In situ microplastic ingestion by neritic zooplankton of the central Mexican Pacific.

Microplastics (MPs) are present across the global ocean and can be encountered by many species, including zooplankton. Although they fall within the size range of zooplankton prey, there are few studies on MPs ingestion carried out in situ. In this study, we analyzed MPs ingestion during two seasons (rainy and dry) of organisms from 5 taxonomic groups of zooplankton from two bays of the Mexican central Pacific: Manzanillo and Navidad. In total, 2643 individuals were analyzed, and of those 23 individuals contained MPs. The ingestion rate by taxonomic group was 1 MP/36 copepods (0.02), 1 MP/30 decapods-mysis (0.03), 1 MP/29 decapods-megalopa (0.03), and 1 MP/200 fish larvae (0.005). No plastics were found in chaetognaths, amphipods, or decapods-zoea. The average length of the ingested particles was 468.1 ± 113.8 µm, with a minimum of 15.6 and a maximum of 647.6 µm. All MPs >300 µm were fibers, with diameters <50 µm. Fragments were the most abundant MPs (54.2%), followed by fibers (34.2%) and spheres (11.4%). Statistical analyses showed no significant differences (p > 0.05) between the bays or seasons. Using RAMAN spectroscopy, it was possible to identify 6 different types of polymers, with poly (ethylene:propylene) being the most abundant (42.8%). This polymer is commonly used to manufacture plastic bags, ropes and fishing nets. The results confirm that certain zooplankton groups are consuming MPs and suggest that omnivorous species are more likely to ingest MPs, possibly due to their capacity for foraging flexibility and opportunistic feeding strategies. However, the ingestion of MPs cannot be attributed to a single factor; it is necessary to consider variables such as the sampling area, feeding strategy, size, and seasonality to understand the dynamics of MPs ingestion by zooplankton groups.

Immune Response to Natural and Experimental Infection of Panulirus argus Virus 1 (PaV1) in Juveniles of Caribbean Spiny Lobster.

Experimental infections have been used to better comprehend the immune system of organisms, and to probe for additives that generate greater resistance and help reduce antibiotic use in aquaculture. We compared the immune response of juveniles of the Caribbean spiny lobster, Panulirus argus, infected naturally with Panulirus argus virus 1 (PaV1) versus organisms infected experimentally, to determine the analogy between both infectious processes. The immunological response was measured by hemagglutination activity, hemocyte count, and total phenoloxidase activity in plasma and hemocytes in 211 individuals that were either naturally infected (110), or had been injected with viral inoculum and followed for six months (101). The samples were classified into the following four groups according to the severity of the infection: 0, uninfected; 1, lightly; 2, moderately; and 3, severely infected), which was determined on the basis of PCR and histological criteria. A permutational MANOVA showed that both the origin (natural and experimental), and the severity of the infection contributed significantly to explain the variation in the immune response of lobsters. The lack of significance of the interaction term indicated that the immunological response changed with the severity of the infection in a similar way, regardless of its origin. The results of the present study suggest that the experimental viral infection of PaV1 produces a defense response similar to the natural pathways of contagion, and provides the bases to validate an immunological challenge protocol for the first time in crustaceans. The discussion includes the perspective of the conceptual models of immune response within an ecological context.

A first glimpse into the transcriptomic changes induced by the PaV1 infection in the gut of Caribbean spiny lobsters, Panulirus argus (Latreille, 1804) (Decapoda: Achelata: Palinuridae).

