Monitoring the occurrence of Zoea Syndrome (ZS) in pacific white shrimp (Litopenaeus vannamei) larval from several hatcheries in East Java, Indonesia

This study aimed to evaluate Pacific white shrimp larval quality from several shrimp breeding centers in East Java. parameters measured include 1) bolitas, 2) abundance of Vibrio spp. (water and shrimp body), and 3) muscle gut ratio (MGR). Larval samples were collected from 8 hatcheries located in East Java. The results showed that the percentage of bolitas in the hepatopancreas organ of shrimp larval in ponds 6, 7, and 8, respectively was <35%. Larval guts were found in all hatcheries, except for the 2nd pond as many as 60%. The highest abundance of Vibrio spp. (yellow colonies) in water samples and fry bodies was found in the 6th pond (26.5 × 103 CFU mL-1) and the 4th pond (76.9 × 103 CFU gr-1). The abundance of Vibrio spp. (green colony) and the highest water sample was found in the 3rd pond (3.8 × 103 CFU mL-1), while the larval was not detected. The larval found the highest percentage of MGR in the 3rd pond (87.5%), and the lowest was in the 7th pond (57.50%). Overall, our results demonstrate the infection of Vibrio spp. has involvement in the incidence of zoea syndrome in pacific white shrimp fry in hatcheries.


Introduction
The aquaculture sector in Indonesia occupies a strategic position in the development of national economy. Shrimp cultivation is one of the most prominent aquaculture industries today [1,2]. White shrimp, Litopenaeus vannamei is one of the most important commodities. The production value of aquaculture including L. vannamei in Indonesia in 2020 was 7.45 million tons and is targeted to 10.32 million tons by 2024 [3]. Management of L. vannamei is also related with its nutritional value such as protein, various types of fatty acids, vitamins, and main minerals for human health [4].
On the other hand, the rapid growth of national shrimp production is followed by major inhibiting factors such as the emergence of various diseases that actually reduce the development of the shrimp industry [5,6]. It was reported that since 1990s there have been various viral and bacterial disease outbreaks in shrimp farming systems such as white spot syndrome virus (WSS), taura syndrome virus IOP Publishing doi: 10.1088/1755-1315/1036/1/012003 2 (TSV), viral covert mortality disease (VCMD), Enterocytozoon hepatopenaei (EHP), Infection of shrimp hemocyte iridescent virus (SHIV), [7][8][9][10][11] and acute hepatopancreatic necrosis disease (AHPND) which have caused losses of up to 1 billion dollars annually [12]. Until one of the serious diseases that often occurs in L. vannamei hatcheries is the case of Zoea Syndrome. There are still lack concrete reports that explain the emergence of this disease, but several cases show that this outbreak is capable of causing mass death in the zoea larval stage of L. vannamei that is characterized by the empty larval digestive tract [13].
This outbreak is also called "Syndrome Zoea-2" and has been found in L. vannamei hatcheries in countries such as India, Ecuador, Mexico, and the United States. Sathish Kumar et al. [14] reported that shrimp larvae infected with this disease experienced a decrease in molting activity which eventually led to premature death. The death of the zoea L. vannamei stage that occurs in hatcheries is also suspected by the involvement of biotic and abiotic factors [14].
This outbreak is also called "Syndrome Zoea-2" and has been found in L. vannamei hatcheries in countries such as India, Ecuador, Mexico, and the United States. Sathish Kumar et al. [14] reported that shrimp larvae infected with this disease experienced a decrease in molting activity which eventually led to premature death. The death of zoea L. vannamei stage that occurs in hatcheries is also suspected by the involvement of biotic and abiotic factors [14].
Environmental and animal health management efforts are very important to prevent and control the spread of disease [15,16]. In this paper, we report the identification of (i) hepatopancreas and intestinal organ bolus, (ii) abundance of Vibrio spp., and (iii) muscle gut ratio (MGR) of Pacific white shrimp larvae reared in hatcheries in East Java, Indonesia. Our research is expected to provide initial information regarding the importance of monitoring the health of Pacific white shrimp larvae, especially for relevant authorities in preventing the spread of Zoea Syndrome in hatcheries.

