Species‐specific microsatellite loci for the European squid (Loligo vulgaris)

A microsatellite dinucleotide‐enriched library was obtained from the European squid (Loligo vulgaris) and five species‐specific dinucleotide markers were optimized. These markers are highly polymorphic with average expected heterozygosity ranging from 0.706 to 0.927 and allele number ranging from 7 to 17. This set of primers is suitable for population genetic studies.     

The locomotory function of the fins in the squid Loligo pealei

Kinematic data of high spatial and temporal resolution, acquired from image sequences of adult long-finned squid, Loligo pealei, during steady swimming in a flume, were used to examine the role of fins and the coordination between fin and jet propulsion in squid locomotion. Fin shape and body outlines were digitized and used to calculate fin wave speed, amplitude, frequency, angle of attack, body deformation, speed, and acceleration. L. pealei were observed to have two fin gait patterns with a transition at 1.4-1.8 mantle lengths per second (L_m s^-1) marked by alternation between the two patterns. Fin motion in L. pealei exhibited characteristics of both traveling waves and flapping wings. At low speeds, fin motion was more wave-like; at high speeds, fin motion was more flap-like and was marked by regular periods during which the fins were wrapped tightly against the mantle. Fin cycle frequencies were dependent on swimming speed and gait, and obvious coordination between the fins and jet were observed. Fin wave speed, angle of attack, and body acceleration confirmed the role of fins in thrust production and revealed a role of fins at all swimming speeds by a transition from drag-based to lift-based thrust when fin wave speed dropped below swimming speed. Estimates of peak fin thrust were as high as 0.44-0.96 times peak jet thrust in steady swimming over the range of swimming speeds observed. Fin downstrokes generally contributed more to thrust than did upstrokes, especially at high speeds.     

Polymorphic microsatellite DNA markers for the Patagonian squid,Loligo gahi (Cephalopoda)

Squid populations are being put under rapidly increasing commercial fishing pressure worldwide. The same species are known to be susceptible to extreme population fluctuations, so detailed knowledge of population substructuring and genetic diversity is essential for rational management. We present a set of microsatellite DNA loci suitable for population genetic analysis of Loligo gahi, the squid species subject to the most detailed monitoring and fishery control (around the Falkland Islands), with the future aim of generating management‐related information to aid conservation of this valuable natural resource.     

The locomotory function of the fins in the squid Loligo pealei

Kinematic data of high spatial and temporal resolution, acquired from image sequences of adult long-finned squid, Loligo pealei, during steady swimming in a flume, were used to examine the role of fins and the coordination between fin and jet propulsion in squid locomotion. Fin shape and body outlines were digitized and used to calculate fin wave speed, amplitude, frequency, angle of attack, body deformation, speed, and acceleration. L. pealei were observed to have two fin gait patterns with a transition at 1.4-1.8 mantle lengths per second (Lm s-1) marked by alternation between the two patterns. Fin motion in L. pealei exhibited characteristics of both traveling waves and flapping wings. At low speeds, fin motion was more wave-like; at high speeds, fin motion was more flap-like and was marked by regular periods during which the fins were wrapped tightly against the mantle. Fin cycle frequencies were dependent on swimming speed and gait, and obvious coordination between the fins and jet were observed. Fin wave speed, angle of attack, and body acceleration confirmed the role of fins in thrust production and revealed a role of fins at all swimming speeds by a transition from drag-based to lift-based thrust when fin wave speed dropped below swimming speed. Estimates of peak fin thrust were as high as 0.44-0.96 times peak jet thrust in steady swimming over the range of swimming speeds observed. Fin downstrokes generally contributed more to thrust than did upstrokes, especially at high speeds.     

