figure 37. requirements to achieve hatchery production goals,

Figure 37. Requirements to achieve hatchery production goals,e Use of sanitary measures

to control bacterial growthreeds that promote larvagrowth and development

Produce tcsrge numbersof flarvoe

~ Umlted or no exposure tocntiblotlcs in the water orfeeds

~ Detailed records of dallyactivities; ancfysls andreview of tank and cycleproduction trends

~ AvallMlllty of high-qucsltynauplii

~ Ade cycle facllNes mdequipment for larval cul-ture. packing and ship-ment of posticxvae

ttestrict the transmission ofshrimp paihogerts

tO shfirn farrnaPostlarvae that grow welt linthe farm envPronrnent

~ Use high-quality nauplll ~ Use specific pathogen free SPF! mmplii for larvalculture

~ Trailned and experiencedpersonnel to run thehatchery ~ Provide adequate quanti-

ties of a nutrNonaliy sounddiet

Cost-effective productionof stlarvae~ Source of high-quality

~er

Constant maintenance ofthe physical andCherniCai environmentsparameters

~ App}y sound businesspractices to hatcheryoperations

~ Malntencmce ofenvironmentalcondNons suitable forlarval Culture

Requirements To Attain Goats

Personnel

The know edge, skill and effort of people are of primaryimportance to the operatIon r!f a.shrimphatchery

Hatchery operations combine the knowledge and skills of technicallbiological organiza-tional/managerial and business areas, as well as staff to perform maintenance, labor and certainroutine tasks. The number of individual s needed to operate a hatchery facility will vary dependingon the production capacity and intensity. An e~~mple of staff for hatchery operations is given inFigure 38. Larval and live food production activities are often carried out in separate departmentswithin the hatchery organization. Larval and live food production managers are usually trainedbiologists. While each typically carries out tasks in one area, tnanagers and assistant managersshould be cross-trained to a sufficient degree and able to run other hatchery operations if needed.

The most productive hatchery operations are staffed by motivated people, These proc}uctionfacilities oAen use incentive and staff development Pi'ograms for personnel management. Quality

Chapter 4: Strategies for Larval Rearing

Small hatchery systems that produce less than 5 million postlarvae per month can be run with amodest staff. At least one per son must be experienced in 1 arval rearing and larval food productiontechtuques. However, a successful large hatchery operation requires a motivated team approach,because no single person will possess all the attributes and skills needed to run a large productionshrimp hatchery. Thus, within the operations group it is important to assemble individuals withcomplementary knowledge and skills. The greater the production capacity of the facility, the morespecialized the daily individual work tasks will be.

long-term work requires a program that recognizes and compensates outstanding individualproductivity, and setting up realistic production goals and reward systems is one successfulapproach that has been applied in shrimp hatcheries

An understaffed hatchery is a system prone to low yields or poor quality postlarvae production.Also, unmotivated, inattentive staff may cut corners to reduce work and in doing so, createcircumstances that lead directly to outbreaks of disease These are usually ditricult problems torecognize, although certain diagnoses may suggest that operating procedures need to be carefullyreviewed.

Facilities and Equipment

Obviously, adequate facilities are necessary for successful hatchery production of postlarvae.Although no hatchery system is purposefully designed improperly, some hatcheries have systemdesign Aaws, which cause or contribute to lower postlarvae production.Hatchery design and layout features should reflect a detailed knowledge of the various tasks thatcontribute to production and distribution of postlarvae. Poorly constructed or under-designedsystems are commonly contributing factors for reoccurring bacterial disease problems. A systemset-up that makes routinetasks more difficult to accomplish is predisposed to production problemsin that workers fail to carry out some procedure according to a predetermined protocol A skimpyalgae production capacity, for example, is prone to larval problems related to marginal or grossunderfeeding of algae.

P. vannamei are usually cultured in the western "intensive-style" hatcheries. Larvae and live feedrearing areas are indoors and the basic system provides seawater, air supply and drainage, algae,Ar emia and larval rearing areas. Seawater is usually stored in large reservoir tanks where it canbe settled and treated. Optional facilities include receiving sites for nauplii and additional tanksfor holding and shipment of postlarvae. Stock maintenance, algae starter cultures, microscope

use and storage, other laboratory equipment and mix-ing of supplemental feeds are provided for in separateFigure 38, Personneh categoriesrooms. Design features should be incorporated toand backgrounds required to

operate a hatchery. facilitate daily larval rearing tasks, enhance sanitationpractices and prevent the transfer of pathogens betweenlarval rearing rooms, or from larval rearing areas intothe live feed culture areas.

Specialized equipment is needed to control hatcheryconditions. For example, sand and micropore filtra-tion, ultraviolet sterilization and heat exchanger unitsare standard items in many systems. An auxiliarypower generator is essential in case of power failure.Vital pumps and air blowers should have back-up unitsinstalled for emergencies.


EnvironmentTable 7. Normal water quality conditions forthe culture of larval P. vannamei.

High quality, consistent environmentalconditions are important for the culture ofP, vannamei larvae. This environmentincludes all aspects of live feed and culturewater, as well as lighting and temperatureconditions in these areas. Some basicrequirements for hatchery water qualityare given in Table 7.

Stable, healthful environmental condi-tions are achieved through adequate facil-ity design, appropriate equipment andproper hygiene and management, Dailymonitoring of water conditions providesdirect feedback on the system operation.