Material and methods
Pacific white shrimp nauplius was obtained from eight shrimp hatcheries in East Java, Indonesia. There were 400 Nauplius taken from hatchery and put into an oxygenated clear plastic bag. Clinical symptoms of Zoea Syndrome (ZS) were observed manually and microscopically using a light microscope. The parameters observed included Bolitas (hepatopancreas and intestines) and the color of larvae that have entered the zoea-1 and 2 stages. The ideal bolity percentage had a value of <30% [17]. The total number of Vibrio spp. (yellow and green colonies) on water samples and Vibrio spp. (yellow colonies) of shrimp larvae were determined by culturing the Thiosulfate-citrate-bile salts-sucrose (TCBS) Agar (Merck) media, then incubated at room temperature for 24 hours. The calculation of the number of grown bacterial colonies is expressed in CFU -1 (water) and CFU -1 (shrimp larvae body) [17,18]. The assessment of digestive tract of shrimp was carried out by observing intestinal peristaltic movements and muscle:intestinal ratio in the last abdominal segment of shrimp larvae (muscle:intestinal ratio was 4:1). The indication of good digestive tract health had a value of >75% [19]. Data were analyzed using SPSS 23.0 software (IBM, USA). To find out the differences in each hatchery, the data were interpreted using the Brown-Forsythe test (P value < 0.0001) . Data was displayed in images and graphs using GraphPad Prism 8.0.1 software.

Bolitas
Bolitas are organ disorder of shrimp larvae in the hepatopancreas and intestines. The percentage of bolitas in the hepatoancreas organ of shrimp larvae showed different values in each pond ( Figure 1). The highest bolus percentage was found in hatchery 2, which was 67.50±17.68%, the lowest percentage was found in hatchery 7 that was 17.50±3.53% (P<0.0001). It is similar to what happened in the hepatopancreas organ, the highest percentage of bolus in the intestines of shrimp larvae was also found in the 2 nd pond that was 60.00±49.50%, the lowest percentage was found in the 7 th hatchery pond that was 20.00±7.07% (P<0.0001).). Our findings showed that larval conditions of Pacific white shrimp in several hatchery centers in East Java experienced bolus. Beside bolitas, the health condition of shrimp larvae can be observed through the necrosis, swelling and cytoplasm. Karunasagar et al. [20] and Austin & Zhang [21] explained that bolitas disease can cause hepatopancreas degeneration, thereby inhibiting the digestive glands due to swelling. The white shrimp culture management approach by observing the development and condition of the hepatopancreas and intestines that can have a profound and positive effect on the shrimp culture system [22]. Studies have also reported that diverse gut bacterial communities have a positive correlation with disease development and shrimp development [23]. The high percentage of bolitas in our findings could be caused by the influence of a less stable hatchery environment that affects the health condition of white shrimp larvae. In addition, natural feed that is not clean or possibly contaminated by toxic agents can also cause this disorder. It should be recalled, that under ideal conditions, the hepatopancreas of the shrimp plays an important role in regulating the innate immune system as a vital organ that is responsible for the digestion and absorption of feed nutrients [24].
Another interesting finding reported that beside the gut, microbiota that can be found in the hepatopancreas organ showed the condition that can respond the variations in environmental and health conditions and closely related to the type of host food [24,25]. Recent findings explained that the health condition of shrimp was indicated by the dominance of the general Didymella and Filobasidium in the intestine and Pyrenochaetopsis in the hepatopancreas organ. However, the sick shrimp group had a higher abundance of the genus Candida and can be used as a bioindicator to assess the health of shrimp larvae [26]. AHPND cases caused by Vibrio parahaemolyticus were also able to affect the bacterial community in the stomach and hepatopancreas of shrimp to be dysbiotic [27].

