Genetic variation and cryptic lineage among the sergestid shrimp Acetes americanus (Decapoda).

The taxonomic status of the sergestid shrimp, Acetes americanus, has been questioned for several decades. No specific study has been performed thus far to resolve the incongruences. This species has a wide geographical range in the western Atlantic and is represented by two formally accepted subspecies: Acetes americanus carolinae, distributed in North America, and Acetes americanus americanus, present in South America. However, there are regions where the coexistence of both subspecies has been reported, such as Central America. This study aimed to genetically compare specimens of A. a. americanus collected in South America with A. a. carolinae sampled in North America to check for possible differences and the existence of more than one subspecies of A. americanus on the Brazilian coast. Based on the sequences of two informative markers, the cytochrome oxidase I region (COI) and 16S rRNA, phylogenetic reconstruction demonstrated well-defined clades with high support values, reinforcing the idea that A. a. americanus is genetically different from A. a. carolinae. Our hypothesis was corroborated as the specimens collected in Brazil were divided into two distinct lineages: the first composed of A. a. americanus sensu stricto (Brazil 1) and the second by Acetes americanus (Brazil 2). The three groups evidenced in the haplotype network were the same as those observed in the phylogenetic tree. The morphometric character (height/length of the thelycum) was effective in distinguishing A. a. Brazil 1 from A. a. carolinae. However, more detailed and conclusive studies comprising other characteristics to propose and describe a possible new entity are necessary. To the best of our knowledge, for the first time, the results of this study provide some insights into the taxonomic status of the sergestid shrimp A. americanus in the western Atlantic.

Post-Disturbance Genetic Changes: The Impact of the 2010 Mega-Earthquake and Tsunami on Chilean Sandy Beach Fauna.

Earthquake/tsunamis can have profound impacts on species and their genetic patterns. It is expected that the magnitude of this impact might depend on the species and the time since the disturbance occurs, nevertheless these assumptions remain mostly unexplored. Here we studied the genetic responses of the crustacean species Emerita analoga, Excirolana hirsuticauda, and Orchestoidea tuberculata to the 27F mega-earthquake/tsunami that occurred in Chile in February 2010. mtDNA sequence analyses revealed a lower haplotype diversity for E. analoga and E. hirsuticauda in impacted areas one month after the 27F, and the opposite for O. tuberculata. Three years after the 27F we observed a recovery in the genetic diversity of E. analoga and E. hirsuticauda and decrease in the genetic diversity in O. tuberculata in 2/3 of sampled areas. Emerita analoga displayed decrease of genetic differentiation and increase in gene flow explained by long-range population expansion. The other two species revealed slight increase in the number of genetic groups, little change in gene flow and no signal of population expansion associated to adult survival, rapid colonization, and capacity to burrow in the sand. Our results reveal that species response to a same disturbance event could be extremely diverse and depending on life-history traits and the magnitude of the effect.

Identification key for the Brazilian species and subspecies of the family Ilyocryptidae (Crustacea, Branchiopoda, Anomopoda)

In recent years, an increase in knowledge about the diversity and biogeography of Brazilian Cladocera fauna has been evident. To keep up with these changes, it is opportune to carry out an update of the taxonomy and biogeography for the main cladoceran groups. Since 2008, some progress has been observed in Ilyocryptidae, with reports of four new taxa. In this study, an updated checklist of Ilyocryptidae from Brazil is provided, with a diagnosis for each cited taxon, as well as an identification key. Some of these taxa are considered rare; however, this issue might be an artifact of sampling. As this is the first in a series of papers compiling current information about Brazilian Cladocera, an identification key to orders and families occurring in Brazil is also provided.(AU)

Identification key for the Brazilian species and subspecies of the family Ilyocryptidae (Crustacea, Branchiopoda, Anomopoda)

In recent years, an increase in knowledge about the diversity and biogeography of Brazilian Cladocera fauna has been evident. To keep up with these changes, it is opportune to carry out an update of the taxonomy and biogeography for the main cladoceran groups. Since 2008, some progress has been observed in Ilyocryptidae, with reports of four new taxa. In this study, an updated checklist of Ilyocryptidae from Brazil is provided, with a diagnosis for each cited taxon, as well as an identification key. Some of these taxa are considered rare; however, this issue might be an artifact of sampling. As this is the first in a series of papers compiling current information about Brazilian Cladocera, an identification key to orders and families occurring in Brazil is also provided.

