Las Bolitas Syndrome in Penaeus vannamei hatcheries in Latin America

Las Bolitas Syndrome in Penaeus vannamei hatcheries in Latin America | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Las Bolitas Syndrome in Penaeus vannamei hatcheries in Latin America Pablo Intriago, Bolivar Montiel, Mauricio Valarezo, Xavier Romero, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4009796/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 12 Jun, 2024 Read the published version in Microorganisms → Version 1 posted You are reading this latest preprint version Abstract Several hatcheries in Latin America reported mortality of zoea stage 2 Penaeus vannamei. In fresh mounts, round structures resembling lipid droplets were observed, reminiscent of a disease called "Las Bolitas Syndrome" first identified in 1987. Closer examination under routine histopathology revealed the presence of detached cells and tissue in the digestive tract, whereas unaffected tanks displayed a typical intestinal content containing algae cells. Polymerase Chain Reaction of diseased and healthy batches of larvae for 22 shrimp pathogens revealed similar test results. The larvae were negative for nineteen pathogens, including AHPND. The detection of Vibrio spp. in both samples of affected zoea 3 (Z3) was the principal difference. Histology of affected zoeas were characterized by tissue degeneration in the hepatopancreas forming spheres that eventually moved into the upper gut, midgut and midgut caeca - a pathology known as Bolitas syndrome (BS). Microbiological analysis showed Vibrio spp. at ≤ 10 5 CFU zoea/g, ≈ 2 orders of magnitude higher than healthy zoea. Isolation of bacteria from healthy and BS affected zoea onto TCBS and CHROMagar™ and consequentially identified by API 20 E revelated six strains of V. alginolyticus. Though fresh mounts resembled the general description for “Las Bolitas Syndrome”, the histopathology differed from the original description. The intestine contained sloughed cells; the lateral lobes constituting the developing hepatopancreas in Z3 could be differentiated by their colour, with sloughed cells inside the peritrophic membrane. PCR and microbiological analyses verified that the origin of Las Bolitas Syndrome is bacterial in nature, with Vibrio playing a significant role. Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION During the late 80’s, 90’s and the first years of the 2000’s, the most predominant pathologies in hatcheries in Latin America were the Las Bolitas syndrome (LBS), zoea 2 syndrome and Mysis molt syndrome (Morales 1992 , Robertson et al. 1998 , Vanderberghe et al. 1999). While during the same period, the main pathologies reported in post larvae were luminescent bacteria (Baticados et al. 1990 , Lavilla-Pitogo et al. 1990 , Song & Lee 1993 , Nithimathachoke et al. 1995 ). After 2015, disease outbreaks with high rates of mortality were more commonly seen in post larval production, e.g., Post-larvae AHPND (PL-AHPND), Translucent Post Larva Disease (TPD), etc (Zou et al. 2020 , Yang et al. 2022 , Intriago et al. 2023, Yang et al. 2023 ).LBS is a condition firstly characterized by a distinctive pathology of the hepatopancreas, where there is a cellular desquamation of the walls of the hepatopancreas forming spheres, which eventually move into the upper gut. At the same time, the larvae become bioluminescent, which is accompanied by changes in behaviour and in a loss of appetite (Morales 1992 , Robertson et al. 1998 ). Although LBS might have had incidence during post larval stages (PLs), massive mortalities (i.e., of up to 90%) were observed in the earlier zoea and mysis stages (Morales 1992 ), and as distinctive characteristic LBS that always went together with the bolitas in the hepatopancreas that eventually moved to the intestine. LBS has been associated with infection of Vibrio spp. (Morales 1992 , Intriago and Jimenez 1998, Robertson et al. 1998 ). Zoea-2 syndrome was only associated with V. harveyi and V. alginolyticus (see Vanderberghe et al. 1999, Wiradana et al. 2022 ), and that associated with mysis syndrome has never been identified. The nocturnal luminescence seen in hatcheries in both Asia and the Americas were typically attributed to luminescent Vibrios (Baticados et al. 1990 , Lavilla-Pitogo et al. 1990 , Song & Lee 1993 , Nithimathachoke et al. 1995 , Robertson et al. 1998 ). In more recent times, outbreaks of sudden and acute mortalities in penaeid shrimp hatcheries typically start in the PL stages, from an active and apparently healthy state to moribund and dead. The speed and virulence at which these massive mortalities occur have been observed in many production facilities, and in most cases have been associated with, or linked to, different strains of Vibrio (see Zou et al. 2020 , Yang et al. 2022 , Intriago et al. 2023). Intriago et al. ( 2023a ) provided evidence that the cause of these rapid mortality events was due to a species of Vibrio carrying the same plasmids as the Vp AHPND reported causing acute hepatopancreatic necrosis diseases (AHPND) in culture ponds elsewhere. The condition was tentatively named post-larvae AHPND (PL-AHPND) to differentiate it from other pathologies affecting penaeid shrimp larvae. In Asia, Translucent Post Larva Disease (TPD) has been the main cause of larval disease and mortality, where the causative agent is a strain of V. parahaemolyticus carrying a hemolysin gene (isolate Vp-JS20200428004-2) (Zou et al. 2020 , Yang et al. 2022 , Yang et al 2023 ). In India, a similar condition referred to as zoea 2 syndrome was described affecting zoea 2 of P. vannamei (see Kumar et al. 2017 ). In September 2023, some culture facilities in the Latin America region experienced high mortalities in zoea stages 2 and 3. Given the high mortality, these tanks were discarded. The causal agent associated with those mortalities has not been reported. Microscopic observation of the larva revealed the presence of “bolitas” (spheres) in the hepatopancreas (Fig. 1 a, b). Generally, the clinical indicators and the macroscopic appearance on wet mounts aligned with what was previously identified as LBS. (Morales 1992 ). The present study reports the microbiological, polymerase chain reaction (PCR) tests and histological findings of healthy and of diseased animals suffering from this this condition. Figure 1 about here MATERIALS AND METHODS Sample collection Samples of Penaeus vannamei larvae from two hatcheries in Latin America were sampled (precise location details are withheld acknowledging the facilities request for confidentiality). Samples for microbiology, PCR and histopathology were taken from two tanks from each hatchery. Hatcheries were selected because one of the hatcheries had reported heavy mortality at zoea 2–3 stage, while the other hatchery, with apparently healthy zoea 3, was selected as the control. It should be noted that shrimp sampled for PCR and histology were different individuals from the same populations. To protect client privacy, the country and the precise location of each culture facility from which the samples were obtained will not be disclosed. Microbiology of the larvae The total concentration of bacteria in the larvae, was determined by first bathing the larvae (i.e., 1 gram or approximately > 2000 zoea stage 3) held in a 200-um nylon sieve for 2–3 min in a solution of 50 ppm active chlorine prepared using 35 ppt seawater. The zoea were then rinsed with excess sterile seawater. The weight of the larvae was recorded using a Metler digital balance accurate to 0.01 gram and then placed in a mortar with approximately 1 gram of autoclaved beach sand and 10 mL of sterile seawater which was used to facilitate the grinding of the bulk of larvae. Once the sample had been ground, the volume and weight were recorded again, then the samples were sequentially diluted in test tubes with sterile seawater to 1 × 10 2 , 10 3 and 10 4 . Then, 100 µL of the relevant dilution was placed per duplicated on Petri dish plates containing either Tryptic Soy Agar (TSA, Difco), thiosulphate-citrate-bile-sucrose agar (TCBS, Difco) or on CHROMagar™ Vibrio and then incubated for 24–48 h at 30 o C. Thereafter, the number of colony-forming units (CFU) on each plate was recorded. Dilutions were based on obtaining > 20 to < 200 colonies per plate. Bacteria identification was performed using an API 20E Kit (Buller 2004 ). PCR methods used DNA was extracted from whole larvae fixed in 90% alcohol using an Omega, Bio-Tek E.Z.N.A tissue DNA kit following the manufacturer’s protocol. In brief, each 1 g sample was ground using a microcentrifuge pestle. Approximately 200 mg of the tissue was then transferred to a clean 1.5 mL Eppendorf tube, then 500 µL of tissue lysis buffer (TL) and 25 µL of Omega Biotek (OB) protease solution were added. The sample was then vortexed and then incubated in a thermoblock at 55°C for approximately 3 h with vortexing every 30 min. RNA was removed by adding 4 µL of RNase A (100 mg/mL), then mixing, then keeping the sample at room temperature for 2 min. The sample was then centrifuged at 13,500 RPM for 5 min, and the supernatant carefully transferred to a new 1.5 mL Eppendorf tube. To this, 220 µL of BL buffer was added, and the mixture was vortexed and incubated at 70°C for 10 mins. Thereafter, 220 µL of 100% ethanol was added and vortexed, and the contents were passed through a HiBind® DNA Mini Column into a 2 mL collection tube. The columns were then