Chapter 6
Luminous Vibrio and the Greenwater Culture of the Tiger Shrimp Penaeus monodon with Tilapia
Gilda D. Lio-Po

Abstract: Luminous vibriosis is a devastating infection of penaeid shrimp larvae and juveniles causing heavy mortalities. To counter the bacterial pathogen, Vibrio harveyi, shrimp farmers in the Philippines modified their growout culture method of the black tiger shrimp, Penaeus monodon, juveniles and developed the greenwater culture technique. This culture method involves the use of pond water of all-male, saline-tolerant Oreochromis hornorum as rearing water for the culture of shrimp juveniles in ponds. Such a modified culture of P. monodon juveniles was found effective in preventing the onset of luminous vibriosis. Basic studies revealed that antiluminous Vibrio factors are inherent in the bacterial, fungal, and microalgal flora of the tilapia water, dermal mucus, and gut that singly or collectively inhibit the growth of V. harveyi, in vitro. The skin mucus studies of other brackishwater fish species showed that the siganids, Siganus guttatus, and red hybrid tilapia (Oreochromis niloticus × Oreochromis mossambicus), as well as sea bass, Lates calcarifer, are promising alternative fish species for this novel shrimp culture method.

A review of pond-simulated studies in tanks and ponds, similarly, confirmed these findings and the impact of the greenwater culture technique on water quality, including its economic benefits to the farmer. The greenwater culture of shrimp can sustain the successful production of shrimp juveniles by inhibition of the luminous Vibrio. This culture method is also currently used in the growout culture of the white shrimp, Litopenaeus vannamei.
Keywords: luminous Vibrio, greenwater culture, black tiger shrimp Penaeus monodon, Litopenaeus vannamei, jewel tilapia, biocontrol, fish mucus, growout culture
Introduction
Aquaculture is a major source of fish and crustacean products worldwide and particularly important in Southeast Asia where penaeid shrimp are cultured not only for local consumption but also for export in the world market. The shrimp commodity has generated substantial income in developing countries. Among penaeid shrimp in Southeast Asia, the

Tilapia in Intensive Co-culture, First Edition. Edited by Peter W. Perschbacher and Robert R. Stickney. © 2017 John Wiley & Sons, Ltd. Published 2017 by John Wiley & Sons, Ltd.

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82 Tilapia in Intensive Co-culture

indigenous black tiger shrimp, Penaeus monodon, commands a high price and therefore is the preferred species for culture.
In the Philippines, the indigenous black tiger shrimp was among the first shrimp species developed for aquaculture. Pond production of shrimp juveniles was low before the 1980s. However, with the development and establishment of shrimp hatcheries, postlarvae became readily available for stocking in growout ponds. Hatchery-bred postlarvae were then stocked as juveniles in growout ponds for 120–150 days until reaching the preferred marketable size of 120–150 g. Shrimp farmers started stocking postlarvae in growout ponds at extensive stocking densities (7.5 shrimp/m2). Encouraged by the profitable returns, stocking densities were increased to semi-intensive (20 shrimp/m2) and intensive (30 shrimp/m2) levels. The aquaculture production of shrimp in the country then increased from 1,805 mt in 1982 to 47,951 mt in 1990 (Corre et al. 2000). By 1992, annual production gradually declined when farmers experienced continued crop failures. The total pond culture area of 47,776 ha in 1991 dropped to 36,658 ha in 1993.
By 1994, the successful harvests of marketable pond-reared P. monodon shrimp could no longer be sustained due to outbreaks of luminous vibriosis (Lio-Po 1998; Lavilla-Pitogo et al. 2000). The development of this infection may be attributed to shrimp farmers venturing into intensive stocking densities of shrimp in the growout ponds. Increasing stocking densities concomitantly resulted in the use of more commercial feeds to support growth of the cultured shrimp. Since not all the feed was actually consumed by the shrimp, portions of the uneaten feed decomposed on the pond bottom thereby increasing the organic load in the pond and reducing available dissolved oxygen needed by the cultured shrimps. At the same