Discovering divergence in the thermal physiology of intertidal crabs along latitudinal gradients using an integrated approach with machine learning.

In intertidal marine crustaceans, phenotypic variation in physiological and life-history traits is pervasive along latitudinal clines. However, organisms have complex phenotypes, and their traits do not vary independently but rather interact differentially between them, effect that is caused by genetic and/or environmental forces. We evaluated the geographic variation in phenotypic integration of three marine crab species that inhabit different vertical thermal microhabitats of the intertidal zone. We studied seven populations of each species along a latitudinal gradient that spans more than 3000 km of the Chilean coast. Specifically we measured nine physiological traits that are highly related to thermal physiology. Of the nine traits, we selected four that contributed significantly to the observed geographical variation among populations; this variation was then evaluated using mixed linear models and an integrative approach employing machine learning. The results indicate that patterns of physiological variation depend on species vertical microhabitat, which may be subject to chronic or acute environmental variation. The species that inhabit the high- intertidal sites (i.e., exposed to chronic variation) better tolerated thermal stress compared with populations that inhabit the lower intertidal. While those in the low-intertidal only face conditions of acute thermal variation, using to a greater extent the plasticity to face these events. Our main results reflect that (1) species that inhabit the high-intertidal maintain a greater integration between their physiological traits and present lower plasticity than those that inhabit the low-intertidal. (2) Inverse relationship that exists between phenotypic plasticity and phenotypic integration of the physiological traits identified, which could help optimize energy resources. In general, the study of multiple physiological traits provides a more accurate picture of how the thermal traits of organisms vary along temperature gradients especially when exposed to conditions close to tolerance limits.

Phylogeography of Petrolisthes armatus, an invasive species with low dispersal ability.

Theoretically, species with high population structure are likely to expand their range, because marginal populations are free to adapt to local conditions; however, meta-analyses have found a negative relation between structure and invasiveness. The crab Petrolisthes armatus has a wide native range, which has expanded in the last three decades. We sequenced 1718 bp of mitochondrial DNA from native and recently established populations to determine the population structure of the former and the origin of the latter. There was phylogenetic separation between Atlantic and eastern Pacific populations, and between east and west Atlantic ones. Haplotypes on the coast of Florida and newly established populations in Georgia and South Carolina belong to a different clade from those from Yucatán to Brazil, though a few haplotypes are shared. In the Pacific, populations from Colombia and Ecuador are highly divergent from those from Panamá and the Sea of Cortez. In general, populations were separated hundreds to million years ago with little subsequent gene flow. High genetic diversity in the newly established populations shows that they were founded by many individuals. Range expansion appears to have been limited by low dispersal rather than lack of ability of marginal populations to adapt to extreme conditions.

Transcriptomic insights on the ABC transporter gene family in the salmon louse Caligus rogercresseyi.

BACKGROUND: ATP-binding cassette (ABC) protein family encode for membrane proteins involved in the transport of various biomolecules through the cellular membrane. These proteins have been identified in all taxa and present important physiological functions, including the process of insecticide detoxification in arthropods. For that reason the ectoparasite Caligus rogercresseyi represents a model species for understanding the molecular underpinnings involved in insecticide drug resistance. METHODS: llumina sequencing was performed using sea lice exposed to 2 and 3 ppb of deltamethrin and azamethiphos. Contigs obtained from de novo assembly were annotated by Blastx. RNA-Seq analysis was performed and validated by qPCR analysis. RESULTS: From the transcriptome database of C. rogercresseyi, 57 putative members of ABC protein sequences were identified and phylogenetically classified into the eight subfamilies described for ABC transporters in arthropods. Transcriptomic profiles for ABC proteins subfamilies were evaluated throughout C. rogercresseyi development. Moreover, RNA-Seq analysis was performed for adult male and female salmon lice exposed to the delousing drugs azamethiphos and deltamethrin. High transcript levels of the ABCB and ABCC subfamilies were evidenced. Furthermore, SNPs mining was carried out for the ABC proteins sequences, revealing pivotal genomic information. CONCLUSIONS: The present study gives a comprehensive transcriptome analysis of ABC proteins from C. rogercresseyi, providing relevant information about transporter roles during ontogeny and in relation to delousing drug responses in salmon lice. This genomic information represents a valuable tool for pest management in the Chilean salmon aquaculture industry.