centrifuged at 13,500 RPM for 1 min, and the filtrate was discarded. Subsequently, 500 µL of HBC buffer (diluted with 100% isopropanol) was added to the column, and the sample was spun at 13,500 RPM for 30 seconds. The filtrate was discarded, the column was washed twice with 700 µL of DNA wash buffer diluted with 100% ethanol, and the sample was centrifuged at 13,500 RPM for 30 seconds. The filtrate was discarded. This step was repeated. The column was then centrifuged at 13,500 RPM for 2 mins to dry it out. The dried column was placed in a new nuclease-free 1.5 mL Eppendorf tube, and 100 µL of elution buffer, which was heated to 70°C, was added to the column. The sample was allowed to sit for 2 min before being centrifuged at 13,500 RPM for 1 min. This elution step was repeated. The eluted DNA was then stored at -20°C until required. RNA was extracted from whole larvae, tissue or organs fixed in 90% alcohol following the manufacturer’s protocol (Omega, Bio-Tek E.Z.N.A. Total RNA Kit (TRK)). Approximately 200 mg of tissue was then moved to a clean 1.5 mL Eppendorf tube. To this, 700 µL TRK Lysis Buffer was added, and the tube was left at room temperature for approximately 3 h with vortexing every 30 minutes. The sample was then centrifuged at 13,500 RPM for 5 mins, and the supernatant was carefully transferred to a new 1.5 mL Eppendorf tube to which 420 µL of 70% ethanol was added. After vortexing to mix thoroughly, the contents were passed through a HiBind® RNA Mini Column into a 2 mL collection tube. The columns were then centrifuged at 13,500 RPM for 1 min, and the filtrate was discarded. Subsequently, 500 µL of RNA Wash Buffer I was added to the column, and the sample was spun at 13,500 RPM for 30 seconds. The filtrate was discarded, and the column was washed twice with 500 µL RNA Wash Buffer II and diluted with 100% ethanol. The column was then centrifuged at 13,500 RPM for 1 min to dry it out. The filtrate was discarded. This step was repeated. The column was then centrifuged at 13,500 RPM for 2 mins to dry it out. The dried column was placed in a new nuclease-free 1.5 mL Eppendorf tube, and 70 µL of nuclease-free water was added to the column. The sample was centrifuged at 13,500 RPM for 2 min. This elution step was repeated. The eluted RNA was then stored at -70°C until needed. The pathogens tested are listed below. PCR analyses were conducted for the following pathogens: DNA viruses: Hepanhamaparvovirus (DHPV), Macrobrachium Bidnavirus (MrBdv), Decapod iridescent virus 1 (DIV1), white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) as both, infectious IHHNV and the EVE form (endogenous viral elements). RNA viruses: Wenzhou shrimp virus 8 (WzSV8) /P. vannamei Solinvivirus (PvSV), Penaeus vannamei nodavirus ( PvNV), Covert mortality Nodavirus (CMNV), Infectious Myonecrosis Virus (IMNV), Yellow Head Virus (YHV), Taura Syndrome Virus (TSV) and Machrobrachium Nodavirus (MrNV). Bacteria and other pathogens of concern: Spiroplasma , Propionigenium , Rickettsia -like bacteria (RLB), necrotizing hepatopancreatitis bacteria (NHP-B), Vibrio spp., acute hepatopancreatic necrosis disease (AHPND), Ecytonucleospora [ Enterocytozoon ] hepatopenaei (EHP), other non-EHP Microsporidia, and Haplosporidia. Histopathology For histological analysis, samples were prepared following the procedures outlined by Bell and Lightner (1988). Briefly, larvae were fixed in Davidson’s alcohol, formalin, acetic acid (AFA) using at least 1 gram of larvae from each tank. These were fixed for at least 24 h before processing for routine tissue embedding and histological sectioning. The 5 µm thick tissue sections were then stained with hematoxylin and eosin (H&E). Additional sections, however, were stained with three different stains: Twort’s Gram stain to differentiate Gram positive from Gram negative bacteria (CP Lab Chemicals, Novato, California USA) and methyl green pyronin Y modified stain was employed to distinguish DNA and RNA (PolyRnDcom, Bay Shore, New York, USA). For each sample of larvae collected from each tank, one blocks were prepared, with each block containing approximately 1-gram zoea stage 2. Four tissue sections were cut from each paraffin block. RESULTS PCR results PCR analysis of both the healthy and affected samples of zoea were negative for seventeen known shrimp pathogens, including DHPV, MrBdv, SHIV, WSSV, PvNV, CMNV, IMNV, YHV, GAV, TSV, MrNV, XSV, Spiroplasma , NHPB, EHP, AHPND and Haplosporidia (Table 1). The apparently healthy zoea, however, were positive for IHHNV EVE (2/2), WzSV8/PvSV (2/2), RLB (1/2) and Microsporidia (1/2). By comparison, the LBS affected zoea were positive for IHHNV EVE (1/2), Vibrio spp. (2/2), RLB (2/2) and Microsporidia (1/2). In summary, the only difference between the two sets of samples were the detection of Vibrios in the zoea affected with LBS and WzSV8 in the healthy zoea. Table 1 about here Microbiology The concentration of total bacteria (TSA) and presumptive Vibrios (TCBS and CHROMagar TM Vibrio ) are shown in Table 2. The concentration of total bacteria in the affected zoea were almost an order of magnitude higher than that determined for the sample of healthy zoea. Presumptive Vibrios were almost two orders of magnitude higher in the affected zoea than in the healthy zoea. Presumptive Vibrios were found to represent 17% and 6% of the total bacteria population in the unhealthy and healthy zoea, respectively. Green colonies (on TCBS) and purple colonies (on CHROMagar TM Vibrio )represented 0.2% and 82% of the those recovered from the affected zoea, while a reverse picture of 56% and 1% was found from the analysis of the healthy zoea (Table 2). Table 2 about here A total of eleven strains were isolated from the LBS affected zoea and three from the healthy zoea (Table 3a). Biochemical profiling of the eleven strains recovered from the affected zoea, identified six as V. alginolyticus , two as V. fluvialis and one as V. vulnificus, one Aeromonas (undetermined) and one Pasteurella (undetermined). Of the three strains isolated from the healthy zoea, one strain was identified as V. alginolyticus, one as V. fluvialis and one as Aeromonas. Interestingly, all 14 strains were positive for Vibrio by PCR, which suggests that 27% of the results returned by the API 20E biochemical profiles were false negatives. Two sequences were used to identify Vibrio bacteria, all except by LBS-4 were positive for both methods. None of the 14 bacteria were positive for V. parahaemolyticus or for ToxR nor for the high virulent protein genes tested (Vhp-1, Vhp-2, Vhp-3) (Table 3a). The strains identified as Aeromonas and V. fluvialis were Pir AB positive (AHPND). The strain H-3 was positive for V. harveyi . Table 3b shows the API 20E characterization of the Vibrio spp. that were isolated. This table is divided between LBS zoea (i.e., Vibrio spp. isolated from affected zoea), V. alginolyticus and Vibrio spp. (with 6 and 5 strains), against the three species of Vibrio isolated from the healthy zoea. The availability of fermentation/oxidation of Arabinose was the main difference between Vibrio spp isolated from affected and the other two groups of bacteria, namely vs V. alginolyticus from LBS affected animals and Vibrio spp from healthy zoeas. On the other hand, V. alginolyticus from LBS affected animals differentiate from the other two groups in been unable to fermentation/oxidation of Amygdalin but with the capability of Acetoin production (Table 3b). None of the 14 strains were ToxR positive or luminescent. Table 3a and 3b about here Histopathology In fresh mounts of normal healthy zoea 2 larvae, algal material was seen in the digestive tract, giving it a brown coloration (Fig. 1a). By comparison, the affected / diseased larvae had an empty digestive tract and the presence of a round-shaped material that appeared as lipid droplets (Fig. 1b), this general clinical sign is the one usually described by shrimp hatchery technicians and biologists for what has been known as “Las Bolitas Syndrome”. Figure 1 about here Eight microscopy slides stained under H&E containing approximately 120 larvae of normal larvae at zoea 3 stage and a similar number from each diseased tank were examined. Normal healthy zoea 3 larvae had algae present in their digestive tract. At this stage the digestive tract is still in formation; the lateral lobes that will eventually develop into a fully functional hepatopancreas could also be seen in the tissue sections (Figs. 2a, b) (Abubakr & Jones 1992). An intact peritrophic membrane was also present and the algal material that had been consumed at this stage was seen as small sized brown cells (Fig. 2c). No sloughed cell material was observed in the lumen of the early forming digestive system of the healthy larvae (Fig. 2d). Figure 2 about here Approximately 60% of the larvae from the affected tanks with mortality had brown-green, hyaline spheres, the detached material within appeared to a B-cell (Figs. 3a & b). In the intestine, sloughed cells and material was observed (Figs. 3c & d) giving a contrasting difference to the intestines of the healthy larvae (Fig. 2c). The folds that constitute the early, developing hepatopancreas had sloughed cells that could be differentiated by their colour (Fig. 3e). Interestingly, some of these sloughed cells were inside the peritrophic membrane (Fig. 3f). The faeces from these larvae contained the same material that was seen within the intestine (Fig. 4a) and was contained within a peritrophic membrane. In the sections stained with Twort’s stain, the presence of what appeared to be bacterial cells was observed both within and outside the faecal strands (Fig. 4b). Figure 3 about here During this study, no bolitas (i.e., spheres) were observed in the histological samples, and none have ever been documented in the literature in the zoeal stages. The preparation of tissue samples for histology requires their processing through several solvents and so it is tempting to suggest that nature of the bolitas is lipidic in nature. Figure 4 about here DISCUSSION The present study describes the histopathological lesions, PCR, and microbiology of P. vannamei zoea samples collected from two different hatcheries, one with an outbreak of LBS, and the other with apparently healthy animals. Of all pathogens that were analyzed by PCR, the only key difference between the LBS and healthy zoea were the detection of Vibrios in the disease zoea. The presence of the WzSV8 virus in the healthy zoea requires more study; this RNA virus has been found in a wide range of different environments and regions including wild broodstock, and its action in penaeid production needs to be the subject of further studies (Intriago et al. 2023b ). Microbiology showed a high microbial load within the affected zoea that was one order of magnitude higher in the total number of culturable bacteria (TSA) and almost two orders of magnitude higher in the number of presumptive Vibrios when compared to the healthy zoea. Presumptive Vibrios represent 17% and 6% of the total bacteria population in the unhealthy and healthy zoea, respectively. Green colonies (on TCBS) and purple colonies presumptive of V. parahaemolyticus (on CHROMagar™ Vibrio) represent 0.2% and 82% of the total Vibrio count in affected zoea, while a reverse was seen in the healthy zoea which had 56% and 1% respectively (Table 2 ). The finding is interesting because the common assumption is that green colonies on TCBS and purple colonies on CHROMagar™ Vibrio typically represent V. parahaemolyticus . However, it is known that this is not always necessarily the case and highlights the need for microbiology-based investigations to be conducted in combination with molecular biology-based studies to gain a clear identification of the pathogens present. It is important to note that all 14 strains were identified by PCR as members of the genus Vibrio , so it can be concluded that API 20E results should be taken with caution. In this study 21% of the strains were false identified at the genus level. While Overman et al. ( 1985 ) stated that the API 20E is a valid system for use in the identification of the more commonly occurring members of the family Vibrionaceae, however, the system has been reported to result in false negatives (Croci et al. 2006, Fabbro et al. 2010 ). API identification is based on biochemical profiles, but it has been found that biochemical profiles and genotype are not necessarily associated with virulence potential (Lydon et al. 2021 ). The relationship between the variation, or differences in the API 20E identifications and the possible virulence- genus variation of each strain is something that requires further exploration. From the above information though it can be concluded that species of Vibrio are the main pathogenic agent(s) causing LBS. It is also important to note that the pathology is note related to AHPND or other high virulent pathologies described for P. vannamei larvae. Table 3 b provides details regarding the characterization of the isolated Vibrios using the API 20E system. This table is divided between LBS Zoeas (Vibrios isolated from affected Zoeas) V. alginolyticus and Vibrio spp (with 6 and 5 strains), against Vibrios (3) isolated from healthy Zoeas. Fermentation/oxidation of Arabinose and Amygdalin was different between the three groups of Vibrio isolated from affected vs healthy zoeas (Table 3 ). Some strains of V. anguillarum strains were separated in distinct groups could be separated mainly based on their reaction on indole production and the fermentation of amygdalin and arabinose (Grisez et al. 1991 ). Acetoin production was the main difference between V. alginolyticus , and other species of Vibrio isolated from healthy and affected zoeas. The ability for acetoin production by V. alginolyticus isolated from LBS affected animals is interesting, generally this metabolite is produced when microorganisms employ the 2,3-butanediol pathway to ferment sugars, this pathway generates less acidic and more neutral end products, such as acetoin and 2,3-butanediol. Because acetoin is a neutral fermentation product and this biosynthetic reaction consumes intracellular protons, bacterial growth can occur on a glucose carbon source without pH decrease (Vivijs et al. 2014 ; Oh et al. 2016 ). In addition, inhibition of acetoin production has been suggested as potential mechanism to control the pathogenic V. cholerae , that are known to be acid sensitive (Oh et al. 2016 ). A comparison between the histology in this study and the first report of LBS described by Morales ( 1992 ) is impossible. Unfortunately, only transmission electron microscopy (TEM) images were presented, and no tissue sections stained in H&E were recorded. The publication by Robertson et al. ( 1998 ) shows the presence of melanized necrotic bundles in the hepatopancreatic tubules (Fig. 2 in Robertson et al. 1998 ). This feature was never seen during the present research. The most likely reason is that the animals used for the histology were not zoea but were least at PL1 stage when comparing the anatomy of those that were presented to other detailed descriptions regarding the larval development of P. vannamei (see Muhammad et al. 2012 ). Important differences between the pathology presented here for LBS and for PL-AHPND (see Intriago et al. 2023a ) is that the peritrophic membrane remains intact (Figs. 3 a, 4ab) and there is no massive sloughing of cells in the hepatopancreas as was described for PL-AHPND. In addition, PL-AHPND was never found or described in zoea (Intriago et al. 2023a ). The presence of the green-brown material in the B-cells of the hepatopancreas (Figs. 3 a, b & f), is intriguing and it is not wrong to suggest that it is a product that could not be processed by the larvae and stored in these cells; its presence also requires further investigation. A similar material present in shrimp larvae reared in China and suffering mortalities during the zoea 2 stage has been described by other authors; in their case this was associated with a strain of V. alginolyticus (see Sun et al. 2019 ). Historically, the Latin American shrimp sector has followed the Ecuadorian model, where post larvae are produced from broodstock obtained from production ponds. Broodstock are selected based on weight, and then transferred to a hatchery to produce the next generation of post larvae. This process gives little to no long-term control over biosecurity (Wolkenfelt and Blonk, 2021 ). In addition, high concentrations of Vibrio spp. can be very common in non-SPF (i.e., specific pathogen free) or non-bio secure penaeid hatcheries, both as free and as attached to the larvae. From hatching through to harvest, the microbiological environment is a soup of bacteria and virus like particles (VLP) (Hameed 1993 , Garcia and Olmos 2007 , Intriago et al. 2018 ). As the larvae transitions from a diet based on algae as zoea to animals that source proteins as mysis, the larvae then undergo a dramatic change in the volume of the hepatopancreas and the biochemistry of the digestive enzymes (Kurmaly et al. 1989 ). In zoea, the filtration of particles is almost indiscriminate. As the zoea stages are exposed to very high concentrations of bacteria, free-living and attached, including Vibrio that are easily ingested and able to pass into the digestive tract (Soto-Rodriguez et al. 2003 ), the detrimental effects of such can be observed depending on several factors such as the larval sub-stage involved, the Vibrio species present, and their concentration (Guzman et al. 2001). Intriago and Jimenez ( 1997 ) replicated bolitas syndrome in P. vannamei zoea using a luminescent strain of V. harveyi at concentrations as low as 10 3 cell/mL. Interestingly, this strain was isolated from diseased farm cultured P. vannamei affected by haemocytic enteritis. They postulated that pathogens could be bouncing back from hatcheries to the broodstock or vice versa and that the differences found in the histopathology between the larvae and adults could have been attributed on one hand due to the differences in the degree of organ development, and on the other hand to the pathogen species, its virulence, and concentration. The key event in the appearance of diseases could be attributed to stress (temperature, salinity, density, toxins, etc.) because of alterations in the environment, this exerts a change in the host-pathogen interaction and the connection of bacteria between species. Such modifications act on pathogens to facilitate their increased transmission between individual hosts, increased contact with new host populations or species, and on the selection, pressure leading to the dominance of pathogen strains adapted to these new environmental conditions (Carella and Sirri 2017 , Reyes et al. 2023 ). Unfortunately, there is no way to compare the histology of this study with that of previous