Presence of Panulirus argus Virus 1 (PaV1) in juvenile spiny lobsters Panulirus argus from the Caribbean coast of Mexico

Abstract

Macroscopic evidence, histological sections, transmission electron microscopy (TEM) evaluation, and PCR analyses of 25 apparently diseased juvenile spiny lobsters Panulirus argus from the reef lagoon of Puerto Morelos, Mexico, showed the presence of Panulirus argus Virus 1 (PaV1). Cowdry Type A intranuclear viral inclusions were observed in histological analyses, icosahedral viral particles were observed by TEM, and PCR using specific primers for PaV1 amplified a fragment of 499 bp. This is the first report of PaV1 infecting P. argus outside the Florida Keys, USA. KEY WORDS: Spiny lobster · Panulirus argus · PaV1 · Cowdry Type A intranuclear inclusionsResale or republication not permitted without written consent of the publisher Dis Aquat Org 79: 153-156, 2008 water) and processed by a routine histological method followed by hematoxylin and eosin (H&E) stains (Lightner 1996). Stained sections were observed under a standard light microscope to examine histopathological changes and the images were captured using a digital camera (Evolution™ LC Color). The severity of infection of each tissue type was estimated using the numerical scale of Bell & Lightner (1987), who assigned infection severity grades from 0 to 4 based on the estimated number of diagnostic Cowdry Type A intranuclear inclusions (CAI) per microscopic field(s) at 40 X: Grade 0 = no CAI observed; Grade 1 = 1-5 CAI/200 fields; Grade 2 = 1-2 CAI/20 fields; Grade 3 = 1-5 CAI/2 fields); and Grade 4 >10 CAI/field.For the TEM examinations, the hepatopancreas of 8 lobsters were first fixed in 2.5% glutaraldehyde with 0.2 M sodium cacodylate buffer, then in 1% osmium tetraoxide and further dehydrated in increased serial dilutions of ethanol. Finally, samples were mounted in Spurr's resin. Ultra-thin sectioned samples placed on grids were processed through a routine lead citrate stain and observed with a Jeol microscope (JEM-1200 EX II) (Reynolds 1963).Fifteen lobsters were used for the PCR analysis. Genomic DNA were extracted from ~25 mg of hepatopancreatic tissue using the wizard ® genomic DNA purification kit (Promega ® ) according to the manufacturer's protocol. The primers specific for PaV1 45aF-TCCAGCCCAGGTACGTATC and 543aR-AACAGA-TT-TTCCAGCAGCGT that amplify a region of 499 bp were used after a modification of the original protocol of Montgomery-Fullerton et al. (2007). All PCR reactions were carried out in a total volume of 25 µl containing ~32.5 ng of DNA, 0.33 µM of each primer, 2.5 mM of MgCl 2 , 1.2× reaction buffer (50 mM KCl, 10 mM Tris-HCl, pH 9.0, 0.1% Triton X-100), 0.4 mM dNTPs mixture (Promega ® ), and 2.5 U of Taq DNA polymerase (Promega ® ). Reactions were run on a thermal cycler (TECHNE TC-312) at 94°C for 10 min, followed by 30 cycles of 94°C for 30 s, 63°C for 30 s, and 72°C for 1 min, with a final extension of 72°C for 10 min. PCR products were electrophoresed on 2% agarose gels and bands were visualized using 0.1% ethidium bromide stain on a UV transilluminator. DNA from lobsters collected in the Reef lagoon of Puerto More...

Review of Panulirus argus virus 1—a decade after its discovery

“…PaV1 prevalence in nature is independent of lobster density over the small spatial scales in which lobsters interact (i.e. 10s of meters) (Behringer 2003, LozanoÁlvarez et al 2008. However, the size and dimensions of a shelter may affect the frequency of shelter cohabitation as healthy lobsters co-occur more frequently with diseased lobsters in large casitas (21.7 to 29.4%) than in smaller natural shelters (3.5%) (LozanoÁlvarez et al 2008).…”

“…10s of meters) (Behringer 2003, LozanoÁlvarez et al 2008. However, the size and dimensions of a shelter may affect the frequency of shelter cohabitation as healthy lobsters co-occur more frequently with diseased lobsters in large casitas (21.7 to 29.4%) than in smaller natural shelters (3.5%) (LozanoÁlvarez et al 2008). Computer simulations using a spatially explicit, individual-based lobster re cruitment model (Butler 2003, Butler et al 2005, Dolan & Butler 2006) altered for modeling benthic disease dynamics in the Florida Keys have also indicated that the avoidance of infected lobsters by healthy lobsters is effective in dampening the prevalence of PaV1 in the population modeled (Dolan 2010).…”

Microsporidiosis in the Caribbean spiny lobster Panulirus argus from southeast Florida, USA

“…Although these 2 reports may have just been coincidental, considering that they came shortly after the discovery of P. argus Virus 1 (PaV1), the first naturally occurring viral pathogen found infecting any lobster (Shields & Behringer 2004), further investigation was warranted. PaV1 has been well described in the Florida Keys (Behringer et al 2006 and along the Caribbean coast of Mexico (Huchin-Mian et al 2008). Given the importance of P. argus to recreational and commercial fisheries and the increased awareness of disease generated by reports of PaV1, we anticipate that fishers will more likely report incidences of microsporidiosis if it is indeed increasing in prevalence and emerging as a pathogen of concern.…”

Landscape Analysis for PaV1 Infection in Lobsters Panulirus argus from the Artisanal Fishery of the Eastern Coast of Yucatan, Mexico

“…However, the discovery of the pathogenic virus Panulirus argus Virus 1 (PaV1) [11], is becoming a major concern, because it could affect negatively P. argus fisheries; this double stranded DNA virus causes a progressive and detrimental infection that ends in death of juvenile lobsters. In experimental infections with P. argus juveniles, PaV1 infected lobsters show lethargy, morbidity, "milky" hemolymph, lack of hemolymph coagulation, and suppression of molt in a range of 30 to 80 days post infection [12]. In the field, PaV1 infected lobsters are recognized by local fishermen as "milky lobsters".…”

“…PaV1 is more prevalent in juveniles. This prevalence decreases in sub-adults and adults and clinical signs characteristic of PaV1 are difficult to evaluate in the field [12]. PaV1 is highly prevalent throughout the Caribbean region and its propagation could impact P. argus fisheries [2] [10] [12]- [15].…”