Antibiotic-resistant vibrios in farmed shrimp.

Antimicrobial susceptibility pattern was determined in 100 strains of Vibrio isolated from the Litopenaeus vannamei shrimp and identified phenotypically. A high antibiotic-resistance index (75%) was observed, with the following phenotypic profiles: monoresistance (n = 42), cross-resistance to ß-lactams (n = 20) and multiple resistance (n = 13). Plasmid resistance was characterized for penicillin (n = 11), penicillin + ampicillin (n = 1), penicillin + aztreonam (n = 1), and ampicillin (n = 1). Resistance to antimicrobial drugs by the other strains (n = 86) was possibly mediated by chromosomal genes. The findings of this study support the conclusion that the cultured shrimps can be vehicles of vibrios resistant to ß-lactam and tetracycline.

Screening of genes related to ovarian development in the swimming crab, Portunus trituberculatus, by suppression subtractive hybridization.

The swimming crab, Portunus trituberculatus, is an important marine animal and is widely cultured in China. In the present study, suppression subtractive hybridization was applied to identify the differentially expressed genes in the ovaries of mature and immature P. trituberculatus. One hundred and seventy six expressed sequence tag (ESTs) were identified, of which 100 were down-regulated, and 76 up-regulated. BLAST analysis identified 51 unigenes, of which 27 were down-regulated, and 24 up-regulated. Quantitative real-time reverse transcriptase polymerase chain reaction results indicated that the SSH technique is valuable in screening genes related to ovarian development. Genes identified in this study encoded proteins corresponding to a wide range of functions and included immune response protein, transcription initiation factor, metabolic proteins, chromosome, histone h3, ovarian development-related protein, and vitellogenin. In addition, 64 metabolic pathways were annotated in differentially expressed ESTs by using the Kyoto Encyclopedia of Genes and Genomes pathway. Four annotated pathways (oxidative phosphorylation, carbon metabolism, fatty acid degradation, and protein digestion and absorption) appeared to be involved in ovarian development. In ontology analysis, 5.83% of the cellular process genes in reverse subtraction cDNA library are involved in reproduction, and 5.88% involved in developmental process. In up-regulated genes, myosin II-expressed polehole-like protein; histone h3; ovigerous-hair stripping substance; peritrophin 48; and ovarian development-related protein appeared to be involved in ovarian development. Identification of differentially expressed genes in the mature and immature ovary of the swimming crab provides new insights for further studies on the mechanism underlying ovarian development in this species.

Cold adaptation, ca2+ dependency and autolytic stability are related features in a highly active cold-adapted trypsin resistant to autoproteolysis engineered for biotechnological applications.

Pig trypsin is routinely used as a biotechnological tool, due to its high specificity and ability to be stored as an inactive stable zymogen. However, it is not an optimum enzyme for conditions found in wound debriding for medical uses and trypsinization processes for protein analysis and animal cell culturing, where low Ca(2+) dependency, high activity in mild conditions and easy inactivation are crucial. We isolated and thermodynamically characterized a highly active cold-adapted trypsin for medical and laboratory use that is four times more active than pig trypsin at 10(°) C and at least 50% more active than pig trypsin up to 50(°) C. Contrary to pig trypsin, this enzyme has a broad optimum pH between 7 and 10 and is very insensitive to Ca(2+) concentration. The enzyme is only distantly related to previously described cryophilic trypsins. We built and studied molecular structure models of this trypsin and performed molecular dynamic calculations. Key residues and structures associated with calcium dependency and cryophilicity were identified. Experiments indicated that the protein is unstable and susceptible to autoproteolysis. Correlating experimental results and structural predictions, we designed mutations to improve the resistance to autoproteolysis and conserve activity for longer periods after activation. One single mutation provided around 25 times more proteolytic stability. Due to its cryophilic nature, this trypsin is easily inactivated by mild denaturation conditions, which is ideal for controlled proteolysis processes without requiring inhibitors or dilution. We clearly show that cold adaptation, Ca(2+) dependency and autolytic stability in trypsins are related phenomena that are linked to shared structural features and evolve in a concerted fashion. Hence, both structurally and evolutionarily they cannot be interpreted and studied separately as previously done.

