petasma and appendix masculine on the first and second abdominal segments with the function of delivering a sperm sac or spermatophore to the female s thelycum, which is located between the bases of the fifth walking legs. This species is known as open thelycum. The open thelycum is not enclosed by the plate, which is normally happening in closed thelycum species. The spermatophore transferring process must happen when the female's exoskeleton is hard (usually within hours of spawning), and the spermatophore must stick to the female s thelycum to ensure a successful fertilization process (Bailey-Brock and Moss, 1992) Habitat and distribution The white leg shrimp live in tropical marine habitats where water temperatures are normally higher than 20 o C throughout the year. Adults move away from the coastal area, spend their life and spawn in the deep sea, while the larvae after hatching start to go inshore to the coastal estuaries, lagoons, or mangrove areas to spend their juvenile, adolescent and sub-adult stages (FAO, 2006). This species is found in waters with the wide salinity range from 1 g.l -1 up to 40 g.l -1 (Davis et al., 2004). In nature, L. vannamei naturally occurs in the Gulf of Panama ranging in the eastern Pacific from Sonora, Mexico, south to Tumbes, Peru (Perez-Farfante and Kensley, 1997). Artificial spawning of this species was first achieved in Florida in 1976, and by the 1980s, this shrimp was adapted for culture in the mainland US, Hawaii, and Central and South America (FAO, 2006). Nowadays, it is the most widely cultured shrimp in the world. It is currently raised in at least 27 countries, with major production operations occurring in the US, Mexico, Central America, tropical South America, China, India, and southeast Asia (Liao and Chien, 2011). Although the chance for them to escape from aquaculture systems by accidences is high and they are regularly found in natural areas in Asia, there have been no reports of breeding populations (Naylor et al., 2000) Life cycle As described above, adult white leg shrimp make their way offshore to clean, stable, and oceanic water. They get mature; the male inserts the spermatophore in the female s thelycum. This spermatophore can be carried with the female until the next spawning or molting. Every single 5

16 egg after spawning passes the sperm sac and is fertilized. The fertilized eggs drift with the sea currents and hatch into tiny larvae. The larval stages of white leg shrimp consist of a nauplius with five substages, protozoea (or Zoea) with three substages and three substages of mysis (Figure 2). Figure 2.3: Penaeid shrimp larvae stages (after Motoh, 1979), Nauplius (N), Zoea (Z), Mysis (M), and Post larvae (PL) Nauplius is the planktonic stage which does not look like the adult shrimp; they are almost round in shape with three pairs of appendages protruding in all directions. These animals swim briefly and then rest, which produces a zig-zag roll. The nauplii are strongly phototropic and swim in the direction of the light source. They are not feeding yet; the nutrients are supplied from their yolk sac. 6

17 Zoea phase is including three substages, in which their body is elongated; the first and second maxillipeds are well developed. The characteristics of the zoea are their continuous feeding on plankton and the long trail of feces. In the mysis stages, their appearance quite looks like the adult shrimp. The pereiopods are developed supporting swimming. The characteristics for these substages are the lowered head, backward direction movement, and less attraction by the light source. The feed is also plankton. Post larvae stage is right after the mysis substage 3 during the first 4-5 days of post larval life, they are still planktonic. Afterward, they can cling to the substrate and/or burrow in the sand. Feeding of postlarvae is strongly supported by the chelate periopods, which is able to grasp and hold food. Pleopods have a complete function in swimming. The body surface of all Crustacea including white leg shrimp is covered by an exoskeleton shell. In order to allow growth and regeneration, this shell has to be exchanged periodically during a cyclic process called molting. Normally, a full molt cycle consists of four recurrent stages: postmolt (metecdysis), inter-molt (anecdysis), premolt (proecdysis) and the moment of the shedding of the old cuticle (ecdysis) (Drach, 1939) Nutrition requirements Although some producers have been able to successfully culture white leg shrimp in pond condition, maximum growth and survival rate are seldom achieved. One of the importance reasons is nutritional requirements which usually play an importance role in the maximum growth of a species. Aquatic animals have a higher protein requirement than terrestrial animals. According to Guillaume (1999), the aquatic food web is more abundant of lipids and proteins than carbohydrates. That is responsible for the common trend of aquatic organisms to use protein as an energy source. In addition, protein is a nutrient essential for the structure and function of all living organisms including shrimp. Since the animal growth and repairing of their tissues are continually using proteins, a continuous supply of proteins or of its components amino acids is needed. D Abramo and Sheen (1994) reported that crustaceans at different ages and/or physiological stages have their own protein requirements in term of quantity and amino acid profile. In the case of L. vannamei, research of Zwilling (1981) indicated that proteins will play 7

18 its role according to the shrimp s dietary level in the range of 10%-50% and depend on the nature of the protein. The protein from the raw food seems to be better than the processes one. Another research of Kureshy and Davis (2002) showed that the maintenance protein requirement of L. vannamei shrimp was mg of dietary protein per gram of body weight per day (mg DP g BW day -1 ) for juveniles and mg DP g BW day -1 for sub-adults. Maximal growth was observed with 46 mg DP g BW day -1 for juveniles and 24 mg DP g BW day -1 for subadults, both fed 32% of dietary protein. Lipids are important nutrients for shrimp by forming a component of cell membranes and being energy reserves. D'Abramo (1989) and Vansagam et al. (2006) proposed that dietary lipids requirement by penaeid shrimp consist of neutral lipids (including essential fatty acids), sterols, and phospholipids. The essential fatty acids enhance the normal growth of shrimp (Liao and Liu, 1989). Although shrimp can synthesize some fatty acids (D Abramo, 1997), most of the lipids required come from their food. Lim et al. (1997) studied the effect of feeding various sources of dietary lipid on weight gain, feed conversion, survival, and fatty acid composition of juveniles of L. vannamei. The results demonstrated that juveniles of L. vannamei need both n-6 and n-3 HUFAs for their best growth, but n-3 fatty acids promote faster growth than n-6 fatty acids. The growth of shrimp is superior when diets contain lipids from marine source rather than the vegetable origin (Kanazawa et al., 1977). Carbohydrates (CBH) are the cheapest, primary and immediate source of energy. The digestible carbohydrate part of formulated feed can meet the energy demand of the farmed aquatic animals, and spare the use of lipids and protein as dietary energy (NRC, 2011). The efficiency of CBH utilization by aquatic animals depends on the nature and complexity of carbohydrates (Bergot, 1979). The monosaccharides are much easier to digest in comparison to complex carbohydrates, which require enzymatic hydrolysis for digestion (Rosas et al., 2000). Many studies about the digestion of carbohydrates in L. vananmei shrimp have been conducted. Cousin et al., (1993) have done a study showing that juveniles and sub-adults of L. vannamei fed at high starch levels around 40% gave an apparent digestive coefficient that reflected an increase in digestibility in compared to lower starch levels. Starch has been shown to be digested either raw or pre-cooked, and this digestive property represents an interesting aspect in relation to the amount of starch that can be used in the diet of L. vannamei. Best results were attained with standard wheat starch, and this is commonly the main starch source in shrimp feeds. 8