published reports, the histology of this study resembles the zoea 2 syndrome reported by Kumar et al. ( 2017 ). Although, the differences in pathology could be the product of different responses by the host to a wide dynamic bacteria genotype and the concentration of bacterial pathogens (Intriago et al. 1999). In addition, two pathogens could also produce the same macroscopic pathology (LBS) but the differences in the damage at the tissue level will depend on all the factors described above. Declarations Author contributions: P.I. conceived the study, directed the research writing, editing and interpretation of the results. K.A. conducted the microbiological-based components of the research. A.M, A.M and J.G. performed the molecular analysis. B.M. and M.V. processed samples for histology and contributed to the histopathological interpretation of the results. X.R. contributed to the histopathological examination and writing. A.P.S. contributed to the writing and editing. Ethical approval All animals studied in the present research came from commercial hatcheries. Funding No funding was received. Study was carried out using South Florida Farming company funds. Competing interests The authors declare no competing interests. References Abubakr MA, Jones DA (1992) Functional morphology and ultrastructure of the anterior mid-gut diverticulae of larvae of Penaeus monodon Fabricius, 1798 (Decapoda, Natantia). Crustaceana 62(2):142-158. doi.org/10.1163/156854092X00712 Aguilera-Rivera D, Prieto-Davó A, Rodríguez-Fuentes G, Escalante-Herrera KS, Gaxiola G (2019) A vibriosis outbreak in the Pacific white shrimp, Litopenaeus vannamei reared in biofloc and clear seawater. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4009796","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":276552498,"identity":"cb51971e-cba1-4bb4-a9af-6c9c60d57aba","order_by":0,"name":"Pablo Intriago","email":"data:image/png;base64,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","orcid":"","institution":"South Florida Farming Corp","correspondingAuthor":true,"prefix":"","firstName":"Pablo","middleName":"","lastName":"Intriago","suffix":""},{"id":276552499,"identity":"68179991-d1ae-4e4c-8e14-3c506b0bf35d","order_by":1,"name":"Bolivar Montiel","email":"","orcid":"","institution":"South Florida Farming Corp","correspondingAuthor":false,"prefix":"","firstName":"Bolivar","middleName":"","lastName":"Montiel","suffix":""},{"id":276552500,"identity":"873a2c2d-17a5-4875-bfe1-c2ff79e1478f","order_by":2,"name":"Mauricio Valarezo","email":"","orcid":"","institution":"South Florida Farming Corp","correspondingAuthor":false,"prefix":"","firstName":"Mauricio","middleName":"","lastName":"Valarezo","suffix":""},{"id":276552501,"identity":"98d3cba7-86d5-407c-9e9e-353b4a5b6dbf","order_by":3,"name":"Xavier Romero","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xavier","middleName":"","lastName":"Romero","suffix":""},{"id":276552502,"identity":"2d1690ff-a309-4d15-9496-0dc8c0c486de","order_by":4,"name":"Kelly Arteaga","email":"","orcid":"","institution":"South Florida Farming Corp","correspondingAuthor":false,"prefix":"","firstName":"Kelly","middleName":"","lastName":"Arteaga","suffix":""},{"id":276552503,"identity":"0fb90169-b5a3-4aef-b280-c4e3f6898e84","order_by":5,"name":"Nicole Cercado","email":"","orcid":"","institution":"South Florida Farming Corp","correspondingAuthor":false,"prefix":"","firstName":"Nicole","middleName":"","lastName":"Cercado","suffix":""},{"id":276552504,"identity":"ea4c8660-995d-4e56-b65c-b91de7471f4e","order_by":6,"name":"Milena Burgos","email":"","orcid":"","institution":"South Florida Farming Corp","correspondingAuthor":false,"prefix":"","firstName":"Milena","middleName":"","lastName":"Burgos","suffix":""},{"id":276552505,"identity":"587f3593-bc2f-42fa-9eb1-d86e527bf865","order_by":7,"name":"Andrew P. 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Scale bar = 100 µm.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4009796/v1/62a1dfaa1b038894935495d7.jpg"},{"id":52064036,"identity":"3e92c620-e978-4857-a21a-ed18dde6858d","added_by":"auto","created_at":"2024-03-06 06:19:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":186332,"visible":true,"origin":"","legend":"\u003cp\u003eHaematoxylin and eosin-stained sections through normal and affected zoea 2 larvae. \u003cstrong\u003ea\u003c/strong\u003e) Normal zoea 2 larvae. \u003cstrong\u003eb\u003c/strong\u003e) Closer look of image a, showing the early formation of the hepatopancreas. Note the algae content of the digestive system and the absence of tissue debris. \u003cstrong\u003ec\u003c/strong\u003e) Lobes that will form the hepatopancreas (thin arrow). Note the intact peritrophic membrane and the algae content (thick arrow). \u003cstrong\u003ed\u003c/strong\u003e) Closer view of the content of the digestive tract with brown microalgae (arrow). Scale bars: a =200 µm; b \u0026amp; c = 50 µm; d = 20 µm.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4009796/v1/9ad506804a7385c24729fd67.jpg"},{"id":52064038,"identity":"d514642c-6dd8-4cec-a850-1b093bec5410","added_by":"auto","created_at":"2024-03-06 06:19:05","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":174871,"visible":true,"origin":"","legend":"\u003cp\u003eHaematoxylin and eosin-stained sections through affected zoea 2 larvae.\u003cstrong\u003e a\u003c/strong\u003e) Detached lipid material with brownish-green granules within (thin arrow). \u003cstrong\u003eb\u003c/strong\u003e) Close up of image \u003cstrong\u003ea\u003c/strong\u003e that shows the presence of lipid droplets with brown-green material within (thin arrow). \u003cstrong\u003ec\u003c/strong\u003e) Intestine of a larva; note the presence of cellular sloughed material within the lumen. \u003cstrong\u003ed\u003c/strong\u003e) Area of the intestine with sloughed material (thin arrow). Note the presence of an intact peritrophic membrane (thick arrow). \u003cstrong\u003ee\u003c/strong\u003e) Hepatopancreas. Note the presence of sloughed material within the lumen (thin arrow). \u003cstrong\u003ef\u003c/strong\u003e) Hepatopancreas and intestinal tract. Note the presence of sloughed material (thin arrow) inside the peritrophic membrane (thick arrow). Scale bars: a \u0026amp; c = 50 µm; b, d-f = 20 µm.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4009796/v1/c9f51457576d1d85d3553baf.jpg"},{"id":52064037,"identity":"8bf7baf3-cd47-4abf-b187-89ccbdefd492","added_by":"auto","created_at":"2024-03-06 06:19:05","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e) Faecal strands with sloughed cellular material (thin arrow). H\u0026amp;E stain. \u003cstrong\u003eb\u003c/strong\u003e) Sloughed cellular material (thin arrow) and bacterial cells (thick arrow). Twort’s Gram stain. Scale bars = 20 µm.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4009796/v1/7e3feb51ae203fbc2c90c5cf.jpg"},{"id":58822451,"identity":"5f8f0d73-6cdf-4558-89c1-fb46830c39a5","added_by":"auto","created_at":"2024-06-21 16:44:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1072691,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4009796/v1/19256e12-ba3d-4c98-a6e4-7d93ab0b90e7.pdf"},{"id":52064039,"identity":"92de2ca8-be7c-4d2b-b071-a67d6702a445","added_by":"auto","created_at":"2024-03-06 06:19:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":619790,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-4009796/v1/59d0b16a9327e184b7d275d4.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Las Bolitas Syndrome in Penaeus vannamei hatcheries in Latin America","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eDuring the late 80\u0026rsquo;s, 90\u0026rsquo;s and the first years of the 2000\u0026rsquo;s, the most predominant pathologies in hatcheries in Latin America were the Las Bolitas syndrome (LBS), zoea 2 syndrome and Mysis molt syndrome (Morales \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Robertson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1998\u003c/span\u003e, Vanderberghe et al. 1999). While during the same period, the main pathologies reported in post larvae were luminescent bacteria (Baticados et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Lavilla-Pitogo et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Song \u0026amp; Lee \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Nithimathachoke et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1995\u003c/span\u003e). After 2015, disease outbreaks with high rates of mortality were more commonly seen in post larval production, e.g., Post-larvae AHPND (PL-AHPND), Translucent Post Larva Disease (TPD), etc (Zou et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Yang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Intriago et al. 2023, Yang et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).LBS is a condition firstly characterized by a distinctive pathology of the hepatopancreas, where there is a cellular desquamation of the walls of the hepatopancreas forming spheres, which eventually move into the upper gut. At the same time, the larvae become bioluminescent, which is accompanied by changes in behaviour and in a loss of appetite (Morales \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Robertson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Although LBS might have had incidence during post larval stages (PLs), massive mortalities (i.e., of up to 90%) were observed in the earlier zoea and mysis stages (Morales \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1992\u003c/span\u003e), and as distinctive characteristic LBS that always went together with the bolitas in the hepatopancreas that eventually moved to the intestine.