Cleaning symbiosis in coral reefs of Jardines de la Reina National Park.

Background: Cleaning symbiotic interactions are an important component of coral reef biodiversity and the study of the characteristics of these interacting species networks allows to assess the health of communities. The coral reefs of Jardines de la Reina National Park (JRNP) are subject to a protection gradient and there is a lack of knowledge about the effect of different levels of protection on the cleaning mutualistic networks in the area. The present study aims to characterize the mutualistic cleaning networks in the reefs of JRNP and to assess the potential effect of the protection gradient on their characteristics. Methods: We visited 26 reef sites distributed along the National Park and performed 96 band transects (50 m × 4 m). Low, medium and highly protected regions were compared according to the number of cleaning stations and the abundance and number of species of clients and cleaners associated with them. Additionally, we built interaction networks for the three regions and the entire archipelago based on a total of 150 minutes' video records of active cleaning stations. We assessed ecological networks characteristics (specialization, nestedness) using network topological metrics. Results: We found a high diversity and complex cleaning interaction network with 6 cleaner species and 39 client species, among them, the threatened grouper Epinephelus striatus was one of the most common clients. No clear effect of the protection level on the density, abundance or diversity of cleaners and clients was detected during this study. However, we found that the network structure varied among regions, with the highly protected region being more specialized and less nested than the other regions. Our research reveals some patterns that suggest the effect of fishing pressure on cleaning symbiosis, as fishing may reduce the abundance and composition of client species, especially those that are targeted by fishers. However, fishing pressure may not be the main factor influencing cleaning symbiosis inside of the National Park, as other factors, such as habitat quality or environmental conditions may have stronger effects on the demand for cleaning services and the interactions between cleaners and clients. Our research provides insights into the factors that influence cleaning symbiosis and its implications for coral reef conservation and management.

Biology and ecology of the lionfish Pterois volitans/Pterois miles as invasive alien species: a review.

The lionfish is an exotic invasive fish native to the Indo-Pacific, which is established in the western Atlantic Ocean and the Caribbean Sea. Lionfish can affect native fishes and invertebrates through direct predation or competition for food. The present review aims to analyze the most relevant characteristics of the biology and ecology of lionfish as an invasive alien species, with an emphasis on Cuba. We provide a current view of the well-known lionfish as a successful invasive fish, and we put in this context the information regarding lionfish in Cuban waters, enriching the background knowledge, and giving novel and relevant information. The compilation of numerous publications on the subject has allowed for a more complete analysis of essential aspects of this invader in the Cuban archipelago. The consulted literature records that the first report of lionfish in Cuba occurred in 2007; subsequently, sightings of lionfish were reported in numerous localities. In 2010, the lionfish was considered an invasive alien species, which currently is established in various habitats, at depths up to 188 m, throughout the Cuban archipelago (e.g., coral reefs, mangroves, seagrass beds, submerged artificial structures). In addition, it has reached very high densities (12.42 ind./100 m2), which exceed those reported in the Indo-Pacific as well as in many locations in the Western Atlantic. It has been confirmed that the lionfish in Cuba also presents numerous characteristics that guarantee its success as an invader, among them: less quantity and diversity of parasites than other Atlantic fishes found in similar environments, a high number of gametes in the gonads, reproductive activity during all year and wide diet. The most important fish families for the lionfish diet in Cuba have been Pomacentridae, Gobiidae, Scaridae, Holocentridae, Mullidae, Labridae and Acanthuridae; and the most important crustacean orders are Decapoda, Mysida, Stomatopoda and Isopoda. In Cuba, as in the entire invaded region, numerous investigations have been directed to evaluate the impact of this invader on ecosystems, and although there is enough information, their results differ. Additional studies are required to assess the impact of lionfish as a predator after several years of invasion on a larger geographic scale in Cuba and other areas of the region. This knowledge will allow the development of more effective control strategies. Periodic lionfish culling have been carried out in Cuban MPAs as a control strategy, and some positive results have been observed, such as the average size reduction; however, further efforts are still required. Due to the importance of the study of lionfish as an invader, this review is a necessity as it provides, for the first time, a comprehensive analysis of lionfish information and results from Cuba, which is adequately contrasted with previous studies of other areas, particularly, from the Greater Caribbean.

Characterization of the populations of upside-down jellyfish in Jardines de la Reina National Park, Cuba.