19 Vitamins are organic compounds necessary for normal growth, reproduction and health of aquatic animals; the animals have to pick them up from exogenous sources (NRC 2011). According to Lim and Akiyama (1995), there are thirteen vitamins essential for shrimp, including three fat-soluble vitamins (vitamin A, D and E) and ten water-soluble vitamins (thiamine, riboflavin, pyridoxine, nicotinic acid, biotin, folic acid, vitamin B12, inositol, choline and vitamin C). The most applied vitamin in aquaculture is vitamin C (ascorbic acid, AA). Merchie et al., (1995) conducted a study on the application of boosting techniques using ascorbyl palmitate (AP) as the vitamin C source enabled the transfer of elevated levels of bioactive vitamin C via the live food (Artemia) into L. vannamei larvae. The highest survival of shrimp was observed at 2000 mg.l -1 AP and lower survival rates for those fed at lower AP concentrations. In shrimp, essential minerals may be obtained from the water exchanged across the gill membrane or ingestion and by absorption across the gut. Nevertheless, it is generally considered that a dietary source of some minerals for growth is necessary because of the repeat of losses of certain minerals during molting. Minerals are not only essential components of hard tissues, soft tissues, vitamins, enzymes, hormones and respiratory pigments but also required for the maintenance of osmotic pressure and acid-base balance (Lim and Akiyama, 1995). At low salinity, L. vannamei would have higher dietary magnesium (Mg) or potassium (K) requirement, although marine species reared in seawater do not require dietary sources Mg and K (Davis et al., 2002). In addition, Davis et al., (1993) indicated that L. vannamei does not require a dietary calcium supplement; and the dietary phosphorus requirement depends upon the calcium content of the diet. In the absence of calcium supplement, 0.35% of phosphorus in the diet is enough to maintain good growth and survival of this shrimp Culture status in Asia The total farmed production of L. vannamei in Asia has increased from 1.27 million tons in 2008 to 2.30 million tons in This was the highest production in the region in history. With the contribution considerably of research into genetics and the application of genetics, seed quality and quantity of L. vannamei in Asia-Pacific has been improving, especially, the domesticated specific pathogen free (SPF) post-larvae to avoid several important diseases during culture period which now play important roles in the continued increase in regional aquaculture 9

20 production. The introduced species L. vannamei is now completely overtaking regional shrimp production that was dominated before by the native shrimp, P. monodon (FAO, 2017). Figure 2.4: Trends in shrimp (P. monodon and L. vannamei) production in Asia-Pacific from 1990 to 2014 (Figure is collated from report of FAO, 2017) The main L. vannamei producers in Asia are China, India, Vietnam, Thailand, Indonesia and Bangladesh. In Vietnam, according to the ministry of agriculture and rural development of Vietnam (2017), in 2016, the total culture area of L. vannamei in Vietnam was thousand ha, increased 11.5% in comparison to that in 2015; and the total production was thousand tons, increased 15.6%. In the first 6 months of 2017, the estimated culture area and total production of white leg shrimp were around 45 thousand ha and 80 thousand tons, respectively. Despite the expressive growth of farming industry in the last decades, several pathogenic diseases have been reported to cause substantial economic losses. To date, more than twenty viral diseases have been reported to affect shrimp and prawns, and five viral pathogens of penaeid shrimp are currently listed by the World Organization for Animal Health (2009). Besides, the slow growth and high consumption of feed leading to massive increases in the cost of production are also challenging the farmers. 10

21 2.1.7 On growing techniques Depending on the stocking density at low, medium, high and extremely high, ongrowing techniques can be sub-divided into four main categories including extensive, semi-intensive, intensive and super-intensive, respectively (FAO, 2006). Extensive is a natural base method, commonly found in Latin American countries, and then spread to other countries. The extensive grow-out of L. vannamei is normally conducted in tidal areas where minimal or no water pumping or aeration. Huge ponds with areas of 5 10 ha (up to 30 ha) and m deep are usually used, but the shape is irregular. Originally, wild seeds enter the pond tidally through the gate or are supplied through purchasing from collectors or hatchery. The stocking density is low around 4 10 inds/m². Shrimp food sources mainly come from natural environment through fertilization activity and a limited amount of low protein feed. Shrimp is harvested once or twice per year at the size of g. The yield in these extensive systems is very low kg/ha/crop. Semi-intensive ponds are typically smaller than extensive ponds (1 5 ha), but the stocking density with hatchery-produced seeds is higher at inds/m². Pond depth is m. Water management and aeration are applied at best minimal. Along with the natural foods enhanced by fertilization in the pond, the supplemented by formulated diets are supplied 2 3 times daily. Production yields in semi-intensive ponds range from kg/ha/crop; and that could be with 2 crops per year. Intensive farms are commonly difference to two farming methods above. The shrimp ponds are good management. Ponds can be completely drained, dried and prepared before each stocking, and are increasingly being located far from the sea in cheaper and low salinity areas. This culture system nowadays is worldwide distribution, common in Asia and in some Latin American farms with high productivity. Ponds are often earthen. Liners are usually used to reduce erosion and enhance water quality. Small ponds ( ha) are often used with the water level higher than 1.5 m. This culture method has high stocking densities range from inds/m² combined with aeration to support for water circulation and oxygenation. Artificial feed is carried out 4 5 times per day. FCRs are This system requires carefully monitoring and management on feed, water exchange/quality, aeration and phytoplankton blooms to enhance the growth and avoid the outbreak of the disease. Production yields of 7,000 20,000 kg/ha/crop, with 2 3 crops per year can be achieved, up to a maximum of 30,000 35,000 kg/ha/crop. 11

