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Specific isolates of Vibrio parahaemolyticus bacteria that cause acute hepatopancreatic necrosis disease (AHPND) colonize and form biofilms on the chitin lining of the shrimp stomach where they release toxins that cause necrosis in the adjacent hepatopancreas. One potential strategy to prevent AHPND is to inhibit biofilm formation in the stomach using feed additives. Thus, we tested ethanolic extracts of ginger (0.2 mg/ml and 2 mg/ml) for their ability to inhibit biofilm formation by an AHPND-causing isolate of V. parahaemolyticus (3HP) using chitosan coated 96-well polystyrene plastic plates to mimic the shrimp stomach lining. The extract significantly (p

Streptococcus agalactiae secrete virulence factors believed to be able of killing host tissues, especially under elevated water temperature. A direct effect of S. agalactiae secretory products on tilapia cells was tested on the tilapia kidney (TK-1) cell culture. The bacteria were cultured under four different temperature levels: 22, 29, 32 and 37°C; the cell-free portion was processed through SDS-PAGE; and distinct bands were identified by LC-MS/MS. At least, three virulence factors were identified, Bsp, PcsB and CAMP factor, with increasing levels as the cultured temperature rose. Expressions of bsp, pcsB and cfb were also up-regulated with the rising of the temperature in S. agalactiae culture. The supernatant from the bacteria cultured under specified temperatures was added into TK-1 cell-cultured wells. Morphological damage and mortality of the cultured cells, as determined by MTT method, were increased progressively from the supernatant treatment according to the rise of temperature in S. agalactiae culture. This study suggests that the production of the three virulence factors of S. agalactiae reported herein is temperature-dependent, and it is likely that CAMP factor directly kills the TK-1 cells since the other two types of protein are involved in S. agalactiae cell division and the bacterial adherence to host tissues.

One of the clinical manifestations of streptococcosis is swimming errors of the infected fish, which is likely caused by lesions in the brain. As most studies described brain histopathology in streptococcosis as meningitis, with a limited description of lesions in the whole brain, the aim of this study was therefore to explore histopathology of the whole brain of red tilapia experimentally infected with Streptococcus agalactiae serotype III. Transcripts relating to motoneuron functions and inflammatory responses were also investigated. In the S. agalactiae-infected fish, the parenchyma of the whole brain and its associated meninx primitiva were found to be markedly infiltrated by mononuclear cells and Gram-positive cocci. Hemorrhage, neuronal necrosis, and localized spongiform histopathology were observed, especially within the midbrain and the cerebellum. The lesion was observed in the medial longitudinal fasciculus and its nucleus. Expressions of the transcripts CD166, GAP43, SMN, and SV2B of the infected fish did not change, while those of IL-1β and TNF-α were significantly upregulated. It is likely that S. agalactiae cause extensive damage to the fish brain, especially in areas that control swimming activities, through both direct invasion of the bacteria and acute inflammatory responses of the brain resident macrophages, or microglia.

Cellular and humoral effectors are imperative for the innate defense mechanisms of invertebrates, including the spotted babylon snail Babylonia areolata, a marine gastropod belonging to the family Buccinidae. In this study, we have investigated the influence of body size [5–8 g (small), 10–12 g (medium) and 15–17 g (large)], and culture waters of varied quality [natural seawater, low salinity, low alkalinity, high total ammonia nitrogen (TAN) and artificial seawater] on some hemato-immunological parameters [total hemocytic counts (THC), hemolymphatic glucose, total protein concentration, phenoloxidase (PO), agglutinating and lysozyme activity] of the snail. Growth performance measures and survival of test snails were also evaluated after rearing them in culture water of varied quality. Body size did not influence most parameters assessed, except that the lysozyme and PO activities of medium and large-size snails were higher than those of the small-size snails. Sub-optimal culture water quality affected hemato-immunological factors, growth and/or survival of the spotted babylon snail. Seawater with low salinity, low alkalinity and a high TAN level caused decreased THC, hemolymphatic glucose, and PO activity; increased lysozyme activity; and clearly retarded growth of the snail. The snails held in artificial seawater could not survive after 4 weeks, suggesting the lack of certain essential factors necessary for their survival.

The hepatopancreas of the shrimp is the target organ of several pathogens. Diagnosis of diseases of the hepatopancreas usually required an isolation of the organ and the process cannot avoid sacrificing the shrimp. To save life of the animal, this study was aimed at taking hepatopancreatic tissue from the shrimp by biopsy process. Thirty-two sexually mature adults of the black tiger shrimp Penaeus monodon were used as the shrimp model and small pieces of their hepatopancreatic tissue were taken by needle biopsy. Specific position of the needle penetration was suggested to avoid damage to the heart and the gills. From the lateral view of the carapace of the shrimp, this position was at one-third from the rostral end of a line drawn from the last dorsal spine of the rostrum to the posterior margin of the carapace. From the top view, this point was located at a medial quarter of a line drawn from the midline of the carapace toward the lateral extremity. The needle was inserted vertically at a depth of 1 cm into the hepatopancreas, and small pieces of the tissue were quickly withdrawn into the syringe. After the operation, the shrimp were released into rearing tanks and observed for any mortality or morbidity for two months, during which all the shrimp survived and remained apparently healthy. The hepatopancreatic tissues taken from the biopsy were processed for polymerase chain (PCR) reaction method, fresh smear and histology. The PCR revealed the presence of Penaeus monodon densovirus (PmoDNV) in 40% of the samples. The fresh smear and histology revealed good quality of cell morphology and histological features, as well as PmoDNV inclusions in the PCR-positive samples. This study suggests that needle biopsy of hepatopancreas to detect pathogens, or to evaluate health status, of this organ in shrimp is possible, at least in P. monodon.

