Shrimp Farmers Manual

Published by BUREAU OF FISHERIES AND AQUATIC RESOURCES PCA Compound, Elliptical Road, Quezon City

ISBN: 978 - 971 - 8722 - 57 - 2

Photos and Cover design by Reyannald Buan of Biosolutions International Corporation Layout artist Argel Bucud

Shrimp Farmers' Manual Sustainable Strategies in Managing Penaeus vannamei

by: Maria Abegail G. Apostol-Albaladejo and Roselyn D. Usero

Bureau of Fisheries and Aquatic Resources Negros Prawn Producers Cooperative 2019

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MESSAGE I commend the Bureau of Fisheries and Aquatic Resources and its institutional partners who may have contributed in publishing this “Shrimp Farmer’s Manual – Sustainable Strategies in Managing Penaeus vannamei”.

Mature farming technologies resulting from long and arduous research works should ultimately benefit our farmers and fisherfolk. We must ensure that these productivity-boosting technologies are properly disseminated and transferred to farming communities through the use of effective information, education and communication (IEC) materials.

This manual is a perfect example of information material which our shrimp farmers can conveniently refer to if they would like to broaden out their knowledge and skills on the various sustainable shrimp farming methods. This also covers food safety standards set by the ASEAN Shrimp Good Aquaculture Practices which can enhance our farmers and processors’ competitiveness in both local and international markets.

The tough challenges posed by climate change and the emergence of diseases compromising shrimp production should be squarely met with scientific measures which can be found in book. As handy reference, its content presents practical information which can aid our shrimp farmers to become more resilient.

I deeply hope that this manual becomes instrumental in increasing the production, at the same time improving the income of our shrimp farmers – “Masaganang Ani at Mataas na Kita” is what we seek for our farmers and fisherfolk.

Mabuhay ang Sektor ng Agrikultura! Mabuhay ang mga Magsasaka at Mangingisdang Pilipino!

WILLIAM D. DAR, Ph D. Acting Secretary

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MESSAGE The Department of Agriculture – Bureau of Fisheries and Aquatic Resources is pleased to present this manual, entitled “Shrimp Farmer’s Manual – Sustainable Strategies in Managing Penaeus vannamei” which is a product of team work and dedication of our technical experts aiming to enhance the knowledge and skills of our shrimp farmers.

This publication is part of our commitment to implement measures that would sustainably protect and conserve the aquaculture industry towards food security. One such measure is implementing the National Shrimp Production Program (NSPP) which provides holistic monitoring and surveillance mechanisms against diseases threatening the shrimp industry. Through the NSPP, we are also able to capacitate our shrimp farmers on sustainable shrimp farming.

Following the goal of the said program, this manual will ensure that our shrimp farmers are given equal opportunity for continued education to improve their productivity, thereby encouraging inclusive growth in the shrimp industry.

May this information material help realize our goal in the Department of Agriculture under the guidance of Secretary William Dar to ensure “Masaganang Ani at Mataas na Kita” for our hero farmers and fisherfolk.

Maraming salamat at Mabuhay ang Sektor ng Pangisdaan!

COMMODORE EDUARDO B GONGONA PCG (Ret) BFAR National Director

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ACKNOWLEDGEMENT

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TABLE OF CONTENTS Title Messages Acknowledgement Tabel of Contents Illustrations Tables Part I: Prevention and Management of Common Infectious and NonInfectious Shrimp Diseases

Page/s i ii-iii iv v v-vii viii ix

Introduction

A. Review of Biosecurity

B. Land Preparation (Earthen Ponds) C. Water Culture D. Fry Quality Screening E. Acclimation and Stocking F. Water Quality Monitoring G. Feeding Management H. Disease Monitoring, Prevention and Control Management Strategies Part II: Prevention and Management of Mixed Infection

2-7 8-14 15-16 16-17 17-18 18-26 27 27-32

Case Specific Experience References About the Authors

ANNEXES

34-39 40-41 57-58

A. Work Instruction in Measuring Some Field Parameters B. Determination of Ammonia Percentage C. Field Monitoring Forms -Negros Prawn Producers Cooperative (NPPC)

Sampling Sheet Form D. Survival Monitoring Sheet Form (NPPC) E. Laboratory Test Results: Plankton Enumeration Form (NPPC) F. Some Beneficial Planktons G. Aquaculture: Volume and Value of Shrimp and Prawn Production For

2010-2018 H. First Responder: Field Test Report (BFAR) I. On-site Test Report: Water Quality for Penaeus vannamei (BFAR) J. Report of Test (Conventional Polymerase Chain Reaction (PCR) in BFAR

laboratory K. Report of Test (Real-time PCR in BFAR laboratory) L. Report of Test (Insulated Isothermal (ii) PCR in BFAR laboratory M. Report of Test (Bacteriology in BFAR laboratory)

1-2

33

42-43 44 45 46 47 48

49-50 51 52 53 54 55 56

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ILLUSTRATIONS

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Page/s 2 1. Repair of bird lines to ward-off birds approaching approaching the farm 2 2. Crab fences to prevent crustacean crawlers to enter the pond 3 3. Flumes covered with nets to prevent entry of birds and crawlers 3 4. Disinfection and cleaning of paddlewheels 3 5. Conditioning of reservoir 3 6-7. Checking of screening system from water source to reservoir 8. Shrimp disease detection using the latest Polymerase Chain Reaction 4

(PCR) technique 4 9. Crustaceans found in canals and waterways should be tested 4 10-11. Car tire bath at the entrance 5 12a-b. Power spray including car tire rim 5 13. Zero visitor policy in production area 14. Security log book to monitor the entry of staff and visitors into and 5

out the farm 5 15a-b. Cast nets for one (1) pond compartment 6 16. Footbath at entrance 6 17. Footbath at the pond bridge use to check feeding feeding trays 18. Hand wash and footbath with disinfectant at the entry of the pond 6

bridge going to the feeding tray 6 19. Pond aides provided with boots 6 20-21. Safe handling of inputs with gloves 22. Demarcation of route as part of the stringent biosecurity and placing

footbath with hand washing disinfectants in all the entry points in 7

the farm 7 23-25. Policy of no animal allowed in the culture premises 8 26. Sun-drying separates muck layer from top soil layer 8 27a-b. Scraped muck placed in sacks 8 28a-b. Sludge pit or central drain 8 29. Dimension of sludge pit 9 30. Dried pond bottom after scraping 9 31. Soil sampling implements 9 32. Vertical slice of soil at pond bottom for sampling 9 33. Soil sampling depth 9 34. For 0.5 to 1.0 hectare (ha) pond sampling points 9 35. Sampling areas for >1.0 hectare pond 10 36. Preparation of a homogenized soil sample 10 37. Vibrio parahaemolyticus in pond side 11 38. pH comparison in terms of degree of acidity and basicity 11 39. pH influence in productivity (FAO, 2003) 12 40. Pond bottom with accumulated organic matter after harvest 12 41. Soil profile indicating organic matter 12 42. Layer of iron accumulation 13 43. Soil condition high in iron before drying and after plowing and drying

44. During the drying season in between plowing 45. Plow layer should be between 10-15 cm 46. 2nd plowing 47. Drying following the 2nd drying 48. 3rd plowing 49. Flooding with fresh water following the 3rd plowing 50. Footbath and hand wash improvised container 51. Paddlewheel should be operational for efficient mixing effect when

applying all water inputs during water culture 52. Proper collection of water 53. Microscopic analysis 54. PCR analyses for WSSV, AHPN, EHP and IN+HHNV detection 55. Fry upon reaching the farm 56. Plastic bags with post-larvae placed on pond water for temperature acclimation 57-58. Post-larvae are gradually released in stocking box and acclimatized

to match the pond salinity before releasing into the ponds 59. Counting of post-larvae to estimate the total number of post-larvae delivered 60. The same salt concentration of pond water and newly molted shrimp body 61. Higher salt concentration of newly molted shrimp than the pond water 62. Lower salt concentration of newly molted shrimp than pond water 63. Green water dominated by Chlorella spp.

64. Muscle necrosis 65. Primary reservoir 66. Negros Occidental Province map of deep-well profile 67a-b. Mixing of feed inputs 15 minutes before feeding 68a-c. Proper feed storage and stacking 69. SEACORP farm 70. Work instructions for pH determination 71. pH scale from 1-14 72. Dissolve oxygen (D.O) meter 73. Immersion of D.O. probe in distilled water for short storage 74. Refractometer 75. Instructions on how to read transparency using the secchi disc

Page/s 13 13 14 14 14 14 14 15 15 16 16 17 17 18 18 20 20 20 21 21 25 25 27 27 34 42 42 43 43 43 43

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TABLES 1. Field parameters important during water culture and culture phase 2. Chemical parameters important during water culture and culture phase 3. Estimation of calcium and magnesium requirement in reference to salinity 4. Microbiological Analysis (done in the laboratory) 5. Interpretation of result (NPPC documented farm date, updated 2018) 6. Physico-chemical analyses of deep-well water of key producing area

based on NPPC laboratory results 7.Suggested monitoring schedule of bacterial and PCR analyses of shrimps 8. Interpretation of bacterial analysis result (NPPC, 2018) 9. Pond inputs to maintain good water quality and corrective measures

when necessary 10. Feed inputs to prevent disease and suggested corrective measures

when necessary 11. Crop performance of ponds infected with WSSV and EHP after

immediate and timely corrective measures 12. Results of soil and crab analyses 13. Results of Polymerase Chain Reaction (PCR) analyses of crustaceans in

the water source and canals 14. Crop performance of the four (4) ponds diagnosed with WSSV and vibriosis 15. Crop performance of six (6) ponds diagnosed with WSSV and vibriosis 16. Crop performance of three (3) ponds diagnosed with WSSV and vibriosis

Graph: Ammonia toxicity increase with increasing pH

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Page/s 18-19 22-23 23 23-24 24 26 28 29

30-31 32 36 36 37 38 38 39

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PART 1

Prevention and Management of Common Infectious and Non-infectious Diseases

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Shrimp Farmers’ Manual: Sustainable Strategies in Managing P. vannamei Farms in the Philippines Introduction

Philippine shrimp farming manifested an increase in the value of production growth rate by 0.94 % from year 2017 to 2018. Which is equivalent to Php 24.8B to Php 25.0B. However, its production volume decreased by 4.53 % from 60, 122 MT to 57, 399 MT. (Source: PSA, 2018). This can be attributed to the strategy of shifting of culture animal from shrimp to finfish, in order break the cycle of dominance by one microorganism which cause disease and to balance the biodiversity in shrimp ponds as part of Good Aquaculture Practices (GAqP), which is currently vastly promoted worldwide.

Good Aquaculture Practices (GAqP) in semi-intensive and intensive shrimp grow-out farms includes rigorous pond preparation, good quality fry, strict implementation of biosecurity measures and stringent water budget to adequately supply the requirement of ponds to minimize risks factors attributable to poor productivity. Myriad of efforts to cope with the need to modify the strategies of established grow-out farm management protocol resulted to successful harvest. New farming techniques were developed to adapt with occurrences of emerging diseases and the changing environment. The challenges brought about by climate change and the detection of Hepatopancreatice microsporidiosis (HPM) in 2017, prompted growers to channel their strength to improve farming practices through scientific approaches to produce better quality shrimps at a higher production volume. Better quality shrimps harvested at large volume will enhance marketability, command higher prices and will gear the industry towards the direction of global competitiveness and better market access. Thus, better socio-economic living standards among those involved in the industry.

This farmer-friendly manual intends to provide the management strategies to growers, farm managers and technicians which were applied and still being applied in successful farms. This also serves as guide on how to operate shrimp farms based on actual experiences applying Good Aquaculture Practices (GAqP), thereby minimizing negative impact to the environment. This includes the documented sustainable practices of farms with earthen ponds including their inputs and protocol on how they operate their farms effectively and efficiently.

This book consists of two parts: Part I contains biosecurity measures to prevent and manage infectious and non-infectious shrimp diseases during adverse environmental conditions. Which showcases evidences of good farm management with production consistency of technically equipped and well-managed farms. It will inform readers on how a farm should maintain a favorable water quality despite the mixed infection of two pathogens, WSSV and EHP in 2017 with several indications of vibriosis. It also mentioned how the application of several inputs during culture period, such as feed additives, immuno-stimulants, probiotics and nutrient mix were able to maintain a consistently high growth rate and survival of shrimps.

