An anticoagulant solution for haemolymph collection and prophenoloxidase studies of penaeid shrimp (Penaeus californiensis)

Vargas‐Albores, Francisco; Guzmán, María-Antonia; Ochoa, J.L.

doi:10.1016/0300-9629(93)90516-7

Cited by 223 publications

(99 citation statements)

References 25 publications

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“…Due to the prevalence of the white spot syndrome virus (WSSV), and the decapod Penstyldensovirus (PstDV-1) in the area, specimens were tested against these pathogens as follows: after acclimation, one volume (400 μL) of hemolymph samples was extracted individually from the base of the fifth pereiopod of each shrimp with a 1-mL syringe containing 400 μL of pre-cooled (4°C) shrimp anticoagulant solution (450 mmol L − 1 NaCl, 10 mmol L − 1 KCl, 10 mmol L −1 Na 2 -EDTA, 10 mmol L −1 HEPES, pH 7.3) (Vargas-Albores et al 1993). Hemolymph was centrifuged at 400×g for 5 min, and the resulting hemocyte pellet was resuspended in 150 μL of lysis buffer (100 mmol L −1 NaCl, 50 mmol L −1 Tris, 10 mmol L −1 EDTA, and 1% SDS) and homogenized over ice with a Teflon disposable pestle.…”

Section: Methodsmentioning

confidence: 99%

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

Götze

Saborowski

Martínez‐Cruz

et al. 2017

Cell Stress and Chaperones

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Crustaceans are intensively farmed in aquaculture facilities where they are vulnerable to parasites, bacteria, or viruses, often severely compromising the rearing success. The ubiquitin-proteasome system (UPS) is crucial for the maintenance of cellular integrity. Analogous to higher vertebrates, the UPS of crustaceans may also play an important role in stress resistance and pathogen defense. We studied the general properties of the proteasome system in the hemocytes of the whiteleg shrimp, Penaeus vannamei, and the European brown shrimp Crangon crangon. The 20S proteasome was the predominant proteasome population in the hemocytes of both species. The specific activities of the trypsin-like (Try-like), chymotrypsinlike (Chy-like), and caspase-like (Cas-like) enzymes of the shrimp proteasome differed between species. P. vannamei exhibited a higher ratio of Try-like to Chy-like activities and Caslike to Chy-like activities than C. crangon. Notably, the Chy-like activity of P. vannamei showed substrate or product inhibition at concentrations of more than 25 mmol L −1 . The K M values ranged from 0.072 mmol L −1 for the Try-like activity of P. vannamei to 0.309 mmol L −1 for the Cas-like activity of C. crangon. Inhibition of the proteasome of P. vannamei by proteasome inhibitors was stronger than in C. crangon. The pH profiles were similar in both species. The Try-like, Chy-like, and Cas-like sites showed the highest activities between pH 7.5 and 8.5. The proteasomes of both species were sensitive against repeated freezing and thawing losing~80-90% of activity. This study forms the basis for future investigations on the shrimp response against infectious diseases, and the role of the UPS therein.

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Section: Methodsmentioning

confidence: 99%

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

Götze

Saborowski

Martínez‐Cruz

et al. 2017

Cell Stress and Chaperones

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“…After an acclimation period of 48 h, spermatophore and haemolymph samples were taken from all animals, snap-frozen in liquid nitrogen and stored at -80°C. Haemolymph (200 µl) was collected by pericardial sinus puncture and placed directly into 500 µl of ice-cold RNase-free (DEPC-treated) anticoagulant solution (Vargas-Albores et al 1993). Haemocytes were pelleted by haemolymph microcentrifugation at 4000 × g for 4 min, snap-frozen in liquid nitrogen and stored at -80°C.…”

Section: Methodsmentioning

confidence: 99%

Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon

Vega¹,

Degnan²,

Hall³

et al. 2004

Dis. Aquat. Org.