Feeds

FU/ Hatchery production of larval P. van-namei relies on live feeds. The produc-tion of live feed comprises a major portion

of the daily labor in hatchery operations. The maintenance of sanitation in live feed preparationand distribution plays a significant role in the prevention of bacterial diseases in larval shrimp.Wyban and Sweeney �991! recommend that the main daily feeding be done in the morning,immediately following the water exchange. Feed levels are monitored throughout the day andadditional algae or Artemia is added if the levels fall below a minimum level.

Most modern hatcheries supplement live feeds with artificial micro-particle foods, The extentand scope of use of the many products currently on the market varies substantially between hatcheryoperations and is based, to a large extent, on the personal preferences and experience of thehatchery operations manager.

Handling

Nauplii should be collected by gentle siphoning and always kept immersed in water rather thancaptured on a net or screen Figure 39!. On the other hand, postlarvae can be netted and held outof water for short periods when transferred from one container to another. During nauplii rinsing,the rate of water flow must be carefully regulated to ensure that the current does not force andtrap the nauplii against the outflow screen.

Chapter 4: Strategies for Larval Rearing48

P. vannamei nauplii and older stage larvae are small, fragile animals that can be easily damagedby rough, inattentive handling, However, their tolerance to handling stress improves as theanimals' progress through successive larval stages. In most instances, handling occurs when theanimals are in the nauplii stage and again when they are in the postlarvae stage. Physicalmanipulations are unusual for the-intermediate larval stages because these animals are rarely movedbetween culture tanks.

Record Keeping

Daily observations, counts and measurements are a means of monitoring pro-gress, productivity and quality control in the hatchery. These measurementsshould be recorded in a systematic way so that the information can be retrievedand easily reviewed to assess production trends and unravel problems Thestorage and analysis of monitoring information in a computer database format isnow widely applied in hatchery operations Examples ofhatchery recordkeepingforms are given in Appendix C.

Pathogen Issues

P, vunnamei larvae can be affected by a variety of pathogenic or potentially pathogenic organisms Table 8!. The majority of these agents are opportunistic and facultative. Several will affect thehealth and survival of larvae, but have little to no effect on juvenile through subadult shrimp.Alternatively, IHHN virus appears to have little to no effect on the larvae or early postlarvaestages, but may affect the shrimp later in life.

VirusesFigure 39. Checking fornauplii in the spawning tank Photo courtesy of theOceanic institute's ShrimpDepartment!.

BacrrIovirrrs penaei BP! is the only virus that has beenconfirmed to cause losses of P. vurrnumei in larval culture Protozoea stage ill and older!. The other viruses Table 8!are either unknown in the larval stages of P. vurrrrumer TSV, HPV, LOVV, Reovirus and RPS! or have not yetshown an impact on growth or survival of larvae IHHNV!Both BP and IHHNV are transferred into growout environ-rnents with infected postlarvae. In addition, previouslycontaminated growout areas e.g, pond sediments! willremain infective between successive crops of shrimp andserve as a reservoir for BP and IHHNV. Circumstantialevidence implies that LOVV may be passed from generationto generation and thus, tnay enter the growout productionphase with I.OVV infected postlarvae.

In the lawal culture of P. vurv~rmei, management andpreventative strategies have been adapted for the control ofviruses, These relationships are outlined in Table 9. Ap-plied approaches either rely on some biological feature ofthe virus or the fact that no discernible negative impact onshrimp survival or growth has been demonstrated for the

49Chapter 4: Strategies for Larval Rearing

During any handling procedure, nauplii, larvae or postlawae should not be exposed to lowdissolved oxygen or rapid, wide changes in temperature, salinity or pH However, because larvalP. vurrrramei can tolerate reasonable handling manipulations, handling is not usually the cause foranimal losses.

Larval Pathogehsand Parasites

Cause Dls~so ihLarval Cultur~

Trahsmitted t< Nursery/Growout via Postlarvae

Viruses

aacufovlrus penoel BP! yesyes

Infectious Hypodermal

and Hematopoietic Necrosis

Virus iHHNV!

Hepatopancreatlc

parvo-like virus H !

Reovirus REO! No

Lymphoid organ vacuollzatlon

virus LOVV! No

R~dovirus of Penaeid Shrimp

RPS! Na

Yes

Various other rod-shaped,

oxidase positive Gram-negative

bacteria Yes

Yes

No

No

Yes variable!

Yes variable!Zoothamn!um spp.

Gregarlnes No

Possible. but has not been well demonstrateThese organisms occur nasally In most n~/P ~«en'" enfs

Table 8, Pathogens Present in P. vanr!~~l larvae

Taura Syndrome Virus TSV!

Sacteria

Vikodosis

Leucothrfx mucor

Fun~i

lagenidlum sp.

&rod"p,

Protozoa

C ap er 4: Strategjes far Larval Rearing

Table 9, Prevention anci management strategies for viruses in larva! cultureof P. vannamei.

Virus Prevention Mana ement

BP 1. SPF broodstock 1. Low stocking density of larvae:reduce stressors ln the cultureenvironment

2, Egg rinsing/disinfection 2. Isolation procedures limit crosstrcmsfer between hatchery tanks

3. Nauplll rinsing 3. Disinfect and discard infected ter@gl'o ups

4. Filtration/disinfectionof Incoming water to thehatchery

5. Maintain constant hightemperature �0-32 C! culturewater environment for Icrvalrearing

lHHNV 1, SPF broodstock 1. None

2. Disinfect and disc<md infected groups2. Use of nauplll from wildgravid spawners e.g., wild nauplii!and avoidance of nauplil derivedby captive reproduction if broodstockare infected with IHHNV

HPV

TSV

SPF broodstock

SPF broodstock

None

Disinfect and discard infected tankgi Q ups

REO

I.OVV

RPS

None

None

None

None

None

None

Chapter 4: Strategies for Larval Rearing 51

agent Interestingly, for IHHNV regional differences exist in the apparent need to exercise acontrol strategy for this agent. That is, in Hawaii, North America and parts of South America,runt-deformity syndrome RDS! has become a recognized problem and is correlated with thepresence of IHHNV. In Central America, RDS has not appeared as a widespread problem,although use of captive reproduction postlarvae with IHHNV is a rather common practice in theregion. The reasons behind these apparent IHHNV regional differences remain undefined

BP was the first virus to be recognized in penaeid shrimp Couch 1974!. The effect of BP on theshrimp host appears to diminish as the shrimp ages. Experimental studies have not confirmed thatBP results in mortality of shrimp much beyond stage 15 postlarvae Overstreet et al. 1988!.