The abundance of Vibrio spp.
ch as rivers, estuaries, seas, and even deep sea waters. In aquaculture activities, water quality parameters and nutrients accumulated in the water column may affect the presence of Vibrio [28]. Our results showed that the abundance of Vibrio spp that varies in each Pacific white shrimp larvae hatchery in East Java. In rearing water, the number of Vibrio spp. The highest yellow color was found in the 6 th pool that was 26.5 × 10 3 CFU mL -1 , while the lowest number of yellow colonies in the water sample was found in the 8 th pool that was 0.9 × 10 3 CFU mL -1 . Furthermore, the highest green-colonies were found in the 3 rd pool water sample that was 3.8 × 10 3 CFU mL-1, and green-colonies were not detected in the 2 nd, 6 th, 7 th pool ( Figure 2). The Vibrio genus is often associated with various species of aquatic animals such as fish, crustaceans, and mollusks. The existence of this genus can affect the development of the larval stage to adult, in various diseases of aquatic organisms [29]. The genus Vibrio was also demonstrated by the formation of biofilms on surfaces submerged using water. In our findings, Vibrio-like bacteria yellow tended to be found more frequently in all water samples in the hatchery when compared to Vibrio-like bacteria green colonies that were only found in three water samples (Figure 2). Selective culture media (TCBS) caused sucrose-positive strains to form yellow colonies, whereas sucrose-negative strains formed green colonies [30].
Generally, the appearance of Vibrio in aquatic crustaceans can be associated with the emergence of infectious disease as a bioindicator with the pathogen. In fact, it is indicated by a large reduction in shrimp production as a consequence of the vibriosis outbreak and the diseases such as acute hepatopancreatic necrosis (AHPND) threatening shrimp farm operations [31]. The appearance of Vibrio-like bacteria yellow can be caused by the high concentration of organic matter in the rearing water column [32,33]. Our results are slightly different from those of Luis-Villasenor et al. [34] who obtained Vibrio-like bacteria in water samples of juvenile Pacific white shrimp cultured with biofloc at 1.57 × 10 4 CFU ml -1 .
In contrast to the appearance of Vibrio-like bacteria yellow found in all water samples, Vibrio-like bacteria green was only found in water samples of n three hatcheries ( Figure 2). Based on the reports from Aryal [35], Vibrio spp. with green appearance on TCBS Agar selective media can be associated with Vibrio parahaemolyticus. The existence of V. parahaemolyticus is currently being highlighted, especially for its potential in reducing aquaculture productivity to human health [36]. Our initial findings suggest that the incidence of Zoea Syndrome demonstrated by the occurrence of Bolitas may be associated with the emergence of this bacterium, the further research is needed to elucidate this.
The abundance of Vibrio spp. on the shrimp larvae body was only highlighted in Vibrio-like bacteria yellow, the Vibrio-like bacteria green was not found in all samples (Figure 3). The highest abundance of Vibrio-like bacteria yellow was found in shrimp bodies from the 4th pond, namely 76.9 × 10 3 CFU gr -1 , while the lowest abundance was found in the 1st and 3rd ponds of 1.3×10 3 CFU gr -1 and 9.1× 10 3 CFU gr -1 . The emergence of Vibrio spp. on shrimp body is normal considering this bacterium is a natural microflora in the body of wild and cultured shrimp. However, when the natural defense mechanisms of shrimp are suppressed due to disease and environmental stressors, these bacteria will become "opportunistic pathogens" for shrimp [37]. The intensive culture systems, both larval and juvenile shrimp species are often exposed to stress conditions due to high stocking densities that lead to the emergence of secondary vibriosis [38]. Thus, our findings can be used as an initial reference in efforts to manage the health of Pacific white shrimp fry which will be cultivated in an intensive system by the relevant authorities.

Muscle gut ratio (MGR)
The highest MGR measurement of Pacific white shrimp larvae was shown in the 3 rd hatchery that was 87.50±3.5%, while the lowest MGR value was statistically shown in shrimp larvae from the 7 th hatchery that was 57.50±38.89%. On average, overall results showed that the MGR values found in shrimp larvae from ponds 1, 6, 7, and 8 were still below the values from ideal larval health conditions (>75%) ( Figure  4). The good MGR of shrimp larvae had a tail muscle condition that covered more than 50% of the tail width in the 6 th segment. For poor tail muscle condition, it was shown by the muscle stretched more than 50% of the tail width in the 6th abdominal segment. Next, overall poor muscle condition was indicated by the occurrence of discoloration and thinness of the tail muscles. Some cases also find the presence of lumpy nematocysts that come out of the digestive tract and can be an indication of the emergence of an infection. In our case, the clot was represented by gregarine appearing in the larval gut.
The condition of the shrimp hatchery in not optimal environment may trigger the decrease in the MGR value. The behavior of shrimp that always exposes the water column may cause the exchange of microflora between the environment and the digestive system [39]. It is possible to cause unfavorable conditions or destabilization of the digestive microflora. Furthermore, the shrimp digestive system that is directly connected to the environment can be an entry point for various chemical or biological pollutants such as protozoa, bacteria, fungi, and viruses that are directly responsible for decreasing the profitability of shrimp production. On the other hand, intestinal disease is also associated with several names such as White Feces Syndrome (WFS) [40], White Gut Disease (WGD) [41], and White Muscle Disease (WMD) [41].
Good water quality management and aquaculture activities need to be carried out to reduce the incidence of Zoea Syndrome case. Some careless mismanagement lead to the emergence of Vibrio as an opportunistic pathogen that is able to delay early moulting for three to four days and zoea death at stage zoea-II in hatcheries with a stocking capacity of 100 million Nauplius [14]. By ensuring good management practices, following by disinfection periods, proper termination between larval production cycles, and reducing the number of stocking days of naupli, and improving the quality of natural feed (microalgae) through series dilutions, ensuring good circulation, and separating units (maturation, spawning) , larval rearing, algal culture, water supply, and labor units) can help reduce the incidence of zoea syndrome in Pacific white shrimp fry [14].

Conclusion
Overall, Zoea Syndrome is also characterized by the empty of larvae intestines due to impaired appetite since the early stage (zoea-1). We expect the involvement of ZS with the abundance of Vibrio spp., although it needs further research to confirm this by using a molecular approach. However, our initial research may provide the important information for the relevant authorities to take policy in monitoring aquatic animals health especially L. vannamei at an early stage.