Part II describes management practices and strategies in cases of complexed mixed infections experienced by consistently successful shrimp growers specifically mixed infection of White Spot Syndrome Virus (WSSV) and Enterocytozoon hepatopenaei (EHP), WSSV and Vibriosis, as well as White Feces Disease (WFD) and WSSV.

This document manifests how successful farms maximized efficiency and productivity without sacrificing the environment. It further reveals how farms were managed to attain competitive advantage based on actual high yields despite the presence of pathogen. As “the new normal,” the shrimps were able to grow into marketable sizes and maintain good quality even with the presence of “diseases.”

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A. Review of Biosecurity

Infectious diseases caused by bacteria, viruses, fungi and parasites are either vertically (from broodstock to larvae) or horizontally transmitted (ingestion of infected animal or through carrier organism) through direct or indirect contact.

Non-infectious diseases are caused by predisposing factors to disease like nutritional defficiency, genetic defects or poor environmental conditions either physical or chemical parameters in the soil or water that measured beyond tolerable values from poor management or animal husbandry, causing stress to the shrimps. One of the challenges encounter is the shrimp's resistance to chronic sub-lethal exposure. This is usually reflected in weight reduction or weakening depending on the degree of the stress factor and exposure period. One strong dose of a single non-infectious disease can cause low dissolved oxygen, very low pH or extremely high pH will result to mortalities. Sometimes, a synergy of two or three non-infectious stress factors in sub-lethal concentrations can be significant in causing mortalities. Non-infectious diseases are localized in the pond to avoid from spreading the adjacent ponds.

Disease prevention includes proper land preparation, implement biosecurity to reduce the risks of exposure to infectious agents prior water culture, stable water parameters prior stocking, stringent fry selection and good water quality management skills during culture to prevent stress to the shrimps that could induce negative shrimp health issues. The pre-stocking procedure has been documented in high yielding earthen ponds giving sustainable and consistent production for near a decade in intensive Penaeus vannamei culture Negros Occidental, Philippines as illustrated in the next part of the manual

The absence of a well-established internal control or biosecurity measures increase the risk of exposure of every farm to viral and bacterial diseases. The biosecurity protocol of a farm and its purpose should be communicated to all staff for understanding and effective implementation.

1. During pond preparation

Illustration 1: Repair of bird lines to ward off birds approaching the farm

Illustration 2: Crab fences to prevent crustacean crawlers to enter the pond during the bottom preparation process

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Illustration 3: Flumes are covered with nets to prevent entry of birds and crawlers

Illustration 4: Disinfection and cleaning of paddlewheels

Illustration 5: Conditioning of the reservoirs (plowing, drying and disinfection)

Illustration 6 and 7: Checking of screening system from the water source to the reservoir and grow out ponds. Filtration can reduce predators.

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Illustration 8: Shrimp disease detection using the latest polymerase chain reaction (PCR) technique

2. During the culture phase

Illustration 9: Crustaceans found in canals and waterways should be tested for WSSV

Sustainable production is achievable when a farm maintains good record keeping and submit to routine laboratory analysis/PRO-ACTIVE MONITORING.

Illustration 10 and 11: Tire bath at the entrance (Seacorp Farm, Sagay City and HP Farm, Bago City, Negros Occidental).

Disinfectant solution used is replaced every 2-3 days during the culture phase.

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Illustration 12: Power spray itnclude the rim of tires

Illustration 13: Zero visitor policy in production area during culture period

Illustration 14: Security log book to monitor entry of staff and visitors into and out of the farm stating name, time, purpose of visit and signature

Illustration 15a and 15b: Cast nets for one (1) pond compartment 5

Illustration 16: Footbath installed at the entrance

Illustration 18: Handwash and footbath with disinfectant platced at the entry of pond

Illustration 17: Foot bath at the pond bridge use to check feeding trays

Illustration 19: Pond aides are provided with boots

Illustration 20 and 21: Safe handling of inputs with gloves 6

Demarcation of routes:

• The farm’s production area can be divided into blocks to demarcate routes in observance with stringent biosecurity specifically during daily monitoring and reading of pond parameters. Suggested rout is from the ponds with less culture days to the oldest culture period.

Illustration 22: Demarcation of route as part of stringent biosecurity and placing footbath and hand washing disinfectants in all the entry points in the farm

Isolation of farm animals away from the production area

Illustration 23, 24 and 25: The policy of no farm animals allowed inside the premises of shrimp farm is ideal but should this be not the case, keeping them away from the production area is a practical measure of biosecurity

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B . Land Preparation (Earthen Ponds) Steps in land preparation:

1. Scraping of the muck layer after drying for 15 days,

Illustration 26: Sundrying facilitates the separation of the dried muck layer from the original top soil layer of the pond bottom.

2. Installation of sludge area per pond

Illustrations 27a and 27b: Scraped muck are placed in sacks for transport to open field away from the ponds.

This can be re-utilized as an organic fertilizer after the rains wash the salt content

This is part of the waste management strategy to improve water quality and prevent self-pollution. This has been among the significant innovation added to the existing protocol in 2017.This is also an effective preventive strategy for Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis Syndrome (AHPNS) by controlling vibrio build up during the culture period.

Illustration 28: Sludge pit (or commonly called “shrimp toilet” or “central drain”)

10 meters in diameter

Slope from pond bottom

Catch basin depth - 20 cm

Depth -1 meter

Illustration 29: Dimensions of sludge pit are area and shape dependent

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3. Soil Analysis Soil Sampling Procedure:

3.1 Sampling should be done preferably when the soil is dry.

3.2 Remove from the surface all weeds/ materials not incorporated with the soil.

3.3 Using a trowel or soil auger, take a sufficient no. of subsamples at properly at 10 random locations per pond (>1.0 hectare).

3.4 Sampling depth is from 2.5 cm (top) to 15 cm, vertical slice for each layer to be represented. Knowing the quality of this layer is advantageous during pond preparation. This is the best time for implementing corrective actions should some important parameters are not within the optimum values. The soil-water interface influences water quality and plays a vital part during culture.

Illustration 30: Dried pond bottom after scraping, ready for soil sampling

Illustration 32: Vertical slice of soil on pond bottom for sampling

Source: Kleinschmidt, 2019

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Illustration 34: For 0.5 to 1. 0 hectare (ha.), get at least 10 sampling points, vertical slice at about 6 inches depth to get representative sample of the plow layer

Illustration 31: Soil sampling implements

Illustration 33: Soil sampling depth

Illustration 35: Sampling area for > 1.0 1.ha. but less than 2.0 ha., with 11 sampling points

• Place in a plastic bag and label using a permanent marker indicating the pond number and name of the farm • For with bacterial analysis, do not air-dry the soil. Proceed with the process in obtaining a composite sample until labelling. Submit immediately after packing.

3.5 Preparation of soil sample for analysis

• For chemical analysis, air-dry the soil by spreading a thin layer in a plastic sheet. Always label the sample as per pond number. AVOID DRYING IN DIRECT SUNLIGHT. • Drying will take about 3-5 days. Breaking the soil particles occasionally will hasten the drying process.

• To secure a homogenous sample representative of the entire area, pulverize and mix all subsample and mix all subsamples and quarter (divide into four (4) equal sizes. About 1 kilo of mixed composite sample should be submitted for analysis.

• =Quartering: -Divide the mixed subsamples into four (4) equal parts.

-Get a pair of opposite sides and discard the rest.

-Continue this until approximately 1 kilo is reached.

Illustration 36: Preparation of a homogenized soil sample

Source: Kleinschmidt, 2019 Important Soil Parameters to be tested:

• Bacterial Profile including Vibrio profile, V. harveyi and V. parahaemolyticus Illustration 37: Vibrio parahaemolyticus in pond soil at 1.9 x 104 CFU/g (Mauve or pinkish in chromatogenic agar)

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Pond conditions in shrimp farms may support the survival and growth of high salt-tolerant pathogenic Vibrio species, such as V. parahaemolyticus, V. harveyi, V. alginolyticus and potentially V. cholera (Noriega-Orozco, L., Acedo-Félix, E., Higuera-Ciapara, I., Jiménez-Flores, R., Cano R., 2007).

The first documented case of APHND in a shrimp farm at the province of Negros Occidental revealed high V. parahaemolyticus in pond bottom soil. In most cases of vibriosis, results of vibrio profile showed high vibrio load dominated by the green colonies in pond soils aside from the water.

• Physico-Chemical Parameters

The chemical properties of the soil are affected by the inputs, stocking density and management techniques overtime.

a. pH

pH is a physico-chemical property. Soil pH is the measurement of the acidity or basicity of the soil ranging from 0-14 on the pH scale.

pH values lower than 7 is classified as acidic, pH above 7 is basic or alkaline. The degree of acidity and basicity is logarithmic. This means that a difference of 1 pH unit either smaller or larger has a 10x difference.

Illustration 38: pH comparison in terms of degree of acidity or basicity (http://www.spectrumanalytic.com)

Figure 38 denotes that pH 7.0 is neutral while pH 6.0 is 10x acidic in reference to pH 7.0, similarly, pH 8.0 is 10x more basic but pH 9.0 is 100x more basic in reference to 7.0 and 10x more basic than pH 8.0.

The availability of major nutrients and micronutrients in the soil is generally dependent on soil pH including bacterial activity. The optimal pH is 7.5–8.0 Boyd (1994).

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Illustration 39: pH’s influence in productivity (FAO, 2003)

b. Organic Matter Content

The pond bottom soils are vital component of the pond system affecting the soil-water dynamics during the culture phase. Some soils have inherently high percentage of organic matter while sandy soils have low organic matter. During the culture phase, the interaction of the soil-water interface is a determining factor in water quality. Soils with high organic matter limits stocking density or holding capacity in terms of yield and culture period.

Excessive organic matter accumulation due to intensive stocking density results to pond bottom deterioration. Anaerobic condition due to microbial decomposition of fresh organic matter coming from waste, metabolic excreta, dead organisms, phytoplankton die-off, etc., results in the production of toxic gases (ammonia, nitrites, hydrogen sulfide).

These toxic substances can cause slow growth due to chronic exposure to sub-lethal levels, lower the resistance of the shrimps Illustration 40: Pond bottom with accumulated eventually leading to increase vulnerability to organic matter after harvest disease infection.

As organic matter increase, the sediment oxygen demand (SOD) in the pond bottom also increases.

SOD is more than 50% of the total shrimp pond oxygen demand (Ellis, 1992). The SOD competes with the dissolve oxygen (DO) requirement of shrimps. It is therefore vital that pond preparation is properly done to avoid high SOD. c. Available Iron and Available Sulfur Very high concentrations of iron are major characteristics of acid sulfate soils.

Illustration 41: Soil Profile indicating the organic matter layer (Ap)

Source: Kleinschmidt, 2019

Illustration 42: The layer of iron accumulation (B)

Problems associated with high iron and sulfate: • Mortalities due to acidity and high iron • Poor fertilizer response • Low natural food production • Phosphates are not in available form • Significant concentrations of free aluminum and its compound in the surface soil make the phosphates unavailable • slow growth due to stress brought about by the iron compounds or soluble iron clogging the gills and

hindering respiration.

• Leaching of iron and aluminum from the dikes during heavy rains can cause stress due to acidity.

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POOR AERATION

GOOD AERATION

Iron is reduced

Iron is oxidized

Fe ++

Fe +++

dull colors (grays, blue)

bright colors (yellow, browns)

poorly drained

well drained

Available sulfur is abundant in acid sulfate soils and in seawater. Presence in excessive concentrations in the action of sulfur reducing bacteria will favor formation of hydrogen sulfide. Hydrogen sulfide is a toxic gas. Toxicity increases with decreasing pH. Illustration 43: Soil condition high in iron before drying and after plowing and drying

Source: Kleinschmidt, 2019

4. Drying (1 week)

The drying process facilitates the decomposition and oxidation of organic matter making most nutrients in available form. This also provide oxygen supply to the bacteria necessary to decompose organic matter and kills pathogens.

Excessive drying will devoid the soil from moisture needed for bacterial activity during the pond preparation process. Boyd (1994) concluded that optimum soil moisture concentration for respiration was 12–20%.

5. 1st Plowing Purpose: • To volatilize toxic gases specifically hydrogen sulfide, ammonia, nitrites and methane gases entrapped from the previous culture.