Effective control strateyes were developed for BP once it was recognized that transmission frombroodstock to eggs could be reduced or interrupted by the application of mechanical rinsing of thespawned eggs and/or nauplii. Adaptation of nauplii rinsing Appendix C! as a standard protocolfor all incoming nauplii that have potentially been exposed from BP-infected broodstock, and use

of nauplii spawned from RP-free broodstock, have resulted in a dramatic reduction of hatcherydisease outbreaks from this agent Figure 40! Research to eliminate the transter of BP betweenculture tanks indicated that low pl I �! and drying �N hrs! would effectively destroy the infectivityof the virus LeBlanc and Overstreet 1990!

In cases where BP disease occurs even though the proper control strategies are used, evaluationshould be undertaken of the incoming water as the possible source for the introduction of the vi«s-If a direct intake pipe from the ocean provides the hatchery water supply, then it is possible thatBP may be introduced from the nearshore sediments In this case, water treatment would beindicated and should include mechanical filtration sand!, settling and disinfection chlorination[10 ppm] and dechlorination!, filtration S~t, lit! and UV disinfection. If the above measures areimplemented and BP disease continues, re-evaluate each step and consult with a disease specialist

Bacterial Diseases

Bacterial infections Table 10! of P. i umiamei larvae can be categorized by appearance.~ filamentous and rod-shaped cuticle

foulingFigure 40, Flow-through washing system fornauplli Photo courtesy of the OceanicInstitute's Shrimp Department!.~ shell disease characterized by ero-

sions and in elanization

~ digestive tract infections foregut,hepatopancreas, midgut!

~ systemic infections bacterial aredisseminated throughout the larvae!

Although light to moderate bacterial in-festations or an occasional small melan-

ized cuticle lesion is characteristic of

normal larval populations, digestive tractor systemic infections oAen result in sig-nificant larval mortality.

Chapter 4: Strategies for Larval Rearing52

Bacterial diseases have had serious economic consequences in the hatchery production of I'.iasrnamei The environmental conditions elevated temperature and high nutrient loading! presentin many penaeid hatchery systems promote prolileration of bacteria. Some of these organismsare more pathogenic to shrimp larvae than others, but these relationships are not clearlyunderstood. Monocul ture blooms of opportunistic pathogens can have catastrophic consequences.Obviously, greater demands are placed on the hatchery resources water, feeds, personnel,equipment, etc ! as the stocking density �00 larvae/liter! and production requirements increase ! 10 million postlarvae/month!. Running a hatchery close to or beyond maximum sustainablecapacity, provides a fruitful arena for devastating bacterial epidemics. Moreover, shrimp larvaecoinpromised by sublethal conditions may fall prey to opportunistic bacterial infections. Forexainple, a disruption in feed availability to larvae may predispose larvae to bacterial infection.At times, bacterial attack is the only recognizable sign of the disease, in part because the effectof the contributing factors may be transient or technically impractical to identify directly,

Hatchery design and sanitation procedures can be effective means for the prevention of bacterialdiseases of shrimp larvae. However, larvae that are compromised by nutritional or environmentalstressors or other diseases will not be spared from bacterial attack by system features or sanitationprocedures. Hatchery design and sanitation procedures that help to mitigate bacterial problemsare covered in more detail later in this section.

Vaccination has recei ved attention in recent years as a means to prevent bacterial disease lossesin larval and early postl arval shrimp Sufficient information on P. vannamei larvae is unavailableto judge if vaccination indeed protects against bacterial attack While companies that produce andmarket vaccines may promote their products as effective for controlling hatchery bacterial diseases,scientific data is apparently not available from other sources to corroborate these claims.

Hatchery probiotics "or the intentional inoculation of healthful" bacteria into larval culture wateris a new, promising means to prevent bacterial diseases in shrimp larvae. In the probiotics scheme,beneficial and non-pathogenic bacteria are inoculated daily into larval rearing tanks after waterexchange and refilling. These pure cultures of selected organisms are added at sufficient density�0' -10 /ml! to dominate the bacterial flora in the culture tank water. This strategy is effective,

Table 10. Appearance and interpretation of bacterial infections of larvalP. vari rT amei.

A~rance and Location of Organisms Inter Action

1. If lnfestallon level remcins low, larval perfomxmce Is not likelyto be affected; no action needed

Fllmnentous and rodMapedcuitde fo Uing

2. At higher levels of Infestatlon, fouling may affect larvae duringmolting and postlcsvae may die as a result of transport stress orscdinlty acclimation, Postpone shipment col/or increaseacdlmatlon protocols as compensatory mecmres.