Co-culture of Nile tilapia, Oreochromis niloticus , with marine shrimp has been an increasing farm practice as evidence has suggested that the co-culture is one of a few ways to mitigate shrimp diseases, especially the acute hepatopancreatic necrosis disease that has affected shrimp culture industry worldwide. Since Nile tilapia are basically freshwater species, having them in co-culture with marine shrimp therefore requires a certain degree of salinity adaptation, in which the fish are readily capable to. The present study was aimed at studying certain hemato-immunological changes of the fish reared under elevated salinity, as well as their susceptibility to pathogens. Nile tilapia (180 g initial BW) were reared under gradually increasing salinity within one month from 0 to 30 ppt at 3 ppt/3 d and maintained for another month in 30-ppt salinity. At the end of the 60-day experimental period, 50% of the fish survived with mortality occurred mainly during the second month when the salinity was at 30 ppt. Their growth rate was retarded to 60% of that of the control fish maintained under freshwater (0 ppt). The fish reared under elevated salinity also had significantly lower hematocrit, higher total white blood cell counts, higher absolute numbers of lymphocytes and thrombocytes and higher serum lysozyme activity, compared to those reared under freshwater. Presence of three representative pathogens: infectious spleen and kidney necrosis virus (ISKNV), Francisella noatunensis and Streptococcus agalactiae ; were determined in the fish reared under freshwater and in those reared under elevated salinity, using polymerase chain reaction (PCR) method. Of the three pathogens, only ISKNV was detected in 1/10 of the freshwater and 6/10 of the elevated-salinity fish. In both groups, the virus was present in the gills, brain, liver, gonads, skin, kidney and spleen, with moderately and severely positive reaction observed in the kidney and spleen. The detection of ISKNV was confirmed by in situ hybridization of the gonads and kidney, using loop-mediated DNA amplification with digoxigenin, which clearly showed dark-brown stain of the positive signals in the PCR-positive samples. These results suggest that a substantial number of O. niltoicus could survive elevated salinity up to full-strength seawater provided that the salinity had been gradually elevated. And under that situation, changes in hemato-immunological functions of the fish did occur, which probably caused lethal proliferation of pre-existing ISKNV that otherwise would remain dormant.

Fishmeal is the main source of protein in the shrimp feed industry and is normally derived from trash fish. As such, the production of fishmeal has an adverse effect on the marine environment by taking away small and juvenile fish, leading to depletion of marine species. There is a need for alternative sources of protein which will substitute fishmeal in the aquaculture industry. This study evaluated the components and nutritional efficacy of bioflocs, which were used to substitute fishmeal protein. The effect of bioflocs diets on growth performance, survival rate, and immune response in shrimp compared to normal fishmeal feed were determined. Bioflocs were harvested from the shrimp ponds (C:N ratio >12:1) at Shrimp Village, Chaiya district, Surat Thani, Thailand. The total protein in bioflocs was about 48% and the total lipid was about 5% (dried weight) and the percentages of essential amino acids (EAA) and fatty acids (EFA) in bioflocs were similar to those of fishmeal feed. Shrimp fed with the different dietary bioflocs feed regimens [% to replace fishmeal; 0% (B0), 25% (B25), 50% (B50), 75% (B75), and 100% (B100)] for 42 days revealed that all growth parameters were almost similar to those of the control shrimp (shrimp fed with normal fishmeal, B0) including final body weight, weight gain, specific growth rate, and feed conversion ratio. Remarkably, the survival rates, the levels of immune parameters, and expression of immune genes (proPO-I, PEN-4 and dicer) were significantly higher in bioflocs fed shrimp, especially in B25 and B50 shrimp. Moreover, B25 and B50 bioflocs fed shrimp showed notably increased survival rates following Vibrio parahaemolyticus (V. parahaemolyticus) infection. In conclusion, the present study demonstrates that shrimp survival and immunity are enhanced by biofiocs substituted fishmeal. Significantly, the bioflocs diets activated the immune response to prevent V. parahaemolyticus infection.

Natural food items like amphipods in bioflocs have been shown to provide a nutrient-rich live feed for aquaculture species. In the present study, we aimed to identify the major species of amphipods in the biofloc technology shrimp ponds at Shrimp Village, Surat-Thani, Thailand, determine its nutritional values, and enrich the amphipods in the biofloc technology system (BFS). Breeder stage amphipods were cultured in BFS with different carbon sources; molasses, rice bran, and corn flour at a carbon: nitrogen (C:N) ratio of 16:1 for 4 weeks. The number and the growth of spawned juveniles, and the nutritional compositions of the amphipods enriched bioflocs were examined. We found that the major species of amphipods in bioflocs produced in the cultured shrimp ponds was Bemlos quadrimanus sp. Proximate analysis revealed that the juvenile stage B. quadrimanus had higher levels of protein and lipid contents than those in other stages. Moreover, their essential amino acid index (EAAI) and ratios of polyunsaturated fatty acid/saturated fatty acid (PUFA/SFA) and docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA) were similar to those values in fishmeal. The bioflocs produced with corn flour enhanced the growth and increased the number of juvenile amphipods greater than in bioflocs produced with the other two carbon sources, and had the highest levels of crude protein and lipid. In summary, this study demonstrates that amphipod B. quadrimanus provide an important source of protein that shows potential to replace fishmeal in shrimp feed, and reveals the benefit of a culture of amphipods with BFS in the aquaculture industry.

Outbreaks of acute hepatopancreatic necrosis disease (AHPND) have caused a decrease in shrimp production in Thailand for years. Previous studies revealed that seaweed proteins, particularly lectins, have antibacterial property. Therefore, this study aimed at evaluating the antibacterial activity of a protein extract from the red seaweed Gracilaria fisheri against Vibrio parahaemolyticus, the causative agent of AHPND. The crude protein extract from G. fisheri (GPE) was evaluated for lectin-like activities. GPE showed hemagglutination activity towards rabbit, sheep, and goose erythrocytes and required Mn2+ for the hemagglutination. GPE agglutinating activity could be inhibited by glycoproteins such as fetuin and porcine stomach mucin. The activity was stable within the temperature range 0–50 °C. GPE caused agglutination of V. parahaemolyticus in a dose dependent manner and inhibited the growth rates of the virulent bacterial strains (MIC50 = 30–38 μg mL−1). LC-MS/MS and BLAST analyses revealed that GPE contained proteins with similar amino acid sequences to lectins and phycobiliproteins from other red seaweeds. Penaeus vannamei fed with GPE showed improved survival rates and more normalized histological features of the hepatopancreas and less AP3 toxin gene expression following AHPND infection. In summary, this study demonstrates that GPE has antibacterial activity against V. parahaemolyticus, and that G. fisheri is a viable source of antibacterial substance that could be used as a feed supplement in shrimp culture to protect against or prevent AHPND.