time, the organic matter in the pond nourished the bacterial populations including those that were pathogenic. In addition, the levels of ammonia, nitrite, hydrogen sulfide, and other toxic chemicals in the pond became elevated and caused stress on the shrimp (Chen and Lei 1990). Increased biochemical oxygen demand, chlorophyll a, total suspended and dissolved solids, and sublethal to lethal levels of hydrogen sulfide were, similarly, observed in P. monodon shrimp ponds cultured to 141 days (Corre et al. 1997). Said culture was stocked with 120,000 postlarvae/ha but yielded low survival rates (18.9–23.2%).
Adverse environmental conditions in the pond enhance the development of bacterial disease outbreaks. The most devastating of these maladies were those attributed to luminous vibriosis. By 1997, almost all shrimp ponds in the Philippines suffered consistent, massive outbreaks of the disease. Subsequent experimental studies on shrimp culture management methods were then conducted to prevent and control the occurrence of the disease in shrimp growout ponds. Among these was the greenwater culture technique.
Luminous Vibriosis
The disease is devastating to penaeid shrimp larvae and juveniles, resulting in mortalities up to 100%. The causative agent is a gram-negative bacterium, Vibrio harveyi (Lavilla-Pitogo et al. 1990; Lavilla-Pitogo et al. 1998; Leaño et al. 1998). The junior synonym of V. harveyi is Vibrio carchariae (Pedersen et al. 1998; Gauger and Gomez-Chiarri 2002). Colonies of this bacterial pathogen cultured in nutrient agar exude luminescence in the dark (Fig. 6.1). Thus, in shrimp hatchery tanks, when larvae are infected with the bacterium, a diagnostic feature

Luminous Vibrio and the Greenwater Culture of the Tiger Shrimp Penaeus monodon 83

Figure 6.1 Luminous colonies of Vibrio harveyi cultured in nutrient agar and photographed in the dark. Photograph by C. Lavilla-Pitogo.

is a greenish glow-in-the-dark phenomenon when observed at night. Hence, some hatchery operators in the Philippines initially called this condition, the “New York, New York Disease,” after the famous American city of lights.
Luminous Vibrio outbreaks also caused mass mortality of larvae and juveniles of P. monodon and other penaeid shrimp in Indonesia (Suranyanto and Mariam 1986; Prayitno and Latchford 1995), Australia (Muir 1987 as cited by Oakey and Owens 2000), Thailand (Ruangpan and Kitao 1991; Jiravanichpaisal et al. 1994), India (Karunasagar et al. 1994), Taiwan (Song and Lee 1993; Liu et al. 1996b), PR China (Xu et al. 1993; Vandenberghe et al. 1998), and Ecuador (Robertson et al. 1998). Experimental infection confirmed the pathogenicity of V. harveyi to P. monodon, Penaeus indicus, Penaeus japonicus, Penaeus orientalis, Penaeus chinensis, and Litopenaeus

vannamei (Lavilla-Pitogo et al. 1992; Prayitno and Latchford 1995; Jiravanichpaisal et al. 1994; Liu et al. 1996a; Robertson et al. 1998).
Affected shrimp larvae are lethargic and become opaque-white. Its hemocoel and internal tissues are densely packed with bacteria. There are no significant external signs of the disease except for the occasional darkening of the cephalothorax region in the area of the hepatopancreas. The bacterial infection route is oral, resulting in the formation of plaques on the mouth apparatus of the infected larvae (Lavilla-Pitogo et al. 1992).
Luminous vibriosis of P. monodon juveniles in growout ponds were observed at 18–32 days of culture in the Philippines (Leaño et al. 1998). Affected shrimp had luminous Vibrio loads of 104 colony forming units (CFU)/hepatopancreas (hp) vis-a-vis 101 CFU/hp in uninfected shrimp.
Infected shrimp juveniles often swim to the pond surface and edges. Diseased shrimp

84 Tilapia in Intensive Co-culture

manifest loss of appetite as a result of severe inflammation in and around the tubules of the hepatopancreas. Melanized lesions were found in the proximal region of the hepatopancreas. These lesions disrupt the digestive function of the organ as the necrotic parts become nonfunctional. Total necrosis and dysfunction lead to death, while partial dysfunction causes slow growth due to poor digestion, absorption, and assimilation of nutrients. Systemic infections result in mortality reaching up to 100%. The occurrence of mortality is preceded by a shift of the bacterial profile in the rearing water, dominated by luminous Vibrio (Lavilla-Pitogo et al. 1998). This phenomenon was also confirmed by Sung et al. (2001) reporting that during the initial 60 days of culture, the composition of the Vibrio community in the pond water remained fairly diverse, but subsequently, decreases in species diversity were observed.
The virulence of V. harveyi has been attributed to its secretion of exotoxins (Liu et al. 1997; Liu and Lee 1999; Montero and Austin 1999; Harris and Owens 1999). In addition, the presence of a bacteriophage was experimentally demonstrated to mediate the virulence of V. harveyi in P. monodon (Ruangpan et al. 1999; Oakey and Owens 2000; Austin et al. 2003). In Thailand, the bacteriophage was identified as a novel siphovirus-like phage 1 (VHS1) (Pasharawipas et al. 2005; Khemayan et al. 2006). On the other hand, in Australia, Oakey and Owens (2000) identified the associated bacteriophage, V. harveyi myovirus-like (VHML). Infection of naïve avirulent V. harveyi strains with VMHL converted them into virulent cultures (Munro et al. 2003).
The luminous Vibrio can be isolated in the laboratory using nutrient agar (supplemented with 2% sodium chloride) or a selective and differential medium, thiosulfate-citrate-bile salt-sucrose (TCBS) agar. Harris et al. (1999)