22 Many studies conducted in the United States of America have focused on growing L. vannamei in super intensive raceway systems enclosed in greenhouses, using no water exchange (only the replacement of evaporation losses) or discharge, stocked with pathogens free PL. They are thus bio-secure, eco- friendly, have a small ecological footprint and can produce cost-efficient, highquality shrimp. The culture area much smaller and higher stocking density with higher than 200 inds/m3 give production level above metric tons/ha/year. 2.2 Apply fertilizers in shrimp pond The application of fertilization programs for chemical fertilizers and manures will increase the primary productivity by phytoplankton and finally increasing the cultured animal production. That could be 2- to 10-fold depending upon species and the natural fertility of water (Boyd, 1990). Fertilization is seldom used alone in shrimp ponds; it is used to enhance the production of natural food organisms for shrimp, along to the primary source of nutrient the feed. The shrimp farmers reported that shrimp production is better if there is a stable and relatively high abundance of phytoplankton. The fertilization programs are in common use in shrimp farming nations because they are effective. Most of the shrimp farmers want a plankton bloom in their ponds. For example in semi-intensive farming, the amount of applied feed for a grow-out period normally do not exceed 30 kg per ha daily in the final weeks. Therefore, fertilizers usually apply in the first half of the growth out period to boost the phytoplankton growth; and the density of phytoplankton above 300,000 to 400,000 cells.ml -1 is normally considered sufficient (Boyd, 1993). The fertilizers application in ponds is not continuously, but at intervals when the turbidity is low or low density of phytoplankton cells in ponds. Boyd (1993) also conducted a research that applied fertilization in shrimp pond with the purpose of increasing the phytoplankton and benthos production, especially the production of diatom. The results showed that shrimp production often reaches 1,000 to 2,000 kg per ha with feeding or feeding plus fertilizers, while kg per ha is the production of shrimp in the fertilized ponds. In addition, a high N:P ratio (30:1 or 15:1) can stimulate the production of diatom in shrimp ponds and also the proportion of diatoms in the phytoplankton community (20 to 30% of the total phytoplankton cells). Moreover, Daniels (1989) supported that composition of silica in the fertilizers can increase the growth of diatoms in brackish water ponds. Besides chemicals fertilizers, manure is sometimes applied to shrimp ponds. Amount vary greatly, but the usual amount of applied fresh manure rarely over 250 kg per ha per week. The 12

23 production of shrimp in ponds used manure is not difference to that in ponds applied chemical fertilizers; and the more manure supplied, the greater shrimp production than chemical fertilizers. However, the amount of manure cannot be increased forever due to its decomposition characteristic and cause oxygen depletion. The ponds with a high amount of manure application should be aerated and exchanged water of 20% pond volume per day (Wyban et al., 1987). In the heavily feed supplied, high production, intensive ponds, the large part of nutrients entering the ponds through the uneaten feed and the feces of shrimp would cause plankton blooms, so the application of fertilizers are not often used in these ponds. However, to increase the number of beneficial algae diatom in shrimp ponds, farmers sometimes apply the high nitrogen fertilizers periodic to increase the proportion of diatom the plankton community. Furthermore, the nitrogen fertilizer can enhance the decomposition of organic matter (Boyd, 1993). Therefore, they often apply in a high concentration of undecomposed materials ponds or in the bottom of the pond when ponds are dry between crops. 2.3 Plankton in shrimp pond Phytoplankton According to Pronob et al. (2012), most of the algae are aquatic; they are bearing chlorophyll and being in nature in unicellular or multi-cellular plants. Besides chlorophyll, some algae content various carotenoid pigments which make them having different in colors. That was used to classify them into three divisions such as Chlorophyta (green algae), Phaeophyta (brown algae) and Rhodophyta (red algae). Brown and red algae are mostly marine species meanwhile green algae are often available in freshwater. Green algae are considered as the primary producers in the food chain in both marine and fresh water ecosystems. Microalgae normally have greater contribution in the extensive and semi-extensive culture ponds where the total shrimp production range from 500 to 6,000 kg per ha per crop than in the intensive culture ponds where the total production is often higher than 7,000 kg per ha per crop; that because of a high shrimp biomass in the intensive culture ponds that consume higher amount of supplying feed (D Abramo and Conklin, 1995). Muller-Feuga (2000) also mentioned that microalgae comprise important parts of the natural productivity of the various aquatic systems play a vital role in the rearing of aquatic animals in which they keep balance and enhance water quality parameters (i.e., water quality, dissolved oxygen, ammonia, alkalinity and ph). Some researchers (Castille and 13