The anti-Vibrio activity of essential oils (EOs) of nine medicinal plants was tested against 28 Vibrio spp. isolated from diseased Fenneropenaeus indicus. EO of Nigella sativa exhibited anti-Vibrio activity against all Vibrio spp. and greater inhibition was noted for the isolate V2 which was identified as Vibrio parahaemolyticus Dahv2. Further, EO of N. sativa effectively inhibited V. parahaemolyticus Dahv2 with an inhibition zone of 23.9mm at 101.2μgml(-1). Moreover, EO of N. sativa revealed anti-biofilm activity at 101.2μgml(-1) against V. parahaemolyticus Dahv2 and inhibited the growth of V. parahaemolyticus Dahv2 at 100μgml(-1).In vivo experimental infection studies showed that the survival of Artemia spp. infected with V. parahaemolyticus Dahv2 at 1×10(3)cfuml(-1) was only 40%. However, the survival of Artemia spp. was significantly increased after challenge with 100μgml(-1) of EO of N. sativa. EO of N. sativa showed higher anti-oxidant potential and total phenol content than other EOs tested. The anti-oxidant activity of EO of N. sativa was highly correlated to their total phenolic contents (r=0.836, P

The present study is the first report of a novel disease called Hahellosis or “red egg disease” that has been affecting tilapia ( Oreochromis spp.) hatcheries in Thailand. Hahella chejuensis , a red pigmented Gram negative bacterium, was recovered from samples of the red egg and identified to species level based on 99.5%–99.7% nucleotide homology to 16S rDNA of the type strain H. chejuensis KCTC 2396. Experimental infection of eggs indicated that H. chejuensis was able to cause red egg disease and also reduce their hatching. PCR protocols were developed for detection of H. chejuensis in tilapia samples including eggs and ovaries and testes of tilapia broodstocks. Positive signals were obtained in the ovaries and testes of tilapia broodstocks with in situ DNA hybridization using probes specific for H. chejuensis , suggesting possible vertical transmission of the red pigmented bacteria from broodstock to eggs. Statement of relevance The authors strongly believe that our manuscript would provide significant knowledge to fish aquaculture especially to that of the tilapia ( Oreochromis spp.) hatcheries.

In this study we examined the effect that a Francisella noatunensis (Fno) infection had on hybrid red tilapia (Oreochromis niloticus × Oreochromis mossambicus) subsquently infected with Streptococcus agalactiae. A variety of hemato-immunological parameters (haematocrit, total red blood cell count, mean corpuscular volume, total white blood and differential cell counts, total plasma protein, plasma lysozyme and plasma peroxidase activities, and respiratory burst and phagocytic activities of head-kidney macrophages) were measured in hybrid red tilapia that had been previously exposed to an Fno outbreak in a tilapia grow-out farm. The head-kidneys of these apparently healthy survivors, when checked by PCR were found to be Fno-positive with hemato-immunological parameters that were similar to fish without an a priori infection. The only exception was the percentage lymphocyte count in the peripheral blood, which was slightly, but significantly, lower in the Fno-infected fish, compared to those without the infection. When experimentally infected with S. agalactiae, the Fno-infected fish died more rapidly and at a significantly higher rate than fish without the infection. During the challenge, the hemato-immunological parameters of both groups of fish were very similar, although the Fno-infected fish, challanged with S. agalactiae expressed significantly higher plasma lysozyme and peroxidase activities, and their head kidney macrophages had significantly higher respiratory burst activity compared to non-Fno-infected fish challanged with S. agalactiae. The only two parameters for which Fno-infected fish showed significantly lower expressions than that of their non-infected counterparts were haematocrit and total red blood cell count. The cause of the rapidity and higher rates of mortality observed in the Fno-infected fish when challenged with S. agalactiae is unknown; but it may be due to a reduced erythropoiesis capability within the head-kidney because of the presence of Fno.

World production of farmed shrimp is focused on a few species. At present, the Pacific whiteleg shrimp Litopenaeus (Penaeus) vannamei tops the list, followed by the black tiger shrimp Penaeus monodon and a few others. The former attains the market size between 15 and 25 g within three months in culture, while P. monodon requires at least four months to reach the marketable size of ~25 g and larger. It is therefore desirable to produce stocks of P. monodon with a fast-growth-rate trait, either through selective breeding or via other modes of scientific invention. Among others, chromosome manipulation of P. monodon that confers three sets of chromosomes (3n) to the shrimp, a condition called triploidy and a feat that was achieved through a non-GMO technique, could be the answer. Thermal chromosome set manipulation for triploid induction is considered as a safe and environmentally friendly technique to produce sterile offspring for genetic protection, and prevent genetic pollution from aquaculture stocks in the wild. The black tiger shrimp can be induced to yield a high percentage of triploid offspring with abnormal reproductive histology. Two studies applying different induction methods briefly at the very early stage of embryo formation—one from Australia (chemical shock) and another from Thailand (cold shock) have reported successful results. The triploid shrimps produced from chemical shock method had reduced growth rate, while that from cold shock displayed higher growth rate compared to shrimp with the natural double set of chromosomes. Gender distribution of the triploid P. monodon induced by cold shock was skewed towards females, the larger size of the two sexes and hence is favored more in aquaculture. Because of the favorable results of the cold shock method, it is currently pursued to produce triploid P. monodon for commercial purposes. The production is accomplished through an automatic cold-shock induction system, consisting of spawning detection and cold shock treatment sections. This triploid induction project is an extension of the ongoing selective breeding program of a specific pathogen -free P. monodon that has already been commercially launched, and is part of an initiative to promote sustainable genetic stock improvement protocols for this shrimp species.