On three new species of Cypretta Vávra, 1895 (Crustacea: Ostracoda) from the Yucatan Peninsula, Mexico.

Three new species of the genus Cypretta Vivra, 1985 are described from Southern Mexico, representing the first record of the genus in the country. Cypretta campechensis n. sp. is closely related to Florida and North Carolina species such as C. nigra Furtos, 1936, C. brevisaepta Furtos, 1934 and C. bilicis Furtos, 1936. Cypretta spinosa n. sp. is related to the North and South American species C. intonsa Furtos, 1936 and C. vivacis Würdig & Pinto, 1993, but also to some Australian and South-East Asian Islands ones, such as C. obfuscata Victor & Fernando, 1981 and C. raciborskii (Grochmalicki, 1915). The last species described herein, Cypretta maya n. sp., is closely related to the South-East Asia islands, Australian and north Indian species, in particular to C. hirsuta Henry, 1923, C. longidactyla Victor & Fernando, 1981, and C. patialensis Battish, 1982.

First isolation and characterization of genomic SSR markers for the giant red shrimp Aristaeomorpha foliacea (Risso, 1827).

Fourteen microsatellite markers were isolated from the giant red shrimp Aristaeomorpha foliacea (Risso, 1827) using the FIASCO protocol (fast isolation by AFLP of sequences containing repeats). Polymorphism was assessed in 30 individuals from two localities of the western Mediterranean basin (N = 20 from Sardinia and N = 10 from Sicily); nine loci showed polymorphism with 2 to 19 alleles per locus (average: 8.9). Polymorphic information content ranged from 0.36 to 0.91, and the observed and expected heterozygosities ranged from 0.50 to 0.97 and from 0.47 to 0.93, respectively. Two loci showed significant deviation from Hardy-Weinberg equilibrium, and evidence of linkage disequilibrium was found for only one locus pair. These loci are the first to be characterized in A. foliacea and could be effective tools for the investigation of genetic diversity, population structure, and demographic connectivity, useful information for the management of this important commercial resource.

Monitoring an alien invasion: DNA barcoding and the identification of lionfish and their prey on coral reefs of the Mexican Caribbean.

BACKGROUND: In the Mexican Caribbean, the exotic lionfish Pterois volitans has become a species of great concern because of their predatory habits and rapid expansion onto the Mesoamerican coral reef, the second largest continuous reef system in the world. This is the first report of DNA identification of stomach contents of lionfish using the barcode of life reference database (BOLD). METHODOLOGY/PRINCIPAL FINDINGS: We confirm with barcoding that only Pterois volitans is apparently present in the Mexican Caribbean. We analyzed the stomach contents of 157 specimens of P. volitans from various locations in the region. Based on DNA matches in the Barcode of Life Database (BOLD) and GenBank, we identified fishes from five orders, 14 families, 22 genera and 34 species in the stomach contents. The families with the most species represented were Gobiidae and Apogonidae. Some prey taxa are commercially important species. Seven species were new records for the Mexican Caribbean: Apogon mosavi, Coryphopterus venezuelae, C. thrix, C. tortugae, Lythrypnus minimus, Starksia langi and S. ocellata. DNA matches, as well as the presence of intact lionfish in the stomach contents, indicate some degree of cannibalism, a behavior confirmed in this species by the first time. We obtained 45 distinct crustacean prey sequences, from which only 20 taxa could be identified from the BOLD and GenBank databases. The matches were primarily to Decapoda but only a single taxon could be identified to the species level, Euphausia americana. CONCLUSIONS/SIGNIFICANCE: This technique proved to be an efficient and useful method, especially since prey species could be identified from partially-digested remains. The primary limitation is the lack of comprehensive coverage of potential prey species in the region in the BOLD and GenBank databases, especially among invertebrates.

Effects of various salinities on Na(+)-K(+)-ATPase, Hsp70 and Hsp90 expression profiles in juvenile mitten crabs, Eriocheir sinensis.