24 Lawrence, 1989; Lawrence and Houston, 1993; Tacon, 1996) stressed that the limited of phytoplankton levels in the shrimp culture ponds will lead to a negative effect on the water quality in the culture system which could be due to the fluctuation of oxygen and carbon dioxide balance; and lacking of microalgae in pond could also be a change for benthic algae to grow which have not much contribution in shrimp pond; thus phytoplankton not only contributes nutrients but also stabilizes the culture system thereby optimizing production costs. Besides good algae, high nutrient level or eutrophicated condition in ponds may cause abundant of some genera of phytoplankton, such as Microcystis, Anabaena, Nostoc and Aphanizomenon, which could lead to problems with hypoxia, toxins and changes in the structure of biological communities (Carmichael, 2001; Chen et al., 2008). Phytoplankton growth in ponds is typically stimulated by nutrient available. Thus understanding the relationship between nutrient concentration and the growth of algae can effectively help in the eutrophication management. Phosphorus (P) and Nitrogen (N) are often considered as the principal limiting nutrients for aquatic algal production. Boyd (1993) reported that the diatoms are preferred by most of the brackish water shrimp culture managers and it can be stimulated by an N:P application ratio of 20:1. On the other hand, the nutritional value of microalgae is varied significantly by species and by different culture conditions (Enright et al., 1986; Brown et al., 1997). Dried algae are composition of 90-95% of protein, carbohydrate, lipids, minerals and vitamins (Brown et al., 1989); and at late-logarithmic growth phase, they typically contain 30 to 40% protein, 10 to 20% lipid and 5 to 15% carbohydrate (Brown et al. 1997). Enrich et al. (1986) and Brown and Jeffry (1992) reported that the optimum nutritional value of microalgae, as aquaculture feed species, is very much influenced by their fatty acid composition of the lipids and, in a lesser extent, by the amino acid composition of the proteins and the composition of the carbohydrates Zooplankton In shrimp pond condition, zooplankton is comprised protozoans, rotifers and the planktonic forms of crustaceans, cladoceran, ostracoda and copepods and their larvae. A study of Cardozo et al. (2007) on the composition, density and biomass of zooplankton in the L. vannamei culture ponds in southern Brazil showed that copepods and cladocerans were the most abundant groups; They suggested that zooplankton grow well in the shrimp ponds and represents a potential food source for the shrimp during the first month of culture. Zooplankton is often required as a first food for many aquatic species; for others it contributes to faster growth and higher survival due 14

25 to its high nutrient content, small in size, and easy digestible; they represent an important food item for the growth of post-larval shrimp during their first days of culture (Anderson et al., 1987; Chen and Chen, 1992). Some zooplanktons are also very useful water quality indicators for the shrimp culture farms. They displayed fairly distinct patterns in the species composition and abundance as the water quality changed spatially. This may be attributed to the fact that the zooplankton community itself responds directly or indirectly to changes in the physicochemical variables and the availability of phytoplankton food (Raymont, 1980), and is therefore less affected by manipulation via farm management processes. A research of Brucet et al. (2010) showed that rotifers and small-sized crustaceans as copepods are the domination of small organisms in the brackish water. Furthermore, Preston et al. (2003) illustrated that the high concentration of chlorophyll a, detritus, uneaten feeds and phytoplankton density in the culture ponds most of the time leads to the abundance of zooplankton. That was in agreement with the study of Barbieri and Ostrensky (2002) which also presented the increased of phytoplankton productivity due to fertilization activities will contribute to the increased in the production of zooplankton in the culture ponds. Grazing zooplankton influences the dynamics of pond phytoplankton (Coman et al., 2003); then the predation on zooplankton by shrimp (Martinez-Cordova et al., 1998) may transfer a significant proportion of the nutrients from natural biota to the shrimp tissue (Anderson et al., 1987), feces, and water. This, together with the nutrient input through shrimp rations, normally cause the nutrient rich environment in shrimp ponds. The outranked of protozoans ciliate, Tintinnopsis spp. and Favella ehrenbergi could be characteristic of this eutrophic systems (Pierce and Turner, 1993). The Protozoans have fast reproduction rates, and short generation times; they also have a high capacity to use a large spectrum of food resources (Urrutxurtu, 2004). Moreover, rotifers are a valuable live food for larval shrimp culture and have also been used as indicators of the trophy (Saksena, 1987). Several characteristics of rotifers, including their nutritional quality, body size, and relatively slow motility, have contributed to their usefulness as good prey for active larvae (Snell and Carrillo, 1984). On the other hand, copepods, other crustaceans, larvae of polychaetes, larvae of insects, molluscs, and ostracods have also been considered as the important sources of food for shrimp in pond condition (Rubright et al., 1981). Nowadays, many studies have stressed that copepods have a high nutritional value which accepted to be very good for shrimp larvae due to their high protein content (44-52%) and a good amino acid profile (Lavens and Sorgeloos, 1996). 15

26 Chapter 3: MATERIALS AND METHODS 3.1 Study location and system The study was carried out from 1 st February to the end of May The samples were collected from the intensive white leg shrimp grow-out ponds at Long Vinh commune, Duyen Hai district, Tra Vinh province, Vietnam. The samples were then transferred to the laboratory which is located in the Department of Hydrobiology, College of Aquaculture and Fisheries at Can Tho University, Vietnam for further analytical steps. On the other hand, the shrimp fatty acids content analysis was conducted in the Laboratory of Aquaculture & Artemia Reference Center of Ghent University, Belgium. The study system included 9 ponds which have an area from 2,000 to 2,500 m 2 each. Figure 3.1: Study location (cited from Google maps, 2017) 3.2 Experimental design The study was conducted to determine the contribution of natural plankton in ponds to the diets of shrimp during the first 20 days after stocking. At the beginning, the ponds were well prepared by the farmers. The preparation procedure included drying the pond bottom, liming, adding filtered brackish water which was at salinity level from 15g.L -1 to 18 g.l -1 and treating water by applying a disinfectant product namely Iodine 99 with a concentration of mg.l -1. Two days later, water parameters as ph, NO - 2, NH + 4, and salinity were checked by using test kits and 16