Flavobacterium columnare and Edwardsiella ictaluri are two major bacterial pathogens threatening catfish aquaculture globally. Earlier studies have focused on the characterization of single bacterial infection. In reality, multiple bacterial pathogens are present in aquaculture systems and are probably responsible for disease outbreaks and considerably outweigh single infection. The objectives of this study, therefore, were to investigate whether single or concurrent bacterial pathogens were involved in naturally diseased striped catfish ( Pangasianodon hypophthalmus ) and subsequently investigate the pathogenicity of single- and dual-infection through experimental challenges. The investigation revealed coinfections of F. columnare and E. ictaluri found in naturally diseased Thai striped catfish exhibiting columnaris and edwardsiellosis diseases. Bacterial identification was confirmed by phenotypic tests, species-specific PCR and 16S rDNA sequence analysis. Molecular data analysis also identified that the infected fish species was P. hypophthalmus . Experimental challenges of striped catfish juveniles with single and dual bacterial species using both immersion (i.m) and injection (i.p) approaches were performed. Injection of two different doses of combined bacteria caused markedly high mortality of 86.7–100%, indicating high virulence of the bacterial isolates. Immersion (i.m.) coinfection of E. ictaluri (2.6 × 10 6 CFU mL − 1 ) and F. columnare (1.0 × 10 4 CFU mL − 1 ) caused significantly high cumulative mortality (96.7 ± 5.8%) compared to i.m. of single infection of E. ictaluri (80.0 ± 20%) or F. columnare (3.3 ± 5.7%) with the same dose of bacteria. Both coinfection challenge routes i.p. and i.m. successfully mimicked typical signs and histopathological manifestations of natural coinfection. This study had fulfilled Koch's postulates through single- or dual-challenged tests to mimic the natural disease case in striped catfish. Statement of relevance The authors strongly believe that our manuscript would provide significant knowledge to fish aquaculture especially to that of the striped catfish P. hypophthalmus .

Neuropeptides are key mediators of physiological processes in animals and a considerable amount of information has been accumulated on their diversity and functions across phyla. However, progress in echinoderm neurobiology has been much slower than others. The sea cucumber Holothuria scabra is an economically important tropical echinoderm species in which a neuropeptide is yet to be identified. This immunohistochemical study utilized antibody raised against GFSKLYFamide neuropeptide together with confocal laser scanning microscopy. GFSKLYFamide-like immunoreactivity was shown to be localized in the stomach and small intestines of H. scabra . This is the first report that provides evidence for the presence of GFSKLYFamide neuropeptide in the digestive tract of this species. Keywords: GFSKLYFamide, SALMFamides, immunohistochemistry, sea cucumber, Holothuria scabra .

Monodon baculovirus (MBV) causes slow growth of the shrimp being due to its infection in the hepatopancreas, the organ producing digestive enzymes and for nutrient storage. It has long been documented that the virus is transmitted from broodstock to offspring via contamination in rearing water by feces of the infected broodstock that contained sloughing-off damaged hepatopancreatic cells containing MBV. As a management practice, the washing of eggs and nauplii with disinfectant has been recommended to eliminate MBV that are attached to their surface and thus prevent the infection at the later development stage of the shrimp. However, we detected the MBV infection in postlarvae and juveniles of the black tiger shrimp Penaeus monodon, even after our repeated attempts to eliminate the virus by washing the eggs and nauplii with povidone iodine as disinfectant. Therefore we used MBV-infected broodstock to identify the cause of this problem by tracing the presence of MBV in hepatopancreas and ovary of the broodstock, eggs, nauplii and postlarvae, using histology with hematoxylin–eosin staining, polymerase chain reaction (PCR), in situ hybridization (ISH) and immunohistochemistry (IHC) specific for polyhedrin, a protein produced by MBV. We found all the ovaries collected from the broodstock, which were detected MBV-positive in the hepatopancreas by histology, PCR and ISH, were also PCR-positive. By ISH, positive signals were detected in the cell membrane, cytoplasm and nuclear membrane of the oocytes. The eggs and nauplii from the MBV-positive broodstock were also positive by PCR, in both “wash” and “no-wash” specimens. By ISH, positive signals were detected in ooplasm and subcuticular region of nauplii, as well as inside its body. Using IHC, positive signals were detected inside the body and appendage of the nauplius. Taken all these together, it is most likely that MBV could be vertically transmitted through trans-ovarian route. Hence, simply washing the eggs and nauplii with disinfectant may not be an adequate procedure to eliminate the MBV infection in offspring from MBV-infected broodstock.

Molt-inhibiting hormone (MIH) is commonly localized in the X-organ sinus gland complex (XOSG) of the crustacean. The present study aimed to elucidate the expression of MIH at the subcellular and cellular levels in the eyestalks and integument of juvenile Penaeus monodon during the molting cycle. Gene expression of Pem-MIH1 in the optic lobes showed a single PCR product at 172 bp, and was restricted only to the eyestalk XOSG but not detected in pleopods, cephalothorax integument, muscle, hepatopancreas or retina. Quantitative analysis of gene expression in the eyestalks demonstrated no significant alteration of Pem-MIH1 mRNA in XOSG during the molt cycle. Immunohistochemistry using antibody against recombinant molt-inhibiting hormone-like peptide (anti-MIH-like) revealed variable staining intensities of individual MTXOs but were most persistently intense in SGs. During the molt cycle, the mean numbers of XO immunoreactive cells were slightly oscillated but not statistically different. The quantitative immunohistochemistry measured from XOSG illustrated minimal fluctuations of the values obtained suggesting the periodical synthesis and release of MIH from XOSG system during the molt cycle. The positive reaction was also detected in the tegumental glands located at the retina and the integument of walking leg, base of eyestalk and cephalothorax of the shrimp. The large variations of immunostaining and amount of reactive tegumental glands in the eyestalk were noted throughout the molt cycle. The numbers of the MIH-like immunoreactive glands obtained from the retina as well as the integument base did not significantly change during the molt cycle but tend to increase during postmolt to intermolt and decrease during premolt. These results suggest that the MIH-like peptides in the tegumental glands are probably released mostly during the late premolt. Our findings thus propose the novel storage site of MIH besides SG of the eyestalk including a new target tissue, an epidermal cell, the role of which may link to the complicated molting regulation and/or the dark-adaptation of shrimp.

The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from Penaeus monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability and viral load were determined. Haemocytes treated with WSSV-SG pre-mix showed decreased CPE, viral load and cell mortality from the viral infection. Solid-phase virus-binding assays revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26 and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from Gracilaria fisheri elicits its anti-WSSV activity by binding to viral proteins that are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for viral binding.

