Virus discovery in cultured portunid crabs: Genomic, phylogenetic, histopathological and microscopic characterization of a reovirus and a new bunyavirus.

Portunid crabs are distributed worldwide and highly valued in aquaculture. Viral infections are the main limiting factor for the survival of these animals and, consequently, for the success of commercial-scale cultivation. However, there is still a lack of knowledge about the viruses that infect cultured portunid crabs worldwide. Herein, the genome sequence and phylogeny of Callinectes sapidus reovirus 2 (CsRV2) are described, and the discovery of a new bunyavirus in Callinectes danae cultured in southern Brazil is reported. The CsRV2 genome sequence consists of 12 dsRNA segments (20,909 nt) encode 13 proteins. The predicted RNA-dependent RNA polymerase (RdRp) shows a high level of similarity with that of Eriocheir sinensis reovirus 905, suggesting that CsRV2 belongs to the genus Cardoreovirus. The CsRV2 particles are icosahedral, measuring approximately 65 nm in diameter, and exhibit typical non-turreted reovirus morphology. High throughput sequencing data revealed the presence of an additional putative virus genome similar to bunyavirus, called Callinectes danae Portunibunyavirus 1 (CdPBV1). The CdPBV1 genome is tripartite, consisting of 6,654 nt, 3,120 nt and 1,656 nt single-stranded RNA segments that each encode a single protein. Each segment has a high identity with European shore crab virus 1, suggesting that CdPBV1 is a new representative of the family Cruliviridae. The putative spherical particles of CdPBV1 measure ∼120 nm in diameter and present a typical bunyavirus morphology. The results of the histopathological analysis suggest that these new viruses can affect the health and, consequently, the survival of C. danae in captivity. Therefore, the findings reported here should be used to improve prophylactic and pathogen control practices and contribute to the development and optimization of the production of soft-shell crabs on a commercial scale in Brazil.

High prevalence of CsRV2 in cultured Callinectes danae: Potential impacts on soft-shell crab production in Brazil.

Crabs can be infected by a variety of pathogenic micro-organisms but the most damaging are viruses. Naturally-occurring Callinectes sapidus reovirus 1 (CsRV1) is thought to contribute to mortality of Callinectes sapidus in soft crab culture in the USA. In Brazil, soft crabs are frequently produced using Callinectes danae, which suffers a similar rate of mortality in culture as C. sapidus. This study investigated whether CsRV1 could be detected in healthy or dead Callinectes danae from Paraná, Brazil and kept in captivity, we also evaluated the relationship between viral infection, and biochemical and behavioral parameters. C. danae from Paranaguá Bay were kept in a recirculation system for 14 days and subjected to weekly biochemical analyses and a reflex action mortality predictors (RAMP) test. RT-qPCR assays for CsRV1 were negative for all samples. However, electrophoretic analysis of extracted RNA from some crabs showed a pattern of 12 dsRNA bands that indicated intense infection by a reovirus with a genome organization different from CsRV1. The banding pattern was indistinguishable from a putative novel reovirus detected in C. sapidus in Rio Grande do Sul, Brazil, provisionally called CsRV2. The prevalence of dsRNA of CsRV2 showed no significant difference between crabs that died and survived. Interestingly, the presence of CsRV2 dsRNA was correlated with a significant reduction in glycogen concentration in hepatopancreas and a decrease in reflex action. The results obtained in this study are an early glimpse of the occurrence of reoviruses in C. danae and their potential effects in soft-shell crab systems in Brazil.

Contamination gradient affects differently carbonic anhydrase activity of mollusks depending on their feeding habits.