Upside-down jellyfish are a group of benthic scyphozoans belonging to the genus Cassiopea, whose members are in symbiosis with dinoflagellates and inhabit tropical and subtropical waters. Although there are some studies of the genus in the Caribbean, these are limited. In Cuba, the group's studies are restricted to reports on taxonomic lists and, as far as we know, no one has performed any analyzes of the densities of these jellyfish in seagrass or mangrove ecosystems in Cuba. In this work, the populations of Cassiopea spp. in Jardines de la Reina National Park (JRNP) were characterized, for the first time for this Marine Protected Area and Cuba. One hundred 1m2 square frames were placed at 14 JRNP sites. For each site, the species, density, size of the individuals and abiotic factors were determined. Density and diameter comparisons were made between sites, zones and regions within the JRNP. The percentage of the benthic cover was determined and a correlation was made between density and diameter. A total of 10,803 individuals were recorded, of which 7,618 belong to Cassiopea xamachana and 3,185 belong to Cassiopea frondosa. Both species share a niche and no evident segregation was detected according to abiotic variables. Significant differences were detected in comparisons of density and size across sites and zones. Density and size in the JRNP were negatively correlated, and higher aggregations of the species were observed at lower sizes. Density mean values ranged from 2.18 to 14.52 ind. /m2 with maximum values of 79 ind. /m2. Cayo Alcatraz was the site found to have the highest density while Cachiboca was the site with the lowest density. The average bell diameter size of the individuals ranged from 9.34 to 15.31 cm for the sampled sites, with minimum and maximum values of 2.5 cm and 32.6 cm. The smallest size was recorded at Cayo Alcatraz while the largest size was reported for Boca de las Anclitas. The environmental factors evaluated showed no significant relationship with the density or diameter of Cassiopea, while the Thalassia testudinum cover was negatively correlated with Cassiopea density at all fourteen sites in the JRNP. The percentage of Cassiopea coverage was higher than those reported in the literature, with four sites exceeding 20% coverage. In general, the populations of Cassiopea spp. in the JRNP did not differ greatly, although a higher density was observed towards the eastern region of the park. It was shown for the first time for the species that density and size have a negative correlation. Future studies are required to quantify the impact of Cassiopea on coastal marine ecosystem processes, and to further determine how anthropogenic changes may be altering the function of these tropical ecosystems.

Feeding ecology of invasive lionfish in the Punta Frances MPA, Cuba: insight into morphological features, diet and management.

Cuba's shelf has been invaded by lionfish (Pterois volitans/Pterois miles), which have become established over the archipelago, including areas of natural importance. The present study aims to evaluate morphometric features of lionfish and to explore the relationship between lionfish size and diet composition in different habitats in the Punta Frances National Park, Cuba. In total 620 lionfish were captured at 29 sites between 2013 and 2016. Lionfish stomachs were removed and their contents were analyzed using frequency and numerical methods. The length-weight allomentric relationship was obtained, and a decrease in lionfish sizes was shown over time, likely due to the extractions carried out. The diet was composed by fishes, crustaceans, mollusks and phytobenthos, with a predominance of fishes. Lionfish caught in seagrass beds tended to be smaller in size and consumed fewer fishes and more crustaceans than those captured in coral reefs. A positive correlation was observed between lionfish body size and gape size; however, no significant correlation was detected between lionfish body size and prey size. Larger lionfish tended to consume more fishes, while crustaceans were more significant in the diet of juvenile lionfish. This is the first study that examines the feeding habits of lionfish in the Punta Frances MPA, and provides valuable information on lionfish inhabiting this MPA across four years of sampling. Furthermore, this research may serve as a baseline for subsequent evaluations of lionfish impact and management actions in the area.

Toward a More Comprehensive View of α-Amylase across Decapods Crustaceans.