The Caribbean spiny lobster, Panulirus argus (Latreille, 1804) supports important fisheries in the Caribbean region. This species is affected by a deadly virus, Panulirus argus Virus 1 (PaV1), the only known pathogenic virus for this species. As infection progresses, the effects of PaV1 on its host become systemic, with far reaching impacts on the host's physiology, including structural injuries to its gastrointestinal organs, such as the hepatopancreas and the gut. This last one becomes highly compromised in the last stages of infection. Since the gut is a key organ for the physiological stability of lobsters, we compared the transcriptomic changes in the gut of juvenile individuals of Panulirus argus naturally infected with PaV1. In the RNA-Seq analysis, we obtained a total of 485 × 106 raw reads. After cleaning, reads were de novo assembled into 68,842 transcripts and 50,257 unigenes. The length of unigenes ranged from 201 bp to 28,717 bp, with a N50 length of 2079, and a GC content of 40.61%. In the differential gene expression analysis, we identified a total of 3405 non redundant differential transcripts, of which 1920 were up-regulated and 1485 were down-regulated. We found alterations in transcripts encoding for proteins involved in transcriptional regulation, splicing, postraductional regulation, protein signaling, transmembrane transport, cytoskeletal regulation, and proteolysis, among others. This is the first insight into the transcriptomic regulation of PaV1-P. argus interaction. The information generated can help to unravel the molecular mechanisms that may intervene in the gut during PaV1 infection.

Alterations in the gut-associated microbiota of juvenile Caribbean spiny lobsters Panulirus argus (Latreille, 1804) infected with PaV1.

The spiny lobster Panulirus argus (Latreille, 1804) is currently affected by an unenveloped, icosahedral, DNA virus termed Panulirus argus virus 1 (PaV1), a virulent and pathogenic virus that produces a long-lasting infection that alters the physiology and behaviour of heavily infected lobsters. Gut-associated microbiota is crucial for lobster homeostasis and well-being, but pathogens could change microbiota composition affecting its function. In PaV1 infection, the changes of gut-associated microbiota are yet to be elucidated. In the present study, we used high-throughput 16S rRNA sequencing technology to compare the bacterial microbiota in intestines of healthy and heavily PaV1-infected male and female juveniles of spiny lobsters P. argus captured in Puerto Morelos Reef lagoon, Quintana Roo, Mexico. We found that basal gut-associated microbiota composition showed a sex-dependent bias, with females being enriched in amplicon sequence variants (ASVs) assigned to Sphingomonas, while males were enriched in the genus Candidatus Hepatoplasma and Aliiroseovarius genera. Moreover, the alpha diversity of microbiota decreased in PaV1-infected lobsters. A significant increase of the genus Candidatus Bacilloplasma was observed in infected lobsters, as well as a significant decrease in Nesterenkonia, Caldalkalibacillus, Pseudomonas, Cetobacterium and Phyllobacterium. We also observed an alteration in the abundances of Vibrio species. Results from this study suggest that PaV1 infection impacts intestinal microbiota composition in Panulirus argus in a sex-dependent manner.

The influence of temperature and salinity on mortality of recently recruited blue crabs, Callinectes sapidus, naturally infected with Hematodinium perezi (Dinoflagellata).

The parasitic dinoflagellate Hematodinium perezi is highly prevalent in juvenile blue crabs, Callinectes sapidus, along the eastern seaboard of the USA. Although the parasite is known to kill adult crabs, the mortality rate of naturally infected juvenile crabs remains unknown. We analyzed the influence of temperature and salinity on the mortality of recently recruited blue crabs that were naturally infected with H. perezi. Over 492 juvenile crabs (infected, n = 282; uninfected controls, n = 210) were held individually in six-well plates and held at six temperatures (4, 10, 15, 20, 25, and 30 °C) or three salinities (5, 15, and 30 psu) for a maximum of 90 days. Mortality of infected crabs was 10 times higher at elevated temperatures (25 and 30 °C) and salinity (30 psu) compared to uninfected control treatments. By contrast, infected crabs exposed to mild temperatures (10, 15, and 20 °C) showed a high survival (>80%), no different than uninfected control treatments. Infected crabs at the lowest temperature (4 °C) exhibited a high mortality, but the intensity of infection was lower than in the other temperature treatments. In addition, this study revealed the optimal temperature (25 °C) and salinity (30 psu) for H. perezi to progress in its life cycle leading to sporulation in juvenile crabs; 31.6% (19/60) of crabs held under these conditions released dinospores of H. perezi after 10 days. Crabs held at other temperatures did not release dinospores over the time course of the experiment. Infected crabs were capable of molting and in most cases molted at the same frequency as uninfected crabs serving as controls. The mortality observed in this study indicates that early benthic juveniles will experience significant mortality due to H. perezi with increasing ocean temperatures and that this mortality may be a significant factor in the recruitment of blue crabs to high salinity regions.