Aquatic organisms that inhabit coastal areas are often exposed to several contaminants. It is known that the bioaccumulation of contaminants can be amplified according to the species feeding habits and contaminant properties. As a consequence, species can experience different effects to contaminant exposure even if they inhabit the same area. The present study aimed to investigate the activities of carbonic anhydrase (CA), Ca2+-ATPase, and Mg2+-ATPase in different tissues (soft tissue, mantle, and gill) of three mollusk species (Lottia subrugosa, Stramonita brasiliensis, and Crassostrea brasiliana) with different feeding habits (herbivore, carnivore, and filter-feeder, respectively) which were sampled within a known contamination gradient at Santos Estuarine System (Southeastern Brazil). From the three enzymes tested, only CA was affected by the presence of contaminants within the contamination gradient evaluated. In general, the CA activity from the three species were lower in contaminated sites when compared to the reference site. The contrasting CA activity response observed in S. brasiliensis compared to L. subrugosa and C. brasiliana could be related to the tissue-specificity of this enzyme activity and species feeding habits (filter-feeders can accumulate more contaminants than herbivores and even carnivores). Results indicated that C. brasiliana mantle is the most suitable tissue for the use of CA analysis as a biomarker.

Evaluation of methods for reducing epibionts during farming of the mangrove oyster Crassostrea gasar (Adanson, 1757).

Due to the competition for food, space, oxygen and due to their role as diseases vector, epibionts can negatively affect oyster farming. We assessed the efficacy of six methods commonly used for the removal of epibionts from oyster shells during farming. The experiment was conducted at an oyster farm on the Paraná coast - South Brazil. Oysters (Crassostrea gasar) were acclimated for 90 d in the cultivation system and later exposed to cleaning treatments: i) freshwater; ii) hypersaline water; iii) sodium hypochlorite solution; iv) quaternary ammonia solution; v) exposure to air; vi) hydroblasting; and vii) no cleaning procedure (control). After treatment, oysters were kept in the cultivation system for 15 and 30 d - when the total incrustation and mortality were measured. Epibionts from nine phyla were identified. The most abundant were Arthropoda (Crustacea) (62.5%), Mollusca (33.8%) and Annelida (3.1%). Freshwater [15 (n = 2263 epibionts) and 30 days (n = 2822 epibionts)] and hydroblasting [15 (n = 1850 epibionts) and 30 days (n = 2389 epibionts)] treatments were the most efficient to reduce epibionts and caused lower rates of oyster mortality [15 (5.0 and 3.33%, respectively) and 30 days (1.67 and 6.67%, respectively)].

Evaluation of different induced molting methods in Callinectes ornatus (Crustacea, Decapoda, Portunidae) as a tool for the commercial production of soft-shell crabs.

We investigated the effects of eyestalk ablation and cheliped autotomy in inducing molting in Callinectes ornatus. The specimens in intermolt stage were divided into two size classes: 1 (30-50 mm) and 2 (51-70 mm) and were further divided into four experimental groups. In the CA group (Cheliped Autotomy, n=76), crabs were submitted to cheliped autotomy; in the UA group (Unilateral Ablation, n=66) and BA group (Bilateral Ablation, n=66) to unilateral and bilateral eyestalk ablation, respectively. The C group (Control, n=70) was used as control. The animals were individually kept in tanks interconnected to a recirculation system for 30 days. The highest frequency of premolt was recorded in the BA group (32%), followed by the CA group (16%). The premolt frequency of class 1 (28%) was significantly higher (p<0.05) than of class 2 (8%). The mean time until molt in the BA group was significantly lower (p <0.05) than other groups tested. The highest mortality rates were 55% and 25% in crabs from the BA and CA groups, respectively. The results indicate bilateral eyestalk ablation and cheliped autotomy are potentially capable of inducing molt in C. ornatus, but these techniques have limited efficiency for the commercial application of large-scale soft-shell crab.

Metagenomic analysis of the bacterial microbiota associated with cultured oysters (Crassostrea sp.) in estuarine environments.