Illustration 44: During the drying process in between plowing

• To reduce excessive organic matter accumulation due to intensive stocking density results to pond bottom deterioration

• Heavy-textured soils (clays and clay loams) will benefit more from tilling than the lighttextured soils (sands, sandy loams, and loams).

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Illustration 45: Plow layer should be between 10-15 cm

• Tilling should be done while bottom soils are still moist, but bottoms should be dry enough to support the weight of the tractor tires and prevent formation of ruts in the pond bottom (Boyd, Claude E., 2003).

• Tilling should only be at the reactive portion of the soil (upper 10-15 cm) to prevent exposure of the high iron layer (true in most areas) that may pose potential problem durng the culture period.

Illustration 46: 2nd Plowing (Regular plowing should be considered as part of a routine pond preparation procedure practice. This has been proven to break life cycles of pathogens)

Illustration 47: Drying following the 2nd plowing

6. Drying ( 1 week) 7. 2nd plowing (15 days) 8. Drying (1 week) 9. 3rd plowing (15 days) 10. Flooding with fresh water (20-30 cm depth) 11. Flushing with levelling using a hand tractor.

12. Liming (agricultural lime or dolomite, dose depends on soil analysis). Aquamin is also an option at 1-2 tons/ha.

13. Drying (5 days) 14. Application of chlorine in the center canal (5 kg) 15. Installation and positioning of paddlewheels and meter stick for monitoring of water level.

Illustration 48: 3rd plowing

16. Wild crustaceans found in the canals and in the surrounding areas were check for presence of pathogens (White spot syndrome virus, APHND/ Early Mortality Syndrome, EHP and bacteria).

When the results revealed not detected, this was part of the signal to start admission of water to commence water culture.

Illustration 49: Flooding with fresh water following the 3rd plowing (Step 11)

17. Application of probiotic for water at 100-200 grams per hectare mixed with 5 kilos of mineral mix.

18. Footbath and handwash disinfectant per pond should be changed every 2-3 days. This biosecurity policy during the entire production period from water culture to harvest.

Provide separate containers with disinfectants

Illustration 50: Footbath and handwash improvised containers (Step 18)

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Provide separate containers with disinfectants for cast nets and other shared equipment.

This will prevent disease occurrences. Having separate equipment (basins, nets, pails, sampling bottles) for each module would help to eliminate the risks of contamination between modules.

Disinfection is an important aspect of disease prevention and control. Routine disinfection decreases the pathogen load in a farm facility, thereby reducing the risk of spreading the pathogen to other ponds and farms.

C. Water Culture

1. Admission of water from the reservoir using a submersible pump at 1.2 meters depth.

2. Application of chlorine at 30 ppm with aeration.

3. After 4 days, application of mineral mix containing sodium nitrate, silicates and trace elements at 10 kg/ha each of rice bran, molasses and yeast soaked for 2 days. This is to promote good plankton growth and desirable water color.

4. Application of probiotic at 100 g/ha. 3 days prior stocking. This is to ensure supply of beneficial bacteria using the competitive exclusion of pathogens principle in which no two bacterial species can co-exist using the same food or resources for survival due to competition. The bio-remediation product should contain at least 1.0 x 1010 CFU/gram total bacterial count.

Ensure adequate aeration when using probiotic or substances that enhances bacterial activity (molasses, sugar, etc) and when applying chemicals specifically during treatments or implemetaing preventive or curative measures to manage the water.

Illustration 51: Paddlewheels should be operational for efficient mixing effect when applying all water inputs during water culture.

5. Collection of water samples for analysis

5.1 How to collect water samples for submission to the laboratory for analysis: • Prepare a clean sterile glass bottle for microbiological and physico chemical analysis.

• Volume should be 250-500 mL

• Take the sample near the gate. Be sure do sampling at the same point or area for consistency.

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Illustration 52: Proper collection of water samples

• Label the sampling bottle properly.

-Farm name and location (if the sample will be submitted in another laboratory) -Pond Number -Sampling Time • Submit samples within 2-4 hours. Place in styrobox with ice.

• Water parameters for laboratory testing 2-3 days prior to stocking • Bacterial analyses, phytoplankton count, bicarbonate alkalinity

D. Fry Quality Screening

The screening procedure follows the criteria for fry selection. At least two (2) analyses should be conducted as a matter of protocol in fry selection. The samples will be tested for presence of pathogens (luminous vibriosis, V. parahaemolyticus, Total Vibrio Count and profile, WSSV, EHP and APHND).

Microscopic test should also be performed to assess for growth and development of fries. All the test results should be compared for consistency before finalizing the booking and stocking schedule.

PCR screening significantly minimizes the risk of introducing the pathogen into the system with the fry. In some cases, the virus may be present at levels below the detection limit of the test method if the percentage of prevalence in the population is low since the sample size is only about 300 pieces.

It is suggested that at least 2-3 tests be done prior stocking. The suggested sampling protocol in the hatchery should be done (Albaladejo MAG and Usero RD, 2017).

Bacterial analyses

In bacterial analyses using chroma agar fry sample should be negative for presence of Vibrio parahaemolyticus, a causative agent of AHPND. In triosulphate-citrate –bilesalts-sucrose (TCBS) media, it should manifest in its vibrio load that the dominance of the yellow colonies against green colonies

Microscopic Analyses

The morphological age is determined based on the rostral development, the ideal ratio of the gut to muscle is 1:4. The average body length is also an important parameter in fry quality assessment.

Illustration 53: Microscopic analysis

Modified Fry Criteria (Penaeus vannamei)

Illustration 54: PCR analyses for WSSV, AHPND, EHP and IHHNV detection

1st check

a. PCR analysis (less than pl <8) WSSV, EHP, EMS

Test Methods Used in PCR Analyses

• IQ 2000 for WSSV, AHPND, IMN, TS (Plasmid and Toxin 1) • IQ Plus for WSSV, AHPND and HPM • Japanese Kit using QIAGEN 4:1 (HPM, WSSV, IHHN, AHPND)

b. 1st microscopic analysis

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c. Bacterial Analysis (Total Vibrio Count, % Green (G), %Yellow (Y), Vibrio parahaemolyticus and Luminous Bacteria)

Methods Used in Bacterial Analysis

Culture Media Used:

• •

Pour Plate Method Serial Dilution Method Nutrient Agar TCBS Agar Chromatogenic Agar

: Luminous Bacteria

: Total Vibrio Count (TVC) : Vibrio parahaemolyticus

2nd Check

a. PCR analysis (less than post-larvae (PL) 9-10)

b. 2st microscopic analysis

c. Bacterial Analysis (TVC, %G, %Y, V.parahaemolyticus and Luminous Bacteria)

If the PL passed in the 1st and 2nd screening, final assessment of quality is made 1-2 days prior to delivery. Results of the two (2) monitoring are then compared for consistency with reference to the criteria for microscopic test and bacterial analysis result. Increasing body length with retrospect to PL age should be noted.

It is important to note that a laboratory screened PL does not guarantee a disease free stock during culture because of possible stress factors that may occur anytime.

E. Acclimation and Stocking

It is beneficial for the pond water and hatchery tank salinity to match before delivery. This can ensure less stress and shorter acclimation time or adjustment to the pond environment.

Parameters to consider during acclimation: • pH of the water with the PL • Temperature • Salinity

17

Illustration 55: Fry upon reaching the farm

These parameters should not be overlooked in reference to distance from the hatchery to the farm and travel time, PL age, packing density and transport facilities.

Illustration 56: The plastic bags with post-larvae placed in the pond water for temperature acclimation.

Illustration 57 & 58: Post-larvae are gradually released in the stocking box and acclimatized to match the pond salinity (when necessary) before releasing into the pond.

F. Water Quality Monitoring

Illustration 59: Counting to estimate the volume of post-larvae delivered and the stocking density

Important Water Parameters

A knowledge and understanding water chemistry’s role in pond dynamics is an important aspect in managing water quality. Successful production is a direct effect of good water chemistry.

Prior to water culture, all monitoring equipment including refractormeters, pH meters and D.O. meters should be checked to ensure precision and result accuracy for better and timely interventions, whenever necessary. The following water paramenters should be monitored regularly and documented properly at pond-side from the start of water culture to the end of the production cylce.

Table 1: Field Parameters important during water culture and culture phase Parameter Equipment Suggested Optimum Comments needed Frequency Level pH

Color

Dissolved Oxygen

Calebrated pH meter using buffer 7.0 and 10.0

Twice 9:00 a.m.

3:00 p.m.

Calibrated D.O meter with probe solution and membrane checked regularly

At least 3x

7.5-8.5

Twice 9:00 a.m.

3:00 p.m.

5:00 a.m.

3:00 p.m.

10:00 p.m

< 4 ppm

Daily fluctuation >0.5 often measured approximately 0.5 m. below the surface Monitor together with most parameters (pH, Transparency, depth, salinity, temperature) Taken in the feeding area nea the pond bottom

18

Table 1: Field Parameters important during water culture and culture phase Parameter Depth Salinity

Equipment needed

Transparency

Optimum Level

Meter stick with verified measurement

2x 9:00 a.m.

3:00 p.m.

<1.0 meter

2x 9:00 a.m.

3:00 p.m.

10-30 ppt

Thermometer with verified temperature reading (>500C with 0.1 degrees increment Secchi disc with verified measurement

2x 9:00 a.m.

3:00 p.m.

29-300C

2x 9:00 a.m.

3:00 p.m.

30-60cm

Calibrated refractometer

Temperature

Suggested Frequency

Weather Conditioning

(Boyd, 1990)

2x 9:00 a.m.

3:00 p.m.

Important facts:

Comments Measurement should be taken in the areas identified to represent the average depth of the pond, not in shallow or deeper portion Aside from the daily routine monitoring in reference to time, salinity should be checked right after water exchange or addition has been done.

Readings shall be taken from points that represents area that is homogeneous, not in points where there is heavy or too low plankton due to the influence of wind action..

Aside from the daily routine monitoring in reference to time, significant changes in weather should be noted specifically during heavy rains (# of hours)

pH

Water pH affects water chemistry, the action of micro-organisms, availability and solubility of nutrients. It is one of the most important parameter in shrimp culture. The daily fluctuation of pH varies depending on the following:

• • • • •

Salinity

Alkalinity Phytoplankton density Shrimp biomass Respiration of the aquatic organisms in the pond ecosystem.

Water source used to refill the grow out ponds

Salinity is an important physical parameter. It is pertains to the dissolved solids (salt) in the water.

Shrimps, having possesses aqueous solutions with semi-permeable membranes, are affected by the osmoregulation process. As the concentration of the salt content in the pond water changes, the osmotic pressure of the shrimps’ body also change.

19

Pond salinity = 15 ppt Incoming water from reservoir = 15ppt Resulting Salinity = 15ppt (±1)

Illustration 60: When the concentration of the salt content in the pond water changes, the osmotic pressure of the shrimps’ body also change. If the body of the newly molted shrimps has the same salinity with the pond water, water will not enter the body of the animal.

Pond salinity = 15 ppt

Resulting Salinity = 10 ppt Low salinity in reservoir, high salinity in pond Incoming water from reservoir = 7ppt

Illustration 61: When the body of the newly molted shrimps have higher salinity than the pond water, greater volume will enter into the body. The body will swell and increase in volume.

Salinity of the reservoir must be conditioned to match more or less the pond salinity but a 2 ppt change during water additions can be tolerated.

Increased osmoregulation process due to abrupt salinity change can stress the shrimps that can lead to weakened resistance to disease pathogens. This change can also affect the plankton population attributable to partial die-offs.

High salinity in reservoir, low salinity in pond Illustration 62: When the body of the shrimp has lower concentration of salt than the pond water, the tendency of the water from the body of the shrimp will shrink.

20

Water color and transparency

Water color is affected by the predominant species of the plankton population in the pond water. Planktons are classified into beneficial and non-beneficial planktons (Appendix B). Green water dominated by Chlorella spp. is well favored due to its stability and good water quality.

Chlorella spp. is reported to control luminous vibrio population because of its inhibitory effect.

The transparency of secchi disc is suggested to be taken at the same time as the dissolved oxygen and pH for these parameters are affected by phytoplankton density. This should be interpreted parallel to the plankton density since Total Suspended Solids (TSS) during heavy rains from the earthen dikes or dead plankton cells after a partial die-off or total die-off can also affect transparency or turbidity reading. The high concentration of TSS can clog the gills, thus, hindering respiration causing stress.