Subtle population structuring within a highly vagile marine invertebrate, the veined squid Loligo forbesi, demonstrated with microsatellite DNA markers

Microsatellite DNA markers were applied for the first time in a population genetic study of a cephalopod and compared with previous estimates of genetic differentiation obtained using allozyme and mitochondrial DNA (mtDNA) markers. Levels of genetic variation detected with microsatellites were much higher than found with previous markers (mean number of alleles per locus=10.6, mean expected heterozygosity ( H _E)=0.79; allozyme H _E=0.08; mtDNA restriction fragment length polymorphism (RFLP) H _E=0.16). In agreement with previous studies, microsatellites demonstrated genetic uniformity across the population occupying the European shelf seas of the North East Atlantic, and extreme genetic differentiation of the Azores population ( R _ST/ F _ST=0.252/0.245; allozyme F _ST=0.536; mtDNA F _ST=0.789). In contrast to other markers, microsatellites detected more subtle, and significant, levels of differentiation between the populations of the North East Atlantic offshore banks (Rockall and Faroes) and the shelf population ( R _ST=0.048 and 0.057). Breakdown of extensive gene flow among these populations is indicated, with hydrographic (water depth) and hydrodynamic (isolating current regimes) factors suggested as possible barriers to migration. The demonstration of genetic subdivision in an abundant, highly mobile marine invertebrate has implications for the interpretation of dispersal and population dynamics, and consequent management, of such a commercially exploited species. Relative levels of differentiation indicated by the three different marker systems, and the use of measures of differentiation (assuming different mutation models), are discussed.     

The ultrastructure and innervation of muscles controlling chromatophore expansion in the squid,Loligo vulgaris

Squid chromatophores are organs of colour change, consisting of a pigment sac opened by contraction of 10–24 radial muscle fibres. The ultrastructure and innervation of these muscle fibres were examined by electron microscopy and diagramatic reconstructions made on the basis of serial ultra-thin sections. At the proximal end of the fibre, nearest the pigment sac a cortical myofilament zone surrounds 2 cores containing mitochrondria; further along the fibre these merge to form one central core. The myofilament zone forms a groove containing a nerve bundle consisting of 2 to 4 axons per muscle fibre. The axons are surrounded by glial cell processes, and either originate from a neighbouring fibre, or join the fibre at some point along its length. Axons twist around each other, forming a series of synapses with the muscle fibre. As many as 6–37 synapses exist along the length of each muscle fibre; the mean synapse interval is 9.05 m, but the largest may be 123 m. At the distal end of the muscles, the nerve is located towards the middle of the fibre, which it penetrates as the muscle splits up. Electron-lucent vesicles are present in all synaptic regions, but electron-dense vesicles are only found towards the distal end of the fibre. There is thus a possibility that more than one neurotransmitter is present in the nerves innervating chromatophores. Electron-lucent and dense-cored vesicles are not colocalised.This work was carried out during the tenure of a BBSRC CASE studentship     

Membrane differentiations in spermatozoa of the squid,Loligo pealeii

Regional differences in the structure of the plasma membrane and acrosome membrane of squid spermatozoa were studied by freeze-fracture and thin section electron microscopy. In regions of close apposition the plasma membrane and acrosome membrane are adjoined to one another by regularly spaced linkages. These linkage sites, overlie a set of fibers located at the inner face of the acrosomal membrane. The acrosomal fibers terminate in a layer of granular material located at the base of the acrosome. Detergent treatment of sperm releases the fibers and granular material as an interconnected complex. Freeze-fracture replicas reveal a random arrangement of intramembranous particles in the plasma membrane over the sperm head and linear aggregates of intramembranous particles in the acrosomal membrane. Several regional differences in the structure of the flagellar plasma membrane are present. The thickness of the glycocalyx is progressively reduced distally along the flagellum. Freeze-fracture replicas show evenly spaced linear arrays of intramembranous particles which extend parallel t o the flagellar long axis. Examination of spermatozoa extracted to disrupt flagellar geometry suggest that the dense fiber-doublet microtubule complexes are attached to the plasma membrane. The possible functional role of these membrane differentiations and their relationship t o membrane structures in mammalian spermatozoa are discussed.     

A scanning electron microscope study of ectodermal differentiations in the caudal mantle epithelium of embryos and juveniles of Loligo vulgaris,Loligo forbesi and Sepia officinalis

Summary

The mantle epithelium of embryos and early juveniles of the squids Loligo vulgaris and Loligo forbesi and the cuttlefish Sepia officinalis was studied using scanning electron microscopy. In embryos of L. vulgaris and L. forbesi, previously undescribed epidermal structures were found. They are missing in S. officinalis embryos. These so-called “extruding structures” are located near Hoyle's organ and first appear at stage XIII of Naef. At the same embryonic stage, Hoyle's organ starts to differentiate and “uniform-type” ciliated cells become visible in the epidermis of both L. vulgaris and L. forbesi. Directly after hatching the epidermis of the species examined starts to slough off and finally the extruding structures, Hoyle's organ and both types of ciliated cells of the mantle epithelium disappear. The function of the extruding structures remains obscure.     