In this work, we identified the bacterial microbiota associated with farmed oystersin estuarine regions of four states in the north eastern region of Brazil. During the drought and rainy seasons, for eight months, twenty oysters were sampled seasonally from seven different marine farms. In the laboratory, DNA extraction, amplification, and sequencing of the 16S rRNA gene were performed to establish the taxonomic units. We identified 106 genera of bacteria belonging to 103 families, 70 orders, 39 classes, and 21 phyla. Out of the total, 40 of the genera represented bacteria potentially pathogenic to humans; of these, nine are known to cause foodborne diseases and six are potentially pathogenic to oysters. The most prevalent genera were Mycoplasma, Propionigenium, Psychrilyobacter, and Arcobacter. The results indicate the need for more systematic monitoring of bacteria of the genus Mycoplasma in oyster farming operations in the Brazilian north eastern region. Currently, Mycoplasma is not one of the microorganisms analysed and monitored by order of Brazilian legislation during the oyster production and/or commercialization process, even though this genus was the most prevalent at all sampling points and presents pathogenic potential both for oysters and for consumers.

Development of a real-time PCR assay for the detection of the golden mussel ( Limnoperna fortunei , Mytilidae) in environmental samples.

The golden mussel, Limnoperna fortunei, is among the most devastating invasive species in freshwater habitats worldwide, leading to severe environmental disturbances and economic losses. Therefore, management efforts would be greatly improved by methods that efficiently detect and quantify the abundance of the golden mussel in freshwater habitats, particularly in early stages of colonization. In this study, we describe a highly-sensitive real-time PCR assay targeting a 100-bp region of the COI mitochondrial gene of the golden mussel. The method was able to detect as little as 0.225 pg of target DNA. This assay represents an important contribution to surveillance methods, as well as to optimize field measures to contain and manage populations of the golden mussel in its introduced range.

Is Marine Dispersion of the Lethargic Crab Disease Possible? Assessing the Tolerance of Exophiala cancerae to a Broad Combination of Salinities, Temperatures, and Exposure Times.

Since 1997, an emergent fungal disease named lethargic crab disease (LCD) has decimated stocks of the edible mangrove land crab Ucides cordatus (Linnaeus, 1763) (Brachyura: Ocypodidae) along the Brazilian coast, threatening the mangrove ecosystem and causing socioeconomic impacts. Evidence from a variety of sources suggests that the black yeast Exophiala cancerae (Herpotrichiellaceae, Chaetothyriales) has been responsible for such epizootic events. Based on the spatiotemporal patterns of the LCD outbreaks, the well-established surface ocean currents, and the range of ecological traits of Exophiala spp., a marine dispersal hypothesis may be proposed. Using in vitro experiments, we tested the survival and growth of E. cancerae CBS 120420 in a broad combination of salinities, temperatures, and exposure times. While variation in salinity did not significantly affect the growth of colony-forming units (CFUs) (P > 0.05), long exposure times visibly influenced an increase in CFUs growth (P < 0.05). However, higher temperature (30 °C) caused a reduction of about 1.2-fold in CFUs growth (P < 0.05). This result suggests that sea surface temperatures either above or below the optimum growth range of E. cancerae could play a key role in the apparent north-south limits in the geographical distribution of LCD outbreaks. In light of our results, we conclude that a fundamental step toward the understanding of LCD epidemiological dynamics should comprise a systematic screening of E. cancerae in estuarine and coastal waters.

Black yeast biota in the mangrove, in search of the origin of the lethargic crab disease (LCD).

Knowledge of natural ecology is essential for a better understanding of pathogenicity and opportunism in black yeast-like fungi. Although etiological agents of diseases caused by these fungi are supposed to originate from the environment, their isolation from nature is difficult. This is probably due to their oligotrophic nature, low competitive ability, and, overall, insufficient data on their natural habitat. We obtained environmental samples from mangrove areas where mortalities by lethargic crab disease (LCD) are reported and areas without disease recorded. Isolation of chaetothyrialean black yeasts and relatives was performed using a highly selective protocol. Species-specific primers were used to determine if these isolates represented Exophiala cancerae or Fonsecaea brasiliensis, two proven agents of LCD, in order to test hypotheses about the origin of the disease. Isolates, identified by morphology as Fonsecaea- or Exophiala-like, were tested specific diagnostic markers for the fungi associated with LCD. Although several black fungi were isolated, the main causative agent of the LCD, E. cancerae, was not found. Molecular markers for F. brasiliensis revealed 10 positive bands for isolates from biofilms on mangrove leaves, branches, and aerial roots, of which four were confirmed by ITS sequencing. The absence of E. cancerae in environmental samples suggests that the species is dependent on the crab, as a genuine pathogen, different from F. brasiliensis, which is probably not dependent on the host species, U. cordatus. However, we did not attempt isolation from the marine water, which may represent the pathway of dispersion of the black yeast species between neighbor mangroves.

