Both d- and l-specific Lactate Dehydrogenases Co-exist in Individual Cephalopods

Both d-lactate-specific and l-lactate-specific lactate dehydrogenases coexist in individual cephalopods, contrary to the commonly held view that invertebrate species may contain one or other, but not both. We describe the tissue distribution of these lactate dehydrogenases and of octopine dehydrogenase, the major pyruvate reductase activity in cephalopods, in three species: common squid (Loligo vulgaris), cuttlefish (Sepia officinalis) and lesser octopus (Eledone cirrhosa). The l-specific lactate dehydrogenase of squid is shown to be a dimer of 36,000 dalton subunits.     

The secret life of the giant Australian cuttlefish Sepia apama (Cephalopoda): Behaviour and energetics in nature revealed through radio acoustic positioning and telemetry (RAPT)

Sepia apama were tagged with acoustic transmitters and monitored on their native House Reef, Boston Bay, South Australia, with a radio acoustic positioning telemetry (RAPT) system. Cuttlefish were tagged with position-only and intra-mantle jet pressure transmitters. New data analyses were developed to handle problem data that arise with an uneven reef environment. Maximum range for the cuttlefish varied from 90 m to 550 m. Cuttlefish home range was between 5300 m² and 23,700 m². S. apama were found to be diurnal as average distance travelled was higher in the day than at night, and cuttlefish were active for 32 days, but only 18 nights. After the cuttlefish settled into reef crevices, activity spectrum and positioning analysis showed foraging behaviour at only 3.7% per day and 2.1% per night. Cuttlefish were found to spend more than 95% of the day resting, which suggests that their bioenergetics are more akin to those of octopus than of squid. The cuttlefish combination of predator avoidance, efficient foraging and quiescent lifestyle allows energy to be channelled into growth and fulfillment of the live-fast-die-young cephalopod philosophy.     

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.     

Factors influencing the embryonic development and hatchling size of the oceanic squid Illex coindetii following in vitro fertilization

Egg masses of oceanic squid accidently collected in the wild have been observed only from a few spawning events in aquaria, and as a consequence, the study of their embryos and hatchlings is very limited. Here, we used in vitro fertilization techniques to understand the abiotic factors that influenced egg development and hatchling performance of the ommastrephid squid Illex coindetii from the Mediterranean Sea. Egg and hatchling sizes of these ommastrephids are close to the minimum for cephalopod species, and therefore, short embryonic developmental periods were expected under a Mediterranean temperature regime. Low incubation densities and the use of antibiotics in the incubation medium resulted in relatively high survival rates to hatchling. Hatching time was inversely proportional to incubation temperature and ranged from 5 to 11 days at 21 °C and 13 °C, respectively. The addition of oviducal jelly to the eggs immediately after fertilization, followed by another addition at the beginning of organogenesis, resulted in better chorionic expansion, with an increase in egg diameter, delayed hatching and higher hatchling weight, which suggests a better use of yolk reserves. Larger hatchlings in length and weight also tended to be produced at low temperature, probably also due to better yolk absorption. As this species spawns all year round, size differences between summer and winter hatchlings may be suspected. In vitro fertilization techniques used in the laboratory proved to be useful in shedding some light on the early life of this oceanic squid. Further research is needed to elaborate on tests to predict gamete quality and to develop methods to avoid premature hatching.     

“Spinner” cephalopods: defects of statocyst suprastructures in an invertebrate analogue of the vestibular apparatus

Summary Individuals of seven species of coleoid cephalopods (three species of octopus, three of squid, and one of cuttlefish), that were cultured and reared in laboratory aquarium systems, had a behavioral defect at hatching which was characterized by an inability to control orientation while swimming. These defective animals were designated as spinners.An examination of statocysts from individuals of five of the affected species revealed abnormalities of the neuroepithelial suprastructures: absence or malformation of the statolith of the gravity receptor system and absence of the cupulae of the angular acceleration receptor systems. The sensory epithelia did not differ from those of normal animals, nor did the synaptic structures and relationships, when examined both with scanning and transmission electron microscopy. The abnormalities were compared with congenital defects of the neuropeithelial suprastructures of the vestibular apparatus (especially in mammals). The defects observed in statocysts of spinner animals are thought to be the result of environmental causes, such as the temperature or chemistry of the seawater in the transportation vessels or rearing systems, rather than genetic causes.     

Growth and tissue composition as a function of feeding history in juvenile cephalopods

We present the results of a series of experiments that examined the effect of feeding history on the growth and tissue composition of juveniles of two tropical cephalopods; the squid Sepioteuthis lessoniana and the cuttlefish Sepia elliptica. Juveniles were reared in individual containers for between 35 and 42 days at different ration levels, three ration levels for the squid and two levels for the cuttlefish. Although differences in ration were sufficient to cause different growth rates, both in body length and mass, the effects on tissue composition were less definitive. Sepioteuthis juveniles on the highest rations had higher concentrations of water, but no difference in lipid, carbohydrate or protein when compared with their lower ration siblings. In the case of juvenile cuttlefish no difference in tissue composition was detected between the two ration levels. RNA:protein ratios were also determined for the juveniles to provide an estimate of instantaneous growth. A significant correlation was found between body size and RNA:protein ratio in the squid; those juveniles that ate more had higher RNA:protein ratios than lower ration individuals. Significantly, the juvenile cuttlefish showed no relationship between growth rate and RNA:protein ratios, which means that we are unable to use this measure to estimate the growth rates of wild individuals. In conclusion, ration level did affect growth rates and food availability is an important factor in modifying growth rates of wild individuals. However, we could not find, at the individual level, an index or measure that could be used to explain the variability of observed differences in growth rates as a function of nutritional history.     