27 a salt meter. According to the farmer, water quality parameters ph around 8, NO - 2 concentration smaller than 5 mg.l -1, NH + 4 concentrations smaller than 1mg.L -1, and salinity g.l -1 should be good to start a new crop. Otherwise, they have to wait for a longer time or take new water and do everything again. Fertilizer was applied to stimulate the growth of primary producer in the ponds, then the zooplankton. The chosen product was the SUPA-stock, a product of Bayer Company. This study has three treatments with different fertilization applications. In the first treatment, the fertilizer was applied daily from four days before stocking up to day 10 after stocking with the dose approximately 1 kg per 1000 m 3. Shrimp was fed by a commercial fine pellet feed 4-5 times per day after 10 days of the stocking. Treatment 2 had the same application and management procedure as treatment 1, but the only difference was the fertilization period, in which fertilizer was applied two days later than in treatment 1. The last treatment (control treatment) was not fertilized, but shrimp was fed right after stocking. Each treatment had three replications. The treatments and the replications were randomly arranged within these nine ponds. Besides, the farmer also supplied probiotic and mineral products to provide essential minerals and maintain good water environment for shrimp. 3.3 Sampling parameters and analytical methods A B C Figure 3.2: samples collection on the farm: (A) collecting zooplankton quantitative samples; (B) collecting qualitative samples; (C) shrimp sampling Water parameters Temperature and ph were measured twice a day at 6 a.m. and 2 p.m. by using a pocket Hanna ph & temperature meter at ponds. Other water parameters as salinity, total nitrogen (TN), and total phosphorus (TP) were sampled every three days at 6 a.m. by taking a water sample into a 17

28 1L bottle and stored at 4 o C awaiting their analysis in the laboratory. TN was analyzed by the digestion and phenate method (Kjeldahl, 1883). TP was analyzed by digestion and SnCl 2. Method (Kjeldahl, 1883). Salinity was measured by a salt meter Plankton samples Plankton samples of phytoplankton and zooplankton were taken every morning using a phytoplankton net with the mesh size of 30 µm and a zooplankton net with mesh size of 60µm. The qualitative samples of phytoplankton and zooplankton in the ponds were collected by drawing the nets along two sides of the ponds. The concentrated samples were then stored in 110 ml bottles and fixed with formalin of 4-6%. The quantitative samples of algae were collected by using the settling method in which water was taken at different points in the pond into 20L buckets and then mixed well before taking a subsample into a 1L bottle. Every sample was fixed with formalin of 4-6%. The quantitative samples of zooplankton were collected by using a filtering method in which 5 buckets of 20L of water taken from 5 points (4 at the corners and 1 at a random point of the pond) were poured through the zooplankton net to be concentrated in a 110mL bottle. Then the sample was fixed with formalin of 4-6%. In the laboratory, phytoplankton and zooplankton qualitative samples analysis was done by pipetting 0.1 ml of the settled material in the bottom of the sample bottle to the slide, and doing observation under the microscope at magnification of 40x, 100x and 400x depending on sizes and characteristics of each species; the plankton species were identified based on the available taxonomic key (Shirota, 1966). Zooplankton species were identified based on available taxonomic keys (Shirota, 1966; Boltovskoy, 1999; Khoi, 2001). These actions were repeated until no more new species were observed. Phytoplankton quantitative sample analysis in which the sample was concentrated using a small tube which was covered by a piece of phytoplankton net to remove the top water layer and to keep the settled material in the bottom of the sample bottle. The volume of the sample after concentrating was then measured by using a graduated cylinder. Densities of algae were determined by using Sedgewick-Rafter counting cells. Before counting, the sample was shaken carefully; then 1 ml of sample was transferred to the counting chamber. A total of thirty cells of the chamber were counted and the number of algae according to their phylum (Bacillariophyta, Dinophyta, Chlorophyta, Cyanophyta, and Euglenophyta) was recorded. A total of 180 cells 18

29 were done by repeating the processes five times. The density of each phylum was determined by the formula below: T * V c * 1000 Y = N * V s Where: Y: Density (cells per milliliter - Cells.mL -1 ). T: Number of counted algae by phylum V c : Volume after concentrating (ml) N: Number of counted cells (180 cells) V s : Volume of sample (ml) Zooplankton quantitative sample was analyzed similarly to the phytoplankton quantitative sample analysis. The densities of zooplankton groups (rotifers, copepods, protozoans, etc.) were also determined by using the Sedgewick-Rafter counting cells to count the number of animals under the microscope at the magnification of 10x. The density of each group was determined by the formulation below: T * V c * 1000 P = * 10 3 N * V s Where: P: Density (individuals per liter inds.l -1 ). T: Number of counted individuals by their group V c : Volume after concentrating (ml) N: Number of counted cells (180 ô) V s : Volume of sample (ml) 19

30 Similarity indexes (Cs) were conducted for comparing the species similarity among two treatments or three treatments in this study. The widely used Sorensen s similarity index (Magurran, 2004) measuring the similarity in species composition for two sites (A and B) and an extending of Sorensen s similarity index formulation (Ola and Frode, 2007) for three sites (A, B and C) were used for this purpose. The formulations for these two are Cs = 2ab / (a + b) and Cs = (ab + ac + bc - abc) / (a + b + c), with a, b and c the numbers of species found in sites A, B and C, respectively, and ab the number of species shared by sites A and B, etc., until abc which is the number of species found in all three sites Shrimp digestive system content analysis Shrimp specimens were collected before applying fertilizer in the ponds every day onward until day 10, and then final shrimp sample was taken on day 20. They were fixed with formalin 8% right after collection. A total of 30 shrimps for each sample were collected for two purposes of digestive system content analysis and fatty acids content analysis of shrimp. The digestive system content of shrimp was determined by the dissection process and the observation process. The dissection process had done in which ten shrimp from each sample were randomly picked out; the total length of shrimp was measured according to the procedure described in Kitani (1986). That is from the base of the antennal flagellum to the tip of the telson. The measurements were done under the stereomicroscope at magnification from 12x to 25x depending on the size of shrimp and a ruler (cm) which was attached to the Petri disk. The gut fullness ratio was measured by microscopically observation at the same time with length measurement, and the gut fullness ratio in percentage was determined by observing the ratio of the length of the gut part which contains materials over the total length of the gut. This ratio was used to understand the feeding situation of the shrimp sample. On another side, the gut content of shrimp was basically digested, and thus mostly unidentifiable. Therefore, dissection of the full digestive system was needed to be done, not only the gut. Before dissecting, shrimp was cleaned by using distilled water to remove the materials attached to the shrimp body. Then the shrimp was transferred to a slide with a small amount of distilled water or filtered seawater to avoid other plankton or suspended matters to get into the sample. Under a stereoscopic microscope, the whole digestive system from the mouth to the stomach and finally to the gut of the shrimp was separated from their body by using two dissecting needles or tweezers. The rests of muscle and 20