White spot syndrome virus (WSSV) is currently the most serious global threat for cultured shrimp production. Although its large, double-stranded DNA genome has been completely characterized, most putative protein functions remain obscure. To provide more informative knowledge about this virus, a proteomic-scale network of WSSV-WSSV protein interactions was carried out using a comprehensive yeast two-hybrid analysis. An array of yeast transformants containing each WSSV open reading frame fused with GAL4 DNA binding domain and GAL4 activation domain was constructed yielding 187 bait and 182 prey constructs, respectively. On screening of ∼28,000 pairwise combinations, 710 interactions were obtained from 143 baits. An independent coimmunoprecipitation assay (co-IP) was performed to validate the selected protein interaction pairs identified from the yeast two-hybrid approach. The program Cytoscape was employed to create a WSSV protein–protein interaction (PPI) network. The topology of the WSSV PPI network was based on the Barabási-Albert model and consisted of a scale-free network that resembled other established viral protein interaction networks. Using the RNA interference approach, knocking down either of two candidate hub proteins gave shrimp more protection against WSSV than knocking down a nonhub gene. The WSSV protein interaction map established in this study provides novel guidance for further studies on shrimp viral pathogenesis, host-viral protein interaction and potential targets for therapeutic and preventative antiviral strategies in shrimp aquaculture.

Immobilized Spirulina mat was co-cultured with Pacific white shrimp (Penaeus vannamei) postlarvae for water quality control in a closed re-circulating system. During the culture period, the experimental Spirulina mat system showed total ammonia nitrogen and nitrate accumulation rates lower by 32.92 and 32.26 %, respectively, compared with the system without the mat. The water exchange rate in the system with the mat was comparatively reduced by 40 %. Moreover, survival rate, growth rate, and size (length) of the co-cultured shrimp larvae were greater than those of the control (P

Production of sterile triploid red tilapia [Oreochromis mossambicus (Mozambique tilapia); Peters, 1852 × Oreochromis niloticus (Nile tilapia); Linnaeus, 1758] is an effective strategy to overcome their prolific breeding. Optimal conditions for cold-shock induction of triploidy in red tilapia were investigated by experimentally examining two variables: appropriate temperature of the shock and duration of shock treatment. A constant time after insemination of 4 min was used to determine the best combination of temperature (6, 7, 8, 9, 11, 13, 15 °C) with different durations of shock (10, 20, 30, 40, 50 min) with resultant ploidy level verified karyotypically. Shock duration for 30 min at a temperature of 9 °C was found most effective in producing maximum triploidy (98.7 %) with higher rates of hatching (63.2 %) and survival up to yolk-sac stage (75.8 %). The chromosome count confirmed that triploid percentages were higher when cold shock was used for longer durations at each temperature; however, hatching rates were generally decreased. The maximum triploid yield (82.1 %) obtained was higher than the yield obtained using heat shock (72.7 %) in red tilapia previously. The application of the results of this study has the potential to greatly improve the production of triploid red tilapia in commercial aquaculture.

The organic matrix proteins of molluscan nacre are known to engage biocompatible and osteoinductive properties. In this study, we aimed to extract Haliotis diversicolor nacreous proteins and assayed their effect on pre-osteoblastic cell differentiation. We found that acid extracted nacreous proteins (AEP) consisted primarily of a major protein band of 25 kDa and three other minor proteins. AEP enhanced alkaline phosphatase (ALP) activity of MC3T3-E1 cells both in time- and concentration-dependent manners. Transcriptional up-regulation of osteogenic markers, including collagen type I (COL-I), osteopontin (OPN) and osteocalcin (OCN), was also apparent in days 7 and 14 upon AEP treatments. At the translational level, higher protein expression of COL-I was evident in AEP treated cells, and the protein was presumably laid down as extracellular matrix. Further de novo sequencing of an AEP major protein revealed a match with the abalone mantle protein sometsuke. Conclusively, we demonstrated that H. diversicolor AEP contains a factor, potentially the mantle protein sometsuke, which may impart in the osteoblastic cell differentiation.

We exploited Artemia as a double-stranded (ds)RNA-delivery system to combat viral diseases in shrimp. First, the transformed Escherichia coli (E. coli) expressing red fluorescent protein (RFP) was tested in the Artemia enrichment process. RFP signals detectable in the gut of Artemia under confocal microscope were evident for the successful encapsulation. Second, the Artemia enrichment process was performed using E. coli producing Laem-Singh virus (LSNV)-specific dsRNA, which has been previously shown to inhibit the viral infection in the black tiger shrimp Penaeus monodon by intramuscular injection and oral administration. The enriched Artemia nauplii were confirmed to contain dsRNA-LSNV by RT-PCR, and were subjected to the feeding test with P. monodon postlarvae. Quantitative RT-PCR indicated that a number of LSNV copies in most of the treated shrimp were, at least, 1000-fold lower than the untreated controls. During 11-17weeks after feeding, average body weight of the treated group was markedly increased relative to the control group. A smaller differential growth rate of the treated group as compared to the control was also noticed. These results suggested that feeding shrimp with the dsRNA-enriched Artemia can eliminate LSNV infection, which is the cause of retarded growth in P. monodon. The present study reveals for the first time the therapeutic effect of dsRNA-enriched Artemia for shrimp disease control.

UNLABELLED Streptococcus agalactiae and Strep. iniae are bacterial pathogens that cause streptococcosis in many fish species. An accelerated colorimetric loop-mediated isothermal amplification (LAMP) assay with pre-addition of calcein was established, and the transmission and detection of Strep. agalactiae and Strep. iniae in tilapia under natural aquatic environment were investigated. A positive reaction was observed by a colour change from orange to green through the naked eyes after completion at 63°C for 30 min with 10 times higher sensitivity than that of nested PCR assays and without cross-amplification with other fish bacterial pathogens. All sample types of Nile and red tilapia (broodstock, fertilized egg, fry) were Strep. agalactiae- and Strep. iniae positive by this new method, implying that they could be vertically transmitted. With its application for screening broodstock and fry before stocking and for monitoring fish health in grow-out ponds, the method would become very useful in fish farming industry. SIGNIFICANCE AND IMPACT OF THE STUDY The application of colorimetric LAMP with pre-addition of calcein offers simple, rapid and sensitive technique with applicability for small field laboratories. This technique explored the possible vertical transmission mode of Strep. agalactiae and Strep. iniae under natural aquatic environment. It could be such preliminary data provided for the screening broodstock before breeding and/or the specific-pathogen-free production.