Temperature

Illustration 63: “Green Water” dominated

by Chlorella spp.

High temperature during the peak of summer can be stressful and can cause white muscle necrosis in synergy with low dissolved oxygen. Increasing water level with adequate aeration can buffer the ill effects of high temperature. Low temperature during the cold months in synergy with a parameter of group of parameters that went off the optimum values are generally the attributable cause of disease outbreaks like WSSV infection. Often, there is a decrease in feed consumption due to the low metabolic rates in the shrimp’s body.

Illustration 64: Muscle Necrosis is a kind of physical stress.Causative factors are high temperature, high salinity and low dissolved oxygen.

Predicting water quality trends is useful in preventing stress to the shrimp. Some growers are already aware of the seasonal pattern of the disease in their farm. They usually lower the stocking densities during these vulnerable months and became more vigilant in their water quality monitoring scheme to identify early the stress factors for timely response time and implementing the appropriate corrective measures. This shortens the exposure of the shrimp to stress. Boosting the feed with additional nutritional and immune enhancer inputs proved advantageous.

21

Table 2: Chemical parameters important during water culture and culture phase

Parameter Equipment Suggested needed Frequency Bicarbonate Alkalinity

Alkalinity Test kit

2 x a week and when necessary

Optimum Level

Comments

< 70 ppm100 ppm bicarbo-nate Alkalinity

Some kits only measure Total Alkalinity but knowing the concentration of bicarbonate alkalinity is important.

The total alkalinity might be high but, if dominated by the hydroxides and carbonate ions, wider fluctuation in pH will be observed.

Five Possible Conditions For Total Alkalinity Computation using test kits or standard method.

P-phenolpthalein Indicator M- Methyl Orange Indicator

1.P = M Hydroxide, [OH-] ppm = P x 20 2.P>½ M Hydroxide, [OH- ] ppm= (2P-M) x 20 Carbonate,[CO3- 2]ppm=2(M-P ) x 20 3.P = ½ M Carbonate, [CO3- ] ppm=M x 20 4.P < ½ M Bicarbonate,[HCO3- ] ppm=(M2P) x 20

Carbonate, [CO3- 2] ppm = 2P x 20 5. P=0 Bicarbonate, [ HCO3- ] ppm = M x 20

*Calcium *Magnesium

Calcium Test Kit CalMag Test Kit

2 x a week and when necessary 2 x a week and when necessary

Ideal range 300-400 ppm Ideal range 600-1200 ppm

Nos. 4 and 5 conditions indicate very good buffering capacity of the water.

Calcium is important during shell formation after molting.

Magnesium is a powerfully bioactive ion, with the activities of some enzymes dependent on specific concentration requirements (Tavabe, Rafiee, Frinsko, Daniels 2013).

Magnesium (Mg) plays an important role in the formation of specific enzyme complexes needed for osmotic and ionic regulation in acclimation of shrimp to lower salinity water and for neuromuscular transmission and skeletal tissue metabolism (Cheng, Hu, Liu, Zheng, Qi, 2006)

Assists in photosynthesis, improving phytoplankton development.

22

Table 2: Chemical parameters important during water culture and culture phase Ammonia

Nitrite

Ammonia Test Kit

DOC 30-40 Once a week

DOC 45 onward – 2 x a week and when necessary

Nitrite Test Kit

DOC 30-40 Once a week

< 0.1 ppm Un-ionized Ammonia

Toxicity of the Un-ionized ammonia increases with increasing pH and temperature.

The unionized form of ammonia is more toxic to aquatic organisms due to its ability to gain entry through the gills (Waikhom, Aanand, Rajeswari, Padmavathy and George, 2018).

The kit gives only the Total Ammonia Nitrogen (TAN), the percentage of the Un-ionized ammonia from the TAN should be computed to get the concentration of the toxic form.

Chronic exposure to sub-lethal concentrations may cause lowered immune resistance and mortalities.

More toxic than ammonia. Toxicity increases with decreasing salinity.

< 0.1 ppm

DOC 45 onward 2 x a week and when necessary

Table 3: Estimation of Calcium and Magnesium Requirement in reference to salinity (Boyd and Thunjai, 2003 and ICAR, 2016) Ions

Seawater as Reference Standard (35ppt)

Factor

Sodium (mg/L or ppm) 10,500

Potassium (mg/L or ppm) 380

Calcium (mg/L or ppm) 400

Magnesium (mg/L or ppm) 1350

304.5

10.7

11.6

39.9

Example: Salinity 15 ppt

Calcium Requirement 15 x 11.6 = 174 ppm Magnesium 15 x 39.9 = 598.5 ppm

Table 4: Microbiological Analysis (done in laboratory)

Parameter

Total Bacterial Count Luminous bacteria

23

Media Nutrient Agar Chronomogenic Agar

Suggested Frequency

Once-twice a week

Table 4: Microbiological Analysis (done in laboratory) Total Vibrio Count % Green colonies % Yellow Colonies Luminous Vibrio Count

TCBS

Once-twice a week

V. parahaemolyticus

Table 5: Interpretation of Results (NPPC Documented Farm Data, updated 2018)

Total Bacterial Count(CFU/ml) Total Vibrio Count (CFU/ml)

Vibrio parahaemolyticus Yellow % Green %

Water (cfu/ml) <1.0 x 104 >1.0 x 104-9.9x104 >9.9 x 104 - 1.0 x 105 >1.0 x 105-1.0x106 <1.0 x 106 <1.0 x 103 >1.0 x 103-9.9x103 >9.9 x 103 - 1.0 x 104 >1.0 x 104-1.0x105 >1.0 x 105

Interpretation Normal slightly high High very high extremely high Normal slightly high High very high extremely high

>60%

Ideal

>30%-70%

Very High

<1.0 x 102

<40%

Ideal

>40%-60% >80%-0

High

>3.9 x 102-4.9 x 102

Luminous Bacterial Count (cfu/ml)

>5.90 x 10 - 1.0 x 10 >1.0 x 104

3

Not Detected at 101 <1.0 x 102

>1.0 x102-3.0x 102

>3.0 x 10 -5.0 x 10 >5.0 x 102-9.9 x 102 2

2

>9.9 x 103-1.0 x 104 >1.0 x 104

Low

4

Extremely High High

Very High

Extremely High Normal Low

Close Monitoring Slightly High High Very High

Extremely High 24

Reservoirs:

Reservoirs should be check (two) 2 days prior to pumping water into the culture ponds. A two stage reservoir is an advantage.

The first stage pumps in water for settling, the second one is for conditioning prior pumping into the grow-out ponds.

Conditioning may mean disinfection of the reservoir water prior use in the grow-out ponds.

Parameters to monitor: • Total vibrio count and profile (% Green and Yellow Colonies, V. harveyi, V. parahaemolyticus) • Salinity • pH

Illustration 65: Primary reservoir (AQUACARDS, Victorias City, Negros Occidental)

Major Water Sources

A better understanding on the quality of the river, creek, seawater or groundwater sources like deep wells and shallow wells is advantageous in water management. It is also important to keep in mind that biosecurity starts at the water source. Knowing the characteristics of the major water sources and implementing the appropriate corrective measures is one tool in crafting management strategies to prevent diseases and profitably harvest the crop.

• Seawater Since the quality of seawater is uncertain due to organic pollutants and pathogen contamination from effluent of disease-stricken neighboring farms, a treatment pond/reservoir is necessary upon its entry into the farm. This is the initial phase of water culutre. Generally, seawater is 35 ppt and is sufficient in macro-minerals, like calcium, magnesium, potassium and sodium. Buffering capacity is also not a problem due to high total alkalinity dominated by bicarbonates.

Since the quality of seawater is uncertain due to organic pollutants and pathogen contamination from effluent of disease-stricken neighboring farms, a treatment pond/reservoir is necessary upon its entry into the farm. This is the initial phase of water culutre. Generally, seawater is 35 ppt and is sufficient in macro-minerals, like calcium, magnesium, potassium and sodium. Buffering capacity is also not a problem due to high total alkalinity dominated by bicarbonates.

• Rivers and Creeks

The risk of disease contamination is higher in shallow creek and river sources adjoined by a number of farms over the years. The following are the concerns:

a. Effluents are shared. Farms intake water and discharge their effluent from the same water source. Organic wastes build up enriches nutrient loads and enhances the population of pathogenic organisms. This condition is more severe during neap tides where wastes are concentrated due to low high tides or when high tides are at their lowest levels.

b. Mineral imbalance due to low salinity especially during wet season.

c. Non-point source contaminants either up stream or downstream.

d. Extremely high salinity during summer, a good breeding condition for vibrios in the presence of high organic load.

25

Illustration 66: Mapping the deep-well profile in Negros Occidental in shrimp culture key producing areas

• Deep-wells

Some farms have access to deep wells with practically zero to brackishwater salinity. Generally, deep well water based on the documented results (NPPC-ADL, 2019) have low mineral contents specifically magnesium, calcium and potassium but high in total alkalinity with the dominance of bicarbonate ions. In 2017, two intensive farms suffered WSSV after a relatively downtrend in magnesium ions were noted as culture progressed despite very frequent application of dolomite. The shrimps were soft-shelled. The molting process is stressful to the shrimps and insufficient supply of minerals in the water to support the molting process in shorter exposure period and normal shell formation can be a factor in the entry of pathogens to the shrimps.

A comparison was made among three (3)croppings. The first and second croppings were successful, whole the thrid (3rd) harvest did not profit. The only difference was the dependency on the use of very low salinity deep-wells to replenish the water loss due to seepage and also used during water exchange. Results of the analysis of the 3rd cropping revealed low in mineral content.

Table 6. Physico-chemical Analyses of Deep-well water of key producing areas based on NPPC lab results Area

Bacolod Talisay

Salinity (ppt)

pH

6

4-8

6.7-7.0

366-394

192-224

177-225

4

0-25

230-560

134-368

125-759

24-72

62-158

6.98-8.80

112-274

145-264

165-600

8

3-10

7.5-7.6

370-372

0-15

7.1-7.3

6.5-7.4

135-172

120-648

62-821

0.2-3

6.3-6.8

140-165

29-801

4

0.5-5.0

6.2-6.5

66-140

8-104

43-317

5-20

6.7-7.6

88-334

80-416

48-336

144-715

1-2

6.5-6.7

142-226

142-226

79-120

2

0.5-1

6.5-6.6

154-189

113-145

145-260

1-2

6.7-6.8

133-167

127-146

162-189

1-5

6.0-7.6

40-248

40-248

153-293

120-360

120-360

62-821

4

Manapla

4

Victorias Cadiz

7

Sagay

11

Himamaylan

2

Bago

Pulupandan Iloilo

Calcium (ppm)

Magnesium (ppm)

# of wells checked

Silay

Saravia

Bicarbonate Alkalinity (ppm)

4

8

1-3

3-10 Batangas 8 7.0-7.4 Figures in red font indicates beyond optimum values.

Total AmmoniaNitrogen (ppm)

0.2

0.65-0.98 0.80-0.85 nil

0.08-0.90 nil-0.95 nil

0.07-.0.42

nil nil nil

0.01-0.66 0.28-0.88

The values of important water parameters highlighted in red font Table 3 revealed the necessity of knowing the physico-chemical characteristics of fresh water sources specifically deep-wells or underground water. Parameters to monitor at least every week since the chemical parameters in the water table is unpredictable:

• • • • • •

Calcium Magnesium pH Salinity Total Alkalinity Ammonia

26

G. Feeding Management

Feed monitoring generally follows the feed supplier’s instruction as a guide but the “art” on the adjustments depends on the existing culture situation and assessment of the farm technician.

The use of immune-enhancer with vitamins incorporated with the feed at 5 grams per kilo of feed administered twice a day at 6:00 a.m. and 6:00 p.m. proved beneficial in a number of farms. Application of the right feed “gut” probiotic in feeds at 2 grams per kilo.

The gut probiotic enhance digestion and has the ability to suppress the population of the non-beneficial bacteria.

The combined effect of both in feeds applied all meals in times of adverse weather condition, vibriosis, white fecal disease and light WSSV infection proved that shrimps had better chances of survival Illustration 67a and 678b: Mixing of feed inputs.