developed a new medium, V. harveyi agar, for isolation and enumeration of V. harveyi that differentiates V. harveyi from other marine and estuarine Vibrio species.
Presumptive detection of luminous Vibrio is based on its bioluminescent colonial characteristics on nutrient agar, green-colored colonies in TCBS agar, and microscopic visualization of gram-negative short rods from pure colonies, or the presence of the bacteria in histological sections of the hepatopancreas of infected shrimp (Lavilla-Pitogo et al. 1998). Standard biochemical tests may be useful for Vibrio identification, but confirmatory identification of Vibrio species will require molecular tests such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), fluorescent amplified fragment length polymorphism (FAFLP), and loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) (Robertson et al. 1998; Conejero and Hedreyda 2003; Oakey et al. 2003; Conejero and Hedreyda 2004; Gomez-Gil et al. 2004; Hernandez and Olmos 2004; Thongkao et al. 2015).
Greenwater Culture
As a result of continuous crop failures in the aquaculture of P. monodon, modifications in its growout culture technique were developed. Greenwater culture was among the most promising. The concept of greenwater culture of P. monodon was initially conceived and developed by shrimp aquaculturists in Negros Occidental, Philippines, in cooperation with the Negros Prawn Producers and Marketing Cooperative, Inc. (NPPMCI) staff, currently known as Negros Prawn Producers Cooperative, Inc. (NPPCI), and the Bureau of Fisheries and Aquatic Resources (BFAR) staff in 1996 (NPPMCI 2000; Paclibare et al. 2001). This novel culture method prevented luminous

Luminous Vibrio and the Greenwater Culture of the Tiger Shrimp Penaeus monodon 85

vibriosis and enhanced growth and survival of the tiger shrimp. Subsequently, this culture technique was adopted and modified by other shrimp aquaculturists in the country (Corre et al. 2000).
The greenwater culture method summarized here is derived from a report that documented the development and established this culture technique (NPPMCI 2000). Basically, the culture method involves the use of tilapia culture water as rearing water for the culture of P. monodon juveniles in growout ponds. The method uses the all-male, saline-tolerant jewel tilapia, Oreochromis hornorum. The fish are stocked 4–5 months in advance in brackishwater ponds. When the tilapia stock grows to a 3–3.5 t/ha biomass, its rearing water is pumped into the shrimp pond 3 days before initial stocking of the shrimp postlarvae. The standard procedure of pond preparation consisting of flushing, drying, tilling, and liming is followed. Two to three tilapia ponds for two shrimp ponds are prepared as tilapia water reservoirs. The tilapia and shrimp ponds are usually 0.5 ha each. In addition, four tilapia net cages (10 m × 10 m × 1.5 m) per 0.5 ha shrimp pond are installed in the middle of the shrimp pond. Tilapia are stocked at 1,000/net cage (6.67/m3) at 100–200 g sizes. The stocking density of shrimp is 18–20 postlarvae/m2. Shrimp postlarvae intended for stocking in the growout ponds are screened for bacterial load, specifically, luminous Vibrio, and virus presence before purchase and stocking. Paddlewheels are operated from late afternoon until the next morning to ensure availability of sufficient dissolved oxygen for the cultured shrimp. Commercial feeds are provided to the shrimp after the natural food is exhausted. Throughout the culture period, quantitative determination of the luminous Vibrio is frequently monitored. Water management is largely dependent on the luminous Vibrio