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eLBS has been associated with infection of \u003cem\u003eVibrio\u003c/em\u003e spp. (Morales \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1992\u003c/span\u003e, Intriago and Jimenez 1998, Robertson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Zoea-2 syndrome was only associated with \u003cem\u003eV. harveyi\u003c/em\u003e and \u003cem\u003eV. alginolyticus\u003c/em\u003e (see Vanderberghe et al. 1999, Wiradana et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), and that associated with mysis syndrome has never been identified. The nocturnal luminescence seen in hatcheries in both Asia and the Americas were typically attributed to luminescent \u003cem\u003eVibrios\u003c/em\u003e (Baticados et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Lavilla-Pitogo et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1990\u003c/span\u003e, Song \u0026amp; Lee \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Nithimathachoke et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1995\u003c/span\u003e, Robertson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In more recent times, outbreaks of sudden and acute mortalities in penaeid shrimp hatcheries typically start in the PL stages, from an active and apparently healthy state to moribund and dead. The speed and virulence at which these massive mortalities occur have been observed in many production facilities, and in most cases have been associated with, or linked to, different strains of \u003cem\u003eVibrio\u003c/em\u003e (see Zou et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Yang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Intriago et al. 2023). Intriago et al. (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) provided evidence that the cause of these rapid mortality events was due to a species of \u003cem\u003eVibrio\u003c/em\u003e carrying the same plasmids as the \u003cem\u003eVp\u003c/em\u003e\u003csub\u003eAHPND\u003c/sub\u003e reported causing acute hepatopancreatic necrosis diseases (AHPND) in culture ponds elsewhere. The condition was tentatively named post-larvae AHPND (PL-AHPND) to differentiate it from other pathologies affecting penaeid shrimp larvae. In Asia, Translucent Post Larva Disease (TPD) has been the main cause of larval disease and mortality, where the causative agent is a strain of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e carrying a hemolysin gene (isolate Vp-JS20200428004-2) (Zou et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2020\u003c/span\u003e, Yang et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Yang et al \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). In India, a similar condition referred to as zoea 2 syndrome was described affecting zoea 2 of \u003cem\u003eP. vannamei\u003c/em\u003e (see Kumar et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn September 2023, some culture facilities in the Latin America region experienced high mortalities in zoea stages 2 and 3. Given the high mortality, these tanks were discarded. The causal agent associated with those mortalities has not been reported. Microscopic observation of the larva revealed the presence of \u0026ldquo;bolitas\u0026rdquo; (spheres) in the hepatopancreas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea, b). Generally, the clinical indicators and the macroscopic appearance on wet mounts aligned with what was previously identified as LBS. (Morales \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). The present study reports the microbiological, polymerase chain reaction (PCR) tests and histological findings of healthy and of diseased animals suffering from this this condition.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e \u003cb\u003eabout here\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cb\u003eSample collection\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eSamples of \u003cem\u003ePenaeus vannamei\u003c/em\u003e larvae from two hatcheries in Latin America were sampled (precise location details are withheld acknowledging the facilities request for confidentiality). Samples for microbiology, PCR and histopathology were taken from two tanks from each hatchery. Hatcheries were selected because one of the hatcheries had reported heavy mortality at zoea 2\u0026ndash;3 stage, while the other hatchery, with apparently healthy zoea 3, was selected as the control. It should be noted that shrimp sampled for PCR and histology were different individuals from the same populations. To protect client privacy, the country and the precise location of each culture facility from which the samples were obtained will not be disclosed.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e \u003cb\u003eMicrobiology of the larvae\u003c/b\u003e \u003c/p\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe total concentration of bacteria in the larvae, was determined by first bathing the larvae (i.e., 1 gram or approximately\u0026thinsp;\u0026gt;\u0026thinsp;2000 zoea stage 3) held in a 200-um nylon sieve for 2\u0026ndash;3 min in a solution of 50 ppm active chlorine prepared using 35 ppt seawater. The zoea were then rinsed with excess sterile seawater. The weight of the larvae was recorded using a Metler digital balance accurate to 0.01 gram and then placed in a mortar with approximately 1 gram of autoclaved beach sand and 10 mL of sterile seawater which was used to facilitate the grinding of the bulk of larvae. Once the sample had been ground, the volume and weight were recorded again, then the samples were sequentially diluted in test tubes with sterile seawater to 1 \u0026times; 10\u003csup\u003e2\u003c/sup\u003e, 10\u003csup\u003e3\u003c/sup\u003e and 10\u003csup\u003e4\u003c/sup\u003e. Then, 100 \u0026micro;L of the relevant dilution was placed per duplicated on Petri dish plates containing either Tryptic Soy Agar (TSA, Difco), thiosulphate-citrate-bile-sucrose agar (TCBS, Difco) or on CHROMagar\u0026trade; \u003cem\u003eVibrio\u003c/em\u003e and then incubated for 24\u0026ndash;48 h at 30\u003csup\u003eo\u003c/sup\u003eC. Thereafter, the number of colony-forming units (CFU) on each plate was recorded. Dilutions were based on obtaining\u0026thinsp;\u0026gt;\u0026thinsp;20 to \u0026lt;\u0026thinsp;200 colonies per plate. Bacteria identification was performed using an API 20E Kit (Buller \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2004\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePCR methods used\u003c/b\u003e \u003c/p\u003e \u003cp\u003eDNA was extracted from whole larvae fixed in 90% alcohol using an Omega, Bio-Tek E.Z.N.A tissue DNA kit following the manufacturer\u0026rsquo;s protocol. In brief, each 1 g sample was ground using a microcentrifuge pestle. Approximately 200 mg of the tissue was then transferred to a clean 1.5 mL Eppendorf tube, then 500 \u0026micro;L of tissue lysis buffer (TL) and 25 \u0026micro;L of Omega Biotek (OB) protease solution were added. The sample was then vortexed and then incubated in a thermoblock at 55\u0026deg;C for approximately 3 h with vortexing every 30 min. RNA was removed by adding 4 \u0026micro;L of RNase A (100 mg/mL), then mixing, then keeping the sample at room temperature for 2 min. The sample was then centrifuged at 13,500 RPM for 5 min, and the supernatant carefully transferred to a new 1.5 mL Eppendorf tube. To this, 220 \u0026micro;L of BL buffer was added, and the mixture was vortexed and incubated at 70\u0026deg;C for 10 mins. Thereafter, 220 \u0026micro;L of 100% ethanol was added and vortexed, and the contents were passed through a HiBind\u0026reg; DNA Mini Column into a 2 mL collection tube. The columns were then centrifuged at 13,500 RPM for 1 min, and the filtrate was discarded. Subsequently, 500 \u0026micro;L of HBC buffer (diluted with 100% isopropanol) was added to the column, and the sample was spun at 13,500 RPM for 30 seconds. The filtrate was discarded, the column was washed twice with 700 \u0026micro;L of DNA wash buffer diluted with 100% ethanol, and the sample was centrifuged at 13,500 RPM for 30 seconds. The filtrate was discarded. This step was repeated. The column was then centrifuged at 13,500 RPM for 2 mins to dry it out. The dried column was placed in a new nuclease-free 1.5 mL Eppendorf tube, and 100 \u0026micro;L of elution buffer, which was heated to 70\u0026deg;C, was added to the column. The sample was allowed to sit for 2 min before being centrifuged at 13,500 RPM for 1 min. This elution step was repeated. The eluted DNA was then stored at -20\u0026deg;C until required.