Assignment of paternity groups without access to parental genotypes: multiple mating and developmental plasticity in squid

We present a novel approach to investigating sibling relationships and reconstructing parental genotypes from a progeny array. The Bayesian method we have employed is flexible and may be applicable to a variety of situations in addition to the one presented here. While mutation rates and breeding population allele frequencies can be taken into account, the model requires relatively few loci and makes few assumptions. Paternity of 270 veined squid (Loligo forbesi) hatchlings from three egg strings collected from one location was assigned using five microsatellite loci. Paternal and maternal genotypes reconstructed for each of the three strings were identical, strongly indicating that a single female produced the strings that were fertilized by the same four males. The proportion of eggs fertilized was not equal between males in all three strings, with male 1 siring most offspring (up to 68% in string 1), through to male 4 siring the least (as low as 2.4% in string 1). Although temperature had a profound effect on incubation time, paternity did not affect this trait at 12 degrees C or 8 degrees C.     

Mating games squid play: reproductive behaviour and sexual skin displays in Caribbean reef squid Sepioteuthis sepioidea

Observation of the sexual interactions of Sepioteuthis sepioidea squid during the short reproductive stage of their lives showed a scramble competition system, with both male and female polygyny. Mature females were faithful to a specific location in the daytime, whereas males moved from group to group and formed short-term consortships with females. Males defended females from other males, particularly with an agonistic Zebra display. Male–female pairs exchanged Saddle-Stripe displays, after which males might display an on–off Flicker. There was considerable female choice. Only if a female responded to this display with a parallel Rocking action would she pair and would the male deposit spermatophores at the base of her arms, and only 50% of the time did females move the spermatophores internally to where sperm might be released and stored in the oviducal gland for later fertilization of eggs. This long-term set of interactions and solitary deposition of hidden egg strings contrasts with the attraction of both sexes to a common ‘egg mop’ laid by many females which was a site of competition in other loliginid squid. Since Sepioteuthis is a primitive genus within the family Loliginidae, it may represent a generalist reproductive strategy that evolved into a specialized localization one.     

Vampire squid: detritivores in the oxygen minimum zone

Vampire squid (Vampyroteuthis infernalis) are considered phylogenetic relics with cephalopod features of both octopods and squids. They lack feeding tentacles, but in addition to their eight arms, they have two retractile filaments, the exact functions of which have puzzled scientists for years. We present the results of investigations on the feeding ecology and behaviour of Vampyroteuthis, which include extensive in situ, deep-sea video recordings from MBARI''s remotely operated vehicles (ROVs), laboratory feeding experiments, diet studies and morphological examinations of the retractile filaments, the arm suckers and cirri. Vampire squid were found to feed on detrital matter of various sizes, from small particles to larger marine aggregates. Ingested items included the remains of gelatinous zooplankton, discarded larvacean houses, crustacean remains, diatoms and faecal pellets. Both ROV observations and laboratory experiments led to the conclusion that vampire squid use their retractile filaments for the capture of food, supporting the hypothesis that the filaments are homologous to cephalopod arms. Vampyroteuthis'' feeding behaviour is unlike any other cephalopod, and reveals a unique adaptation that allows these animals to spend most of their life at depths where oxygen concentrations are very low, but where predators are few and typical cephalopod food is scarce.     

Myosin-linked calcium regulation in squid mantle muscle. Light-chain components of squid myosin.