Eriocheir sinensis is a euryhaline crab migrating from sea to freshwater habitats during the juvenile stage. We used quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the gene expression profile of Na(+)-K(+)-ATPase, Hsp70 (heat shock protein 70) and Hsp90 in megalopa exposed to salinities of 0, 2, 5, 10, and 15 parts per thousand. Both low and high salinities markedly stimulated expression of Na(+)-K(+)-ATPase, Hsp70 and Hsp90 genes of Chinese mitten crab megalopa; salinity had different effects on Na(+)-K(+)-ATPase, Hsp70 and Hsp90 levels depending on the duration of salinity stress, implying that Na(+)-K(+)-ATPase, Hsp70 and Hsp90 may play an important role in salinity tolerance in this crab species.

Different processes lead to similar patterns: a test of codivergence and the role of sea level and climate changes in shaping a southern temperate freshwater assemblage.

BACKGROUND: Understanding how freshwater assemblages have been formed and maintained is a fundamental goal in evolutionary and ecological disciplines. Here we use a historical approach to test the hypothesis of codivergence in three clades of the Chilean freshwater species assemblage. Molecular studies of freshwater crabs (Aegla: Aeglidae: Anomura) and catfish (Trichomycterus arealatus: Trichomycteridae: Teleostei) exhibited similar levels of genetic divergences of mitochondrial lineages between species of crabs and phylogroups of the catfish, suggesting a shared evolutionary history among the three clades in this species assemblage. RESULTS: A phylogeny was constructed for Trichomycterus areolatus under the following best-fit molecular models of evolution GTR + I + R, HKY + I, and HKY for cytochrome b, growth hormone, and rag 1 respectively. A GTR + I + R model provided the best fit for both 28S and mitochondrial loci and was used to construct both Aegla phylogenies. Three different diversification models were observed and the three groups arose during different time periods, from 2.25 to 5.05 million years ago (Ma). Cladogenesis within Trichomycterus areolatus was initiated roughly 2.25 Ma (Late Pliocene-Early Pleistocene) some 1.7-2.8 million years after the basal divergences observed in both Aegla clades. These results reject the hypothesis of codivergence. CONCLUSIONS: The similar genetic distances between terminal sister-lineages observed in these select taxa from the freshwater Chilean species assemblage were formed by different processes occurring over the last ~5.0 Ma. Dramatic changes in historic sea levels documented in the region appear to have independently shaped the evolutionary history of each group. Our study illustrates the important role that history plays in shaping a species assemblage and argues against assuming similar patterns equal a shared evolutionary history.

Cloning, partial sequence, and single-nucleotide polymorphism of the ryanodine receptor gene of the Pacific white shrimp Litopenaeus vannamei (Penaeidae).

Ryanodine receptor/calcium release channel is a large protein that plays an essential role in muscle contraction; mutations in the ryanodine receptor gene affect sensitivity to stress. As a first step towards investigating the relationship between the ryanodine receptor and shrimp cramped muscle syndrome, we cloned, partially sequenced, and examined single-nucleotide polymorphisms (SNPs) of the ryanodine receptor gene of the Pacific white shrimp (Litopenaeus vannamei). The nucleotide sequence of a 15.06-kb L. vannamei genomic DNA segment containing a partial ryanodine receptor gene sequence was determined (deposited in GenBank nucleotide database: HM367069). Direct sequencing of PCR-amplified ryanodine receptor exons with their intron-flanking regions in 10 cramped muscle syndrome shrimp and 10 healthy shrimp, revealed seven SNPs. Five of them (1713A/G, 1749T/C, 1755T/C, 3965G/A, and 8737C/T) are located in exons; however, they appear to be neutral (synonymous), since they do not alter the encoded amino acid. The other SNPs (1553C/T and 13337A/G) are in introns. The SNPs identified in the ryanodine receptor gene could be useful for association studies aimed at determining the physiological role of the ryanodine receptor in cramped muscle syndrome of shrimp.

The post-embryonic development of Remipedia (Crustacea)--additional results and new insights.

The post-embryonic development of a species of the enigmatic crustacean group Remipedia is described in detail for the first time under various aspects. Applying a molecular approach, we can clearly prove the species identity of the larvae as belonging to Pleomothra apletocheles. We document the cellular level of several larval stages and the differentiation of segments, limbs, and the general body morphology applying the techniques of confocal laser scanning microscopy and scanning electron microscopy. In addition, we document the swimming behavior and the peculiar movements of the naupliar appendages. A comparison of our results with published data on other Crustacea and their larval development tentatively supports ideas about phylogenetic affinities of the Remipedia to the Malacostraca.