Mixing of feed probiotic and immuno-enhancers with vitamin thoroughly at least 15 minutes prior to feeding

The storage area for feeds, probiotics and other feed inputs should be safe from pests, clean and well ventilated to avoid moulds and othe contaminants.

Figure 68a, 68b and 68c: Proper feed storage and stacking in pallets up to 5 to 7 layers above the concrete floor and with a distance of 30 cm between layers. Feeds should be at least 30-50 cm away from the concrete walls for effective ventillation.

To prevent spoilage, practice the “first in, first out” withdrawal basis.

H. Disease Monitoring Prevention and Control Management Strategies

1. Shrimp Health Monitoring

Improvements in technology focusing on the prevention and management of diseases by early identification of stress factors and timely implementation of the appropriate corrective measures dramatically enhance the production of shrimp farms.

27

Table 7. Suggested Monitoring Schedule of Bacterial and PCR Analyses of Shrimps Days of Culture (DOC) 30

45

60

75

Tests Bacterial Analysis Total Bacterial Count Vibrio Profile % Green % Yellow Luminous Vibrios V. parahaemolyticus

PCR WSSV APHND / EMS EHP

Total Bacterial Count Vibrio Profile % Green % Yellow Luminous Vibrios V. parahaemolyticus

WSSV APHND / EMS EHP (when slow growth is suspected)

Total Bacterial Count Vibrio Profile % Green % Yellow Luminous Vibrios V. parahaemolyticus

WSSV

Total Bacterial Count Vibrio Profile % Green % Yellow Luminous Vibrios V. parahaemolyticus

WSSV APHND / EMS EHP (when slow growth is vsuspected)

Vibrios are opportunistic bacteria but not all are pathogenic or disease causing. V. harveyi or the luminous vibrios and Vibrio parahaemolyticus are the potential pathogenic species that commonly infect the shrimps in the Philippines. The latter is responsible in the Acute Hepatopancreatic Necrosis Disease or AHPND. Generally, vibrio bacteria sets in when the shrimps are stressed or weakened. Stress factors like poor nutrition, water quality or sudden changes in environmental conditions trigger infection in farms with high stocking density and warmer water temperature.

White Spot Syndrome Virus (WSSV) infection has been the bottleneck of most growers. Higher disease outbreaks were mostly experienced during rainy season when temperature low despite keenness in biosecurity.

Enterocytozoon hepatopenaei (EHP) is a microsporidian disease that does not cause significant mortalities but seriously impacts growth. The target organ is the hepatopancreas.

28

Table 8: Interpretation of Bacterial Analysis Results (NPPC, 2018) Fry Total Bacterial Count

Total Vibrio Count

Juvenile

(cfu/fry)

<1.0g(cfu/hp)

>1.0g(cfu/hp)

>1.0x104- 9.9 x 104 >1.0 x 105 - 9.9 x 105 >9.9x104 -1.0 x 105 >9.9 x 105 - 1.0 x 106 >1.0x105 -1.0 x 106 >1.0 x 106 - 1.0 x 107 >1.0x105 -1.0 x 106 <1.0 x 107

>1.0 x 106 - 9.9 x 106 >9.9 x 106- 1.0 x 107 >1.0 x107 - 9.9 x 107 <9.9 x 107

<1.0 x 104

<1.0 x 105

slightly high high

very high

extremely high

<5.0 x 104

normal

1.0 x 104

>5.5 x 104 - 9.9x >9.9 x 104 - 1.0 x high 4 5 10 10 4 5 >9.9 x 10 -1.0x10 >1.0 x 105-9.9x106 very high

>9.9x103

>1.0 x 104-5.5x104 >5.0x 104-9.9x104

>9.9 x 104 >60 %

>1.0 x 105

>9.9 x 106

<50%-40%

<50%-40%

>60 %

<50%-40% <40%-30%

slightly high High

very high

<30%-20%

<20%-0

V. parahaemolyticus Not detected

Ideal

<40%-30%

<30%-20%

extremely high

<20%-0

Not detected

slightly high

extremely high

>60 %

<40%-30%

<20%-0

Not detected

Ideal

not detected at 101 not detected at 102 not detected at 103 Normal <1.0 x 102

<1.0 x 102

Low

<1.0 x 103

>1.0 x 102-9.9x102 >1.0 x 102-1.0x103 >1.0 x 103-9.9x103 close >1.0 x 10 -1.0x10 2

3

>1.0 x 10 -9.9x10 3

3

>1.0 x 103-9.9x103 >9.9 x 103 - 1.0 x 104 >9.9 x103 - 1.0x >1.0 x 104-9.9x105 104 >1.0 x 104 >1.0 x 105

29

normal

<1.0 x 106

<1.0 x 104

<30%-20%

Luminous Bacterial Count

Interpretation

<9.0 x 103

>1.0 x 104-9.9x104

% Yellow Vibrio Colonies

Adult

>9.9 x 10 -1.0x10 3

4

monitoring Slightly high

>1.0 x 104 - 1.0 x High 105 >1.0 x 105-9.9x106 Very high >1.0 x 106

extremely high

2. Disease Prevention and Management Strategies

Most farms now follow the "closed system" approach. This potentially eliminates many of the obvious failures by operating a more environmentally "friendly" practice. The use of inputs like probiotics or the use of commercially available bacteria to rehabilitate and maintain the beneficial water microflora for shrimp farming proved advantageous.

Most disease problems can be avoided with strict biosecurity, good feeding and water quality management. Diagnosis, disease prevention, control and management depend on a thorough understanding of the system. The degree and the combined effect of the stress factors if not identified early and corrected might trigger infections and may result to disease outbreaks. Early detection and timely implementation of corrective or remedial measures to reduce the degree of stressors and the application of additional inputs to the shrimps’ resistance proved to be an effective management strategy in preventing outbreaks.

Table 9. Pond inputs to maintain good water quality and corrective measures when necessary Parameter Low Alkalinity (<70 bicarbonate alkalinity), wider pH fluctuation (>0.5) a.m and p.m.

Inputs

Purpose

Dose and frequency

Bicarbonate containing compounds

•Water conditioner, adjust water pH and enhances availability of nutrients.

Dolomite

•Increase bicarbonate alkalinity twice as much as agri lime and sodium bicarbonate and improves buffering capacity of the water preventing drastic fluctuation in pH CaMg(CO3)2 + H2O + CO2 ↔ Ca2+ + Mg2+ + 2HCO3-+ CO32-

5-10 ppm or50-100 kg/ha, 1 meter depth Dilute in water or broadcast directly into the pond water.

Sodium Bi-carbonate

200-250 kg/ha. oncetwice a week and when necessary

Mix first in water to dissolve fine particles before broadcasting on the pond water. This could facilitate •Provides calcium and magnesium ions faster reaction in water. needed during the molting process •Based on the reaction, dolomite competes with phytoplankton for CO2 and possibly reduce photosynthetic rates if applied during daytime

•Increases bicarbonate alkalinity with 100-200 kg/ha diluted a slight increase in pH due to the OH- in in water once a week and the chemical reaction, also expensive when necessary

Agricultural Lime •Increase buffering capacity of the 200-300 kg/ha twice a

water but does not dissolved quickly. week and when necessary.

•Provides calcium and bicarbonate ions by reaction with pond water.

Possibly reducet photosynthetic rates if applied at day time.

CaC03 + H20 + C02 -----> Ca2+ + HCO3-

30

Parameter High pH (>8.5) due to dense phytoplanktonbloom

High Ammonia and Nitrite

High Vibrio load (>1.0x104 CFU/mL) High Luminous Bacterial (>1.0x103 CFU/mL) High V. parahaemolyticus (>1.0x102CFU/mL)

Inputs

Purpose

Dose and frequency

Agricultural Lime

•Can compete with photosynthesis if 200-300 kg/ha. 2x a week applied at daytime by using carbon and when dioxide as shown in the chemical necessary reaction •Can be applied after draining (1015 cm) and refilling the water. This is done to dilute the phytoplankton concentration. Be sure to refill a less dense treated water from the reservoir.

Water Disinfectant

•Aquaculture disinfectant and 0.3 ppm or 3 kg/ha. at 1.0 virucide, can also be used to reduced meter depth phytoplankton density provided other parameters are within optimum levels including temperature. Care should be done including full paddlewheel operation.

Water Probiotic

Water Probiotic

•Reduced feeding when necessary while correcting the phytoplankton bloom.

•Beneficial bacterial consortium especially formulated to degrade and mineralized organic matter. Pro-W also controls the build-up of anaerobic decomposition products specifically ammonia, nitrites and hydrogen sulfide.

Preventive dose: 50-100 grams per hectare every 14 days or weekly.

The bacterial species and unique strains are efficient in displacing the non-beneficial bacterial population by competitive exclusion. Some have antimicrobial activity against pathogenic vibrios.

Preventive dose: 50-100 grams per hectare every 14 days or weekly.

In cases of high vibrio count, high luminous bacteria or high V. parahaemolyticus:

In cases of un-ionized ammonia toxicity and increasing trend in nitrite: 1st application - 300-500 g/ha following 10-15% water change and 2nd application - 200-300 g/ha after 3 days 3rd application-300-500 g/ ha. once a week and when necessary

The total bacterial count is 5.0 x 1010 CFU/g.

1st application - 500 g/ha 2nd application - 200-300 g/ha after 3 days 3rd application-300-500 g/ ha. once a week and when necessary Lower salinity gradually.

31

Table 10. Feed inputs to prevent disease and suggested corrective measures when necessary Pathogen High Vibrio load, majority green colonies •V. parahaemolyticus (>1.0x103CFU/mL

Inputs Feed Probiotic

ImmunoEnhancer

Purpose The new enhanced formulation had a high concentration of total bacterial count at 20,000,000,000 CFU/g or 2.0 x 1010CFU/g. This probiotic is made to protect the shrimps from colonization of pathogenic organisms in the gut.

Pro-2 together with Top S also aids in the development and increase the resistance the shrimps’ immune system.

Dose/frequency/Corrective Measures Preventive dose:

Feed Probiotic –- 2g/kg of feed once a day for 45 days from stocking.

Imuno-enhancer – 5g/kg of feed once a day for 45days from stocking.

Immuno enhancer is the most widely used feed additive in shrimp Disease management measure in cases of grow-out from the early stage up to WSSV, APHND, EHP and vibriosis harvestable sizes.

1. Submission of samples to the laboratory for It is a complete topdressing for disease diagnosis. shrimp feeds that provides optimal 2. Identification of stress factor/s attributable disease and stress resistance to weaken the shrimps. under a wide range of production 2. Timely implementation of corrective meacircumstances. It increases the sures. overall growth and survival rates 3. Boosting the shrimps resistance to combat for all species of farmed shrimp, the stress factors identified while implementfrom post-larvae until the final ing the appropriate corrective measures. harvest stage.

• Feed Probiotic -- all meals for 15 days at 2 g/kg of feed and when necessary • Immuno-enhancer – all meals for 15 days and when necessary at 5g/kg of feed.

4. Monitoring of shrimp health (submission of samples to determine disease status).

32

PART 2

Prevention and Management of Mixed Infection

33

Case Specific Experiences

SEACORP Farm, Sagay City, Negros Occidental

WSSV and EHP (2017 1st crop, 4 ponds) WSSV (2017 2nd crop, 4 ponds) Vibriosis (V. parahaemolyticus, 2017 2nd crop 6 ponds) WSSV (2018, 6 ponds) WSSV (2019, 3 ponds)

Site Characteristics:

Soil Texture Soil pH Organic Matter Content Available Iron Salinity

Farm Data

: Clay Loam : 7.9-8.6 : Low : Low :15-17 ppt

Illustration 69: SEACORP Farm

Number of Productive Ponds – 26 Mean Survival – 95-100% Stocking Density range (pcs/sq. meter) – 90-100 Mean Days of Culture – 120 days

The farm embraced the concept of entirely closed system to prevent the entry of White Spot Syndrome Virus (WSSV) and other pathogenic agents that could pose threats to the culture species.

Probiotics, vitamins and use of dis-infectants are indispensable part of its biosecurity protocol.