Triploidy could reduce breeding activity in tilapia without the use of hormones. In this study, the effect of triploidy on survival, growth, and gender of a line of red hybrid tilapia (Oreochromis mossambicus X Oreochromis niloticus) was assessed relative to the performance of diploid siblings. Triploidy was induced by preventing second polar body extrusion by applying either heat or cold shock. Growth was similar for both ploidies during the first 90 days of culture. However, at the age of 120 days, the average body weight of triploids produced by heat shock (215.5 ± 3.61 g) was significantly higher than that of cold shock (192.7 ± 2.6 g) and the diploid control (191.9 ± 1.74 g). Survival among triploids was inferior to diploids. Percentage of males in the triploid population was 82.9% in the heat-shocked treatment group, 54.8% in the cold-shock treatment, and 50% in the diploid control. Maximum attainable weight of red tilapia was calculated by applying the Ford-Walford growth plot: 650 g (heat-shocked ...

Viral inhibition by double-stranded (ds)RNA is a potential therapeutic approach for controlling shrimp viral diseases. Here, we describe the successful oral application of dsRNA targeting Laem-Singh Virus (LSNV) to diminish monodon slow growth syndrome (MSGS) in Thai Penaeus monodon. Shrimp feed formulated with bacterially expressed LSNV-dsRNA was given to shrimp for 9 weeks. RT-PCR results revealed that all control shrimp were LSNV-positive at the end of experiment, while the shrimp that received dsRNA-feed exhibited 20–60% LSNV reduction. The average body weight of treated shrimp (number of shrimp = 100) was significantly higher than that of the control group. Such increase is likely due to the elimination of MSGS caused by LSNV, as size variation of the treated group is much lower than that in the control group. This study demonstrates for the first time that feed with LSNV-specific dsRNA promotes the overall growth of P. monodon and relieves MSGS condition in LSNV-infected shrimp. The work reaffirms the potential of dsRNA application for controlling viral disease in shrimp farming.

Marine invertebrates respond to fluctuations in their environment throughout development and growth. Homeostatic adaptation is mediated in part through the regulation of Na+-K+ ATPase enzymes and members of this class of enzymes are localized to the antennal gland of many crustaceans. In this study, we investigated the morphological and biochemical changes of the antennal glands in eight families of cross-breed post-larval shrimp subjected to hypotonic (10 ppt) and isotonic (20 ppt) conditions. The greatest changes in the antennal glands were detected in the hypotonic condition with families two and five cross-breed shrimp exhibiting the highest tolerance to this diluted salinity. The changes detected in hypotonic tolerant shrimp included enlargement of coelomosac and labyrinth spaces, the presence of many dense cytoplasmic globules in podocytes as well as highly wavy apical architecture of labyrinth cells. Several alterations were also observed at the subcellular level such as widening of the intercellular spaces between podocytes, organelle destruction, and decreased basal membrane in-folding in labyrinth cells. In addition, shrimp exposed to hypotonic conditions displayed decreased expression of both α and β subunits of Na+-K+ ATPase and decreased enzymatic activity. The changes observed in animals upon exposure to hypotonic stress are discussed.

A sulfated galactans (SG) supplemented diet was evaluated for the potential to stimulate immune activity in shrimp Penaeus vannamei (P. vannamei). Shrimp given the SG supplemented diet (0.5, 1 and 2% w/w) for 7 days showed enhanced expression of the downstream signaling mediator of lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and immune related genes including p-NF-κB, IMD, IKKβ and IKKe, antimicrobial peptide PEN-4, proPO-I and II. Following immersion with Vibrio parahaemolyticus (V. parahaemolyticus) for 14 days, the shrimp given the SG supplemented diet (1 and 2% w/w) showed a decrease in bacterial colonies and bacterial toxin gene expression, compared to shrimp given a normal diet, and they reached 50% mortality at day 14. However, shrimp given the normal diet and challenged with the bacteria reached 100% mortality at day 6. SG-fed shrimp increased expression of immune genes related to LGBP signaling at day 1 after the bacterial immersion compared to control (no immersion), which later decreased to control levels. Shrimp on the normal diet also increased expression of immune related genes at day 1 after immersion which however decreased below control levels by day 3. Taken together, the results indicate the efficacy of the SG supplemented diet to enhance the immune activity in shrimp which could offer protection from V. parahaemolyticus infection.

Conventional isolation and identification based on phenotypic characteristics is challenging with the highly fastidious, intracellular bacterium Francisella noatunensis subsp. orientalis (Fno). Here, we developed a duplex PCR method for simultaneous detection of the Francisella genus and Fno in one PCR reaction and an in situ hybridization method for paraffin section based diagnosis of Fno. The PCR results showed genus- and species-specific bands (1140 and 203 bp) from Fno but only one genus-specific band (1140 bp) from F. noatunensis subsp. noatunensis. Sensitivity of the duplex PCR assay revealed a detection limit of 20 to 200 fg genomic DNA (~10 to 100 genome equivalents) depending on DNA template extraction methods. The newly developed duplex PCR assay could be used to detect Fno from clinically sick fish exhibiting signs of visceral granulomas and would also be able to detect Fno infection in naturally diseased fish without symptoms of francisellosis, indicating potential application for diagnosis of field samples. The in situ hybridization assay using Fno species-specific probe revealed positive signals in multiple organs including the spleen, liver, kidney, gills and intestine of infected fish.

Aims Infectious spleen and kidney necrosis virus (ISKNV) has recently been recognized as a causative agent of serious systemic disease in tilapia. Our objective was to establish a new colorimetric loop-mediated isothermal amplification (LAMP) assay with pre-addition of hydroxynapthol blue (blue-LAMP) to investigate ISKNV transmission in tilapia. Methods and Results The blue-LAMP, targeting a major capsid protein gene of ISKNV, was conducted at 65°C for 45 min, allowing unaided visual detection of the pathogen based on colour change without cross-amplification of other known fish pathogens tested. Comparison of blue-LAMP and PCR assays revealed a higher detection level for blue-LAMP assay (41·33%) in a population of farmed tilapia infected with ISKNV. The investigation of ISKNV transmission pattern in farmed red tilapia using the blue-LAMP revealed a possible matroclinical form. The presence of ISKNV in the gonad samples was confirmed by in situ LAMP assay. Positive signals only appeared in ovarian follicles, and not in oocytes. Moreover, tissue tropism assay revealed that the brain was the main target organ in both farmed red tilapia (40%) and Nile tilapia (20%). Conclusions The developed blue-LAMP assay has the potential to be used as a viable tool for screening covert and natural infections of ISKNV in tilapia. The evidence of vertical transmission of ISKNV infection in tilapia indicates the seriousness of this disease and will require a close attention and collaboration between tilapia hatcheries and disease experts in order to find a solution. Significance and Impact of the Study The new blue-LAMP assay is a time-saving and economically viable detection tool, which allows unaided visual detection for ISKNV in tilapia, and it could be applicable for field applications. Evidence on the vertical transmission of ISKNV in farmed tilapia suggests a need for developing farm management practices to control the spread of virus in aquaculture industries.