Phylogeography of an Island endemic, the Puerto Rican Freshwater Crab (Epilobocera sinuatifrons).

The endemic Puerto Rican crab, Epilobocera sinuatifrons (Pseudothelphusidae), has a freshwater-dependant life-history strategy, although the species has some capabilities for terrestrial movement as adults. In contrast to all other freshwater decapods on the island (e.g., caridean shrimp), E. sinuatifrons does not undertake amphidromous migration, and is restricted to purely freshwater habitats and adjacent riparian zones. As Puerto Rico has a dynamic geologic history, we predicted that both the life history of E. sinuatifrons and the geological history of the island would be important determinants of phylogeographic structuring in the species. Using a fragment of the cytochrome c oxidase subunit 1 mtDNA (mitochondrial DNA) gene, we tested for deviations from panmixia among and within rivers draining Puerto Rico and used statistical phylogeography to explore processes that may explain extant patterns of genetic variation in the species. While populations of E. sinuatifrons were significantly differentiated among rivers, they were likely to be recently derived because nested clade analysis (NCA) indicated evolutionarily recent restricted gene flow with isolation by distance (IBD) and contiguous range expansion at various spatial scales. Ongoing drainage rearrangements associated with faulting and land slippage were invoked as processes involved in sporadic gene flow among rivers throughout the Pleistocene. Patterns of genetic differentiation conformed to IBD and population demographic statistics were nonsignificant, indicating that although recently derived, populations from different rivers were in drift-mutation equilibrium. A shallow (0.6 million years ago), paraphyletic split was observed in the haplotype network, which NCA indicated arose via allopatric fragmentation. This split coincides with an area of high relief in central Puerto Rico that may have experienced relatively little drainage rearrangements. Shallow but significant genetic isolation of populations of E. sinuatifrons among Puerto Rican rivers suggests phylogeographic patterns that are intermediate to terrestrial habitat specialists (highly divergent populations) and other freshwater biota, such as amphidromous species and insects with aerial adult dispersal (highly connected populations).

Differential expression of duplicated opsin genes in two eyetypes of ostracod crustaceans.

In the first molecular study of ostracod (Crustacea) vision, we present partial cDNA sequences of ostracod visual pigment genes (opsins). We found strong support for differential expression of opsins in ostracod median and compound eyes and suggest that photoreceptor specific expression may be a general phenomenon in organisms with multiple receptors. We infer that eye-specific expression predates the divergence of the two species examined, Skogsbergia lerneri and Vargula hilgendorfii, because eye-specific opsin orthologs are present in both species. We found multiple opsin loci in ostracods, estimating that at least eight are present in Skogsbergia lerneri. All opsins from both ostracod species examined are more closely related to each other than to any other known opsin sequences. Because we find no evidence for gene conversion or alternative splicing, we suggest the occurrence of many recent gene duplications. Why ostracods may have retained multiple recent opsin gene duplicates is unknown, but we discuss several possible hypotheses.

Molecular insights into Cumacean family relationships (Crustacea, Cumacea).

Cumaceans are a diverse order of small, benthic marine crustaceans. Phylogenetic hypotheses for the eight currently recognized cumacean families have not been formally proposed. However, based on external morphological traits and Linnean classification, a few conflicting hypotheses of relatedness have been proposed. Family definitions rely on morphological characters that often overlap and diagnoses are based on a combination of non-unique characters. Morphological analysis does not provide a well-resolved phylogeny. In the present study, we use amino acid sequences from the mitochondrial cytochrome oxidase I gene to produce a molecular phylogenetic hypothesis for the families of Cumacea. Phylogenetic analyses at the amino acid level were performed under Bayesian, likelihood, and parsimony methods. Results strongly suggest that families lacking an articulated telson form a monophyletic group. This pleotelson clade, composed of the families Bodotriidae, Leuconidae, and Nannastacidae, is the most derived within the Cumacea. Within this group, the Bodotriidae resolve paraphyletically, with Leuconidae and Nannastacidae embedded within it. Comparison of the molecular phylogeny with that based on morphology suggests that many "diagnostic" characters are homoplasious.