Panulirus argus virus 1 detected in oceanic postlarvae of Caribbean spiny lobster: implications for disease dispersal.

Panulirus argus virus 1 (PaV1), a pathogenic virus that specifically attacks Caribbean spiny lobsters Panulirus argus, was recently detected in newly settled postlarvae of P. argus. As PaV1 appears not to be vertically transmitted, infected postlarvae likely acquire PaV1 from the water, but whether this can occur in oceanic waters where the planktonic larvae (phyllosomata) metamorphose into nektonic postlarvae remains unknown. Late-stage phyllosomata and postlarvae of P. argus were collected at distances of 2 to 100 km from the Caribbean coast of Mexico in 2 oceanographic cruises. Most postlarvae were caught in the upper meter of water, usually along with masses of floating Sargassum algae. A PaV1-PCR assay was used to test 169 phyllosomata (stages VI-X) and 239 postlarvae. All phyllosomata tested negative, but 2 postlarvae, 1 from each cruise, tested positive for PaV1. These postlarvae were collected at 55 and 48 km offshore over depths of 850 and 1800 m, respectively, suggesting that postlarvae can acquire PaV1 in offshore waters. We hypothesize that floating Sargassum may be an environmental reservoir for PaV1. The PaV1 allele (460 pb) found in an infected postlarva was more closely related to PaV1 alleles found in lobsters from Puerto Rico than in lobsters from any other location (including Mexico), suggesting high gene flow and long-distance dispersal of PaV1, consistent with previous studies of high genetic connectivity across the Caribbean.

Panulirus argus virus 1 (PaV1) infection prevalence and risk factors in a Mexican lobster fishery employing casitas.

In Bahía de la Ascensión in Mexico, the Caribbean spiny lobster Panulirus argus fishery is based on extensive use of artificial shelters (casitas) that can harbor both juveniles and adults of this highly gregarious species. There is concern that the use of casitas might increase contact transmission of Panulirus argus virus 1 (PaV1). However, a previous study found no evidence for lobster crowding within casitas influencing the prevalence of clinical PaV1 disease, although differences in clinical prevalence were noted between different bay environments. To investigate this more closely, 683 lobsters were sampled from casitas in 2 zones in this bay (Vigía Chico, a shallow low-vegetation zone, and Punta Allen, a deeper dense-vegetation zone) previously found to have the lowest and highest prevalence, respectively, of observed clinical signs. When hemolymph collected from these lobsters was tested by PCR, the prevalence of PaV1 infection was found to be significantly lower in Vigía Chico relative to Punta Allen irrespective of season or the size, sex, or presence of shell injuries on lobsters. Among 714 large commercial-catch lobsters collected throughout the bay, the prevalence of infection was low irrespective of year or sex. For all lobsters tested, the sensitivity (0.510) at which PaV1 infection was detected by observed clinical signs was about half that determined by PCR, but the specificity of clinical signs was absolute (1), indicating that a simple 2× correction factor can be used to accurately estimate PaV1 infection prevalence based on more easily conducted visual assessments of lobsters.

Physiological and immunological characterization of Caribbean spiny lobsters Panulirus argus naturally infected with Panulirus argus Virus 1 (PaV1).