This study seeks to determine the best method for preventing the second body (PBII) extrusion as a means to induce triploidy in the black tiger shrimp Penaeus monodon . Chemical (cytochalasine-B and 6-dimethylaminopurine) and temperature (heat and cold) shocks were applied to newly fertilized eggs. Cold shock that was administered at 8 °C, for 10 min at 8 min post-spawning, was the best method for PBII triploidy induction, as evidenced by the highest percentage of forming three pronuclei in syngamy. Therefore, cold shock induction was employed to further explore the advantage of triploid over diploid shrimp. After hatching of the cold-shocked eggs, the larvae were allowed to reach juvenile and adult levels of development. The number and amount of chromosomes were determined in juveniles and adult stages, using Fluorescence Activating Cell Sorting methods, by which the shrimp were divided into diploid and triploid groups. At day 150 in culture, the average body weight of the triploid females (35.2 ± 3.2 g) and triploid males (31.5 ± 3.5 g) was significantly higher ( P P. monodon , and its feasibility for commercial production.

Monodon Slow-Growth Syndrome (MSGS), a pathological condition in the black tiger shrimp Penaeus monodon, is associated with Laem–Singh virus (LSNV) infection. Infected shrimp grow slowly when the virus invades the part of the retina called the zona fasciculata. Since molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH) are synthesized in the optic lobe and are related to molting activity and growth of the shrimp, the purpose of this study is therefore to determine the levels of these hormones and related parameters in MSGS shrimp. P. monodon juveniles were sampled from normal and MSGS ponds and were divided into small-negative, large-negative, small-positive and large-positive groups, depending on the size of the shrimp and whether they were LSNV-negative or LSNV-positive. Individual shrimp were measured for duration of each molt stage and molt interval. Levels of MIH1 and CHH1 transcripts were determined from the optic lobe, using real-time reverse transcriptase–polymerase chain reaction. The results revealed that the small-positive and the small-negative shrimp did not differ in durations of molt stages or molt intervals, as well as on the levels of MIH1 transcript in the optic lobe, MIH1 peptide in the optic lobe and hemolymph, or ecdysteroids in the hemolymph. Differences in molting activities and related transcript and hormones were observed between the small- and large-sized shrimp, but not in the status of LSNV infection. While levels of CHH1 transcript in the optic lobe of both small-negative and small-positive shrimp did not differ, levels of CHH1 peptide, as well glucose, in the hemolymph of the small-positive shrimp were significantly lower than those of the small-negative group. Levels of glycogen in the hepatopancreas of the small-positive shrimp were also significantly higher than that of the small-negative ones. The results suggest that growth retardation in MSGS shrimp is related to the suppression of the release of CHH1 peptide by LSNV invasion in the zona fasciculata, consequently causing decreased hepatopancreatic glycogenolysis and persistent hypoglycemia, resulting in growth stunting.

The aim of this study was to reveal the process of hatching envelope (HE) formation in eggs of the black tiger shrimp Penaeus monodon, using fluorocytochemistry with fluorescein isothiocyanate (FITC)-labelled lectins and transmission electron microscopy (TEM) with mouse monoclonal anti-FITC-conjugated gold-lectin labelling. Following lectin binding screening tests, Concanavalin A (Con A) and wheat germ agglutinin (WGA) were chosen to trace movements of specific sugar-associated components of the HE. This revealed that both Con A and WGA-binding components migrated from the ooplasm to the HE. Using TEM, it was revealed that membranous materials in the ooplasm were released at the time of spawning, that these became associated with granular structures outside the oocyte and that they together developed into an outer layer of the HE. Contents of flocculent vesicles and dense vesicles in the ooplasm were exocytosed and formed the inner layer of the HE. The TEM with gold-labelled Con A and WGA revealed that the dense and flocculent vesicles and the inner layer of the HE contained components associated with mannose (sugar affinity to Con A) and N-acetyl-β-d-glucosamine (sugar affinity to WGA).

A legume-type lectin (L-Lectin) gene of the red algae Gracilaria fisheri (GFL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of GFL was 1714 bp and contained a 1542 bp open reading frame encoding 513 amino acids with a predicted molecular mass of 56.5 kDa. Analysis of the putative amino acid sequence with NCBI-BLAST revealed a high homology (30-68%) with legume-type lectins (L-lectin) from Griffithsia japonica, Clavispora lusitaniae, Acyrthosiphon pisum, Tetraodon nigroviridis and Xenopus tropicalis. Phylogenetic relationship analysis showed the highest sequence identity to a glycoprotein of the red algae Griffithsia japonica (68%) (GenBank number AAM93989). Conserved Domain Database analysis detected an N-terminal carbohydrate recognition domain (CRD), the characteristic of L-lectins, which contained two sugar binding sites and a metal binding site. The secondary structure prediction of GFL showed a beta-sheet structure, connected with turn and coil. The most abundant structural element of GFL was the random coil, while the alpha-helixes were distributed at the N- and C-termini, and 21 beta-sheets were distributed in the CRD. Computer analysis of three-dimensional structure showed a common feature of L-lectins of GFL, which included an overall globular shape that was composed of a beta-sandwich of two anti-parallel beta-sheets, monosaccharide binding sites, were on the top of the structure and in proximity with a metal binding site. Northern blot analysis using a DIG-labelled probe derived from a partial GFL sequence revealed a hybridization signal of (approx.) 1.7 kb consistent with the length of the full-length GFL cDNA identified by RACE. No detectable band was observed from control total RNA extracted from filamentous green algae.