Non-Invasive Methods for Assessing the Welfare of Farmed White-Leg Shrimp (Penaeus vannamei).

Gradually, concern for the welfare of aquatic invertebrates produced on a commercial/industrial scale is crossing the boundaries of science and becoming a demand of other societal actors. The objective of this paper is to propose protocols for assessing the Penaeus vannamei welfare during the stages of reproduction, larval rearing, transport, and growing-out in earthen ponds and to discuss, based on a literature review, the processes and perspectives associated with the development and application of on-farm shrimp welfare protocols. Protocols were developed based on four of the five domains of animal welfare: nutrition, environment, health, and behaviour. The indicators related to the psychology domain were not considered a separate category, and the other proposed indicators indirectly assessed this domain. For each indicator, the corresponding reference values were defined based on literature and field experience, apart from the three possible scores related to animal experience on a continuum from positive (score 1) to very negative (score 3). It is very likely that non-invasive methods for measuring the farmed shrimp welfare, such as those proposed here, will become a standard tool for farms and laboratories and that it will become increasingly challenging to produce shrimp without considering their welfare throughout the production cycle.

From egg to slaughter: monitoring the welfare of Nile tilapia, Oreochromis niloticus, throughout their entire life cycle in aquaculture.

The primary aim of this study was to comprehensively evaluate the welfare of Nile tilapia (Oreochromis niloticus) throughout their entire life cycle within aquaculture, spanning from reproduction to slaughter. The methodology was structured to identify welfare indicators closely aligned with the principles of animal freedoms defined by the Farm Animal Council, encompassing environmental, health, nutritional, behavioral, and psychological freedom. Notably, psychological freedom was inherently considered within the behavioral and physical analyses of the animals. To accomplish this, an integrative systematic literature review was conducted to define precise indicators and their corresponding reference values for each stage of tilapia cultivation. These reference values were subsequently categorized using a scoring system that assessed the deviation of each indicator from established ideal (score 1), tolerable (score 2), and critical (score 3) ranges for the welfare of the target species. Subsequently, a laboratory experiment was executed to validate the pre-selected health indicators, specifically tailored for the early life stages of tilapia. This test facilitated an assessment of the applicability of these indicators under operational conditions. Building on the insights gained from this experimentation, partial welfare indices (PWIs) were computed for each assessed freedom, culminating in the derivation of a general welfare index (GWI). Mathematical equations were employed to calculate these indices, offering a quantitative and standardized measure of welfare. This approach equips tilapia farmers and processors with the tools necessary for the continuous monitoring and enhancement of their production systems and stimulate the adoption of more sustainable and ethical practices within the tilapia farming.

Osmoregulatory responses in the neotropical fish species Astyanax lacustris, exposed to single and combined microplastics, polycyclic aromatic hydrocarbons, and their mixture.