Cuticle eroslons and mel~lzatlon A more seitous form of bacterial infection, Systemic Infection andmortality may oc~r. If Infections ere prevalent In postlarvae,anticipate lower survival during shipping and hanc9ing,

Dgestive tract Infections Grave flnclng: heavy larval mort&lty is characteristic; re~ totreatment Is unsaNsfactoty, Surviving animals will be of lowquality. Disinfect and disap affected tanks.

These infections cs e teimi~, Disinfect and disap affected tanks.

Chapter 4; Strategies for Larval Rearing

Historically, and in some modern hatchery facilities, antibiotics have been usedto control bacterial diseases Appendix C! The principal biological weaknessof this practice is antibiotic resistance. Populations of marine aquatic bacteriamay be highly susceptible to a particular antibiotic, but will have the occasionalindividual that is naturally resistant. When the antibiotic is repeatedly appliedto water with these organisms, the drug inhibits those bacteria that are notresistant. The only individuals that can multiply successfully are those that have natural resistanceto the antibiotic compound. This provides a selective advantage for the resistant bacteria whichmultiply at normal rates and, eventually, increase in number and may become the dominantbacterial Aora of the hatchery Thus, antibiotic use has the disadvantage ofbeing only a short-termcontrol solution for the problem of bacterial disease

presumably because the proliferation of potentially pathogenic strains of bacteria is inhibitedThus pathogens, if present, remain at minimal and no consequence levels in the culture water

FUngoi Diseases

Historically, the fungi I~>ge»radium sp and iiirr>lpiditim sp.!, which cause larval inycosis, were aserious threat to commercial production of I'. vu»>atmci postlarvae. Culture crashes in tanks werecharacteristic and when an outbreak occurred in a rearing tank, operators would disinfect anddump the tank ln the early 1980s, Lio-Po et al. �982! discovered that trifuralin trade nameTreflan, an herbicide! electively inhibited the motile zoospores the infective stage of the fungus!associated with larval mycosis. Trifuralin provided hatchery operators with a prophylactic meansof preventing larval mycosis As trifuralin use has become a common practice, the frequency andseverity of larval mycosis has declined Today, the disease is a sporadic problem, althoughIwgeniditrm sp and,'Y>rr>II>idium sp are probably still present in hatchery environments.

I'. vu>mamei larvae inl'ected with I cg;e»idi»m sp. or,'ii rr>II>i di t>m sp can be easily recognized instandard wet-mount preparations Appendix 8! because the fungal hyphae invading the tissues ofthe shrimp are large and easily observed While I~@;enidi»m sp. usually attacks nauplii or earlyprotozoea and Sirr>Ip>chum sp. attacks late protozoea to mysis stages, identification of the specificfungus is not necessary to control the disease

Persistent outbreaks of larval mycosi s in a shrimp hatchery implies a problem with routine hygieneand sanitation, nauplii quality, lack ol' trifuralin use, age or application protocol for trifuralin orsome combination of these factors Trifuratin is unstable in water Williams et al. 1986! andcontinuous or frequent dosage is advised to keep the compound at therapeutic levels in the water Williams and Lightner 1988!. Routine use tif standard concentrations of chlorine as a hatcherydisinfection practice does not necessarily control Ixq;enid'»m sp. outbreaks Lightner 1988!. Highchlorine levels e.g, 500 ppm! and prolonged exposure �4 hours! are needed to kill the funguscompletely Lightner 1988!

Protozoan s

The peritrich protozoans associated with epicommensal fouling disease in I'. vaonamei larvalculture include Zi>oihamni»m sp., Voriiceila sp and less frequently, I jris ylis sp. If infestationsare high, larvae and postlarvae can be adversely affected by impaired swimming and possibly,hindered mol ting

Peritrich protozoans should not become established in well managed, intensive shrimp hatcherysystems. Their occurrence in mysis or early postlarval stages indicates a breakdown in thehatchery sanitation and hygiene procedures. Filtration and water sanitation measures should beevaluated as well as mechanical rinsing procedures for incoming nauplii, Some protozoans maycolonize inanimate surfaces and bottom detritus. These areas can be sampled and examinedmicroscopically for protozoans to determine the organism's distribution in the system, If theincoming water supply is the source, the protozoans will be distributed in the algae cultures aswell as the larval rearing areas. However, if these protozoans enter with incoming nauplii, thentheir distribution should be more limited e.g., in the larval rearing tanks and effluent lines!,


Table l l, Helpful system features in the prevention and control of hatchery diseases,

BenefitsFeature Location

Removes larger detrital, organicparticulates. which are a substratefor bacteria

Filtration Scud! For incoming water to reservoir tank

on open Intake or shallow beach

well systems. fvlay not be needed

It saltwater comes from a deep well

Allows one tank to be drained and

cleaned without disruption of water

availability in the hatchery

Double reservoir

tanks

Between water source and

hatchery operations

Allows for 10VIt water exchange 2-3

times to control bacterial disease

Large reservoir

capacity

Between water source and

hatchery operatl ons

Retains progressively smaller particles.including most protozoa. fungalspores and many bacteria

On Incoming lines to algae, Arfemia

and larval rearing areas

Filtration �n. 1nu

and 0.45ii!

Ultraviolet disinfection of bacteria and

other microorganisms

On incoming lines to algae, ArfemJa


UV disinfection

Improves the reliability of routine

cleaning and sanitation

Easy access to

water distribution

lines for cia<ning

and disinfection

For incoming lines to reservoirs, algae,

Arfemia and larval rearing areas

Disinfection of one set of pipes can be

carried out without disruption of the

normal routine

Double piping for

water transfer lines

From reservoir to algae, Arfemia


Double main air

distribution lines

Air blower to algae. Arfemia


Disinfection of one set of the airlines

can be carried out without disruption of

the normal routine

Physical barriers

between areas

where algae.