Proprotein convertase subtilisin/kexin 4 (PCSK4) is implicated for sperm fertilizing ability, based on studies using Pcsk4-null mice. Herein we demonstrated proprotein convertase (PC) activity in intact sperm and acrosomal vesicles. To determine whether this activity was important for sperm fertilizing ability, a peptide inhibitor was designed based on PCSK4 prodomain sequence (proPC475–90), which contains its primary autocatalytic cleavage site. ProPC475–90 inhibited recombinant PCSK4's activity with a Ki value of 5.4 µM, and at 500 µM, it inhibited sperm PC activity almost completely. Treatment of sperm with proPC475–90 inhibited their egg fertilizing ability in a dose dependent manner. Correlation between sperm PC activity and fertilizing ability showed a high co-efficient value (>0.9), indicating the importance of sperm PC activity in fertilization. In particular, sperm PC activity was important for capacitation and zona pellucida (ZP)-induced acrosome reaction, since proPC475–90-treated sperm showed markedly decreased rates in these two events. These results were opposite to those observed in Pcsk4-null sperm, which contained higher PC activity than wild type sperm, possibly due to overcompensation by PCSK7, the other PCSK enzyme found in sperm. ADAM2 (45 kDa), a sperm plasma membrane protein, involved in sperm–egg plasma membrane interaction, was also processed into a smaller form (27 kDa) during capacitation at a much reduced level in proPC475–90-treated sperm. This result suggested that ADAM2 may be a natural substrate of sperm PCSK4 and its cleavage by the enzyme during acrosome reaction may be relevant to the fertilization process. J. Cell. Physiol. 226: 2817–2826, 2011. © 2011 Wiley-Liss, Inc.

Vibriosis is a common bacterial disease that can cause high mortality and morbidity in farmed shrimp. Since compounds from seaweed have been reported to have anti-bacterial and immunostimulant activity, this study was conducted to determine whether solvent extracts from the red seaweed Gracilaria fisheri might be a possible alternative for prevention and treatment of shrimp vibriosis caused by Vibrio harveyi. Seaweed extracts prepared using ethanol, methanol, chloroform and hexane were evaluated for anti-V. harveyi activity by the disc-diffusion method. The ethanol, methanol and chloroform extracts showed activity against a virulent strain of V. harveyi with potency (minimal inhibitory concentrations in the range of 90-190 μg ml(-1)) equivalent to the antibiotic norfloxacin. The ethanol extract was not toxic to the brine shrimp Artemia salina when it was fed to them for enrichment prior to their use, in turn, as feed for postlarvae of Penaeus monodon. Postlarvae fed with these enriched Artemia gave significantly lower mortality than control postlarvae after challenge with V. harveyi. In addition, P. monodon juveniles injected with the ethanol extract showed a significant increase in the total number of haemocytes and an increased proportion of semi-granulocytes and granulocytes when compared to control shrimp. The activities of phenoloxidase and superoxide dismutase were also increased, with an accompanying increase in superoxide anion production. When these juvenile shrimp were challenged with V. harveyi, mortality was markedly reduced compared to that of control shrimp. The results indicated that ethanol extracts of G. fisheri had immunostimulant and antimicrobial activity that could protect P. monodon against V. harveyi.

Brains from ovariectomised (ovx) rats can display features similar to those observed in menopausal women with Alzheimer's disease (AD), and oestrogen seems to play a key role. Preliminary studies on young coconut juice (YCJ) have reported the presence of oestrogen-like components in it. The aim of the study was to investigate the effects of YCJ on the AD pathological changes in the brains of ovx rats. Rat groups included sham-operated, ovx, ovx+oestradiol benzoate (EB) and ovx+YCJ. Brain sections (4 μm) were taken and were immunostained with β-amyloid (Aβ) 1–42, glial fibrillary acidic protein (GFAP) (an intermediate neurofilament of astrocytes) and Tau-1 antibodies. Aβ 1–42, GFAP and Tau-1 are considered as reliable biomarkers of amyloidosis, astrogliosis and tauopathy (neurofibrillary tangles), respectively, which in turn are characteristic features associated with AD. The serum oestradiol (E2) level was measured using a chemiluminescent immunoassay technique. YCJ restored the serum E2 to levels significantly (P P P

Litopenaeus vannamei is one of the most important species of farmed shrimp. The females have an ‘open’ thelycum. Mating is accomplished by attaching the male spermatophore onto the surface of the thelycum 4–6 h before spawning. During this period, sperm may have to undergo morphological changes associated with a capacitation process that has been described for other shrimp species. The objective of this research was to extend research on sperm capacitation in L. vannamei by ultrastructural and biochemical means. The sperm of L. vannamei were divided into those freshly prepared from the spermatophore (S-sperm), extracted from the male gonopores, and those extracted from the female thelycum (T-sperm). Under transmission electron microscopy, ultrastructural differences were detected between the S- and the T-sperm in the nuclear material, the filamentous meshwork and the cytoplasmic particles. Under scanning electron microscopy, the difference was observed in the cap and spike regions. Immunofluorescence using confocal microscopy to detect tyrosine phosphorylated proteins revealed different distribution patterns between S- and T-sperm. The location of phosphorylation activity changed from the spike in S-sperm, to the filamentous meshwork in the T-sperm. These morphological and biochemical changes confirm that capacitation of L. vannamei sperm takes place following mating.

A recent study showed Thalassiosira weissflogii to be a diatom containing suitable nutrition for larviculture of the black tiger shrimp, Penaeus monodon. Accurate and practical identification of this diatom species is therefore important for commercial hatcheries. The purpose of this study was to establish a DNA-based method of identification to supplement morphological examination, avoiding confusion with other Thalassiosira sp. Primers, 18SF/28SR1, specific for ribosomal DNA genes (3′-end of 18S rDNA through 5′-end of 28S rDNA, covering two internal transcribed spacers), were employed as a first-step polymerase chain reaction, followed by a second nested amplification using specifically designed primers, ITS1-F-D/ITS1-R-D. The nested-PCR result revealed specificity in the detection, distinguishing T. weissflogii from T. pseudonana, Cyclotella meneghiniana, and Chaetoceros sp., and the PCR fragment of the amplified region had a sequence that was 99% identical to the T. weissflogii sequence held by GenBank.