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eRNA was extracted from whole larvae, tissue or organs fixed in 90% alcohol following the manufacturer\u0026rsquo;s protocol (Omega, Bio-Tek E.Z.N.A. Total RNA Kit (TRK)). Approximately 200 mg of tissue was then moved to a clean 1.5 mL Eppendorf tube. To this, 700 \u0026micro;L TRK Lysis Buffer was added, and the tube was left at room temperature for approximately 3 h with vortexing every 30 minutes. The sample was then centrifuged at 13,500 RPM for 5 mins, and the supernatant was carefully transferred to a new 1.5 mL Eppendorf tube to which 420 \u0026micro;L of 70% ethanol was added. After vortexing to mix thoroughly, the contents were passed through a HiBind\u0026reg; RNA Mini Column into a 2 mL collection tube. The columns were then centrifuged at 13,500 RPM for 1 min, and the filtrate was discarded. Subsequently, 500 \u0026micro;L of RNA Wash Buffer I was added to the column, and the sample was spun at 13,500 RPM for 30 seconds. The filtrate was discarded, and the column was washed twice with 500 \u0026micro;L RNA Wash Buffer II and diluted with 100% ethanol. The column was then centrifuged at 13,500 RPM for 1 min to dry it out. The filtrate was discarded. This step was repeated. The column was then centrifuged at 13,500 RPM for 2 mins to dry it out. The dried column was placed in a new nuclease-free 1.5 mL Eppendorf tube, and 70 \u0026micro;L of nuclease-free water was added to the column. The sample was centrifuged at 13,500 RPM for 2 min. This elution step was repeated. The eluted RNA was then stored at -70\u0026deg;C until needed. The pathogens tested are listed below. PCR analyses were conducted for the following pathogens:\u003c/p\u003e\u003cp\u003eDNA viruses: Hepanhamaparvovirus (DHPV), \u003cem\u003eMacrobrachium\u003c/em\u003e Bidnavirus (MrBdv), Decapod iridescent virus 1 (DIV1), white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) as both, infectious IHHNV and the EVE form (endogenous viral elements).\u003c/p\u003e\u003cp\u003eRNA viruses: Wenzhou shrimp virus 8 (WzSV8)\u003cem\u003e/P. vannamei\u003c/em\u003e Solinvivirus (PvSV), \u003cem\u003ePenaeus vannamei\u003c/em\u003e nodavirus \u003cb\u003e(\u003c/b\u003ePvNV), Covert mortality Nodavirus (CMNV), Infectious Myonecrosis Virus (IMNV), Yellow Head Virus (YHV), Taura Syndrome Virus (TSV) and \u003cem\u003eMachrobrachium\u003c/em\u003e Nodavirus (MrNV).\u003c/p\u003e\u003cp\u003eBacteria and other pathogens of concern: \u003cem\u003eSpiroplasma\u003c/em\u003e, \u003cem\u003ePropionigenium\u003c/em\u003e, \u003cem\u003eRickettsia\u003c/em\u003e-like bacteria (RLB), necrotizing hepatopancreatitis bacteria (NHP-B), \u003cem\u003eVibrio\u003c/em\u003e spp., acute hepatopancreatic necrosis disease (AHPND), \u003cem\u003eEcytonucleospora\u003c/em\u003e [\u003cem\u003eEnterocytozoon\u003c/em\u003e] \u003cem\u003ehepatopenaei\u003c/em\u003e (EHP), other non-EHP Microsporidia, and Haplosporidia.\u003c/p\u003e\u003cp\u003e \u003cb\u003eHistopathology\u003c/b\u003e \u003c/p\u003e\u003cp\u003eFor histological analysis, samples were prepared following the procedures outlined by Bell and Lightner (1988). Briefly, larvae were fixed in Davidson\u0026rsquo;s alcohol, formalin, acetic acid (AFA) using at least 1 gram of larvae from each tank. These were fixed for at least 24 h before processing for routine tissue embedding and histological sectioning. The 5 \u0026micro;m thick tissue sections were then stained with hematoxylin and eosin (H\u0026amp;E). Additional sections, however, were stained with three different stains: Twort\u0026rsquo;s Gram stain to differentiate Gram positive from Gram negative bacteria (CP Lab Chemicals, Novato, California USA) and methyl green pyronin Y modified stain was employed to distinguish DNA and RNA (PolyRnDcom, Bay Shore, New York, USA). For each sample of larvae collected from each tank, one blocks were prepared, with each block containing approximately 1-gram zoea stage 2. Four tissue sections were cut from each paraffin block.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003ePCR results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePCR analysis of both the healthy and affected samples of zoea were negative for seventeen known shrimp pathogens, including DHPV, MrBdv, SHIV, WSSV, PvNV, CMNV, IMNV, YHV, GAV, TSV, MrNV, XSV, \u003cem\u003eSpiroplasma\u003c/em\u003e, NHPB, EHP, AHPND and Haplosporidia (Table 1). The apparently healthy zoea, however, were positive for IHHNV EVE (2/2), WzSV8/PvSV (2/2), RLB (1/2) and Microsporidia (1/2). By comparison, the LBS affected zoea were positive for IHHNV EVE (1/2), \u003cem\u003eVibrio\u003c/em\u003e spp. (2/2), RLB (2/2) and Microsporidia (1/2). \u0026nbsp;In summary, the only difference between the two sets of samples were the detection of \u003cem\u003eVibrios\u003c/em\u003e in the zoea affected with LBS and WzSV8 in the healthy zoea.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1 about here\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicrobiology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentration of total bacteria (TSA) and presumptive \u003cem\u003eVibrios\u003c/em\u003e (TCBS and CHROMagar\u003csup\u003eTM\u003c/sup\u003e \u003cem\u003eVibrio\u003c/em\u003e) are shown in Table 2. The concentration of total bacteria in the affected zoea were almost an order of magnitude higher than that determined for the sample of healthy zoea. Presumptive \u003cem\u003eVibrios\u003c/em\u003e were almost two orders of magnitude higher in the affected zoea than in the healthy zoea. Presumptive \u003cem\u003eVibrios\u003c/em\u003e were found to represent 17% and 6% of the total bacteria population in the unhealthy and healthy zoea, respectively. Green colonies (on TCBS) and purple colonies (on CHROMagar\u003csup\u003eTM\u003c/sup\u003e \u003cem\u003eVibrio\u003c/em\u003e)represented 0.2% and 82% of the those recovered from the affected zoea, while a reverse picture of 56% and 1% was found from the analysis of the healthy zoea (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2 about here\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA total of eleven strains were isolated from the LBS affected zoea and three from the healthy zoea (Table 3a). Biochemical profiling of the eleven strains recovered from the affected zoea, identified six as \u003cem\u003eV. alginolyticus\u003c/em\u003e, two as \u003cem\u003eV. fluvialis\u003c/em\u003e and one as \u003cem\u003eV. vulnificus,\u0026nbsp;\u003c/em\u003eone\u003cem\u003e\u0026nbsp;Aeromonas\u0026nbsp;\u003c/em\u003e(undetermined) and one\u003cem\u003e\u0026nbsp;Pasteurella\u003c/em\u003e (undetermined). Of the three strains isolated from the healthy zoea, one strain was identified as \u003cem\u003eV. alginolyticus,\u0026nbsp;\u003c/em\u003eone as\u003cem\u003e\u0026nbsp;V. fluvialis\u0026nbsp;\u003c/em\u003eand one as\u003cem\u003e\u0026nbsp;Aeromonas.\u0026nbsp;\u003c/em\u003eInterestingly, all 14 strains were positive for \u003cem\u003eVibrio\u003c/em\u003e by PCR, which suggests that 27% of the results returned by the API 20E biochemical profiles were false negatives.\u0026nbsp;Two sequences were used to identify Vibrio bacteria, all except by LBS-4 were positive for both methods. None of the 14 bacteria were positive for \u003cem\u003eV. parahaemolyticus\u003c/em\u003e or for ToxR nor for the high virulent protein genes tested (Vhp-1, Vhp-2, Vhp-3) (Table 3a). The strains identified as \u003cem\u003eAeromonas\u003c/em\u003e and \u003cem\u003eV. fluvialis\u003c/em\u003e were Pir AB positive (AHPND). The strain H-3 was positive for \u003cem\u003eV. harveyi\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eTable 3b shows the API 20E characterization of the \u003cem\u003eVibrio\u0026nbsp;\u003c/em\u003espp. that were isolated. This table is divided between LBS zoea (i.e., \u003cem\u003eVibrio\u0026nbsp;\u003c/em\u003espp. isolated from affected zoea), \u003cem\u003eV. alginolyticus\u003c/em\u003e and \u003cem\u003eVibrio\u0026nbsp;\u003c/em\u003espp. (with 6 and 5 strains), against the three species of \u003cem\u003eVibrio\u003c/em\u003e isolated from the healthy zoea.\u003c/p\u003e\n\u003cp\u003eThe availability of fermentation/oxidation of Arabinose was the main difference between \u003cem\u003eVibrio spp\u003c/em\u003e isolated from affected and the other two groups of bacteria, namely vs \u003cem\u003eV. alginolyticus\u003c/em\u003e from LBS affected animals and \u003cem\u003eVibrio\u003c/em\u003e spp from healthy zoeas. On the other hand, \u003cem\u003eV. alginolyticus\u003c/em\u003e from LBS affected animals differentiate from the other two groups in been unable to fermentation/oxidation of Amygdalin but with the capability of\u0026nbsp;Acetoin production (Table 3b).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNone of the 14 strains were ToxR positive or luminescent.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3a and 3b about here\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn fresh mounts of normal healthy zoea 2 larvae, algal material was seen in the digestive tract, giving it a brown coloration (Fig. 1a). By comparison, the affected / diseased larvae had an empty digestive tract and the presence of a round-shaped material that appeared as lipid droplets (Fig. 1b), this general clinical sign is the one usually described by shrimp hatchery technicians and biologists for what has been known as “Las Bolitas Syndrome”.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1 about here\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEight microscopy slides stained under H\u0026amp;E containing approximately 120 larvae of normal larvae at zoea 3 stage and a similar number from each diseased tank were examined. \u0026nbsp;Normal healthy zoea 3 larvae had algae present in their digestive tract. At this stage the digestive tract is still in formation; the lateral lobes that will eventually develop into a fully functional hepatopancreas could also be seen in the tissue sections (Figs. 2a, b) (Abubakr \u0026amp; Jones 1992). An intact peritrophic membrane was also present and the algal material that had been consumed at this stage was seen as small sized brown cells (Fig. 2c). No sloughed cell material was observed in the lumen of the early forming digestive system of the healthy larvae (Fig. 2d).