Phylogeography of the veined squid, Loligo forbesii, in European waters.

The veined squid, Loligo forbesii Steenstrup, 1856, occurs at the European Shelf areas including the Azores and represents a valuable resource for the European commercial fishery in the North East Atlantic. However, very little is known about its population structure and phylogeography. This lack of knowledge also impedes the development of sustainable fishery management for this species. The present study combined the use of two types of markers that retrieve patterns of gene flow in different time spans; the analysis of 16 nuclear microsatellites and sequencing of the mitochondrial cytochrome oxidase subunit I (COI). Whereas the high mutation rate of microsatellites allows the description of recent patterns of connectivity in species, the lower mutation rate of COI provides phylogeographic patterns on a longer timescale. A total of 347 individuals of L. forbesii were investigated from nearly the entire distribution range of the species, including the North East Atlantic Shelf, the Azores and the Mediterranean. Individuals from the Western and Eastern Mediterranean Sea have never been included in a genetic study before. We were able to analyse COI sequences from all 12 sampling areas and define three clades of L. forbesii. Due to our large sampling area, we are presenting 13 COI-haplotypes that were previously unknown. The microsatellite analysis does not include the Azores but three main clades could be identified at the remaining 11 sampling sites. Low FST values indicate gene flow over large geographical distances. However, the genetically significant differences and an additional slight grouping in the microsatellite structure reveal that geographical barriers seem to influence the population structure and reduce gene flow. Furthermore, both markers provide strong evidence that the observed phylogeographic pattern reflects the geographical history of the Azores and the Mediterranean Sea.

Disulfide bridge formation to increase thermostability of DFPase enzyme: A computational study.

Organophosphate compounds bioremediation by use of organophosphorus degradation enzymes such as DFPase is a developing interest in industry and medicine. The most important problem with the bio-catalytic enzymes is their instability on high temperatures. This work carried out to find suitable locations for introducing disulfide bridges in DFPase enzyme. We employed some computational approaches to design the disulfide bridges and evaluate their roles in the enzyme structural thermostability. According to the in silico results, mutant 6 (V24C, C76) increased the enzyme thermostability relative to wild-type.

Multiple Mating, Paternity and Complex Fertilisation Patterns in the Chokka Squid Loligo reynaudii.

Polyandry is widespread and influences patterns of sexual selection, with implications for sexual conflict over mating. Assessing sperm precedence patterns is a first step towards understanding sperm competition within a female and elucidating the roles of male- and female-controlled factors. In this study behavioural field data and genetic data were combined to investigate polyandry in the chokka squid Loligo reynaudii. Microsatellite DNA-based paternity analysis revealed multiple paternity to be the norm, with 79% of broods sired by at least two males. Genetic data also determined that the male who was guarding the female at the moment of sampling was a sire in 81% of the families tested, highlighting mate guarding as a successful male tactic with postcopulatory benefits linked to sperm deposition site giving privileged access to extruded egg strings. As females lay multiple eggs in capsules (egg strings) wherein their position is not altered during maturation it is possible to describe the spatial / temporal sequence of fertilisation / sperm precedence There were four different patterns of fertilisation found among the tested egg strings: 1) unique sire; 2) dominant sire, with one or more rare sires; 3) randomly mixed paternity (two or more sires); and 4) a distinct switch in paternity occurring along the egg string. The latter pattern cannot be explained by a random use of stored sperm, and suggests postcopulatory female sperm choice. Collectively the data indicate multiple levels of male- and female-controlled influences on sperm precedence, and highlights squid as interesting models to study the interplay between sexual and natural selection.

Complete mitochondrial genome of the Loligo beka.

In this study, we determined the complete mitochondrial genome of Loligo beka. The genome was 17 483 bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and three main non-coding regions. The overall base composition of L.beka is A 40.35%, T 32.53%, C 18.53%, and G 8.58%, with a highly A + T bias of 72.89%. All the three control regions (CR) contain termination-associated sequences and conserved sequence blocks. Here we describe a phylogenetic analysis of 11 species cephalopoda based on the complete mitochondrial genome, the result showed that the Loliolus uyii is most closely related to L. beka. This mitogenome sequence data would play an important role in the investigation of phylogenetic relationship, taxonomic resolution and phylogeography of the cephalopoda.

Complete mitochondrial genome of the Loligo opalescence.

In this study, we determined the complete mitochondrial genome of the Loligo opalescence. The genome was 17,370 bp in length and contained 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 3 main non-coding regions. The composition and order of genes, were similar to most other invertebrates. The overall base composition of L. opalescence is A 38.62%, C 19.40%, T 32.37% and G 9.61%, with a highly A + T bias of 70.99%. All of the three control regions (CR) contain termination-associated sequences and conserved sequence blocks. This mitogenome sequence data would play an important role in the investigation of phylogenetic relationship, taxonomic resolution and phylogeography of the Loliginidae.

Complete mitochondrial genome of the Loligo duvaucelii.

In this study, we determined the complete mitochondrial genome of the little squid (Loligo duvaucelii). The genome is 17,413 bp in length, containing 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and 3 main non-coding regions. The overall base composition of L. duvaucelii is 40.01% A, 32.33% T, 19.14% C and 8.52% G, with a high A + T bias of 72.34%. All of the three control regions (CR) contain termination-associated sequences and conserved sequence blocks. Here, we describe a phylogenetic analysis of 10 species of Cephalopoda based on the complete mitochondrial genome, the result showed that the Loliolus uyii is most closely related to L. duvaucelii. This mitogenome sequence data would play an important role in the investigation of phylogenetic relationship, taxonomic resolution and phylogeography of the Cephalopoda.