Inputs from December 2013 to date

• Disinfectant – Chlorine • Probiotics Water – to improve and maintain good bacterial flora Feeds – to improve and maintain beneficial gut flora • Vitamins/Immuno-Enhancer • Other inputs

Mineral Mix – for beneficial phytoplankton development

Agricultural Lime – for pH correction

Molasses to improve water transparency during water

Rice Bran during water culture

Yeast

34

Crop Performance History • • • • •

• •

2013 – WSSV (Dec) later part of the year (satisfactory crop performance) 2014 – None (Good Crop Performance) 2015 – None (Better Crop Performance) 2016 – None (Best Crop Performance) 2017 – January WSSV and EHP (4 ponds, 1st crop) June-July WSSV (4 ponds, 2nd crop) 2/24 Ponds DOC 23-30 2/24 Ponds DOC 41-48 High Vibrio Parahaemolyticus 6/24 Ponds 2 weeks DOC 56-71 2018 – 6/26 ponds infected with WSSV (July 2018) 2019– 3/7 ponds infected with WSSV (Feb 2019)

Technology before the mixed infections:

The farm practiced the concept of entirely closed system to prevent the entry of White Spot Syndrome Virus (WSSV) and other pathogenic agents that could pose threats to the culture species.

Probiotics, vitamins and use of disinfectants are part of its biosecurity protocol.

2017 Ponds with dual infection (EHP+WSSV) 1st crop

Concern: ‘Mixed infection” of WSSV and EHP pathogens. Slow Growth and significantly varied sizes were experienced. Mixed infection with WSSV could trigger significant mortalities and early termination of stocks. The concern on how to prevent re-infection was addressed using the modified interventions.

Considering that this farm has no previous history of having experienced Enterocytozoan hepatopenaei (EHP) microsporidian infection, over-reliance on quality fry stocked without proper screening procedures can be a causative factor for the pathogen to find its way into the pond Corrective actions implemented specific to the problem: Disease management strategy applied in the 4 ponds infected with WSSV and EHP • • •

• • • •

Prevention of further stress or minimizing stress Low Salinity Extended use of feed probiotic and immune-enhancer in feeds applied in all meals up to harvest.

Use of probiotic in water Regular laboratory analysis at least once a week and timely feedback system Effective communication between the owner and the farm team.

Dedicated and responsive farm staff.

Advantage of the interventions:

• The relatively high survival despite of the documented incidence of EHP and light + WSSV infection in the four ponds stocked last January 2017 proved that the management strategy adapted in the farm is sustainable and successful.

• The incorporation of the probiotic and Vitamins/immune enhancer in the protocol since Dec. 2013 had proven cost efficient inputs and resulted to good yields. The dose and response to manage mixed infections has been successfully validated and verified in other farms provided early detection and timely implementation of appropriate measures.

• The use of “mineral mix” managed to directly maintain beneficial phytoplankton density, ionic balance and suitable pond environment.

35

Table 11. Crop performance of ponds infected with WSSV and EHP after immediate and timely corrective measures done.

WSSSV + and EHP + Ponds Area (ha.)

Pond 14 0.66

Production Data

Pond 15 0.66

Pond 16

Pond 17

0.59

0.59

Fry Source

Dumaguete Dumaguete Dumaguete Dumaguete

Number of pieces

710,000.0

Date Stocked

Stocking Density (psd/sq.m)

Feeds

Date Harvested Date Culture

Average Body Weight (g) Biomass (kg)

Feed Conversion Ratio (FCR)

% Survival

1/7/2017

1/7/2017

108.58

107.58

INVIVO

710,000.0

1/13/2017 1/13/2017 618,800.00 529,800.00 104.74

CP

INVIVO

67

60

105.96

Total

2,568,600.00

Mean

0.63

642,150.00

CP

3/17/2017 3/16/2017 3/13/2017 3/16/2017 63 10

10.26

63

8.9

11.85

7,469.95

7,717.18

5,230.5

5817.8

106.27

105.94

96.39

92.66

1.47

1.44

1.38

1.28

Per ha. Basis

106.72

1.00

1,027,440.00

106.26

63.25 26,235.43

10.25

6,558.86 1.39

10,494.17

100.32

Preventive and management measures implemented in the 2nd CROP of 2017

Modification in the management strategy after the harvest of EHP and WSSV infected ponds:

Pond preparation procedure

•Soil Analysis •Admission of water (20 cm), application of Chlorine at 60 ppm (6:00pm) •After 3 days, re-chlorination at 30 ppm.

•After 7 days, the water was flushed out.

•Application of Class A Hydrated Lime at 5 tons/ha •Analysis of Crustaceans •Drying for 15 days •1st Plowing using hand tractor (1 week) •Drying (7 days) •2nd Plowing (1 week) •Drying 7 days.

•3rd Plowing (1 week) •Drying 7 days •4th Plowing (7 days) •Drying 7 days •Analysis of crustaceans for presence of WSSV and EHP •Admission of water at 5-10 cm and levelling (1 day) •Drying for 7 days

Specimen crabs

Pond Number Irrigational Canal 1

WSSV/ EHP Test Result Not Detected

soil

9B

Not Detected

crabs soil soil soil

Irrigational Canal 2 6

10 23

crabs

Irrigational Canal 1

soil

1

crabs crabs soil soil

Irrigational Canal 2 Irrigational Canal 3 8

20

Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected Not Detected

Table 12: Results of soil and crab analyses

36

Summary of Pond Preparation Drying Period – 50 days Chlorination – 60 ppm 1st dose; 30 ppm 2nd dose Hydrated Lime Application – 5 tons/ha. (Class A) Plowing – 4 times Total Number of days : 80 days Water Culture Procedure

•Water admission at 100 cm •

•Chlorination at 30 ppm after 24-48 hours

(4 units 2 Hp paddlewheels with 4 blades)

•Addition of water at 20 cm

•Water Culture

-molasses 10 kg 8:00 a.m. daily for 5

- rice bran 10 kg days

-Mineral Mix 10 kg

•Inputs wo days before stocking

-probiotic in water 200g/ha.

-molasses at 10 kg/ha.

Pond Number 17

EHP Test Result Not Detected

18

Not Detected

19

Not Detected

20

+

21

Not Detected

1

+

8

+

2

Not Detected

3

Not Detected

4

Not Detected

5

Not Detected

6

Not Detected

7

Not Detected

8

Not Detected

9A

Not Detected

9B

Not Detected

10

Not Detected

11

Not Detected

12

Not Detected

23

Not Detected

Table 13: Result of PCR analyses of crustaceans in the water source and

Fry Selection and Sourcing using the Modified Fry Criteria (P. vannamei) 1st check

-PCR analysis (less than pl <8) WSSV, EHP, EMS, IHHNV) -1st microscopic analysis -Bacterial Analysis (TVC, %G, %Y, V.p and Luminous Bacteria)

-PCR analysis (less than pl 9-10) -2nd microscopic analysis -Bacterial Analysis (TVC, %G, %Y, V.p and Luminous Bacteria)

2nd Check

37

2017-2018 Ponds with dual infection (Vibriosis+WSSV) 2nd crop Table 14. Crop performance of the 4 ponds diagnosed with WSSV and vibriosis (2017) 2017 Ponds with WSSV

Date Stocked

FCR

2nd crop

Area (ha.)

10

9A

0.81

1.67

121

9B

0.80

6/31/2017

0.80

6/6/2017

1.71

143

23

Average

6/5/2017

1.72

0.80 5/31/2017

1.75

0.80

171

DOC at Survival harvest (%)

142

125

133

79.93

Date Harvested

ABW (g)

9/28/2017

30.50

93.55

10/25/2017

91.41

10/2/2017

95.90

90.20

10/15/2017

DOC diagnosed with WSSV

Light + 30

35.36

31

31.20

30

34.89

32.99

30

Observation

no mortalities, high V.p

30

Management strategy implemented:

The mortality is chronic and there is a chance to continue the culture, monitoring was intensively and rigidly done. Since the PCR results showed WSSV light infection and V. parahaemolyticus level in the animal was <1.0 x 103 CFU/hp (juvenile), there was a strong determination to continue.

a. Feed probiotic and immune-enhancer applied all meals everyday until DOC 60

b. Laboratory analysis weekly (water and shrimps)

c. Early identification of stress factors, predicting water quality trends and rapid response through implementation of appropriate actions.

d. Regular use of probiotics in water.

Table 15. Crop performance of the 6 ponds diagnosed with WSSV and vibriosis (2018) 2018 Ponds with WSSV

Area (ha.)

Date Stocked

FCR

28

0.50

3/17/2018

1.43

93

14

0.66 8/16/2018

1.62

89

16

0.59 8/25/2018

1.77

0.62

1.61

34 19 5

Average

0.50 7/16/2018

1.34

0.50 8/20/2018

1.83

0.96 7/26/2018

1.64

DOC at Survival harvest (%) 91

Date Harvested

ABW (g)

6/17/2018

24.43

11/12/2018

20.13

12/11/2018

97

122

10/20/2018

109

71

12/6/2018

107

87

109

101

79

67

86

11/9/2018

DOC diagnosed with WSSV

Light + 30

22.1

68

26.56

54

Observation no mortalities

with 1-2 pcs mortalities in check trays

58

on and off for 2 weeks

25.94

51

very few mortalities

24.08

56

25.32

76

very few mortalities

no mortalities

38

Table 16. Crop performance of the 3 ponds diagnosed with WSSV and vibriosis (2018) 2019 (ponds left from 2018 stocking) Ponds with WSSV

25

Area (ha.)

0.5

Date Stocked 12/20/2018

FCR

1.35

DOC at Survival harvest (%)

59

90%

ABW (g)

Date Harvested

ongoing

12.00

DOC diagnosed with WSSV

52

2pcs mortalities in check trays

26

0.5

12/20/2018

1.21

63

100%

ongoing

on and off for 10 days, no dead shrimp in the center mortalities 12.10 56

28

0.5

12/15/2018

1.21

68

100%

ongoing

12.50

none in 10 days observation

66

It is important to identify various forms and aspects of stressors in our grow-out to be able to implement corrective measures. Pro-active monitoring and good record keeping is a vital component of farm management that could guide crafting management strategies for the improvement of the next crop.

39

References: Boyd CE (1990). Water quality in ponds for aquaculture. Alabama Agricultural Experiment Station.

Auburn University, Alabama.

Boyd, C.E., Pippopinyo, S. (1994). Factors affecting respiration in dry pond bottom soils. Aquaculture 120,283–293.

Boyd CE, Thunjai T (2003) Concentrations of major ions in waters of inland shrimp farms in China, Ecuador, Thailand, and the United States. Journal of the World Aquaculture Society 34: 524–532.

Cheng KM, Hu CQ, Liu YN, Zheng SX, Qi XJ (2006). Effects of dietary calcium, phosphorus and calcium/ phosphorus ratio on the growth and tissue mineralization of Litopenaeus vannameireared in lowsalinity water. Aquaculture. 2006; 251:472-483. de la Peña LD, Cabillon NAR, Catedral DD, Amar EC , Usero RC and others (2015) Acute hepatopancreatic necrosis disease (AHPND) outbreaks in Penaeus vannamei and P. monodon cultured in the Philippines.

Dis Aquat Org 116:251-254. https://doi.org/10.3354/dao02919

Kamran R.T., Gholamreza R., Frinsko, M., Daniels H. (2013). Effects of different calcium and magnesium concentrations separately and in combination on Macrobrachium rosenbergii (de Man) larviculture.

Aquaculture. 2013.07.023 Kleinschmidt, A., McClure, Andy Don (2009) Introduction to Soil.Van Wert County NRCS-USDA. https://www.slideshare.net/andykleinschmidt/introduction-to-soil-science-presentation

FAO Training Series: Simple methods for aquaculture (2003). Inland Water Resources and Aquaculture Service of the Fishery Resources Division, FAO Fisheries. Department.http://www.fao.org/fishery Lazur, Andrew (2007). JIFSAN Good Aquacultural Practices Program Growout Pond and Water Quality Management. https://articles.extension.org.

Muralidhar, Dr. M. Saraswathy, Dr. R., Raja, Kumara Dr. P., Suvana. C. and Nagavel Dr. A.(2016). Application of minerals in shrimp culture system. ICAR - Central InstItute of Brackishwater Aquaculture (Indian Council of Agricultural Research)s. 2016.,.http://www.ciba.res.in/Books/Brochure%204.pdf.