31 shell were removed out of the slide. In case the digestive system was broken due to the dissection activity, the removed part was washed by one drop of distilled water to decrease the ability of plankton attaching on the removed part. Most tissues were carefully removed as much as possible using a dissecting needle, as there is a possibility of mistaking the fragment of tissue as stomach contents. Under the binocular stereoscopic microscope, the hepatopancreas which covers around the beginning of the gut was removed by two dissecting needles or tweezers. Then the stomach and gut were separated to two sides of the slide. The stomach was opened and all of its content was removed. The rest of the stomach was washed by one drop of distilled water and removed from the slide. Then the gut was also opened and combined with the material from the stomach. Often the diet contents of shrimp are destroyed by the mandible and spines of the stomach inner wall. The agglomerated stomach content was unravelled slowly and gently so as not to destroy it. When the stomach was dissected in water, the content was relatively easy to diffuse. The slide was transferred to a microscopy; and the content was identified under the magnification of 100x and/or 400x and measured width and length by the eyepiece micrometer for the further calculation. The identified prey items were with a focus on the zooplankton and phytoplankton, which was identified up to the genus or family level. The artificial feed content and other materials in the digestive system of shrimp were not considered in this study. The species composition and densities of algae and zooplankton in the intestine of shrimp were determined by the commonly used method of frequency and size of feed in the intestine. The dietary content was recorded using the following methods: The occurrence method (Hynes, 1950): Each occurred food item is listed as a percentage of number of shrimp in which that food item is found, over the total number of shrimp examined. The following steps had been done: Step 1: Each food item of each shrimp is marked in the list of occurring food items. Step 2: The number of shrimp for each available food item is counted. Step 3: The ratio of shrimps (expressed as a percentage) with a specific food item to the total number of sampled shrimps was calculated to express the occurrence of that food item by the following formulation: 21

32 J i O i = * 100% P Where: O i : Occurrence (%) of a food item (i) in the digestive system of shrimp P: Total number of sampled shrimps J i : Total number of shrimp with food item (i) available in the digestive system. The points method (Hynes, 1950): The list of occurred food items as the previous method was used for this method as well. The size of the organisms, as well as their abundance, was taken on the basis of rough counts and judgement by eye and by their volume which calculated by their width and length of above measurement (i.e. one large organism counted as much as a large number of small ones). Each category was then allotted a number of points and all the points gained by each food item were used to calculate the percentage of that food item in the total amount of identifiable food in the digestive system of shrimp. Step 1: Each food item was allotted points. The smallest occurring food item was allotted one point (called a). Other food items were allotted points according to that smallest food item. Step 2: The points of item (a) were multiplied according to the number of occurrences (n) in the digestive system of one shrimp, and the result is called A. (A i = n i x a i ) Step 3: A is multiplied by the frequency (f) of that food item in the number of shrimp with food available in the digestive system and the results are called B). B i = f i x A i Step 4: The total of all B values of all food items is calculated as follows: B = B i + B ii + B iii B n Step 5: The percentage of each food item is calculated as B i / B, expressed as percentage Indices of electivity: the degree of selection of the prey items as found in the diet was expressed as the index of electivity E, calculated by the formulation of Ivlve (1961): (r i p i ) E = (r i + p i ) 22

33 Where: r i : percentage of a food item (i) in the total number of the food items available in the shrimp digestive system p i : percentage of that food item (i) in the total number of food items available in the water environment The E value oscillates from -1 to +1. A positive E value shows the selection of shrimp on that food item, a negative E value shows the non-selection of shrimp on that food item, and an E value equal to 0 means that the availability of that food item in the shrimp digestive system was by accident. A B C A part of gut D A part of gut Stomach Figure 3.3: some pictures of shrimp dissection process: (A) dissecting; (B) full shrimp before dissection; (C) separating the gut; (D) Digestive system parts after removed all other tissues 23

34 3.3.4 Determination of fatty acid methyl esters (FAME) of shrimp larvae Shrimp samples were weighed for the wet weight and dried weight (dried at 100 o C for 4 hrs). Before FAME analysis, dried shrimps in each sample were homogenized by using a pounder. A Modified method based the improving recovery of fatty acids through direct transesterification without prior extraction, as formulated by Lepage and Roy (1984), was followed by the FAME analysis. 3.4 Data analysis Data processing and counting of the average numbers and the standard deviations were done using Microsoft excel The one-way analysis of variance ANOVA method was applied to compare means of data (the water parameters, FAME data, and shrimp growth) in the three treatments at a sampling day by using Minitab 16.0 software, at a significant level of

35 Chapter 4: RESULTS 4.1 Water parameters Water temperature ( O C) and ph during the days sampled were not statistically different (P<0.05) among three treatments in both the morning and the afternoon (Figure 4.1 & 4.2). Temperature levels in the morning ranged from 25.0 o C (in treatment 1) to 28.5 o C (in treatment 2) whereas temperature was from 27.8 o C (in treatment 1) to 31.1 o C (in treatment 2) in the afternoon. Levels of ph in all treatment were not much fluctuating on over the time and between morning and afternoon. The lowest and highest ph levels recorded in the morning were 8.0 in treatment 1 and 8.6 in treatment 3; the lowest and highest ph levels recorded in the afternoon were 8.0 in treatment 1 and 8.7 in treatment ph Temperature( o C) T1 T2 T3 T1 T2 T * 2* 3* 4* Sampling period Figure 4.1: Variation of ph (bar) and temperature (line) at 6 A.M. over time during the sampling period (data in the graphs is mean ± SD; n=3; the days with * symbols belong to the before stocking phase) ph Temperature( o C) T1 T2 T3 T1 T2 T3 1* 2* 3* 4* Sampling period Figure 4.2: Variation of ph (bar) and temperature (line) at 2 P.M. over time during the sampling period (data in the graphs is mean ± SD; n=3; the days with * symbols belong to the before stocking phase) 25