Decapod crustaceans are a very diverse group and have evolved to suit a wide variety of diets. Alpha-amylases enzymes, responsible for starch and glycogen digestion, have been more thoroughly studied in herbivore and omnivore than in carnivorous species. We used information on the α-amylase of a carnivorous lobster as a connecting thread to provide a more comprehensive view of α-amylases across decapods crustaceans. Omnivorous crustaceans such as shrimps, crabs, and crayfish present relatively high amylase activity with respect to carnivorous crustaceans. Yet, contradictory results have been obtained and relatively high activity in some carnivores has been suggested to be a remnant trait from ancestor species. Here, we provided information sustaining that high enzyme sequence and overall architecture conservation do not allow high changes in activity, and that differences among species may be more related to number of genes and isoforms, as well as transcriptional and secretion regulation. However, recent evolutionary analyses revealed that positive selection might have also occurred among distant lineages with feeding habits as a selection force. Some biochemical features of decapod α-amylases can be related with habitat or gut conditions, while less clear patterns are observed for other enzyme properties. Likewise, while molt cycle variations in α-amylase activity are rather similar among species, clear relationships between activity and diet shifts through development cannot be always observed. Regarding the adaptation of α-amylase to diet, juveniles seem to exhibit more flexibility than larvae, and it has been described variation in α-amylase activity or number of isoforms due to the source of carbohydrate and its level in diets, especially in omnivore species. In the carnivorous lobster, however, no influence of the type of carbohydrate could be observed. Moreover, lobsters were not able to fine-regulate α-amylase gene expression in spite of large changes in carbohydrate content of diet, while retaining some capacity to adapt α-amylase activity to very low carbohydrate content in the diets. In this review, we raised arguments for the need of more studies on the α-amylases of less studied decapods groups, including carnivorous species which rely more on dietary protein and lipids, to broaden our view of α-amylase in decapods crustaceans.

Crustacean Proteases and Their Application in Debridement.

Digestive proteases from marine organisms have been poorly applied to biomedicine. Exceptions are trypsin and other digestive proteases from a few cold-adapted or temperate fish and crustacean species. These enzymes are more efficient than enzymes from microorganism and higher vertebrates that have been used traditionally. However, the biomedical potential of digestive proteases from warm environment species has received less research attention. This review aims to provide an overview of this unrealised biomedical potential, using the debridement application as a paradigm. Debridement is intended to remove nonviable, necrotic and contaminated tissue, as well as fibrin clots, and is a key step in wound treatment. We discuss the physiological role of enzymes in wound healing, the use of exogenous enzymes in debridement, and the limitations of cold-adapted enzymes such as their poor thermal stability. We show that digestive proteases from tropical crustaceans may have advantages over their cold-adapted counterparts for this and similar uses. Differences in thermal stability, auto-proteolytic stability, and susceptibility to proteinase inhibitors are discussed. Furthermore, it is proposed that the feeding behaviour of the source organism may direct the evaluation of enzymes for particular applications, as digestive proteases have evolved to fill a wide variety of feeding habitats, natural substrates, and environmental conditions. We encourage more research on the biomedical application of digestive enzymes from tropical marine crustaceans.

Carbohydrates digestion and metabolism in the spiny lobster (Panulirus argus): biochemical indication for limited carbohydrate utilization.

As other spiny lobsters, Panulirus argus is supposed to use preferentially proteins and lipids in energy metabolism, while carbohydrates are well digested but poorly utilized. The aim of this study was to evaluate the effect of dietary carbohydrate level on digestion and metabolism in the spiny lobster P. argus. We used complementary methodologies such as post-feeding flux of nutrients and metabolites, as well as measurements of α-amylase expression and activity in the digestive tract. Lobsters readily digested and absorbed carbohydrates with a time-course that is dependent on their content in diet. Lobster showed higher levels of free glucose and stored glycogen in different tissues as the inclusion of wheat flour increased. Modifications in intermediary metabolism revealed a decrease in amino acids catabolism coupled with a higher use of free glucose as carbohydrates rise up to 20%. However, this effect seems to be limited by the metabolic capacity of lobsters to use more than 20% of carbohydrates in diets. Lobsters were not able to tightly regulate α-amylase expression according to dietary carbohydrate level but exhibited a marked difference in secretion of this enzyme into the gut. Results are discussed to highlight the limitations to increasing carbohydrate utilization by lobsters. Further growout trials are needed to link the presented metabolic profiles with phenotypic outcomes.

Panusin represents a new family of ß-defensin-like peptides in invertebrates.

Beta_defensin have been solely found in vertebrates until ß-defensin-like peptides were described as transcript isoforms in two species of Panulirus genus. They were considered as putative antimicrobials since their biological activity have not been demonstrated. Here we purified and characterized a defensin-like peptide from the hemocytes of spiny lobster P. argus, hereafter named panusin. Structurally, panusin presents a cysteine-stabilized α/ß motif, and is prone to form homodimers. Biological activity of panusin showed broad-spectrum antimicrobial activity, characterized for being strikingly salt-resistant. Panusin did not showed hemolytic activity but was demonstrated its binding capacity to different lipid membrane models, indicating amphipathicity of ß-sheet core as driving force for its antimicrobial activity. Panusin is considered a new kind of arthropod defensin which share structural and biological features with beta-defensin from vertebrates. The presence of beta-defensin like peptides in crustacean might suggest the emergence of the evolutionary relationship of ß-defensins from vertebrates.