Microplastic (MP) pollution poses a significant environmental threat. These MPs can adsorb toxic compounds such as polycyclic aromatic hydrocarbons (PAH), which are highly lipophilic and carcinogenic. To assess the potential effects of virgin MP, PAH, and MP+PAH in association with osmoregulation and energetic substrate, we conducted experiments with the tetra cardinal Astyanax lacustris. The environmentally relevant concentration of MP (10 mg L-1) and 20 % of the LC50-96 h of crude oil for A. lacustris (2.28 µg L-1) were used during the 96-h exposure. Fish were exposed to virgin MP, PAH, MPC (MP loaded with PAH), PAH+MP (PAH and MP in association), and the control without (CT) and with handling (CH). After 96 h, blood was collected for osmoregulatory parameters (plasma osmolality; Na+, K+, Cl-, Mg2+; glycose and lactate); gills for osmoregulatory enzyme activities (Na+, K+ ATPase, H+ ATPase, and carbonic anhydrase); and white muscle samples were used to determine glycogen as an energetic substrate. The low molecular weight PAH was not detected in PAH-loaded MP (MPC) and PAH in combination with MP (PAH+MP). The PAH concentration of the MPC and PAH+MP was similar and low compared to other works. Virgin MP, PAH, MPC, and PAH+MP were able to cause muscle glycogen depletion. The activity of v-type H+ ATPase and plasma Na+ concentrations were lower in PAH with MP (MPC). However, the hydromineral balance (K+, Mg2+, Cl-, and osmolality) was not affected by any treatment. In this sense, we can conclude that the MPC caused osmoregulatory disturbances not seen in the MP associated with PAH (MP+PAH). However, this seems unrelated to the PAH leaking from the MPC or the PAH absorption to the virgin MP once the PAH concentrations from the MPC and PAH+MP were similar.

Response of Oreochromis niloticus (Teleostei: Cichlidae) exposed to a guanitoxin-producing cyanobacterial strain using multiple biomarkers.

Changes in environmental conditions in aquatic ecosystems caused by anthropic actions can modify the composition of primary producers, promoting the excessive proliferation of cyanobacteria. These organisms can form cyanobacterial blooms, which directly affect aquatic life. The present study investigated the mutagenicity of the cyanobacterium Sphaerospermopsis torques-reginae (strain ITEP-024), guanitoxin-producing (natural organophosphate), and sublethal effects on fish in relevant environment concentrations. For this, the Ames test (Salmonella/microsome) was performed as a mutagenic assay for extracts of the ITEP-024 strain. Specimens of Oreochromis niloticus (Teleostei: Cichlidae) were subjected to acute 96 h exposure to different concentrations of aqueous extract of the strain: C = control group; T1 = 31.25 mg/L; T2 = 62.5 mg/L; T3 = 125 mg/L; and T4 = 250 mg/L. Genotoxic, biochemical, osmoregulatory, and physiologic biomarkers were analyzed. Our results showed that the cyanobacterium had a weak mutagenic response for the TA102 strain of Salmonella with and without metabolic activation by S9. Strains TA98 and TA100 were not affected. Fish from treatments T3 and T4 showed changes in oxidative stress (CAT, SOD, and GST enzymes), inhibition of the enzyme acetylcholinesterase activity, micronucleus formation, and osmoregulatory disorders. No guanitoxin accumulation was detected in the different tissues of O. niloticus by LC-MS/MS. Our results showed unprecedented mutagenicity data of the guanitoxin-producing cyanobacteria by the Ames test and biochemical, osmoregulatory, and genotoxic disorders in fish, providing efficient aquatic contamination biomarkers. Despite the great concern related to the presence of guanitoxin in blooms in freshwater ecosystems, its concentration is not yet regulated, and thus there is no monitoring agenda in current legislation.

Postglacial north-south expansion of populations of Rhizophora mangle (Rhizophoraceae) along the Brazilian coast revealed by microsatellite analysis.