Cuttlefish Sepia officinalis Preferentially Respond to Bottom Rather than Side Stimuli When Not Allowed Adjacent to Tank Walls

Cuttlefish are cephalopods capable of rapid camouflage responses to visual stimuli. However, it is not always clear to what these animals are responding. Previous studies have found cuttlefish to be more responsive to lateral stimuli rather than substrate. However, in previous works, the cuttlefish were allowed to settle next to the lateral stimuli. In this study, we examine whether juvenile cuttlefish (Sepia officinalis) respond more strongly to visual stimuli seen on the sides versus the bottom of an experimental aquarium, specifically when the animals are not allowed to be adjacent to the tank walls. We used the Sub Sea Holodeck, a novel aquarium that employs plasma display screens to create a variety of artificial visual environments without disturbing the animals. Once the cuttlefish were acclimated, we compared the variability of camouflage patterns that were elicited from displaying various stimuli on the bottom versus the sides of the Holodeck. To characterize the camouflage patterns, we classified them in terms of uniform, disruptive, and mottled patterning. The elicited camouflage patterns from different bottom stimuli were more variable than those elicited by different side stimuli, suggesting that S. officinalis responds more strongly to the patterns displayed on the bottom than the sides of the tank. We argue that the cuttlefish pay more attention to the bottom of the Holodeck because it is closer and thus more relevant for camouflage.     

Gradients of strain and strain rate in the hollow muscular organs of soft-bodied animals

The cylindrical shape of soft-bodied invertebrates is well suited to functions in skeletal support and locomotion, but may result in a previously unrecognized cost—large non-uniformities in muscle strain and strain rate among the circular muscle fibres of the body wall. We investigated such gradients of strain and strain rate in the mantle of eight long-finned squid Doryteuthis pealeii and two oval squid Sepioteuthis lessoniana. Transmural gradients of circumferential strain were present during all jets (n = 312); i.e. for a given change in the circumference of the outer surface of the mantle, the inner surface experienced a greater proportional change. The magnitude of the difference increased with the amplitude of the mantle movement, with circular muscle fibres at the inner surface of the mantle experiencing a total range of strains up to 1.45 times greater than fibres at the outer surface during vigorous jets. Differences in strain rate between the circular fibres near the inner versus the outer surface of the mantle were also present in all jets, with the greatest differences occurring during vigorous jetting. The transmural gradients of circumferential strain and strain rate we describe probably apply not only to squids and other coleoid cephalopods, but also to diverse soft-bodied invertebrates with hollow cylindrical or conical bodies and muscular organs.     

Feeding habits of the Atlantic bluefin tuna, Thunnus thynnus (L. 1758), in the central Mediterranean Sea (Strait of Messina)

The study of feeding habits of the Atlantic bluefin tuna was carried out in 123 specimens, ranging from 115 to 222 cm fork length (FL) and collected during spring seasons of 2010 and 2011 in the central Mediterranean Sea (Strait of Messina). The analysis of stomach contents allowed us to identify 91 taxa of prey items, mainly belonging to Teleostea (54), Cephalopoda (20) and Crustacea (13). The percentage of index of relative abundance (IRI) shows the highest values for the myctophid Hygophum benoiti (%IRI = 22.854) and the stomiid Chauliodus sloani (%IRI = 15.124), followed by the oegopsid squid Illex coindetii (%IRI = 14.316). The broad spectrum of prey items could suggest a generalist behavior of this predator, with several species that occasionally occurs in its diet. However, if prey are grouped into food categories, the importance of mesopelagic and benthopelagic fishes can be appreciated (54.41 % of %IRI). The assessment of the hypothetical foraging rhythm of the Atlantic bluefin tuna highlighted that its feeding activity is concentrated on diel migrating fauna during night and on larger preys upon daylight. The predation on the high-energetic food as mesopelagic and bathypelagic fishes during the pre-spawning and the spawning period may bring an energetic advantage in tuna metabolism and gonadal maturation     

Cuttlefish dynamic camouflage: responses to substrate choice and integration of multiple visual cues

Prey camouflage is an evolutionary response to predation pressure. Cephalopods have extensive camouflage capabilities and studying them can offer insight into effective camouflage design. Here, we examine whether cuttlefish, Sepia officinalis, show substrate or camouflage pattern preferences. In the first two experiments, cuttlefish were presented with a choice between different artificial substrates or between different natural substrates. First, the ability of cuttlefish to show substrate preference on artificial and natural substrates was established. Next, cuttlefish were offered substrates known to evoke three main camouflage body pattern types these animals show: Uniform or Mottle (function by background matching); or Disruptive. In a third experiment, cuttlefish were presented with conflicting visual cues on their left and right sides to assess their camouflage response. Given a choice between substrates they might encounter in nature, we found no strong substrate preference except when cuttlefish could bury themselves. Additionally, cuttlefish responded to conflicting visual cues with mixed body patterns in both the substrate preference and split substrate experiments. These results suggest that differences in energy costs for different camouflage body patterns may be minor and that pattern mixing and symmetry may play important roles in camouflage.     