Molecular, Biochemical, and Dietary Regulation Features of α-Amylase in a Carnivorous Crustacean, the Spiny Lobster Panulirus argus.

Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.

Trypsin isozymes in the lobster Panulirus argus (Latreille, 1804): from molecules to physiology.

Trypsin enzymes have been studied in a wide variety of animal taxa due to their central role in protein digestion as well as in other important physiological and biotechnological processes. Crustacean trypsins exhibit a high number of isoforms. However, while differences in properties of isoenzymes are known to play important roles in regulating different physiological processes, there is little information on this aspect for decapod trypsins. The aim of this review is to integrate recent findings at the molecular level on trypsin enzymes of the spiny lobster Panulirus argus, into higher levels of organization (biochemical, organism) and to interpret those findings in relation to the feeding ecology of these crustaceans. Trypsin in lobster is a polymorphic enzyme, showing isoforms that differ in their biochemical features and catalytic efficiencies. Molecular studies suggest that polymorphism in lobster trypsins may be non-neutral. Trypsin isoenzymes are differentially regulated by dietary proteins, and it seems that some isoenzymes have undergone adaptive evolution coupled with a divergence in expression rate to increase fitness. This review highlights important but poorly studied issues in crustaceans in general, such as the relation among trypsin polymorphism, phenotypic (digestive) flexibility, digestion efficiency, and feeding ecology.

A holistic view of dietary carbohydrate utilization in lobster: digestion, postprandial nutrient flux, and metabolism.

Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2-6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6-12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients.

Lobster (Panulirus argus) hepatopancreatic trypsin isoforms and their digestion efficiency.

It is well known that crustaceans exhibit several isoforms of trypsin in their digestive system. Although the number of known crustacean trypsin isoforms continues increasing, especially those derived from cDNA sequences, the role of particular isoenzymes in digestion remains unknown. Among invertebrates, significant advances in the understanding of the role of multiple trypsins have been made only in insects. Since it has been demonstrated that trypsin isoenzyme patterns (phenotypes) in lobster differ in digestion efficiency, we used this crustacean as a model for assessing the biochemical basis of such differences. We demonstrated that the trypsin isoform known to be present in all individuals of Panulirus argus has a high catalytic efficiency (k(cat)/K(m) ) and is the most reactive toward native proteinaceous substrates, whereas one of the isoforms present in less efficient individuals has a lower k(cat) and a lower k(cat)/K(m), and it is less competent at digesting native proteins. A fundamental question in biology is how genetic differences produce different physiological performances. This work is the first to demonstrate that trypsin phenotypic variation in crustacean protein digestion relies on the biochemical properties of the different isoforms. Results are relevant for understanding trypsin polymorphism and protein digestion in lobster.

Dietary protein quality differentially regulates trypsin enzymes at the secretion and transcription level in Panulirus argus by distinct signaling pathways.

The effects of pelleted diets with different protein composition (fish, squid or soybean meals as main protein sources) on trypsin secretion and expression were studied in the lobster Panulirus argus. Trypsin secretion was shown to be maximal 4 h after ingestion. At this time, fish- and squid-based diets induced trypsin secretion, as well as up-regulation of the major trypsin isoform at the transcription level. While fish- and squid-based diets elicited a prandial response, soybean-based diet failed to stimulate the digestive gland to secrete trypsin into the gastric fluid or induce trypsin expression above the levels observed in fasting lobsters. In vitro assays showed that intact proteins rather than protein hydrolysates stimulate trypsin secretion in the lobster. However, the signal for trypsin transcription appears to be different to that for secretion and is probably mediated by the appearance of free amino acids in the digestive gland, suggesting a stepwise regulation of trypsin enzymes during digestion. We conclude that trypsin enzymes in P. argus are regulated at the transcription and secretion level by the quality of dietary proteins through two distinct signaling pathways. Our results indicate that protein digestion efficiency in spiny lobsters can be improved by selecting appropriated protein sources. However, other factors like the poor solubility of dietary proteins in dry diets could hamper further enhancement of digestion efficiency.