36 The variations of TP, TN, and salinity during the sampled period are presented in table 4.1. The results showed that treatment 3, which was not applying fertilizer had higher levels of TP and TN at the beginning of the study in comparison to the other two fertilized treatments and always higher than the other two in the next sampling times. The TP and TN levels in treatment 3 ranged from 10.3 mg.l -1 to 13.9 mg.l -1 and from 5.1 mg.l -1 to 11.9 mg.l -1, respectively. However, TP and TN values were not significantly different among treatments during the study at most of the sampling times, except for TP values at day 4 where the TP level of treatment 3 was significantly higher than the other two treatments. The two fertilized treatments (treatment 1 and 2) showed an increasing trend of TP and TN levels, in which TP values at treatment 1 ranged from 2.6 mg.l -1 to 8.6 mg.l -1, TP values at treatment 2 ranged from 3.0 mg.l -1 to 8.0 mg.l -1, TN values at treatment 1 ranged from 2.0 mg.l -1 to 9.8 mg.l -1, and TN values at treatment 2 ranged from 0.8 mg.l -1 to 7.9 mg.l -1. Salinity levels of the three treatments were not significantly different; differences in salinity levels were due to taking difference sources of water at the beginning. The salinity levels of all treatments were rather stable. The salinity difference within pond over time was less than 2 g.l -1. The highest salinity levels of treatment 1, 2, and 3 were 18.7 g.l -1, 17.7 g.l -1, and 15.0 g.l -1, respectively. Table 4.1 Variation of TP, TN, and salinity in ponds during the sampling period Sampling period 1* 3* TP (mg.l -1 ) T1 2.6± ±0.7 a 7.7±2.0 a 6.6±0.9 a 8.6±2.0 a 8.4±1.4 a 8.4±0.7 a T2 3.0±0.5 a 7.6±1.8 a 7.9±1.3 a 8.0±2.2 a 8.0±1.6 a 7.8±1.5 a T3 11.7±3.7 a 10.3±1.7 b 10.7±1.7 a 13.9±4.5 a 8.9±3.7 a TN (mg.l -1 ) T1 2.0± ±1.4 a 4.0±0.4 a 4.2±2.2 a 9.8±0.8 a 8.8±2.6 a 6.4±2.2 a T2 0.8±0.2 a 2.8±0.2 a 4.3±0.6 a 7.9±0.4 a 4.9±1.7 a 5.7±1.5 a Salinity (g.l -1 ) T3 5.1±2.4 a 7.0±3.9 a 10.1±5.1 a 11.9±6.0 a 9.7±2.6 a T1 18.3± ±2.9 a 18.3±2.9 a 18.0±3.5 a 18.3±2.9 a 18.7±4.9 a 17.7±4.0 a T2 17.0±2.6 a 17.3±2.5 a 17.3±2.5 a 17.7±2.5 a 17.0±2.6 a 17.3±2.5 a T3 14.0±1.0 a 14.3±0.6 a 14.3±0.6 a 14.0±1.0 a 15.0±1.0 a All values are mean ± SD (n=3). Values with different superscripts in a column of each parameter differ significantly (P<0.05). The days with * symbols belong to the before stocking phase. 26

37 4.2 Plankton compositions in ponds Phytoplankton A total of 126 species, representing five divisions of algae (Bacillariophyta (or Diatom), Dinophyta, Chlorophyta, Cyanophyta, and Euglenophyta), were identified from the treatments throughout the study period. The highest species diversity was found in treatment 3 with 110 species followed by treatment 1 with 108 species and treatment 2 with only 90 species. Overall, the most commonly observed genera were Amphora, Amphiprora, Coscinodiscus, Navicula, Nitzschia, Thalassiosira, and Pleurosigma belong to group of Bacillariophyta; Alexanderium, Goniodoma, Gymnodinium, Peridinium, and Sphaerodinium belonging to the group of diatoms; and Chlorella, Oscillatoria, and Euglena being the most common genera of Chlorophyta, Cyanophyta, and Euglenophyta, respectively. a b c d e f g h i Figure 4.3: Some common phytoplankton species found in the white leg shrimp ponds (a) Melosira sp. (b) Thalassiosira sp. (c) Pleurosigmasp. (d) Coscinodiscus sp. (e) Goniodoma sp. (f) Peridinium sp. (g) Ceratium sp. (h) Oscillatoria sp. (i) Phacus sp. The Bacillariophyta appeared to be the predominant algae division followed by Dinophyta. The other three algae divisions had very small percentages of the total species. The phytoplankton compositions in percentage are shown in figure 4.4 below. 27

38 Treatment 1 Treatment 2 Treatment 3 4% 3% 3% 3% 4% 6% 3% 3% Bacillariophyta 15% 17% 30% Dinophyta Chlorophyta 75% 70% 64% Cyanophyta Euglenophyta Figure 4.4 Percentage compositions of phytoplankton divisions found in three treatments throughout the sampling period 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% Treatment 1 Treatment 2 Treatment 3 0% 1* 2* 3* 4* * 2* 3* 4* * 2* 3* 4* Sampling period Sampling period Sampling period Euglenophyta Cyanophyta Chlorophyta Dinophyta Bacillariophyta Figure 4.5: Daily percentage of phytoplankton groups in three treatments (the days with * symbols belong to the before stocking phase) The numbers of phytoplankton species found daily in the three treatments ranged from in treatment 1, in treatment 2, and in treatment 3. Bacillariophyta and Dinophyta 28