Noriega-Orozco, L., Acedo-Félix, E., Higuera-Ciapara, I., Jiménez-Flores, R., Cano R. (2007). Pathogenic and non pathogenic Vibrio species in aquaculture shrimp ponds, Vol. 49, Nos. 3-4 July – September 2007; October - December 2007 pp. 60 – 67. Retrieved from http://www.medigraphic.com, March 4, 2019.

Riley, Reynard. Water Quality Factors: Temperature, Dissolved oxygen (DO), pH, Turbidity, https:// slideplayer.com and http://www.spectrumanalytic.com/support/library/ ff/ Soil_ph_Management.htm

Sribidya W., Aanand S., Rajeswari C.P., and George Rosalind (2018). Ammonia and nitrite toxicity to pacific white-leg shrimp Litopenaeus vannamei. I n t e r n a t i o n a l J o u r n a l of A p p l i e d R e s e a r c h. 2 0 18; 4(7):1 8 2-18. www.allresearchjournal.com.

Tendencia, EA, Bosma, RH, Usero, RC, Verreth, JAJ. Effect of rainfall and atmospheric temperature on the prevalence of the white spot syndrome virus in pond cultured Penaeus monodon. Aquac. Res. 2010;41:594–597.

40

Usero and Albaladejo MAGA RD (2017). Fry Quality Assessment Criteria for Shrimp Culture in the Philippines. ISBN 978-971-8722-51-0. Bureau of Fisheries and Aquatic Resources. Quezon City, Metro Manila, Philippines.

Usero RD and Albaladejo MAGA (2015). Philippine shrimp Grow-out Farm Management Practices against Acute Hepatopancreatic Necrosis Disease with emphasis to Green Water Technology. ISBN 9788722-40-4. Bureau of Fisheries and Aquatic Resources, Quezon City, Metro manila, Philippines.

Usero RD, Albaladejo JD and Albaladejo MAGA (2006). Best Farm Practices to Resduce the Rish White Sot Syndrome Virus (WSSV) Infection in Shrimp Culture. ISBN 978-971-8722-41-0. Bureau of Fisheries and Aquatic Resources

41

ANNEXES

ANNEX A

Work instructions in measuring some field parameters using measuring equipment:

• pH

www.sainsmart.com

Interpretation of result:

Illustration 70: Work Instruction for pH determination

Lower warning limit

Death Zone

IDEAL RANGE

Upper warning limit

Death Zone

Illustration 71: pH scale 7.5-8.5 is the ideal range which matches the pH values of the shrimp’s hemolymp but morning and afternoon fluctuation should be less than 0.5

42

• Dissolved Oxygen

Work operational instructions differ depending on the brand and model but the principle is the same so it is best to follow the manual of the instrument.

Unit: Dissolved Oxygen - ppm (parts per million)

Figure 72: Models of D.O meters or mg/L (milligram per liter). commonly used in shrimp farms

Temperature - °C and should have a 0.1°C

resolution preferably Prefer brands or models with salinity adjustments for precise measurements in brackish or saline conditions.

Cleaning and storage of probe with membrane

After use, rinse in clean distilled water, wipe with soft tissue. Be careful not to scratch the membrane. For short term storage, leave the probe connected to the meter. Immerse the probe tip in clean distilled water.

Illustration 73: Immersion of D.O probe in distilled water for short term storage

• Salinity

After measuring, use a lint-free tissue to wipe the sample off of the prism surface. If the sample contains oils or fat, use isopropyl alcohol and water to clean the prism surface. When not in use, keep the refractometer in the storage case at room temperature (20°C). Avoid storing the unit in direct sunlight or in amoist environment and do not subject the unit to strong shocks or vibrations. The ambient temperature at which you can safely store the units is between 0 and 40°C

• Transparency

43

Illustration 74: Refractometer: After using, rinse the prism surface with distilled water and wipe using a clean soft tissue.

Illustration 75: Instruction on how to read transparency using a secchi disc

ANNEX B Determination of ammonia percentage

Table 16. Percentage of Un-ionized Ammonia (toxic form) pH value 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00 8.10 8.20 8.30 8.40 8.50 8.60

% UnNH3) 32 0 1.00 1.40 1.50 1.80 2.10 2.22 2.26 2.27 3.80 5.50 8.10 10.8 13.2 16.00 19.50 23.00 27.70

Computation:

pH value

8.70 8.80 8.90 9.00 9.10 9.20 9.30 9.40 9.50 9.60 9.70 9.80 9.90 10.00 10.10 10.20

% UnNH3) 32 0 0.325 0.378 0.430 0.490 0.541 0.604 0.657 0.707 0.755 0.793 0.835 0.859 0.886 0.906 0.925 0.938

Graph 1: Ammonia toxicity increases with increasing pH

ppm un-ionized ammonia (toxic form) = Total Ammonia (reading from the color standard)

x % un-ionized ammonia from the table in

Example:

reference to pH of the water.

2.0 ppm ( reading from color chart)

Water pH = 8.7

ppm Un-ionized ammonia = 2.05 ppm x 0.325 (value of un-ionized ammonia in decimal at pH 8.1)

= 0.65 ppm (toxic form)

44

APPENDIX C

ANNEX C

Field Monitoring Forms

Field Monitoring Forms SAMPLING SAMPLING SHEET SHEET Pond No. Pond Crop No. Crop Cycle: Date: Days of Cuture: GM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

TALLY

FREQ

pieces Soft Shell Broken Antennae Algal Growth Tail Rot

Blue Color Chronic No. of laya

T.WT.

%

GM 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

TALLY

Antenae scale rot: Brown gills: Black gills: Bamboo:

Total No. Sampled: Total Weight: Average Body Weight: Total No. of Castnets: Sides: Middle: FREQ

T.WT.

Pieces

%

GM 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

TALLY

FREQ.

T.WT.

Remarks (compare w/previous sampling)

Bruise: Others:

51

45

ANNEX D ANNEX D

Farm ____________________ Site _____________________ Quantity______________

Farm: Site:

Pond No. __________ Area ____________ Stocking Date _______ Fry Source _____ pL age _____________ Density ____________

Survival Monitoring Sheet

Week

Date DOC ABW (g) Weight gain (g) Last Feeds (kg) Weekly Feeds(kg) Total Feeds(Com) Biomass %Survival(est'd) %Survival(dmd) FCR(periodic smplng) FCR (Culture period) Monitoring Time Average per cast net Total Weight No. of pieces Average per cast net

Date of stocking: Stocking Density: 1 2 3 4

Pond#: 5 6

7

8

Source: 9 10

Pl age: 11

12

13

14

15

16

Remarks:

Sampled by:

Form 2

52

46

ANNEX E ANNEX E NEGROS PRAWN PRODUCERS COOPERATIVE ANALYTICAL AND DIAGNOSTIC LABORATORY Door No.1 & 2., NOLK Bldg., 6th Street., Brgy. 7, Bacolod City

Tele/Fax 034-4332131 email address nppc_adl@yahoo.com.ph CLIENT ___________________________ Lab. Number: ___ __________________

SITE: ______________ Date Submitted: ____________Date Reported: _______ LABORATORY TEST RESULTS PLANKTON ENUMERATION Beneficial Phytoplankton Non-Beneficial Phytoplankton Cells/ml % Cells/ml 1. Chlorella 1. Nitzchia 2. Cyclotella 2. Rhizosolena 3. Oocystic 3. Euglena 4. Conscinodiscus 4. Scenedesmus 5. Gramatophora 5. Chrococcus 6. Fragillaria 6. Gleocaspa 7. Navicula 7. Synedra 8. Protococcus 8. Bidulpha 9. Planktonella 9. Chodatella 10. Pleurisigma 10. Bacteriastrum 11. Skeletonema 11. *Pelagothrix 12. Chaetoceros 12. *Trichodesmium 13. Melosira 13. *Oscilliatoria 14. Diatom 14. *Spirulina 15. Licmophora 15. *Anabaena 16. Thalasionema 16. *Dinoflagellates 17. Thalassiothrix 17. *Paramesium 18. Thalasiosera 18. *Closterium 19. Amphora 19. Microcystis 20.

20. Chlamydomonas TOTAL TOTAL

%

Total Phytoplankton Count (cells/ml)_________________________ Remarks:_______________________________________________

Note: *Filamentous Blue–green algae are toxic to fry. When swallowed and digested, may release TOXIN that can cause severe necrosis of the midgut membrane. This affects nutrient absorption and makes the host shrimp susceptible to secondary infection.

Results are based only on samples submitted.

CONDUCTED BY:

APPROVED BY: 53

47

ANNEX F

Some Beneficial Planktons as viewed from the microscope

Trichodesmium

Nitchia

Cyclotella

Gramatophora

Chlorella 48

ANNEX G Aquaculture: Volume and Value of Shrimp and Prawn Production 2011-2018 1. Aquaculture: Volume of Shrimp and Prawn Production 2011-2018 Shrimp & Prawn Species Total

2010

2011

2012

2013

2014

2015

2016

2017

2018

55,901.77

54,342.98

56,416.11

59,697.66

58,449.96

60,878.28

61,683.77

62,422.72

59,575.02

(2.79)

3.81

5.82

(2.09)

4.15

1.32

1.20

(4.56)

48,161.94

47,494.68

48,196.64

49,466.92

47,843.44

49,527.33

49,139.48

46,067.65

44,780.43

689.43

689.82

778.45

757.20

1,150.63

950.34

635.80

644.03

520.64

2,076.50

1,974.05

1,878.60

1,870.99

1,826.66

1,645.89

1,673.69

1,744.29

1,758.89

2.80

2.86

4.53

5.75

3.44

2.51

1.63

1.36

0.99

4971.0991

4,181.57

5,557.89

7,596.80

7,625.79

8,752.21

10,233.17

13,965.39

12,514.07

% Change of Total Tiger Prawn Endeavor Prawn White Shrimp Freshwater Prawn P. vannamei

Volume of Shrimp and Prawn Production, 2011-2018 8.00

60,000.00

6.00

Production in MT

50,000.00

4.00

40,000.00

2.00

30,000.00

0.00

20,000.00

(2.00)

10,000.00 -

(4.00) 2011

Source: Philippine Statistics Authority (PSA)

49

2012

2013

2014

2015

2016

2017

Tiger Prawn

Endeavor Prawn

White Shrimp

Freshwater Prawn

P. Vannamei

% Change of Total

2018

(6.00)

2. Aquaculture: Value of Shrimp and Prawn Production 2010-2018 (in million pesos at current prices) Shrimp & Prawn Species

2011

2012

2013

2014

2015

2016

2017

2018

19,890.82

20,619.88

21,893.55

21,578.24

23,261.51

23,776.01

25,298.75

25,527.41

1.28

3.67

6.18

(1.44)

7.80

2.21

6.40

0.90

18,545.62

18,968.36

19,718.92

19,299.13

20,788.88

20,925.98

21,459.70

21,745.82

Endeavor Prawn

143.99

154.26

154.33

214.90

185.28

152.85

154.69

130.63

White Shrimp

309.65

322.45

309.88

321.41

278.45

282.74

344.79

364.97

0.85

1.13

1.84

1.10

0.79

0.50

0.42

0.29

890.73

1,173.67

1,708.58

1,741.70

2,008.11

2,413.95

3,339.16

3,285.71

Total % Change of Total Tiger Prawn

Freshwater Prawn P. vannamei

Value of Shrimp and Prawn Production, 2010-2018 10.00

Value in million pesos

25,000.00

8.00

20,000.00

6.00 15,000.00 4.00 10,000.00

2.00

5,000.00 -

-

1

2

3

4

5

6

7

Tiger Prawn

Endeavor Prawn

White Shrimp

Freshwater Prawn

P. Vannamei

% Change of Total

8

(2.00)

Source: Philippine Statistics Authority (PSA)

50

ANNEX H Annex G

Department of Agriculture

Bureau of Fisheries and Aquatic Resources ǡ ǡ ǡ Ǥ ͻʹͻǦ9597 • 929ǦͺͲ͹Ͷ

F i r s t R es p o n d er : F i el d T es t R ep o r t

R e port No. __ ____ N ame of Client N ame of Farm F a rm Address A c c reditation No. Date of Field Visit Date Release

: :

: : : :

AC U T E H E P AT O P AN C R E AT I C N E C R O S I S D I S E AS E (AH P N D ) T e st Code

R e f erence:

S a m p le C o d e

D a y s o f C u lt u re

S p ecies

R es u lt

Insulated Isothermal PCR (iiPCR) Chang, et al, 2012 Tsai et al 2012

A n a lyzed by :

Field Analyst Designation

Field Analyst Designation

N o t e d by:

_ _ _ _ _ _ _ __ __ __ __ __ __ __ __ __ _ __ _ Focal Person For Shrimp

Note: Result(s) as per sample received and analyzed. This report may not be reproduced except in full and with written consent from BFAR Aquaculture Division.