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2 about here\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 60% of the larvae from the affected tanks with mortality had brown-green, hyaline spheres, the detached material within appeared to a B-cell (Figs. 3a \u0026amp; b). In the intestine, sloughed cells and material was observed (Figs. 3c \u0026amp; d) giving a contrasting difference to the intestines of the healthy larvae (Fig. 2c). The folds that constitute the early, developing hepatopancreas had sloughed cells that could be differentiated by their colour (Fig. 3e). Interestingly, some of these sloughed cells were inside the peritrophic membrane (Fig. 3f). The faeces from these larvae contained the same material that was seen within the intestine (Fig. 4a) and was contained within a peritrophic membrane. In the sections stained with Twort’s stain, the presence of what appeared to be bacterial cells was observed both within and outside the faecal strands (Fig. 4b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 3 about here\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuring this study, no bolitas (i.e., spheres) were observed in the histological samples, and none have ever been documented in the literature in the zoeal stages. The preparation of tissue samples for histology requires their processing through several solvents and so it is tempting to suggest that nature of the bolitas is lipidic in nature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 4 about here\u003c/strong\u003e\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study describes the histopathological lesions, PCR, and microbiology of \u003cem\u003eP. vannamei\u003c/em\u003e zoea samples collected from two different hatcheries, one with an outbreak of LBS, and the other with apparently healthy animals. Of all pathogens that were analyzed by PCR, the only key difference between the LBS and healthy zoea were the detection of \u003cem\u003eVibrios\u003c/em\u003e in the disease zoea. The presence of the WzSV8 virus in the healthy zoea requires more study; this RNA virus has been found in a wide range of different environments and regions including wild broodstock, and its action in penaeid production needs to be the subject of further studies (Intriago et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMicrobiology showed a high microbial load within the affected zoea that was one order of magnitude higher in the total number of culturable bacteria (TSA) and almost two orders of magnitude higher in the number of presumptive \u003cem\u003eVibrios\u003c/em\u003e when compared to the healthy zoea. Presumptive \u003cem\u003eVibrios\u003c/em\u003e represent 17% and 6% of the total bacteria population in the unhealthy and healthy zoea, respectively. Green colonies (on TCBS) and purple colonies presumptive of \u003cem\u003eV. parahaemolyticus\u003c/em\u003e (on CHROMagar\u0026trade; \u003cem\u003eVibrio)\u003c/em\u003e represent 0.2% and 82% of the total \u003cem\u003eVibrio\u003c/em\u003e count in affected zoea, while a reverse was seen in the healthy zoea which had 56% and 1% respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The finding is interesting because the common assumption is that green colonies on TCBS and purple colonies on CHROMagar\u0026trade; \u003cem\u003eVibrio\u003c/em\u003e typically represent \u003cem\u003eV. parahaemolyticus\u003c/em\u003e. However, it is known that this is not always necessarily the case and highlights the need for microbiology-based investigations to be conducted in combination with molecular biology-based studies to gain a clear identification of the pathogens present.\u003c/p\u003e \u003cp\u003eIt is important to note that all 14 strains were identified by PCR as members of the genus \u003cem\u003eVibrio\u003c/em\u003e, so it can be concluded that API 20E results should be taken with caution. In this study 21% of the strains were false identified at the genus level. While Overman et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) stated that the API 20E is a valid system for use in the identification of the more commonly occurring members of the family Vibrionaceae, however, the system has been reported to result in false negatives (Croci et al. 2006, Fabbro et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). API identification is based on biochemical profiles, but it has been found that biochemical profiles and genotype are not necessarily associated with virulence potential (Lydon et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The relationship between the variation, or differences in the API 20E identifications and the possible virulence- genus variation of each strain is something that requires further exploration. From the above information though it can be concluded that species of \u003cem\u003eVibrio\u003c/em\u003e are the main pathogenic agent(s) causing LBS. It is also important to note that the pathology is note related to AHPND or other high virulent pathologies described for \u003cem\u003eP. vannamei\u003c/em\u003e larvae.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb provides details regarding the characterization of the isolated Vibrios using the API 20E system. This table is divided between LBS Zoeas (Vibrios isolated from affected Zoeas) V. alginolyticus and Vibrio spp (with 6 and 5 strains), against Vibrios (3) isolated from healthy Zoeas. Fermentation/oxidation of Arabinose and Amygdalin was different between the three groups of \u003cem\u003eVibrio\u003c/em\u003e isolated from affected vs healthy zoeas (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Some strains of \u003cem\u003eV. anguillarum\u003c/em\u003e strains were separated in distinct groups could be separated mainly based on their reaction on indole production and the fermentation of amygdalin and arabinose (Grisez et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1991\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAcetoin production was the main difference between \u003cem\u003eV. alginolyticus\u003c/em\u003e, and other species of \u003cem\u003eVibrio\u003c/em\u003e isolated from healthy and affected zoeas. The ability for acetoin production by \u003cem\u003eV. alginolyticus\u003c/em\u003e isolated from LBS affected animals is interesting, generally this metabolite is produced when microorganisms employ the 2,3-butanediol pathway to ferment sugars, this pathway generates less acidic and more neutral end products, such as acetoin and 2,3-butanediol. Because acetoin is a neutral fermentation product and this biosynthetic reaction consumes intracellular protons, bacterial growth can occur on a glucose carbon source without pH decrease (Vivijs et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Oh et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In addition, inhibition of acetoin production has been suggested as potential mechanism to control the pathogenic \u003cem\u003eV. cholerae\u003c/em\u003e, that are known to be acid sensitive (Oh et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eA comparison between the histology in this study and the first report of LBS described by Morales (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1992\u003c/span\u003e) is impossible. Unfortunately, only transmission electron microscopy (TEM) images were presented, and no tissue sections stained in H\u0026amp;E were recorded. The publication by Robertson et al. (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) shows the presence of melanized necrotic bundles in the hepatopancreatic tubules (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e in Robertson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). This feature was never seen during the present research. The most likely reason is that the animals used for the histology were not zoea but were least at PL1 stage when comparing the anatomy of those that were presented to other detailed descriptions regarding the larval development of \u003cem\u003eP. vannamei\u003c/em\u003e (see Muhammad et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Important differences between the pathology presented here for LBS and for PL-AHPND (see Intriago et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e) is that the peritrophic membrane remains intact (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, 4ab) and there is no massive sloughing of cells in the hepatopancreas as was described for PL-AHPND. In addition, PL-AHPND was never found or described in zoea (Intriago et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). The presence of the green-brown material in the B-cells of the hepatopancreas (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea, b \u0026amp; f), is intriguing and it is not wrong to suggest that it is a product that could not be processed by the larvae and stored in these cells; its presence also requires further investigation. A similar material present in shrimp larvae reared in China and suffering mortalities during the zoea 2 stage has been described by other authors; in their case this was associated with a strain of \u003cem\u003eV. alginolyticus\u003c/em\u003e (see Sun et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eHistorically, the