load. Only tilapia water is pumped into the shrimp pond whenever water exchange is required. Adherence to the greenwater culture method protocol consistently maintains low levels of luminous Vibrio and helps in the successful production of P. monodon juveniles (NPPMCI 2000).
Subsequent modifications of the greenwater culture method were reported. Corre et al. (2000) described two methods to produce greenwater for the culture of P. monodon juveniles. One is by stocking all-male, saline-tolerant tilapia in a pond and pumping its water into the shrimp pond. The second is by stocking all-male, saline-tolerant tilapia in cages within the shrimp pond. Alternative fish species to O. hornorum is the Mozambique tilapia (Oreochromis mossambicus) if the former species is unavailable. Details of the stocking density of the tilapia, feeds and feeding scheme, and water and wastewater management protocol are described, which are largely similar to the original method (Corre et al. 2000; NPPMCI 2000).
Antiluminous Vibrio Factors in Greenwater Culture
The following information is based on a series of basic studies that investigated the factors inherent in the greenwater culture of the tiger shrimp with tilapia conducted from 1999 to 2005. The sampling sites were the greenwater growout culture ponds of the tiger shrimp in Negros Occidental, Philippines. Research involved identification of the bacterial, fungal, and phytoplankton flora of the rearing water, P. monodon gut, as well as the tilapia gut and skin mucus that were inhibitory to V. harveyi. The results demonstrated the sources of antiluminous Vibrio factors in the greenwater culture technique that explained why greenwater

86 Tilapia in Intensive Co-culture

culture was effective in preventing outbreaks of luminous vibriosis among P. monodon juveniles in growout ponds.
Bacteria
Bacteria associated with the greenwater culture of P. monodon were isolated from the rearing water, shrimp gut, and O. hornorum gut and mucus. The isolates were screened for antiluminous Vibrio metabolites. Among the 87 bacteria tested, 55 (62%) of the isolates caused inhibition (+∼+++) of V. harveyi after 24-h co-cultivation in vitro. Growth inhibition rates of less than 50% (+), 50–80% (++), and more than 80% (+++) were exhibited by 14, 13, and 28 isolates, respectively, 24 h after co-cultivation assays. However, 22 of the isolates lost their antiluminous Vibrio properties 48 h after exposure. The majority of the isolates that inhibited the luminous Vibrio were obtained from tilapia mucus and gut. Eight isolates consistently sustained maximum inhibition (+++/+++) of luminous Vibrio 24–48 h after exposure (Lio-Po et al. 2005b). A combination of three to four of these bacterial isolates when inoculated into the rearing waters of shrimp juveniles in tanks, yielded promising results in the biocontrol of the luminous Vibrio (Lio-Po and Villa-Franco 2005). In addition, this bacterial combination has probiotic potential (Lio-Po et al. 2007; Lio-Po 2010a).
Fungi
Fungi isolated from the greenwater culture system were Rhodotorula sp., Saccharomyces sp., Candida sp., Mycelia sterilia, Penicillium sp., and an unidentified fungus (Leaño et al. 2005). These isolates exhibited slight (+) to moderate inhibition (++) of V. harveyi. Among the 20 yeast isolates, four showed

intracellular metabolites inhibitory to luminous Vibrio. Among the 45 filamentous fungal isolates, 5 had intracellular metabolites while 3 of 41 isolates had extracellular metabolites that were inhibitory to the luminous Vibrio (Lio-Po et al. 2005b).
Phytoplankton
Microalgae sampled from the greenwater culture water were identified as Nannochlorum sp., Leptolyngbia sp., Navicula sp., Nitzschia sp., Thalassiosira sp., Skeletonema sp., Anabaena sp., and Chaetoceros sp. (Guanzon et al. 2004). The Nannochlorum sp. dominated the microalgal population followed by Leptolyngbia sp. Similarly, Cremen et al. (2007) reported the predominance of the diatom Nannochloropsis sp. in greenwater culture of P. monodon in Iloilo, Philippines.
Co-culture of Chaetoceros sp. and Nitzschia sp. with V. harveyi demonstrated complete inhibition of the luminous Vibrio 24 and 48 h postexposure. Luminous Vibrio population was reduced from 104 down to 100 CFU/ml 24 and 48 h postexposure, while that of the controls without the microalgae increased to 105 CFU/ml. Leptolyngbia sp. caused a 94–100% reduction of the luminous Vibrio population, from 104 to 101 CFU/ml 24 h postexposure of the bacterial pathogen, which was sustained for 10 days. In contrast, Nannochlorum sp. yielded fluctuating inhibition of V. harveyi during the 7-day exposure. Skeletonema sp. did not result in a significant reduction of luminous Vibrio. However, when mixed cultures of Nannochlorum sp., Chaetoceros sp., Skeletonema sp., and Leptolyngbia sp. were co-cultured with the pathogen, a drastic reduction of the population of V. harveyi was observed after 3 days exposure (Fig. 6.2) (Lio-Po et al. 2002).