The present study compares 13 physiological and immunological variables between a group of healthy Panulirus argus lobsters and a group of lobsters naturally infected with Panulirus argus Virus 1 (PaV1). Viral infection was determined through histopathology and PCR. Ten of the 13 variables differed significantly between the 2 groups. Using these variables, a principal component analysis yielded 2 separate clusters: one corresponding to the healthy group and the other corresponding to the infected group. In particular, infected lobsters exhibited significantly lower levels of osmotic pressure, total hemocyte counts, plasmatic proteins, and total phenoloxidase (PO) activity in plasma, as well as significantly higher levels of cholesterol and acylglycerides. These features are consistent with metabolic wasting, hyperlipidemia, and presumed immune suppression. Infection with PaV1 appears to increase the susceptibility of lobsters to some other opportunistic pathogens, as 61.1% of infected lobsters presented infestations of ciliate epibionts (Epystilis and Zoothamniun) in the gill chamber compared with 11.5% lobsters in the healthy group. Infected lobsters also showed significantly higher levels of total PO activity in degranulated hemocytes and trypsin inhibitor activity, potentially indicating activation of immune response by the PO system during the systemic infection with PaV1.

Detection of Panulirus argus Virus 1 (PaV1) in exported frozen tails of subadult-adult Caribbean spiny lobsters Panulirus argus.

The Caribbean spiny lobster Panulirus argus is a valuable fishing resource and the trade in frozen lobster tails is an important industry. However, the presence of the pathogenic virus Panulirus argus Virus 1 (PaV1), which causes systemic infection in P. argus and is particularly lethal to juvenile individuals, has not been previously examined in imported/exported lobster products. We used PCR assays to determine the presence of PaV1 in abdominal muscle tissue of 22 frozen P. argus tails exported from Belize to Mexico. Based on their size, the tails belonged to subadult-adult lobsters. Using specific primers targeted for PaV1 resulted in 11 tails showing a specific 499 bp band. The sequence of positive amplified fragments showed a high similarity to PaV1 (95% identity with GenBank accession no. EF206313.1). Although the pathogenicity of PaV1 was not evaluated in the present study, our results provide the first evidence of PaV1 in frozen lobster tails exported in the seafood industry as well as the first molecular evidence of PaV1 in adult lobsters.

Prevalence of Panulirus argus Virus 1 (PaV1) and habitation patterns of healthy and diseased Caribbean spiny lobsters in shelter-limited habitats.

Caribbean spiny lobsters Panulirus argus are socially gregarious, preferring shelters harboring conspecifics over empty shelters. In laboratory trials, however, healthy lobsters strongly avoided shelters harboring lobsters infected with the highly pathogenic Panulirus argus Virus 1 (PaV1). Because PaV1 is transmitted by contact, this behavior may thwart its spread in wild lobsters. In a field experiment conducted from 1998 to 2002 in a shelter-poor reef lagoon (Puerto Morelos, Mexico), densities of juvenile P. argus increased significantly on sites enhanced with artificial shelters (casitas) but not on control sites. Because PaV1 emerged in this location during 2000, we reexamined these data to assess whether casitas could potentially increase transmission of PaV1. In 2001, PaV1 prevalence was 2.5% and the cohabitation level (percentage of healthy lobsters cohabiting with diseased lobsters) was similar between natural shelters (3.5%) and casitas (2.4 %). The relative lobster densities in casita sites and control sites did not change significantly before (1998-1999) or after (2001-2002) the disease emergence. In late 2006, data from casita sites showed a significant increase in prevalence (10.9%) and cohabitation level (29.4%), but no significant changes in lobster density. In May 2006, casitas were deployed on shelter-poor sites within Chinchorro Bank, 260 km south of Puerto Morelos. In late 2006, prevalence and cohabitation level were 7.4 and 21.7%, respectively. Our results are inconclusive as to whether or not casitas increase PaV1 transmission, but suggest that across shelter-poor habitats, lobsters make a trade-off between avoiding diseased conspecifics and avoiding predation risk.

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

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.