As reported by Kendrick-Jones et al. (1976), myosin from squid mantle muscle contains two types of light-chain components, different in size but similar in net charge. We were able to separate the two types of light chains by a five-step procedure, yielding LC-1 (17,000 daltons) and LC-2 (15,000 daltons). It was found that squid mantle LC-1 and LC-2 function exactly like SH-light chains and EDTA-light chains of scallop adductor myosin, respectively. In functional tests, we used "desensitized" myosin of scallop adductor muscle, simply because "EDTA washing" removed neither LC-1 nor LC-2 from squid mantle myosin. The removal and recombination of light chains were examined by gel electrophoresis, and Ca or Sr sensitivity was determined by measuring the Mg-ATPase activity of skeletal acto-scallop or squid myosin. It was found that EDTA washing readily released the EDTA-light chains of scallop myosin completely, and that the EDTA-washed scallop myosin was capable of regaining its full content of EDTA-LC as well as its full sensitivity to calcium. We also found that as regards combining with, and conferring calcium sensitivity on the EDTA-washed myosin of scallop adductor, squid mantle LC-2 could effectively replace scallop adductor EDTA-LC. In addition, calcium or strontium ions were found to induce changes in the UV absorption spectrum of scallop adductor EDTA-LC, although the apparent binding constants estimated from the difference spectrum were too low to account for the Ca or Sr sensitivity of scallop actomyosin-ATPase. The divalent cations also induced changes in the UV absorption spectrum of squid LC-2, and the apparent binding constants estimated from the difference spectrum were sufficiently high (1.5 X 10(5) M-1 for Ca binding, and 1.6 X 10(3) M-1 for Sr binding) to account for the Ca and Sr sensitivities of squid mantle myosin B-ATPase. The findings with scallop adductor myosin are in conflict with those reported by Kendrick-Jones et al., and must be accounted for in formulating the molecular mechanism of myosin-linked calcium regulation in molluscan muscles.     

Comparative expression and tissue distribution analyses of astacin-like squid metalloprotease in squid and cuttlefish

Astacin-like squid metalloprotease (ALSM) is a member of the astacin family of metalloproteases. In the present study, we investigated the expression and tissue distribution of ALSM in bigfin reef squid (Sepioteuthis lessoniana) and golden cuttlefish (Sepia esculenta). Myosin heavy chain hydrolysis tests showed ALSM-I-like activity in both species. We isolated partial cDNA clones showing high sequence similarity to ALSM-I and -III, suggesting that ALSM is common to squid and cuttlefish. Phylogenetic analysis showed that ALSMs are classified into two clades: ALSM-I forms one clade, and ALSM-II and -III form the other. ALSM was expressed in several tissues in bigfin reef squid, though expression was confined to the liver in cuttlefish. ALSMs are distributed in digestive organs but not in mantle muscle of squid and cuttlefish. Immunofluorescence analysis further showed that cellular localization of ALSM is evident not only in hepatic cells but also in pancreatic cells of bigfin reef squid. Thus, ALSM is commonly expressed in squid and cuttlefish, but its expression levels and distribution are distinct.     

The journey of squid sperm

Sperm storage is common in internally fertilizing animals, but is also present in several external fertilizers, such as many cephalopods. Cephalopod males attach sperm packets (spermatangia) to female conspecifics during mating. Females of eight externally fertilizing families comprising 25% of cephalopod biodiversity have sperm-storage organs (seminal receptacles) in their buccal area, which are not in direct physical contact with the deposited spermatangia. The mechanism of sperm transmission between the implantation site and the storage organ has remained a major mystery in cephalopod reproductive biology. Here, jumbo squid females covering almost the entire life cycle, from immature to a laboratory spawned female, were used to describe the internal structure of the seminal receptacles and the process of sperm storage. Seminal fluid was present between the spermatangia and seminal receptacles, but absent in regions devoid of seminal receptacles. The sperm cellular component was formed by spermatozoa and round cells. Although spermatozoa were tracked over the buccal membrane of the females to the inner chambers of the seminal receptacles, round cells were not found inside the seminal receptacles, suggesting that spermatozoa are not sucked up by the muscular action of the seminal receptacles. This finding supports the hypothesis that spermatozoa are able to actively migrate over the female skin. Although further experimental support is needed to fully confirm this hypothesis, our findings shed light on the elusive process of sperm storage in many cephalopods, a process that is fundamental for understanding sexual selection in the sea.     

Mastigoteuthis–the whip-lash squid

Features of the brain of this oceanic squid have been investigated and related, as far as possible, to its habits and mode of life. The body and arms are much vacuolated for buoyancy and the animal probably lives with the head upwards. The very long whip-like tentacles are not vacuolated and perhaps hang downwards. They are covered by numerous minute pedunculated suckers, perhaps providing a sticky surface. A special nerve running outside the brain carries signals from the arms and tentacles to the magnocellular lobe, which is very large and of complex structure. However, there are no giant cells and the mantle is weak. Propulsion is mainly by the large fins, which are controlled from the magnocellular lobe, presumably using the information from the arms and tentacles.     