Arfemia and larvae

Distinct algae, Arfemia and

larval culture areas

Prevention of bacterial contamination

from Arfemia or larval culture areas into

the al gae culture environment

are grown

Assessment of Nauplii Quality


Nauplii quality greatly influences hatchery production. Healthy nauplii are vigorous, stronglyphototactic and free of physical deformities. The quality of nauplii can be determined byassessment of the swimming and phototactic response and microscopic examination for structuraldeforinities. Avoid using nauplii that do not swim up to a light, that have a high prevalence ofindividuals with structural deformities or that are spawned from captive females with a highprevalence of IHHN-virus infected individuals. I'. vannamei nauplii should come from TSV-freebroodstock. These nauplii may go on to display RDS when stocked as young juveniles on growoutfarms. Upon receipt at the hatchery, nauplii should be thoroughly rinsed see Appendix C! toeliminate BP, if the nauplii are derived from broodstock known or possibly infected by this virus.

Table 12. Useful sanitation measures for controlling bacterial diseases,

Location Fra uencMeasure Benefits

Reservoir tanks Bi-weekly Disrupts heavy col onlzatlon

of internal tank surfaces

Water distribution

pipesBi-weeklyDrain and fill with

chlorine solutionDisinfection of Internal

scr face of pipes

Bi-weeklyMain air dlstrlbuffon

linesFill with chlorine

solution

Algae, Affemla

and larval tanksScrub and wash

with chlorine solutionAfter each Scxface disinfection

Algae dlstribution

hoses/pipesAffer each Surface disinfection

Larval rearing

tanks

All equipment After each Surface disinfection

Every other

Bl-weeklyI, Back flushSand fiffers

Disinfechon of sand

Microporefilters

Replace ccxtrtdgesor liners

corrffrr Mecl

Chapter 4: Strategies for Larval Rearing 57

In-tank alriines

and airstones in

larval rearingtanks

Drain, scrub and

wash down with

chlorine soluffon

Wash in chlorine

solution. rinse in

fresh water and

place on drying rack

Wipe down exposedtank walls when

water is lowered

during exchange

Clean. Immerse

Into chlorine

solution. rinse In fresh

water and place on

drying rack

Replace with

clean, dry lines

and stones

2. Take off line.

fili with chlorine

solution overnight

and back flush

thoroughly

Prevents build-up ofbacteria if moistcse

accumulates in airlines

Removal of bacteria

plaques from internal

tank surfaces

Prevents these surfaces

from becoming sites fora build-up of bacterial

plaques

Removal of trapped

particulates. redlsNbution

of sand to reduce

channeling

Prevents these surfaces

from becoming accum-

uiation sites for bacterial

plaques

Table 12, continued!,

Benefitslocation Measure Fre uenc

promotes efficient unit

operationl. Inspect and cleanU.V. bulbs Weekly

promotes efficient unit

operations

2. Peplace bulbs

Lowers in/tlal bacterial

count In culture waterPrior lo each

Art em/a

cysts

Ch/orine

decarx'ulatton

Every batch

prior to hatching

Arfem/a

nauprll

inhibits bacteria proliferationUve Arfem/a

holding

Concentrate and

storage at 4 Co

Every batch

Every batch I owers bacterial count

prior to stocking with incoming nauplii,into larval tanks BP removal

P. vanname/

nauplil

Larval culture

water

Lowers the bacterial count

in the larval culture water

Unthrifty nauplii will usually die before or during the protozoea l-Il stages Thus, if high lossesare encountered in young larvae, poor quality nauplii is the most likely cause


Algae cVture

and Arfem/a

hatching water

Disinfect with

chlorine/

dechlorlnate

Vigorous

rinsing In clean

saltwater

Vigorous

rinsing in clean

saltwater

"Toilet flush"

exchange and

flusNng

Every 6 months

or mare often,

if needed

Every batch

prior to

feeding

Dependent

on larval

stage and

disease status

Lowers Initial bacterial count

In the hatching tank water

Lowers bacterial count

Into larval culture water

with Arfem/a feeding

Figure 41, Troubleshooting larvalproduction problems.

Mortality and Low Yields in Naupliithrough Pastlarvae

Apparent low survival and mortality may be aproblem in the larval stages of P. »crattamei. Theexplanation for the observed lower yields varywith the larval stage Figure 41!.

Nauplii through Protozoea I-II

Ovefview

The following factors need to be evaluated todetermine the cause of mortality or apparent lowsurvival for the nauplii through protozoea 11 stages Tables 13, 14, and 15!: broodstock quality,presence of infections, environmental conditions,handling and transport methods, larval deformityand unaccounted losses.

Comments on Diagnosis ancfManagement

Broodstock 0uaii ty

Broodstock quality may impact nauplii quality iffemale age and/or diet factors are problems Diagnosis of poor nutritional condition ofbroodstockis problematical because there are no specific tests. However, several circumstances suggest thatthere may be a problem with broodstock quality. They include:

~ nauplii derived from a controlled reproduction system

~ low nauplii yields have occurred in the past in this controlled reproduction system

~ in addition to mortality, deformities absent or disfigured appendage or body segment! ofearly stage larvae are also present

e change the nauplii source

~ evaluate and improve broodstock diet, as needed

~ cull and replace broodstock, if broodstock have been in production for an extended period

Infections

» BP Lb'sease

BP is not a cause for nauplii mortality and rarely has this virus been found to cause mortality ofprotozoea I-II larvae. However, BP is easy to diagnose and can be readily ruled out in a differential

59Chapter 4: Strategies for Larval Rearing

If poor broodstock condition is determined as the cause for early larval mortality, managementoptions include:

Table 13. Causes and actions for larval infections in nauplii or protozoea stages.