39 groups always had very high percentages in the ponds. The combined percentages of these two groups are always higher than 86% in treatment 1, 76% in treatment 2, and 84% in treatment 3 all over the time of the study (figure 4.5). The similarity indexes for pairs of treatments and for all three treatments were calculated to measure similarity in species composition for these treatments by using the Sorensen s equations. The Sorensen similarity indexes are shown in table 4.2. The results demonstrated that all of the indexes are smaller than 0.5 which means that the similarity of these pairs of treatments and these three treatments were low. The similarity indexes between treatment 1 and 2 were lowest (0.2<Cs<0.3) during the sampled period. The highest similarity indexes were found in the pair of treatment 2 and 3 with Cs ranging from followed by the pair of treatment 1 and 3 with Cs ranging from over the study period. When comparing the similarity of all three treatments, higher similarity indexes were observed ( ) than when comparing pairs of treatments. The shared similar phytoplankton species of three treatments in this study was always less than half of the total number of species observed on each sampling day. Table 4.2: Sorensen similarity indexes (Cs) of phytoplankton composition in pairs of treatments and in all three treatments Sampling period T1-2 T1-3 T2-3 T T1-2, T1-3, and T2-3 are comparing between treatment 1 and 2, treatment 1 and 3, and treatment 2 and 3, respectively. T1-2-3 is comparing three treatments. 29

40 The fluctuations of phytoplankton densities in treatments are presented in figure 4.6. Phytoplankton showed very low density at all treatments with the initial densities around 9 cells.ml -1 in treatment 1 on day 1*, 18 cells.ml -1 in treatment 2 on day 3*, and 76 cells.ml -1 in treatment 3 on day 1. The densities of phytoplankton in three treatments increased quickly from day 1, especially in treatment 3. The highest densities recorded in treatment 1, 2 and 3 were 287 cells.ml -1 on day 8, 296 cells.ml -1 on day 7, and 469 cells.ml -1 on day 5, respectively. Later in the study period, the phytoplankton densities in all treatments decreased but still maintained a density around 100 cells.ml -1. The growth in density of phytoplankton in treatment 3 was not that much fluctuating as in the treatments 1 and 2. Density T1 T2 T3 1* 2* 3* 4* Sampling period Figure 4.6: Phytoplankton density (cells/ml -1 ) in treatments during the study period; data in the graph is the mean values (n=3); the days with * symbols belong to the before stocking phase Zooplankton composition The diversity of zooplankton did not differ markedly between treatments in the present study. The major groups of zooplankton encountered were protozoans, copepods (copepodites and nauplii), rotifers, cladocerans, and the group of other taxa in the investigated ponds. Protozoans and copepods were the dominant groups in all culture ponds of all treatments. The total numbers of all zooplankton species found in this study were 47 species. The numbers of species found in treatment 1, 2, and 3 were 47, 47, and 40 species, respectively. Protozoans were dominated by Tintinnopsis spp., Codonella spp., Euplotes spp., Difflugia sp., Bursaridium sp., Zoothamnium, and Foraminifera. Copepods were represented by Acartia spp., Apocyclops sp., Calanopia sp., 30

41 Diacyclops sp., Hemicyclops sp., Oithona spp., Euaugaptilus sp., Lapidocera sp., Laophonte sp., Pseudochirella sp., and Schmackeria sp. Rotifers were represented by Brachionus plicatilis and Colurella uncinata. Cladocerans had only one representative of Diphasonoma sp. The group of other taxa was including gastropod larvae, bivalve larvae, Chironomidae, polychaetes, nematodes, and insects. a b c d e f g h i Figure 4.7: Some common zooplankton found in the white leg shrimp ponds (a) Euplotes sp. (b) Difflugia sp. (c) Copepod nauplius. (d) Diphasonoma sp. (e) Acartia sp. (f) Oithona sp. (g) Brachionus plicatilis (h) Polycheata prototroch (i) Hydrozoa A total of 20 species of protozoans, 17 species of copepods, 2 species of rotifers, one species of cladocerans, and 7 other species were found in treatment 1 and 2. Treatment 3 had 3 copepods species and 4 protozoan species less than the other two. The percentages of each zooplankton group are presented in figure 4.8 below. Treatment % 42.55% Protozoans Rotifers Cladocerans Copepods Others Treatment 2 Treatment % 17.50% 42.55% 40.00% 36.17% 36.17% 35.00% 5.00% 2.50% 2.13% 4.26% 2.13% 4.26% Figure 4.8: Percentage compositions of zooplankton groups found in three treatments throughout the sampling period 31

42 The daily numbers of zooplankton species were different between treatments. Treatment 1 showed more diversity of zooplankton than the other treatments with the daily numbers of zooplankton species ranging from 16 to 25 followed by treatment 2 and 3 with the daily numbers of zooplankton species ranging from 14 to 20. Copepods and Protozoans were always the main components with very high percentages at all time. The numbers of copepods and protozoans species in treatment 1 and 2 were similar at most of the sampling days, and they had high percentages of total species with 28-53%. However, copepods were the most abundant group in treatments 3 with very high percentages of 33-54% followed by protozoans with 13-27%. The fluctuations of number of species of zooplankton groups, expressed as percentage of all species found, are shown in the figure below. Treatment 1 Treatment 2 Treatment 3 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% 1* 2* 3* 4* * 2*3* 4* * 2*3* 4* Sampling period Sampling period Sampling period Others Copepods Cladocerans Rotifers Protozoans Figure 4.9: Daily percentage of zooplankton groups in three treatments (The days with * symbols belong to the before stocking phase) Similarity comparison of zooplankton compositions of the paired treatments and of the three treatments was done for this study. The Sorensen s similarity indexes of these comparisons are shown in Table 4.3. Calculations of Sorensen s similarity indexes gave mostly Cs values higher than 0.6, which indicate high similarity of all treatments most of the time. The pair of treatment 1 and 2 showed highest similarity in zooplankton composition with Cs values from 0.7 to 1 after 32