Highly Variable Microsatellites in the California Market Squid Loligo opalescens

Attempts to study the genetic population structure of cephalopods are impeded by the low levels of genetic variation in these species. We have developed polymerase chain reaction (PCR) primers for six hypervariable microsatellite markers in order to analyze the molecular population structure in the Californian market squid Loligo opalescens. Each of these genomic loci has been cloned and fully sequenced. Here we report the sequence and properties of the six PCR primer sets for the amplification of hypervariable microsatellites. Heterozygosity levels in six squid samples from different locations are high for all loci tested. Received February 17, 1999; Accepted March 10, 1999     

Characterization of microsatellite markers in the squid,Loligo bleekeri (Cephalopoda: Loliginidae)

Loligo bleekeri has a long spawning season and the size of mature males changes during the season: dimorphic (large/small) early in the spawning season and monomorphic (small) later in the spawning season. To understand how copulatory behaviours relate to the dimorphism, we developed five polymorphic microsatellite loci in L. bleekeri. The level of polymorphism ranged from 10 to 22 alleles with expected heterozygosities ranging from 0.79 to 0.93, suggesting that the novel polymorphic loci should be useful for parentage analysis of L. bleekeri.     

Isolation and characterization of highly polymorphic microsatellites in the chokka squid, Loligo reynaudii

The chokka squid, Loligo reynaudii, is the target of a commercially valuable fishery in South Africa, but little information is available on population structure and mating system. We developed 11 polymorphic microsatellite markers using both standard nonenrichment and enrichment protocols. Numbers of alleles per locus ranged from 11 to 30, and levels of expected heterozygosity ranged between 0.77 and 0.98. Three loci developed using an enriched library displayed significant departure from Hardy-Weinberg genotype proportions suggesting high frequencies of null alleles. The remaining loci should prove useful for population and parentage studies.     

VMS-based fishing effort and population demographics for the European squid (Loligo vulgaris) off the Portuguese coast

This study presents a 1-year synopsis of the trawl fishery for the European squid Loligo vulgaris in Portuguese waters, integrating length-structured landings with corresponding geo-referenced fishing activities. From vessel monitoring system (VMS) data, landings and biological sampling, a “status-report” was obtained for 2003. Fishing pressure was found to be most intense in inshore areas of the northwest and the south coasts. Population size structure, classified into three categories, was not uniform throughout the fishing areas. Larger squid are found offshore in the northwest (International Council for the Exploration of the Sea—ICES rectangle 10E0) and in the south (rectangles 2E1 and 3E1), whereas all inshore western coast rectangles showed larger proportions of small squid relative to the southern rectangles. The analysis carried out in this study provides an insight into the possible impact of the fishing intensity pattern on the population structure in various zones. The results demonstrate the benefits of combining geo-referenced fisheries information with landings and size data, to produce explicit spatio-temporal information that can contribute to integrated planning and management for the sustainable exploitation of this resource.     

Intense ultrasonic clicks from echolocating toothed whales do not elicit anti-predator responses or debilitate the squid Loligo pealeii

Toothed whales use intense ultrasonic clicks to echolocate prey and it has been hypothesized that they also acoustically debilitate their prey with these intense sound pulses to facilitate capture. Cephalopods are an important food source for toothed whales, and there has probably been an evolutionary selection pressure on cephalopods to develop a mechanism for detecting and evading sound-emitting toothed whale predators. Ultrasonic detection has evolved in some insects to avoid echolocating bats, and it can be hypothesized that cephalopods might have evolved similar ultrasound detection as an anti-predation measure. We test this hypothesis in the squid Loligo pealeii in a playback experiment using intense echolocation clicks from two squid-eating toothed whale species. Twelve squid were exposed to clicks at two repetition rates (16 and 125 clicks per second) with received sound pressure levels of 199-226 dB re1 microPa (pp) mimicking the sound exposure from an echolocating toothed whale as it approaches and captures prey. We demonstrate that intense ultrasonic clicks do not elicit any detectable anti-predator behaviour in L. pealeii and that clicks with received levels up to 226 dB re1 microPa (pp) do not acoustically debilitate this cephalopod species.     