Table 14. Causes and actions for larval deformities in nauplii or protozoea stages,

Cause Action

iEnvironmental stressorstemperature extremes Continuous temperature measurements e.g.. data logger! and

improve conditions

Spot check DO readings and improve as necessary

E DTA treatment of spawning, hatching and larval rearing tankwaters

low dissolved oxygen

water:rnlneral Imbalance

pH. salinity, ammonia, etc. Water quality testing

Table 15, Probable causes. identification procedures and corrective actions for lowsurvival of nauplii to the protozoea il stage.

identification ProcedureProbable Causes Corrective Action

Environment& stressors Measure physical andchemical conditions

Improve if needed

Water analysisDecfinfng water qualityduring sh pment andholding

Reduce denaty and/or decreaseshipment time

Transport or handlingtrauma

Microscopic demonstrationof multiple animals withflexed or fractured setae orruptured bodies

improve shipment or handlingpr'ace dures

Fewer animals thanexpected without theappecxance of dead Icrvae

Unaccounted losses ofkrrvae or counting errors

Improved counting proceduresor repair broken screen etc.

Chapter 4: Strategies for Larva! Rearing

Poor broodstock nutrition Assess nutritional quality of feeds and Improve if necessaryExcessive age of fernale broodstock 1! Determine age of the broodstock, and restock with younger

animals �0-15 ! months>, If available

2! fvleasure the average weight of females in the population ~drestock with younger animals �0-15 months!, If available

3! Ascertain the time period broodstock have been in thereproduction system and restock with younger animals �0-15

months!, It available

diagnostic problem solving ptan Figure 4'i!. BPdisease can be identified by microscopic examina-tion of larval wet-mounts. Infected larvae willhave the typical pyramidal occlusion bodies in thehepatopancreas Mortality will be progressive,with heavy losses continuing into the mysis andearly postlarval stages.

In terms of management, if BP infection is recog-nized in the early protozoea stages, affected tankpopulations should be destroyed by low pll dis-infection acidify water to pH 3 and let contami-nated water stand for 5 hours!. fn addition, thesource for the BP infection should be determined.

The detection of BP in the nauplii, or occurrenceof BP disease in the early protozoea stages, sug-gests a substantial BP-contamination problem.Consideration should be given to changing thesource of the nauplii used in the hatchery or thesource of the broodstock acquired to produce thenauplii. Change the broodstock spawning~hatch-ing system configuration e g, change to a singlefemale broodstock pcr spawning tank with eggcollection immediately after spawning!; iinpl-ment nauplii rinsing procedures or significantimprovements to the existing procedure Appen-dix C!, or iinprove disinfection of the incominglarval rearing water, especially if the water origi-nates from an open ocean intake in an area whereBaculovlrir.v penaeI is endemic..

!> IHHN Ii'nr.i

Although IHHNV may be present in naupliithrough early postlarval stage, this virus is not aknown cause of nauplii or larval mortality. Ifconfirmed SPF spawners are used and postlarvaeare found to be infected with IHHNV, the sourcefor the infection could potentially be the sourcewater or, possibly, the unprocessed feeds used inthe reproduction system that supplied the nauplii.Strategies to mitigate either vertical transmissionor water/wet-feed introduction of IHFINV are

outlined in Figure 43.

Figure 42. Possible causes of BP andstrategies to avoid introducing thevirus in larval shrimp systems.

Figure 43. Possible causes of IHHNVand strategies to avoid introducingthe virus in larval shrimp systems.


Tatcra Syndrome Virrts TSV!

Taura syndrome virus TSV! is not known to be present or cause disease in early to late larval P.vannamei, Research is needed to determine if TSV is transmitted from broodstock to offspringand the impact, if any, of this virus on P. iannamei larval health.

» Bactena1 Disease

Bacterial disease in early larvae signals a major problem in one or more areas of the hatcherysystem While it is fairly easy to recognize significant bacterial involvement in early larvalmortality based on microscopic wet-mount findings of abundant bacterial attack of the larvae!,finding the contributing factors may be difficult. However, identification of these factors isimportant because management changes must be made to mitigate them, or effective, long-termcontrol for the problem is unlikely

» Larva1 81ack Stomach 1>cpa»i t»

Protozoea larvae occasionally have a dark brown to black mass in the stomach Figure 44!. Thecause for the deposits or their significance to the health of larvae seems to be unknown. Additionalinformation on this syndrome is given in Chapter 6.

» larval Mycosis

A known cause of early larval mortality is larval mycosis caused by lagenidr'umsp. The diagnosis of this problem is simple because the fungal hyphae are easilyseen in microscopic wet-mount preparations of dead and dying larvae affected bythis disease. Lagenidt'um sp. is a primary pathogen of nauplii. P. vannameiprotozoea and contributing factors need not be involved in cases of larval mycosis.

However, the occurrence of this disease indicates potential problems with sanitation and/or thequality of application of Trifuralin Treflan!. Evaluation of these issues becomes important to theresolution of a larval mycosis problem.