Integrative omics analysis reveals differentially distributed proteins in dimorphic euspermatozoa of the squid, Loligo bleekeri.

In the coastal squid Loligo bleekeri, each male produces one of two types of fertilization-competent spermatozoa (eusperm) that exhibit morphological and behavioral differences. Large "consort" males produce short-tailed spermatozoa that display free-swimming behavior when ejaculated into seawater. Small "sneaker" males, on the other hand, produce long-tailed spermatozoa that exhibit a self-swarming trait after ejaculation. To understand the molecular basis for adaptive traits employed by alternative male mating tactics, we performed the transcriptome deep sequencing (RNA-seq) and proteome analyses to search for differences in testicular mRNAs and sperm proteins, respectively. From mature male testes we identified a total of 236,455 contigs (FPKM ≧1) where 3789 and 2789 were preferentially (≧10-fold) expressed in consort and sneaker testes, respectively. A proteomic analysis detected 4302 proteins in the mature sperm as post-translational products. A strongly biased (≧10-fold) distribution occurred in 55 consort proteins and 61 sneaker proteins. There was no clear mRNA-protein correlation, making a ballpark estimate impossible for not only overall protein abundance but also the degree of biased sperm type expressed in the spermatozoa. A family encoding dynein heavy chain gene, however, was found to be biased towards sneakers, whereas many enzymes involving energy metabolism were heavily biased towards consort spermatozoa. The difference in flagellar length matched exactly the different amount of tubulins. From these results we hypothesize that discrete differential traits in dimorphic eusperm arose from a series of innovative alterations in the intracellular components of spermatozoa.

Decoding mechanism of non-universal genetic codes in Loligo bleekeri mitochondria.

Non-universal genetic codes are frequently found in animal mitochondrial decoding systems. In squid mitochondria, four codons deviate from the universal genetic code, namely AUA, UGA, and AGA/AGG (AGR) for Met, Trp, and Ser, respectively. To understand the molecular basis for establishing the non-universal genetic code, we isolated and analyzed five mitochondrial tRNAs from a squid, Loligo bleekeri. Primary structures of the isolated tRNAs, including their post-transcriptional modifications, were analyzed by mass spectrometry. tRNA(Met)(AUR) possessed an unmodified cytidine at the first position of the anticodon, suggesting that the AUA codon is deciphered by CAU anticodon via non-canonical A-C pairing. We identified 5-taurinomethyluridine (τm(5)U) at the first position of the anticodon in tRNA(Trp)(UGR). τm(5)U enables tRNA(Trp) to decipher UGR codons as Trp. In addition, 5-taurinomethyl-2-thiouridine (τm(5)s(2)U) was found in mitochondrial tRNAs for Leu(UUR) and Lys in L. bleekeri. This is the first discovery of τm(5)U and τm(5)s(2)U in molluscan mitochondrial tRNAs.

In vitro and in vivo efficacy of PEGylated diisopropyl fluorophosphatase (DFPase).

Highly toxic organophosphorus compounds that irreversibly inhibit the enzyme acetycholinesterase (AChE), including nerve agents like tabun, sarin, or soman, still pose a credible threat to civilian populations and military personnel. New therapeutics that can be used as a pretreatment or after poisoning with these compounds, complementing existing treatment schemes such as the use of atropine and AChE reactivating oximes, are currently the subject of intense research. A prominent role among potential candidates is taken by enzymes that can detoxify nerve agents by hydrolysis. Diisopropyl fluorophosphatase (DFPase) from the squid Loligo vulgaris is known to effectively hydrolyze DFP and the range of G-type nerve agents including sarin and soman. In the present work, DFPase was PEGylated to increase biological half-life, and to lower or avoid an immunogenic reaction and proteolytic digest. Addition of linear polyethylene glycol (PEG) chains was achieved using mPEG-NHS esters and conjugates were characterized by electrospray ionization--time of flight--mass specrometry (ESI-ToF-MS). PEGylated wildtype DFPase and a mutant selective for the more toxic stereoisomers of the agents were tested in vivo with rats that were challenged with a subcutaneous 3x LD(50) dose of soman. While wildtype DFPase prevented death only at extremely high doses, the mutant was able keep the animals alive and to minimize or totally avoid symptoms of poisoning. The results serve as a proof of principle that engineered variants of DFPase are potential candidates for in vivo use if substrate affinity can be improved or the turnover rate enhanced to lower the required enzyme dose.

The role of protein assembly in dynamically tunable bio-optical tissues.

Cephalopods are nicknamed the "masters of disguise" for their highly evolved camouflage mechanisms, including the hallmark ability to rapidly change the color and reflectance of their skin. Previously, reflectin proteins were identified as the major biomaterial component of iridosomes [1], specialized light-reflecting architectures that contribute intense structural color to squid skin, eyes, and organs [2-5]. Supramolecular assembly of reflectin has been recognized as a key property in the protein's function [6]. Here, we report the first cloning and expression of a specific reflectin protein found in the responsive iridophore cells of the squid Loligo pealeii, which are unique in their ability to switch on/off and change color. We demonstrate that these iridophores can be chemically tuned to reflect the entire visible spectrum. By examining recombinant reflectin, we show that this dynamic optical function is facilitated by the hierarchical assembly of nanoscale protein particles that elicit large volume changes upon condensation. These findings provide insight into the design and synthesis of biomaterials for complex, responsive function in optical applications.