IFADHipon-F-02 Rev. No.00 55

51

ANNEX I Annex H

Department of Agriculture

Bureau of Fisheries and Aquatic Resources ǡ ǡ ǡ Ǥ ͻʹͻǦ9597 • 929ǦͺͲ͹Ͷ

O n - s i t e T e s t R e p o r t W a t e r Q u a l i t y T e s t f o r Penaeus vannamei

R e p or t No.

N ame of Client N ame of Farm F a rm Address Date of Field Visit Date Release FR C o d e

Samp l e Code

pH

U n i o n i x ed Amo n ia N i t r o g en 0 .1 - 0 .5 p p m

: :

N itrite N i t r o g en 0 .1 - 1 p p m

: : : N i t rate N i t r o g e n S a l in ity <6 0 p p m ) 5 - 2 5 p pt

D i s s o lve d Oxy g e n 5 .0 - 9 .0 p p m

Al k al i n i t y 50-150 ppm

C al c i u m 100 ppm

M a g n esi u m 50 ppm

METHODOLOGY: Lamotte® Smart Aquaculture Colorimetric Test Kit Source: Van Wyk and Scrapa, 1999

I m p r ession: _ _ _ _ _ _ _ __ __ __ __ __ __ __ __ __ _ __ __ __ __ __ __ __ __ __ __ __ __ _ __ __ __ __ __ __ __ __ __ __ __ __ _ __ __ __ __ __ __ __ __ __ _ _ _ _ _ _ _ _ __ __ __ __ __ __ __ __ __ _ __ __ __ __ __ __ __ __ __ __ __ __ _ __ __ __ __ __ __ __ __ __ __ __ __ _ __ __ __ __ __ __ __ __ __ _ _ _ _ _ _ _ _ __ __ __ __ __ __ __ __ __ _ __ _ A n a lyzed by : A n a lyst Designation

A n a lyst Designation

N o t e d by: F o c al Pe rson for Shrimp Note: Result(s) as per sample received and analyzed. This report may not be reproduced except in full and with written consent from BFAR Aquaculture Division IFADHipon-F-03 IFADHipon-F-03a Rev. No.00 No. 00 Rev.

56

52

ANNEX Annex I J

National Fisheries Laboratory Division ͺ͸Ͳ Ǥǡ ǡ Ǥ Ǥ ͲʹǦ͵͹͵ǦͲͲ͹͸

B F AR

PAB ACCREDITED TESTING LABORATORY PNS ISO/IEC

Test Report No. DFHL-YY-CODE Page 1 of 1

REPORT OF TEST ǣ ǣ ǣ ǣ ǣ

ǣ

LAB CODE:

CUSTOMER’S CODE:

Test Parameters

ȋ Ȍ

ȋ Ȍ ȋ Ȍ Ȁ ȋ Ȁ Ȍ ȋ Ȍ ȋ Ȍ ȋ Ȍ

Ǧ ȋ Ȍ Ȁ ȋ Ȁ Ȍ Penaeus vannamei ȋ Ȍ Macrobrachium rosenbergii ȋ Ȍ

Ǥ

Test Method

ʹͲͲͲ

ʹͲͲͲ

ʹͲͲͲ

ʹͲͲͲ Ȁ ͳ

ʹͲͲͲ

ʹͲͲͲ

ʹͲͲͲ

ʹͲͲͲ

ʹͲͲͲ Ȁ

Result

ʹͲͲͲ

ʹͲͲͲ

ǣ ͳǤ Ǥ ʹǤ Ǥ ͵Ǥ Ǥ DATE ISSUED: Analyzed by:

_________________________________

Ana lys t

Certified by:

ǡ

Approved by:

Form No.: SD-AD-QF17 Revision No.: 3 Effectivity Date: 07/01/2019

57

53

ANNEX J K ANNEX

National Fisheries Laboratory Division ͺ͸Ͳ Ǥǡ ǡ Ǥ Ǥ ͲʹǦ͵͹͵ǦͲͲ͹͸

B F AR

PAB ACCREDITED TESTING LABORATORY PNS ISO/IEC

REPORT OF TEST

ǣ ǣ ǣ ǣ ǣ ǣ

LAB CODE: CUSTOMER’S CODE:

Test Parameters

ȋ Ȍ

ȋ Ȍ ȋ Ȍ Ȁ ȋ Ȁ Ȍ ȋ Ȍ ȋ Ȍ ȋ Ȍ ȋ Ȍ

Ǥ

Test Method

Ȁ ͳ ȋ ͷȌ

Result

ǣ ͳǤ Ǥ ʹǤ Ǥ ͵Ǥ Ǥ DATE ISSUED:

Analyzed by: _________________________________ Analyst

Certified by:

ǡ

Approved by:

Form No.: SD-AD-QF17 Revision No.: 3 Effectivity Date: 07/01/2019

58

54

ANNEX L ANNEX K

National Fisheries Laboratory Division ͺ͸Ͳ Ǥǡ ǡ Ǥ Ǥ ͲʹǦ͵͹͵ǦͲͲ͹͸

B F AR

PAB ACCREDITED TESTING LABORATORY PNS ISO/IEC

REPORT OF TEST

ǣ ǣ ǣ ǣ ǣ ǣ LAB CODE:

CUSTOMER’S CODE:

Test Parameters

ȋ Ȍ

ȋ Ȍ ȋ Ȍ ȋ Ȍ ȋ Ȍ ȋ Ȍ ȋ Ȍ

Ǧ ȋ Ȍ ȋ Ȍ

͵Ǥ

Ǥ

Test Method

Analyzed by: _________________________________

Ana lys t

Certified by:

ǡ

Form No.: SD-AD-QF17 Revision No.: 3 Effectivity Date: 07/01/2019

Approved by:

59

55

Result

Ǥ

Ǥ Ǥ

DATE ISSUED:

ͳ

Penaeus vannamei ȋ Ȍ ȋ Ȍ

ǣ ͳǤ ʹǤ

ANNEX ANNEX L M

B F AR

National Fisheries Laboratory Division ͺ͸Ͳ Ǥǡ ǡ Ǥ Ǥ ͲʹǦ͵͹͵ǦͲͲ͹͸

PAB ACCREDITED TESTING LABORATORY PNS ISO/IEC

REPORT OF TEST

ǣ ǣ ǣ ǣ ǣ ǣ Analysis

Test Method

Total Vibrio Count (CFU/mL)

Quantitative Bacteriology

ȋ Ȍ

LAB CODE:

CUSTOMER’S CODE:

STANDARD BACTERIAL PROFILE Total Vibrio Count

% Yellow

Total Luminous Count (CFU/mL)

RLA No.

NORMAL Slightly High High

Very High

2.2 x 10 3 - 1.3 x 10 4 1.5 x 10 4 - 6.0 x 10 4 7.5 x 10 4 - 9.5 x 10 4 1.0 x 10 5 - 5.5 x 10 5 <2.0 x 10 2

Luminous Bacterial Count

% Green

ǣ ͳǤ Ǥ ʹǤ Ǥ ͵Ǥ Ǥ

DATE ISSUED:

Analyzed by: ___________________________________

Certified by

ǡ

Approved by:

Ǥ

Form No.: SD-AD-QF17 Revision No.: 3 Effectivity Date: 07/01/2019

60

56

About the Authors

Roselyn dela Cruz-Usero and Maria Abegail Apostol- Albaladejo

Roselyn “Rose” dela Cruz-Usero Manager and Laboratory Head, Negros Prawn Cooperative (NPPC)

Ms. Roselyn D. Usero is an invaluable gem in the chemical industry and a game changer in shrimp culture and environmental science in the Visayas region. She is the current manager and laboratory head of the Negros Prawn Producers Cooperative (NPPC), a major contributor to the development of the prawn industry in Negros province. She holds a BSChemistry degree from the Colegio San Agustin and a master’s degree in environmental engineering from the University of St. La Salle, Bacolod City. She specializes in analytical chemistry, environmental engineering, industrial microbiology, aquaculture, and shrimp culture. She has written manuals on the successful shrimp culture techniques in Negros Island and developed a fry quality assessment criteria for the country’s shrimp culture.

She also made significant contributions to the Cooperative. As its manager, the NPPC Analytical and Diagnostic Laboratory became the first and only private laboratory in the Western Visayas region duly recognized by the Department of Natural Resources’ Environmental Management Bureau and became the first laboratory in Negros island to be given an ISO/IEC 17025 accreditation. To cater to the needs of the community, Ms Usero utilized NPPCADL to provide high-quality analytical services to individuals or groups conducting research projects in Bacolod and nearby cities.

She was awarded by the Philippine Federation of Chemistry Societies (PFCS) as its 2018 PFCS Shimadzu Awardee for Chemical Industry and bestowed by the Professional Regulation Commission as the Outstanding Chemist of 2019.

57

For more than three decades in the aquaculture industry and two decades of which, where in government service, Maria Abegail Apostol-Albaladejo or fondly called Abby is presently the Chief of the Fisheries Planning and Economics Division (FPED) of the Bureau of Fisheries and Aquatic Resources (BFAR). She holds a Bachelor of Science Degree Major in Inland Fisheries from the UP Diliman Quezon City and a Master Degree in Fisheries Technology thru a distant education program of BFAR – Iloilo State College of Fisheries (ISCOF). She was trained on: (a) Shrimp Health Management at Maria Abegail “Abby” Apostol- Albaladejo, MFT, CPro Aquatic Animal Health Research Institute, Bangkok Thailand; (b) Shrimp Pathology Course at the Aquaculture Pathology Laboratory, University of Arizona, USA and Minh Phu Mekong Laboratory, Vietnam; (c) Fish Pathology and Environment Management at the National Fisheries University, Yoshimi, Yamaguchi Japan; and (d) Strengthening Aquatic Animal Health Capacity and Biosecurity by AusVet at Singapore and Vietnam. Abby is the first and only (to date) BFAR employee who successfully completed the Leadership and Management Certification Program (CPro) of the Civil Service Institute – Civil Service Commission, a course which adopts measures to promote morale, efficiency, integrity, responsiveness, and courtesy in the civil service.

Aside from her designation as the National Coordinator and Focal Person for the National Shrimp Program of the Bureau of Fisheries and Aquatic Resources since 2008. She supervised the operation of Shrimp Diagnostic Laboratory of the bureau for almost 10 years and worked in the laboratory for 18 years. She was also designated as the National Coordinator of the Seaweed Health Management Program from 1998-2017 where she pioneered the establishment and operations of seaweed culture laboratories/indoor nurseries through branch culture and microprogation (tissue calli production) and spore/sporeling culture) of farmed Eucheumatoids nationwide.

During the 6th Asia-Pacific Algal Biotechnology, she bagged an award for her work entitled, “Development of Histology of Farmed Seaweeds in the Philippines.” She also won an award on her presentation entitled, ”Aquaculture Potentials of Lake Gawaan, Lake Lenneng and Lake Danao at Mt, Province” during the Zonal Research Review of the Department of Science and Technology. She co-authored several publications such as “Best Farm Practices to Reduce Risk of White Spot Syndrome Virus (WSSV) Infection in Shrimp Culture,” “Philippine Shrimp Grow-out Farm Management Practices Against Acute Hepatopancreatic Necrosis Disease (AHPND) with Emphasis on Green Water Technology,” and “Fry Quality Assessment Criteria for Shrimp Culture in the Philippines.”

Mrs. Albaladejo is currently serving her 3rd term as an elected Board Member of the Philippine Phycological Society (PPSI). She is also member in various organizations such as the Asosasyon ng mga Propesyonal sa Pangisdaan ng Pilipinas (APPP), Philippine Shrimp Industry, Inc., (PHILSHIRMP), Seaweed Industry Association of the Philippines (SIAP), and Philippine Society of Microbiologists (PSM).

58

NOTES

61

ISBN: 987-971-8722-57-2