Figure 44. Larvae with black deposits in thestomach,Environmental Condition» or Hcmdhng

and Transportati on Methods

Deterioration of water quality duringshipping or transfer of nauplii and physi-cal trauma are other potential causes formortality of early larvae. Extreme envi-ronmental changes, such as exposure tolow or high temperature, low dissolvedoxygen, elevated unionized ammonia orhigh bacterial count in the water can havean adverse effect on shrimp. Trauma tonauplii may occur during collection,packing or rinsing. If water conditions

Management of larval mycosis has been achieved by regular water treatment with low levels ofthe herbicide Trifuralin Lightner I988!. Dosage may be started in the spawning tank andcontinued through hatching, transfer andlarval rearing.

Chapter 4: Strategies for Larval Peanrig

deteriorate in shipping bags, animals will be exposed to unhealthy environmental conditions.Depending on the agent, the amplitude and the duration of contact with adverse conditions, exposedlarvae may either live or die, but are subject to extreme stress Animals exposed to sublethalconditions may develop other problems prior to the onset of losses.

Adverse water quality conditions can be demonstrated using standard water analysi s methods e.g.,meters, probes or test kit systems!. However, unless a specific effort is made, other activitiesoAen preclude the timely collection of water specimens for later analysis or measurement ofvariables when shipping bags are first opened More oAen, unhealthy conditions are visuallynoted.

The first step in the management of larvae exposed to poor water quality is to transfer the animalsinto clean water. If shipment and holding tank water differ by more than l C, however,temperature acclimation must not be neglected. After stocking, survival estimates can be madeat 24 and 48 hours to reAect the actual survival for the group.

Traumatized larvae may have broken appendages and though alive, may be unable to swimnormally. Using the phototactic response, healthy nauplii should be separated from the affectedanimals and this latter group discarded. Survival estimates should be based on those that continueto be active 24 hours after stocking

Inrval DejbrmI ty

Appendage deformity is a common larval problem The condition is recognized by microscopicexamination of larvae and demonstration of loss, bending or disruption of bilateral syinmetry oflarval appendages. The effect on individual larvae is variable but reAects the extent of impairmentto swimming or locomotor function or inhibition of molting. The cause of appendage deformitiesin younger stage larvae are scarcely documented and poorly understood. Some suspected causationfactors and remedial actions are listed in Table 14

I Inacconnled l.oxses

The total number of larvae will be reduced if larvae are lost through holes in the outflow screens.In these cases, the remaining larvae appear normal and the dead animals are absent. Carefulexamination of outAow screens should be undertaken as well as consideration of the possibilitythat the tank may have been accidentally drained or have overAowed.

Alternatively, the total number of larvae may be reduced due to errors in counting. Countingerrors can arise from use of an improper counting technique or misapplication of a suitableprocedure.

Protozoea Ill through Post larvae

Overvjee

Bacterial infections are the principal cause of mortality in mid-stage larval and postlarval P.vannamei Figure 45!. The effects of poor nutrition and declining water quality conditions tendto be contributing factors, but not the principal cause of death in these shrimp Toxic waterconditions, due to red tide blooms, contaminants from agricultural run-off and leaching from pipescan be problems but, in our experience, are uncommon events.

Chapter 4; Strategies for Larval Peanng

A! though BP can be associated with incoming nauplii, infection from this agent is usually detectedin the mysis or early postlarval stages. The impact of this disease may be variable in older larvaland postlarvai stages. In many instances, losses are minimal and the animals will performreasonably in growout production. The impact of BP on shrimp health declines as the shrimpincrease in age and size LeBianc and Overstreet 1990!

In some cases, BP outbreaks may reoccur in a hatchery system even when rigorous egg and naupliirinsing procedures are being used Optimally, use of BP-free spawners should correct theproblem In situations where BP-free nauplii are unavailable and nauplii rinsing procedures arenot always effective, an alternative course of action is to identify and eliminate factors that maypredispose larval populations to the expression of BP disease. Circumstances and conditions toevaluate include, but are not limited to the following

i Determine if larval populations are exposed to diurnal temperature changesgreater than 2 C. A practical means to do this is to use portable, continuousirnrnersion temperature data logger. If temperature fluctuation is found,improve temperature control to minimize this variation. Ideally, from thestandpoint of BP prevention, larval rearing water temperature should bemaintained between 30 to 320C

~ Assess the water and larval feeds for the presence of heavy metal or pesticidecontaminants It is well known that latent BP may be activated if shrimp areexposed to sublethal concentrations of certain types of toxicants. Submitappropriate samples of incoming water and the larval feeds e.g., Artemia cystsand any artificial feed formulations! foranalysis for the following metals orgroups of synthetic organic compounds:

> Metals: Cadmium, copper,nickel and lead


Commenfs on Diagnosis and Managemenf

Inji'err ons

» BI' Di,seave

S th t 0 ' Polychlori-nated biphenyls PCBs!, chlorin-ated hydrocarbons andorgan ophosphates

Figure 45, Suspected reasons formortality or low survival of P, vort-namei protozoea III throughpostlarvae.

If feed contamination is found, the food should be removed from use in the hatchery and BPoccurrence monitored. If contaminants are recovered from the source water, the source watermust either be changed or a system set up to remove or reduce the contaminant exposure to theshrimp larvae.

A decision about the appropriate management response to BP infection will be based on severalfactors including:

~ The prevalence of BP-atTected larvae

~ The presence of larval mortality

~ The current market acceptance of BP infected postlarvae

~ The BP infection status of the intended nursery or growout site e.g., Is BP already presenton the farm?!