Luminous Vibrio and the Greenwater Culture of the Tiger Shrimp Penaeus monodon 87

1.0E+7

Luminous Vibrio Nannochlorum + Leptolyngbia + Luminous Vibrio Nannochlorum + Chaetoceros + Luminous Vibrio Nannochlorum + Skeletonema + Luminous Vibrio Chaetoceros + Skeletonema + Luminous Vibrio Chaetoceros + Leptolyngbia + Luminous Vibrio Skeletonema + Leptolyngbia + Luminous Vibrio Nannochlorum + Chaetoceros + Skeletonema + Leptolyngbia + luminous Vibrio

1.0E+6

1.0E+5

Luminous Vibrio (cfu/ml)

1.0E+4

1.0E+3

1.0E+2

1.0E+1

1.0E+0 012345 Day
Figure 6.2 Mixed cultures of microalgae and their effect on Vibrio harveyi.

6

7

Fish Mucus
Results revealed that the luminous Vibrio was not detected from the skin mucus of O. hornorum. Moreover, dermal mucus scraped from O. hornorum and co-cultured with V. harveyi inhibited the growth and killed the pathogen 6–48 h after exposure (Lio-Po et al. 2005a; Lio-Po et al. 2005b).
To determine the potential of other fish species cultured in brackishwater ponds

as substitute species for O. hornorum in the greenwater culture of P. monodon, comparative assays on the presence of antiluminous Vibrio properties of skin mucus were conducted with red hybrid tilapia (Oreochromis niloticus × O. mossambicus), milkfish (Chanos chanos), Malabar grouper (Epinephelus malabaricus), rabbitfish or goldlined spinefoot (Siganus guttatus), seabass (Lates calcarifer), and red snapper (Lutjanus argentimaculatus) and compared with

88 Tilapia in Intensive Co-culture

O. hornorum (Lio-Po et al. 2005a). Comparative exposure of the luminous Vibrio to the epidermal mucus of rabbitfish, jewel tilapia, red hybrid tilapia, seabass, red snapper, and milkfish showed that the inoculated bacterial pathogen was inhibited or reduced (Table 6.1). As in the mucus of the jewel tilapia, the mucus of rabbitfish had no resident luminous Vibrio flora. In fact, the skin mucus of rabbitfish exerted the most rapid bactericidal effect on the 105 CFU/ml population of the test bacteria, in less than 3 h. The skin mucus of red hybrid tilapia, seabass, and jewel tilapia induced a rapid reduction in less than 3 and 6 h, 6 and 24 h, and 6 and 48 h when inoculated with 103 or 105 CFU/ml V. harveyi, respectively. Red snapper and milkfish mucus inhibited the luminous Vibrio in 24 and 48/96 h, respectively. Only the skin mucus of grouper did not show any antagonistic effect on the test Vibrio. Thus, different fish species secrete varying potencies of antiluminous Vibrio substances on their skin mucus. The data provide a guide

to the choice of fish to use in the greenwater growout culture of P. monodon in case of the unavailability of jewel tilapia.
Tank/Pond Studies
As an offshoot of the foregoing basic studies on the greenwater culture of P. monodon in growout ponds, tank-simulated and pond studies were conducted by other researchers. The use of the greenwater culture system in concrete tanks using saline-tolerant tilapia, seabass, siganids, snapper, grouper, and milkfish were also reported to prevent luminous bacterial disease (Tendencia et al. 2004, 2006a, 2006b). The microalgae Chlorella sp. and O. hornorum were also shown to be effective in controlling the growth of luminous bacteria in a simulated shrimp polyculture system (Tendencia et al. 2005). In addition, Tendencia et al. (2015) confirmed that water quality in the greenwater culture pond improved as it passed through the tilapia

Table 6.1 Detection of luminous Vibrio on the skin mucus of different fish species and reduction of V. harveyi from 103 and 105 CFU/ml after exposure to skin mucus of the same fish species (Lio-Po et al. 2005a).