PREMISE OF THE STUDY: Red mangrove (Rhizophora mangle) dominates tropical tidal areas along both sides of the Atlantic, yet little is known about its degree of population differentiation over large geographical scales. Information on the genetic variability of mangrove species along the Brazilian coast is important not only for understanding the recent gene flow dynamic between populations, but also to evaluate models of evolutionary diversification and develop effective strategies for conservation. We investigated the genetic variability of the red mangrove along the Brazilian coast. METHODS: Eight microsatellite loci were used to genotype 145 individuals across 10 populations spanning more than 4500 km of coast line. We estimated the genetic variability and structure of the populations and the historical gene flow between them. KEY RESULTS: The level of genetic variability was low, with only 27 different alleles being detected and allele richness between 1.25 and 2.75. On the other hand, there was substantial population differentiation (R(st) = 0.48; P < 0.001), especially between the northern and southern populations. The populations from Pará and Maranhão had significantly greater genetic variability than did the remaining locations. CONCLUSIONS: This difference might reflect the older age of the northern mangroves, which likely remained stable during the Quaternary glaciations. The lowest variability observed in the southern populations of the red mangrove most likely reflects their recent age, associated with allelic reduction, resulting from the consecutive founder events that followed subsequent colonization of estuaries during the gradual warming by the end of the last glacial period.

Fulfilling Koch's postulates confirms the mycotic origin of Lethargic Crab Disease.

In the northeast region of the Brazilian coast, a disease has been causing massive mortalities of populations of the mangrove land crab, Ucides cordatus (L.) since 1997. The clinical signs of this disease, which include lethargy and ataxia, led to the disease being termed Lethargic Crab Disease (LCD). Evidence from a variety of sources indicates that there is an association between LCD and a new species of black yeast, Exophiala cancerae de Hoog, Vicente, Najafzadeh, Badali, Seyedmousavi & Boeger. This study tests this putative correlation through in vivo experiments. Disease-free specimens of U. cordatus were experimentally infected with Exophiala cancerae (strain CBS 120420) isolate. During the 30-day experimental period, only a single death was observed within the control crabs. However, at the end of this period, crabs that were inoculated once or three-times with mycelial elements and hyphae of E. cancerae had a 60% and 50% mortality rates, respectively (n = 6 and n = 5). These results support that the fungal agent is pathogenic and is the causative agent of LCD. Species-specific molecular markers confirm the presence of E. cancerae (strain CBS 120420) in recovered colonies and tissue samples from the infected animals. The experimentally infected crabs manifested signs (lethargy, ataxia and tetany) that were consistent to LCD-affected animals in the environment. These results fulfil Koch's postulates and the hypothesis that the tested strain of Exophiala cancerae is a causative agent of LCD is accepted.

Specific primers for the detection of the black-yeast fungus associated with lethargic crab disease (LCD).

Lethargic crab disease (LCD) is an emerging infirmity that has been causing extensive mortalities in populations of the mangrove land crab Ucides cordatus (Ocypodidae) along the Atlantic coast of Brazil. Previous studies have indicated that LCD is associated with a dematiaceous fungus, Exophiala cancerae de Hoog et al. In the present study, we sequenced the internal transcribed spacer (ITS) of the rDNA region of this black yeast species and developed species-specific PCR primers. Sensitivity tests indicated that the developed protocol is capable of detecting very small amounts of target DNA. Also, the application of the protocol to a variety of other dematiaceous fungi did not generate any false positives. The specific primers provided in the present study represent an important tool for rapidly surveying a large number of crab individuals, as well as environmental samples. Such knowledge will be instrumental in understanding the epidemiological dynamics of LCD.

Shell form and enzymatic alterations in Lottia subrugosa (Gastropoda, Lotiidae) transplanted to a contaminated site.

Studies have shown that shell morphology and enzymatic activities in mollusks are affected by contaminants exposure. However, the correlation between enzymatic activities and the biomineralization process are not fully understood. The present study used a transplant bioassay and field sampling to evaluate shell measurements and the activities of carbonic anhydrase, Ca2+-ATPase, and Mg2+-ATPase in Lottia subrugosa sampled in Brazilian sites under different contamination levels. Results showed that, in general, shells from the reference site (Palmas) were more rounded than the ones from the contaminated site (Balsa). Effects in enzymatic activities in specimens from transplant bioassay were attributed to the known high contaminant levels present at Balsa. While the lack of enzymatic activity alterations during field sampling was attributed to physiological adaptation to contaminants exposure. Enzymatic activities were not correlated to shell biometric parameters in field sampling, indicating that these enzymes were not related to shell alterations detected in the present study.