» Bacterial Disease

As with the younger larval stages, bacterial disease can cause severe losses ofmid-stage larvae and postlarvae. Diagnosis of bacterial involvement is simple andinvolves microscopic observation of bacteria invading moribund larvae A searchfor possible contributing factors, especially problems with sanitation and hygiene,may yield long term benefits. The application of probiotics has shown promise asa means to mitigate bacterial diseases in shrimp larvae. Antibiotics can be usedto reduce mortality due to bacterial diseases Appendix C!. These compounds maybe subject to governinent regulation and their use is prohibited in some countries

» Iwrval Mj:enosis

,'iirolpidium sp. may be more often involved in larval mycosis of older P. vannameilarvae, while Lcq;enid ttm sp, is usually the agent of larval mycosis in younger stageanimals. Diagnosis is made by identifying the characteristic hyphae and modifiedhyphal reproductive structures. See Chapter 6 for information on prevention andcontrol,

Larval Deformity

Several types of deformiti es may be encountered in mid-stage larvae or postlarvae: Loss of distalregions of the pereiopods or pleopods or randomly distributed loss of appendages on affectedanimals. Affected shrimp may or may not survive if appendage damage affects locomotorfunction, such as feeding ability. Other syndromes recognized in postlarvae include a rigid flexureof the abdomen to form a "hook" or the stiff convex extension of postlarvae. These syndromesare often fatal and their cause is unknown.

Urtaccourtterj Losses

As with nauplii and early protozoea stages, the total number of larvae will be reduced if larvaeare lost through holes in the outflow screens or due to errors in counting The same steps shouldbe taken to determine if this factor is responsible for the loss.

Chapfer 4: Strcitegles for Larval Rearing

Table 16, Information included in a postlarval fitness examination report

~ Source of nauplll

~ Age of posnarvae

~ Date of shipment

~ Person s! performing the evaluation

~ Stocking and harvesting density In the larval rearing tank

~ Pate of survival

Summary of a visual assessment graded as good, fair or poor a-c! and estimated prevalence d. e!of the postlmvae ln terms of:

» activity

color

feeding behavior

moltingdeformed postlarvae

~ Summary of wet-mount microscopic examination findIngs on a random sample of 30 postiarvae In-cludes prevalence and grade of:

bacterial fouling

protozoan foulingcuticle black spot

BP infectionlipid levels ln the hepatopancreas [average of numerical assessment!gut to muscle ratio �th abdominal segment!postlarvae with bacterial sepsis

pcstlarvae with lcsval myc osis

appendage or body deformities

method and survival rate for a stress test~ Description of

~ If shipped, time packing started and was completed

+ Temperature of water used ln the shipment

Assessment of Postlarval Quality

An assessment of postlarval condition is an important quality control step in hatchery productionAppraising the health and soundness of postlarvae populations provides a practical means to assigncrop value. The appraisal criteria and methods should be straight forward and clearly understoodby hatchery personnel, as well as prospective seed buyers

Health and fitness evaluations should be carried out for each shipment of postlarvae that isdistributed from the hatchery on a given day to a particular destination The evaluationsobservations, findings and interpretations of these results should be documented in a standardizedform and a copy of the completed evaluation included with the shipment. These data provjde arecord of the animals' condition at the titne of hatchery discharge Table 16! The informationcan be used to compare with observations and findings on postlarvae fitness at the time pf receiptduring acclimation or in the nursery should problems be encountered Additionally an on-farmpostlarvae fitness examination is encouraged when postlarvae are received for acclimation and

Chapter 4: Strategies for Larval gearing

stocking A quality control documentation of postlarvae fitness should include some or all of theinformation listed in Table 16.

References

Couch, J.A. 1974 Free and occluded virus similar to Baculovirus in hepatopancreas of pinkshrimp Nalure 247�438!. 229-231

LeBlanc, B.D. and R.M. Overstreet. 1990. Prevalence of Baculovinis penaei in experimentallyinfected white shrimp Penaetr» vattnamei! relative to age. Aquactditcre 87:237-242

Lightner, D, V. 1988. Diseases of cultured penaeid shrimp in the Americas In. Sinderrnann,C.J. and D.V, Lightner Eds.!. Disease Diagnosis and Control in North American MarineAquaculture. Second Edition. Elsevier Scientific Publishing Co., Amsterdam. pp. 8-113.

Lightner, D.V. 1993. Diseases of Cultured Penaeid Shrimp. In: McVey, J.P. Ed.!. CRCHandbook of Mariculture Crustacean Aquaculture. Vol I, Second Edition. CRC Press,Boca Raton, FL. pp. 393-486.

Lio-Po, G.D., M.E.G. Sanvictores, M.C. Baticados and C.R. Lavilla 1982, In virro effect offungicides on hyphal growth and sporogenesis of lmgenidrnm spp. isolated from I'enaensmonrxlon larvae and Ã~yl/a»errala eggs..I. I i »h I!i». 5,97-112

Overstreet, R.M., K C. Stuck, R.A. Krol and W.E. Hawkins. 1988. Experimental infectionswith Baculovirn» penaei in the white shrimp, Penireu» vamtamei Crustacea; Decapoda!, asa bioassay. I. Wc!rid Aquaculi. 5oc. 19 17%-187

Williams, R. R. and D. V Lightner. 1988 Regulatory status of drugs and chemotheraputants forpenaeid aquaculture in the United States..l. World Aquacwlt Sr~c. 19�!: 188-196

Williams, R.R., T A. Bell and D V Lightner. 1986 Degredation oftrifluralin in seawater whenused to control larval mycosis in penaeid shrimp culture. J World AqnacNlt. 5oc 17�-4! 8-12.

Wyban, J A. and J.N. Sweeney. 1991. Intensive Shrimp Production Technology - The OceanicInstitute Shrimp Manual, Oceanic Institute, Honolulu, HI. 158 pp,


figure 37. requirements to achieve hatchery production goals,

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