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Gill-associated virus (GAV) of the black tiger prawn Penaeus monodon has been implicated as a cause of periodic production losses in Australia since 1996. We report here the development of a real-time quantitative RT-PCR (qRT-PCR) for GAV. A dilution series of in vitro transcribed RNA was used to determine the sensitivity limit of the qRT-PCR and as a standard for GAV quantification. A linear relationship between cycle threshold (Ct) values and input RNA was obtained over a wide concentration range between 4.86 × 10 9 and 0.5 template copies per reaction, the latter being the test detection limit. The qRT-PCR was used to follow the progression of GAV levels in a group of 15 adult male P. monodon with chronic GAV infections that were super-infected by intramuscular injection of an inoculum containing high levels of GAV. By Day 9 post-injection, cumulative mortalities reached 100% (15/15) in the GAV-injected prawns and 40% (2/5) in placebo-injected prawns. Spermatophores were collected at the beginning, and together with other tissues, at the end of the trial. Prawns were also bled at regular intervals to collect circulating haemocytes. The qRT-PCR revealed that GAV loads increased significantly in haemocytes collected from both the control and super-infected prawns (p = 0.010). This increase was significantly higher in the super-infected prawns (p = 0.047). The rapid increase in GAV levels in super-infected P. monodon was expected. However, the increase in the control prawns was not, and indicates that repetitive bleeding and handling stress can stimulate GAV proliferation in chronically infected P. monodon. KEY WORDS: Penaeus monodon · Gill-associated virus · Quantitative RT-PCR (qRT-PCR)Resale or republication not permitted without written consent of the publisher Dis Aquat Org 59: 195-203, 2004 2003, Tang et al. 2002, Callinan et al. 2003. Sequence comparisons of viruses from many healthy and moribund P. monodon indicate that they are genetically inseparable and are now regarded as the same virus (Cowley et al. 2000 and unpubl. data).GAV is a positive-strand ssRNA nidovirus closely related to the yellow head virus (YHV) from Thailand in genome sequence and organisation (Cowley et al. 1999. GAV and YHV have recently been classified in a new genus (Okavirus) and family (Roniviridae) within the Nidovirales (Mayo 2002). Both viruses have rod-shaped enveloped particles and cause similar histopathology and cytopathology (Spann et al. 1995, 1997, Tang & Lightner 1999, Tang et al. 2002. GAV diagnosis based on clinical signs and histopathology is not particularly specific as other viruses can cause similar effects. More precise diagnostic methods include transmission electron microscopy (TEM), in situ hybridisation (ISH) (Tang et al. 2002) and RT-nested PCR (Cowley et al. 2000).There is no detailed quantitative data directly linking GAV infection levels to the manifestation of disease symptoms. However, histological and ISH data on healthy and moribund Penaeus monodon and P. esculentus experimentally infec...

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“…Haemolymph was collected using sterile 24 G needle and syringe, in sterile anticoagulant containing 450 mm NaCl, 10 mm KCl, 10 mm EDTA. Na 2 and 10 mm HEPES, pH 7.3 (Vargas-Albores, 1992; Vargas- Albores et al, 1993) and animals were aseptically dissected after anesthetising on ice. Tissues, mainly gills, 6 th abdominal muscle and lymphoid organs (LO) were aseptically collected separately and immediately processed for RNA extraction.…”

Section: Collection Of Tissue Samplesmentioning

confidence: 99%

Surveillance for infectious myonecrosis virus in Indian shrimp aquaculture

et al. 2017

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Disease surveillance programmes provide information on diseases that mitigate aquaculture production. Infectious myonecrosis virus (IMNV) has emerged recently as one of the OIE (Office International des Epizooties, the World Organisation for Animal Health) listed viral diseases in penaeid shrimps, caused by dsRNA virus. The present study was conducted to investigate the prevalence of IMNV along the Indian coastal states. Samples of the Pacific whiteleg shrimp Penaeus vannamei and the green tiger shrimp Penaeus monodon were collected from 21 randomly selected active shrimp farms located along the coast using a risk-based two stage random sampling survey. Screening of the sampled shrimps based on nested RT-PCR diagnostic tool, did not detect IMNV in any of the samples examined indicating absence of IMN or IMNV incidence in shrimp aquaculture along Indian coastal states.

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An anticoagulant solution for haemolymph collection and prophenoloxidase studies of penaeid shrimp (Penaeus californiensis)

Cited by 223 publications

References 25 publications

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

Proteasome properties of hemocytes differ between the whiteleg shrimp Penaeus vannamei and the brown shrimp Crangon crangon (Crustacea, Decapoda)

Quantitative real-time RT-PCR demonstrates that handling stress can lead to rapid increases of gill-associated virus (GAV) infection levels in Penaeus monodon

Surveillance for infectious myonecrosis virus in Indian shrimp aquaculture

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