Characterization of the phosphorylation sites of the squid (Loligo pealei) high-molecular-weight neurofilament protein from giant axon axoplasm

Axonal caliber in vertebrates is attributed, in part, to the extensive phosphorylation of NFM and NFH C-terminal tail domain KSP repeats by proline-directed kinases. The squid giant axon, primarily involved in rapid impulse conduction during jet propulsion motility, is enriched in squid-specific neurofilaments, particularly the highly phosphorylated NF-220. Of the 228 serine-threonine candidate phosphate acceptor sites in the NF-220 tail domain (residues 401-1220), 82 are found in numerous repeats of three different motifs SAR/K, SEK/R, K/RSP, with 62 of these tightly clustered in the C-terminal repeat segment (residues 840-1160). Characterization of the in vivo NF-220 phosphorylated sites should provide clues as to the relevant kinases. To characterize these sites, proteolytic digests of NF-220 were analyzed by a combination of HPLC, electrospray tandem mass spectrometry and database searching. A total of 53 phosphorylation sites were characterized, with 47 clustered in the C-terminal repeat segment (residues 840-1160), representing 76% (47/62) of the total acceptor sites in the region. As in mammalian NFH, approximately 64% of the K/RSP sites (14/22) in this region were found to be phosphorylated implicating proline-directed kinases. Significantly, 78% of serines (31/40) in the KAES*EK and EKS*ARSP motifs were also phosphorylated suggesting that non proline-directed kinases such as CKI may also be involved. This is consistent with previous studies showing that CKI is the principal kinase associated with axoplasmic NF preparations. It also suggests that phosphorylation of large macromolecules with multiple phospho-sites requires sequential phosphorylation by several kinases.     

Microsatellites reveal high levels of gene flow among populations of the California squid Loligo opalescens

Information on the extent of genetic differentiation among populations of the squid Loligo opalescens is crucial for the conservation of this commercially utilized species. We analysed six highly variable microsatellite loci in 11 collections of L. opalescens from different locations and spawning seasons to estimate the relative influence of two major evolutionary forces, gene flow and genetic drift. Microsatellite allele frequency patterns suggest that gene flow prevents population differentiation in L. opalescens. Tests for genetic differentiation showed homogeneity of the samples with an overall FST/RST of 0.0028/-0.0013. Genetic uniformity among samples from different year classes indicates that allele frequency patterns in L. opalescens are relatively stable over time. However, a more complete and detailed picture of fine-scale allele frequency shifts in this species will require a systematic microsatellite analysis of local populations over consecutive spawning cycles.     

The isolation and characterization of the major glutathione S-transferase from the squid Loligo vulgaris

1. The major glutathione S-transferase (GST) from the common squid Loligo vulgaris has been purified and shown to be a homodimer of subunit molecular mass 24,000 and pI 6.8. 2. It has high activity towards 1-chloro-2,4-dinitrobenzene, p-nitrobenzyl chloride, 4-hydroxynon-2-enal and linoleic acid hydroperoxide, low activity with 1,2-dichloro-4-nitrobenzene and no activity with ethacrynic acid, trans-4-phenyl-3-buten-2-one and 1,2-epoxy-3-(p-nitrophenoxy)propane. 3. The L. vulgaris GST did not cross-react with any of the available polyclonal antibodies raised against mammalian GSTs. 4. Forty amino acids of its N-terminal sequence have been determined. 5. Its activities and primary structure are compared with related proteins from other species.     

A periodic structure in the 'pen' chitin of the squid Loligo vulgaris

The organization of the chitin-proteoglycan in Loligo vulgaris pen was examined ultrastructurally and related to the molecular order indicated by X-ray diffraction. There is a centrosymmetric striated repeat of 22 nm in the system which is based upon dark and light bands of unequal width. The banding is orientated perpendicular to the direction of the major molecular axis of the chitin fibres. The chitin molecules are laid down in sheets with a mutual, though irregular, twist to produce a laminated 'plywood' material.