Monitoring fish communities through environmental DNA metabarcoding in the fish pass system of the second largest hydropower plant in the world.

The Itaipu Hydroelectric Power Plant is the second largest in the world in power generation. The artificial barrier created by its dam imposes an obstacle for fish migration. Thus, in 2002, a fish pass system, named Piracema Channel, was built to allow fish to access areas upstream of the reservoir. We tested the potential of environmental DNA metabarcoding to monitor the impact of both the dam and associated fish pass system in the Paraná River fish communities and to compare it with traditional monitoring methods. Using a fragment of the 12S gene, we characterized richness and community composition based on amplicon sequence variants, operational taxonomic units, and zero-radius OTUs. We combined GenBank and in-house data for taxonomic assignment. We found that different bioinformatics approaches showed similar results. Also, we found a decrease in fish diversity from 2019 to 2020 probably due to the recent extreme drought experienced in southeastern Brazil. The highest alpha diversity was recorded in the mouth of the fish pass system, located in a protected valley with the highest environmental heterogeneity. Despite the clear indication that the reference databases need to be continuously improved, our results demonstrate the analytical efficiency of the metabarcoding to monitor fish species.

Acute exposure to the water-soluble fraction of gasoline (WSFG) affects oxygen consumption, nitrogenous-waste and Mg excretion, and activates anaerobic metabolism in the goldfish Carassius auratus.

Contamination of aquatic environments by petroleum and its products (e.g. gasoline) is a hazard for aquatic organisms as a result of the potential toxicity of monocyclic aromatic hydrocarbons (BTEX) and polycyclic aromatic hydrocarbons (PAH). Our goal was to evaluate the acute effects of the water-soluble fraction of gasoline (WSFG) on nitrogen excretion, osmoregulation, and metabolism of goldfish Carassius auratus. We first chemically characterized the WSFG and then tested its effects on these physiological aspects of C. auratus, in several different exposure scenarios (0, 0.25, 5, 10 and 25% of WSFG). The WSFG contained high concentrations BTEX (toluene 70% and benzene 17%) relative to PAH (<1%), and low levels of several metals (Al, Fe, Zn, Sr). Routine O2 uptake rate (MO2) of goldfish was inhibited by exposure to 5% WSFG, and during post-exposure recovery, MO2 increased in a dose-dependent fashion. Ammonia excretion was not affected by exposure to WSFG, but urea-N excretion increased progressively with the WSFG concentration. The same pattern of dose/response was observed for net Mg2+ loss rates and steadily increasing plasma lactate concentrations. Loss rates of Na+, Ca2+, K+ and Cl-, and plasma concentrations of Mg2+ and urea-N were not significantly altered. We propose that acute exposure to WSFG inhibits aerobic metabolism and activates anaerobic metabolism, breaking down ATP such that bound Mg2+ is liberated and the purine ring component is metabolized to urea-N, both of which are subsequently excreted.

The organization of the mitochondrial control region in 2 Brachyuran Crustaceans: Ucides cordatus (Ocypodidae) and Cardisoma guanhumi (Gecarcinidae).

The control region (CR) is the largest noncoding segment of the mitochondrial DNA and includes the major regulatory elements for its replication and expression. In addition, the high level of intraspecific genetic variability found in the CR favors its use in phylogeographical and population genetic studies of a variety of organisms. However, most of the work on the structure of the CR has focused on vertebrates and insects, and little is known about the evolution of the CR in other taxa. In this study, we sequenced the entire CR of several individuals of 2 crab species: Ucides cordatus (Ocypodidae) and Cardisoma guanhumi (Gecarcinidae). There were neither large conserved regions in the CR of either species nor any similarity among species at the nucleotide level. However, the spatial pattern of genetic variability on the CR was similar among species. In addition, interesting similarities were found in the formation of stable secondary structures and in the position of regulatory elements. These results indicate that the evolution of CR in crustaceans is a remarkably dynamic process, with most homology among species being found at the secondary level.