Latin American shrimp sector has followed the Ecuadorian model, where post larvae are produced from broodstock obtained from production ponds. Broodstock are selected based on weight, and then transferred to a hatchery to produce the next generation of post larvae. This process gives little to no long-term control over biosecurity (Wolkenfelt and Blonk, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, high concentrations of \u003cem\u003eVibrio\u003c/em\u003e spp. can be very common in non-SPF (i.e., specific pathogen free) or non-bio secure penaeid hatcheries, both as free and as attached to the larvae. From hatching through to harvest, the microbiological environment is a soup of bacteria and virus like particles (VLP) (Hameed \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, Garcia and Olmos \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, Intriago et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). As the larvae transitions from a diet based on algae as zoea to animals that source proteins as mysis, the larvae then undergo a dramatic change in the volume of the hepatopancreas and the biochemistry of the digestive enzymes (Kurmaly et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). In zoea, the filtration of particles is almost indiscriminate. As the zoea stages are exposed to very high concentrations of bacteria, free-living and attached, including \u003cem\u003eVibrio\u003c/em\u003e that are easily ingested and able to pass into the digestive tract (Soto-Rodriguez et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), the detrimental effects of such can be observed depending on several factors such as the larval sub-stage involved, the \u003cem\u003eVibrio\u003c/em\u003e species present, and their concentration (Guzman et al. 2001).\u003c/p\u003e\u003cp\u003eIntriago and Jimenez (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) replicated bolitas syndrome in \u003cem\u003eP. vannamei\u003c/em\u003e zoea using a luminescent strain of \u003cem\u003eV. harveyi\u003c/em\u003e at concentrations as low as 10\u003csup\u003e3\u003c/sup\u003e cell/mL. Interestingly, this strain was isolated from diseased farm cultured \u003cem\u003eP. vannamei\u003c/em\u003e affected by haemocytic enteritis. They postulated that pathogens could be bouncing back from hatcheries to the broodstock or \u003cem\u003evice versa\u003c/em\u003e and that the differences found in the histopathology between the larvae and adults could have been attributed on one hand due to the differences in the degree of organ development, and on the other hand to the pathogen species, its virulence, and concentration.\u003c/p\u003e\u003cp\u003eThe key event in the appearance of diseases could be attributed to stress (temperature, salinity, density, toxins, etc.) because of alterations in the environment, this exerts a change in the host-pathogen interaction and the connection of bacteria between species. Such modifications act on pathogens to facilitate their increased transmission between individual hosts, increased contact with new host populations or species, and on the selection, pressure leading to the dominance of pathogen strains adapted to these new environmental conditions (Carella and Sirri \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, Reyes et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Unfortunately, there is no way to compare the histology of this study with that of previous published reports, the histology of this study resembles the zoea 2 syndrome reported by Kumar et al. (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Although, the differences in pathology could be the product of different responses by the host to a wide dynamic bacteria genotype and the concentration of bacterial pathogens (Intriago et al. 1999). In addition, two pathogens could also produce the same macroscopic pathology (LBS) but the differences in the damage at the tissue level will depend on all the factors described above.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions:\u003c/strong\u003e P.I. conceived the study, directed the research writing, editing and interpretation of the results. K.A. conducted the microbiological-based components of the research. A.M, A.M and J.G. performed the molecular analysis. B.M. and M.V. processed samples for histology and contributed to the histopathological interpretation of the results. X.R. contributed to the histopathological examination and writing. A.P.S. contributed to the writing and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animals studied in the present research came from commercial hatcheries. \u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding was received. Study was carried out using South Florida Farming company funds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbubakr MA, Jones DA (1992) Functional morphology and ultrastructure of the anterior mid-gut diverticulae of larvae of \u003cem\u003ePenaeus monodon\u003c/em\u003e Fabricius, 1798 (Decapoda, Natantia). 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C\u0026aacute;mara Nacional de Acuacultura. 25- 28 October 2018, Guayaquil-Ecuador.\u003c/li\u003e\n\u003cli\u003eYang F, Xu L, Huang W, Li F (2022) Highly lethal \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e strains cause acute mortality in \u003cem\u003ePenaeus vannamei\u003c/em\u003e post larvae. Aquaculture 548:737605. doi.org/10.1016/j.aquaculture.2021.737605\u003c/li\u003e\n\u003cli\u003eYang F, You Y, Lai Q, Xu L, Li F (2023) \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e becomes highly virulent by producing Tc toxins. Aquaculture 576:739817. doi.org/10.1016/j.aquaculture.2023.739817.\u003c/li\u003e\n\u003cli\u003eZhang Q, Xu TT, Wan , Liu ,Wang , Li X, Dong X, Yang B, Huang J (2017) Prevalence and distribution of covert mortality nodavirus (CMNV) in cultured crustacean. Virus Res 233:113-119. dx.doi.org/10.1016/j.virusres.2017.03.013\u003c/li\u003e\n\u003cli\u003eZou Y, Xie G, Jia T, Xu T, Wang C, Wan X, Li Y, Luo K, Bian X, Wang X, Kong J, Zhang Q (2020) Determination of the infectious agent of Translucent Post-Larva Disease (TPD) in \u003cem\u003ePenaeus vannamei\u003c/em\u003e. Pathogens 9:741. doi:10.3390/pathogens9090741\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4009796/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4009796/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSeveral hatcheries in Latin America reported mortality of zoea stage 2 \u003cem\u003ePenaeus vannamei.\u003c/em\u003e In fresh mounts, round structures resembling lipid droplets were observed, reminiscent of a disease called \"Las Bolitas Syndrome\" first identified in 1987. Closer examination under routine histopathology revealed the presence of detached cells and tissue in the digestive tract, whereas unaffected tanks displayed a typical intestinal content containing algae cells. Polymerase Chain Reaction of diseased and healthy batches of larvae for 22 shrimp pathogens revealed similar test results. The larvae were negative for nineteen pathogens, including AHPND. The detection of \u003cem\u003eVibrio\u003c/em\u003e spp. in both samples of affected zoea 3 (Z3) was the principal difference. Histology of affected zoeas were characterized by tissue degeneration in the hepatopancreas forming spheres that eventually moved into the upper gut, midgut and midgut caeca - a pathology known as \u003cem\u003eBolitas\u003c/em\u003e syndrome (BS). Microbiological analysis showed \u003cem\u003eVibrio\u003c/em\u003e spp. at \u0026le;\u0026thinsp;10\u003csup\u003e5\u003c/sup\u003e CFU zoea/g, \u0026asymp;\u0026thinsp;2 orders of magnitude higher than healthy zoea. Isolation of bacteria from healthy and BS affected zoea onto TCBS and CHROMagar\u0026trade; and consequentially identified by API 20 E revelated six strains of \u003cem\u003eV. alginolyticus.\u003c/em\u003e Though fresh mounts resembled the general description for \u0026ldquo;Las Bolitas Syndrome\u0026rdquo;, the histopathology differed from the original description. The intestine contained sloughed cells; the lateral lobes constituting the developing hepatopancreas in Z3 could be differentiated by their colour, with sloughed cells inside the peritrophic membrane. PCR and microbiological analyses verified that the origin of Las Bolitas Syndrome is bacterial in nature, with \u003cem\u003eVibrio\u003c/em\u003e playing a significant role.\u003c/p\u003e","manuscriptTitle":"Las Bolitas Syndrome in Penaeus vannamei hatcheries in Latin America","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-06 06:19:00","doi":"10.21203/rs.3.rs-4009796/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d609c05-c666-4992-8ac9-fba46e1ecf5d","owner":[],"postedDate":"March 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-21T14:57:41+00:00","versionOfRecord":{"articleIdentity":"rs-4009796","link":"https://doi.org/10.3390/microorganisms12061186","journal":{"identity":"microorganisms","isVorOnly":true,"title":"Microorganisms"},"publishedOn":"2024-06-12 14:57:41","publishedOnDateReadable":"June 12th, 2024"},"versionCreatedAt":"2024-03-06 06:19:00","video":"","vorDoi":"10.3390/microorganisms12061186","vorDoiUrl":"https://doi.org/10.3390/microorganisms12061186","workflowStages":[]},"version":"v1","identity":"rs-4009796","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4009796","identity":"rs-4009796","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}