Fish species

Vibrio (CFU/ml) Presence of luminous

Reduction time (h) Of V. harveyi to undetectable levels (CFU/ml)
103 105

Rabbitfish (Siganus gutatus) Jewel tilapia (Oreochromis urolepis hornorum hybrid) Red tilapia (Oreochromis niloticus × Oreochromis mosambicus) Seabass (Lates calcarifer) Red snapper (Lutjanus argentimaculatus) Milkfish (Chanos chanos) Grouper (Epinephelus malabaricus)

None None
Not tested
101 103 102, 104 104

<3 <3 6 48
<3 6
6 24 24 24 48 96 to >96 >96 >96

Luminous Vibrio and the Greenwater Culture of the Tiger Shrimp Penaeus monodon 89

reservoir and that soil sulfur level was lower and water qualities were better in the greenwater ponds compared to the non-greenwater ponds. Furthermore, Joyni et al. (2011) that bioturbation can be caused by the swimming activity of tilapia in the reservoir pond, thereby improving soil and water quality by exposing organic matter to oxygen.
The economic advantage implications of the greenwater culture technology have been confirmed (NPPMCI 2000; Bosma et al. 2012; Bosma and Tendencia 2014). The latter specifically reported that the mean harvested shrimp biomass was higher in greenwater than in non-greenwater ponds. Hence, analyses of the financial data from four greenwater culture farms and three non-greenwater culture farms revealed mean annual net profits of US$6,760/ha/yr in the greenwater farms and US$3,900/ha/yr in the non-greenwater farms.
Current Status of Greenwater Culture
With the availability of specific pathogen-free white shrimp, L. vannamei, a faster growing species than P. monodon, many shrimp farmers in the Philippines shifted to the culture of the white shrimp. However, this exotic species is also susceptible to the fatal effects of luminous vibriosis (Robertson et al. 1998). Fortunately, the greenwater culture method is also effective in preventing the disease in white shrimp. Hence, shrimp farmers readily used the greenwater culture technology with the additional application of commercial probiotics, and, to date, majority of shrimp farms in the Philippines are using greenwater culture technology for L. vannamei juvenile production (Roselyn Usero, NPPMCI, personal communication May 2015). In a survey of more than 60 farms in the Philippines, only 17% were practicing

greenwater culture in 2008. That increased to 75% by 2009 (Bosma and Tendencia 2014). Moreover, an integrated mangrove greenwater (MGW) culture of shrimps may improve water quality (Tendencia et al. 2012). Bosma et al. (2012) further concluded that the integrated MGW can sustain additional ecosystems services such as fish breeding and nursing grounds and coastal protection.
Adoption of the greenwater culture technology in other countries can be hindered by unavailability of saline-tolerant O. hornorum. However, other fish species such as red hybrid tilapia, S. guttatus, and L. calcarifer may be considered as alternative species in brackishwater ponds (Lio-Po et al. 2005a). In Vietnam, where Early Mortality Syndrome (EMS) or Acute Hepatopancreatic Necrosis (AHPN) was endemic and caused by Vibrio parahaemolyticus, farm trials of shrimp grown in greenwater induced by tilapia O. niloticus showed better survival of the Pacific white shrimp L. vannamei juveniles (Tran et al. 2014).
A few years after the first outbreak of luminous vibriosis, another more devastating infection caused by the White Spot Syndrome Virus (WSSV) affected shrimp juveniles. The first outbreak of WSSV in the Philippines occurred among greenwater cultured P. monodon juveniles in 1999. The disease can cause much higher mortalities of P. monodon, L. vannamei, and other penaeid shrimp species. In addition, the virus can be carried by other penaeid species and aquatic invertebrates such as crabs, crayfish, lobsters, Acetes, freshwater prawns (Macrobrachium rosenbergii), and Artemia (Lio-Po 2010). Hence, WSSV disease is much more difficult to prevent, contain, and control. The downside of greenwater culture is its ineffectiveness in preventing WSSV infection in penaeid shrimp species. As such, biosecurity measures now include

90 Tilapia in Intensive Co-culture

the stocking of WSSV-free and luminous Vibrio-free postlarvae in growout ponds.
Acknowledgments
Appreciation is due to Roselyn Usero for comments on the greenwater culture and for updates on the use of greenwater culture technology in the Philippines; Valeriano Corre, Jr. for comments on the greenwater culture technology; and Celia Lavilla-Pitogo for sharing the luminous Vibrio culture photo and to the SEAFDEC/AQD Library staff for assistance in retrieving scientific literatures.
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