Methylmercury: Effects on electrical properties of squid axon membranes

At low concentrations (25–100 μM) methylmercury chloride caused a steady increase in the threshold for excitation and on eventual block of action potentials without changing the resting membrane potential in squid giant axons. In the axons exposed to 25 μM methylmercury chloride, peak transient and steady-state conductances were decreased by 58.8 ± 5.1% and 35.9 ± 4.3% (mean ± SEM, 4 axons), respectively and leakage conductance increased to about five times of the control value. Higher concentrations of methylmercury chloride decreased the resting membrane potential. A concentration of 0.5 mM depolarizing the nerve membrane by 16 ± 2 mV (mean ± SEM, 3 axons) in 40 minutes. These changes in ionic conductances and membrane potential were irreversible on washing the axon with drug-free sea water.     

Automated Resonance Assignment of Proteins: 6 DAPSY-NMR

The 6-dimensional (6D) APSY-seq-HNCOCANH NMR experiment correlates two sequentially neighboring amide moieties in proteins via the C′ and C^α nuclei, with efficient suppression of the back transfer from C^α to the originating amide moiety. The automatic analysis of two-dimensional (2D) projections of this 6D experiment with the use of GAPRO (Hiller et al., 2005) provides a high-precision 6D peak list, which permits automated sequential assignments of proteins with the assignment software GARANT (Bartels et al., 1997). The procedure was applied to two proteins, the 63-residue 434-repressor(1–63) and the 115-residue TM1290. For both proteins, complete sequential assignments for all NMR-observable backbone resonances were obtained, and the polypeptide segments thus identified could be unambiguously located in the amino acid sequence. These results demonstrate that APSY-NMR spectroscopy in combination with a suitable assignment algorithm can provide fully automated sequence-specific backbone assignments of small proteins.Francesco Fiorito and Sebastian Hiller - Both authors contributed equally to this work     

The squid accessory nidamental gland: Ultrastructure and association with bacteria

The structure of the accessory nidamental gland of the female squid, Loligo pealei, has been investigated using transmission and scanning electron microscopy. The accessory gland has many of the structural features of a secretory organ. The basic structural unit is a tubule composed of a single layer of epithelial cells containing ordered arrays of rough endoplasmic reticulum and a lumenal surface covered with microvilli, cilia, and structural specializations presumed to be involved in secretion. The lumen of each tubule is filled with a dense population of bacteria. During sexual maturation of the squid, the accessory gland changes in color from white to mottled red. The accessory gland of the sexually mature squid has a mixture of red, white, and yellow tubules ; in each case, the color of the tubule is due to the bacterial population occupying the tubule. Since the red color of the gland is due to the pigmentation of the bacteria, the bacteria must be responsive to the sexual state of the host, possibly through a change in the nature of the material secreted into the tubule lumen. The bacteria can be cultured easily, but in culture they fail to synthesize the red pigment.     

Anaesthetics for the squid Sepioteuthis sepioidea (mollusca: cephalopoda)

1. Specimens of Sepioteuthis sepioidea were exposed to different reagents commonly used as anaesthetics for cold blooded animals.2. Although some reagents affected the specimens, the anesthesia state was only achieved when using ethanol, magnesium sulfate or magnesium chloride.3. The optimal ranges of concentrations necessary to achieve the anesthesia were: 1 to 3% for ethanol, 1.5 to 2% for magnesium chloride and 3 to 4% for magnesium sulfate.     

Magnesium efflux in dialyzed squid axons

The efflux of Mg++ from squid axons subject to internal solute control by dialysis is a function of ionized [Mg], [Na], [ATP], and [Na]o. The efflux of Mg++ from an axon with physiological concentrations of ATP, Na, and Mg inside into seawater is of the order of 2-4 pmol/cm2s but this efflux is strongly inhibited by increases in [Na]i, by decreases in [ATP]i, or by decreases in [Na]o. The efflux of Mg++ is largely independent of [Mg]i when ATP is at physiological levels, but in the absence of ATP reaches half the value of Mg efflux in be presence of ATP when [Mg]i is about 4 mM and [Na] 40 mM. Half-maximum responses to ATP occur at about 350 micronM ATP into seawater with Na either present or absent. The Mg efflux mechanism has many similarities to the Ca efflux system in squid axons especially with respect to the effects of ATP, Nao, and Na on the flux. The concentrations of free Mg and Ca in axoplasm differ, however, by a factor of 10(5) while the observed fluxes differ by a factor of 10(2).