Differences in the Pathogenicity of the p.H723R Mutation of the Common Deafness-Associated SLC26A4 Gene in Humans and Mice

Mutations in the SLC26A4 gene are a common cause of human hereditary hearing impairment worldwide. Previous studies have demonstrated that different SLC26A4 mutations have different pathogenetic mechanisms. By using a genotype-driven approach, we established a knock-in mouse model (i.e., Slc26a4^{tm2Dontuh/tm2Dontuh} mice) homozygous for the common p.H723R mutation in the East Asian population. To verify the pathogenicity of the p.H723R allele in mice, we further generated mice with compound heterozygous mutations (i.e., Slc26a4^{tm1Dontuh/tm2Dontuh}) by intercrossing Slc26a4^+/tm2Dontuh mice with Slc26a4^{tm1Dontuh/tm1Dontuh} mice, which segregated the c.919-2A>G mutation with an abolished Slc26a4 function. Mice were then subjected to audiologic assessments, a battery of vestibular evaluations, inner ear morphological studies, and noise exposure experiments. The results were unexpected; both Slc26a4^{tm2Dontuh/tm2Dontuh} and Slc26a4^{tm1Dontuh/tm2Dontuh} mice showed normal audiovestibular phenotypes and inner ear morphology, and they did not show significantly higher shifts in hearing thresholds after noise exposure than the wild-type mice. The results indicated not only the p.H723R allele was non-pathogenic in mice, but also a single p.H723R allele was sufficient to maintain normal inner ear physiology in heterozygous compound mice. There might be discrepancies in the pathogenicity of specific SLC26A4 mutations in humans and mice; therefore, precautions should be taken when extrapolating the results of animal studies to humans.     

Assessment of the exposure pathway in the uptake and distribution of americium and cesium in cuttlefish (Sepia officinalis) at different stages of its life cycle

Laboratory radiotracer experiments were performed to study the uptake, assimilation and retention of americium (²⁴¹Am) and cesium (¹³⁴Cs) by the common cuttlefish Sepia officinalis. Uptake and loss kinetics of the radionuclides were measured following exposure through sediments, seawater and food at different stages of the animal's life cycle. Sediment was found to be a minor uptake pathway for both radionuclides in juveniles. Following a short seawater exposure, cuttlefish accumulated ²⁴¹Am and ¹³⁴Cs, but only to a limited extent (whole-body CF < 2). Among the cuttlefish organs, branchial hearts and their appendages displayed the highest degree of uptake for ²⁴¹Am (CF = 42 and 16, respectively), but these tissues contained low percentage of total ²⁴¹Am due to their relatively small contribution to whole organism weight. The major fraction of incorporated radionuclides was associated with muscular tissues (viz. 65% and 82% of total ²⁴¹Am and ¹³⁴Cs, respectively). Whole-body loss of ²⁴¹Am and ¹³⁴Cs was relatively rapid (T_b½ = 14 and 6 days, respectively). After dietary exposure, around 60% and 30% of ingested ²⁴¹Am was assimilated into the tissues of juvenile and adult cuttlefish, respectively. However, assimilated ²⁴¹Am was more strongly retained in adults than in juveniles (T_b½ = 28 vs. 5 days, respectively), suggesting that different mechanisms govern ²⁴¹Am elimination at both ages. Ingested ¹³⁴Cs was assimilated to a similar extent in juveniles (29%) and adults (23%), but the depuration rate was four times faster in adults. Our results strongly suggest that these two radionuclides follow different excretion pathways and that the mechanisms can vary with age for a given radionuclide.     

Quantification of cuttlefish (Sepia officinalis) camouflage: a study of color and luminance using in situ spectrometry

Cephalopods are renowned for their ability to adaptively camouflage on diverse backgrounds. Sepia officinalis camouflage body patterns have been characterized spectrally in the laboratory but not in the field due to the challenges of dynamic natural light fields and the difficulty of using spectrophotometric instruments underwater. To assess cuttlefish color match in their natural habitats, we studied the spectral properties of S. officinalis and their backgrounds on the Aegean coast of Turkey using point-by-point in situ spectrometry. Fifteen spectrometry datasets were collected from seven cuttlefish; radiance spectra from animal body components and surrounding substrates were measured at depths shallower than 5 m. We quantified luminance and color contrast of cuttlefish components and background substrates in the eyes of hypothetical di- and trichromatic fish predators. Additionally, we converted radiance spectra to sRGB color space to simulate their in situ appearance to a human observer. Within the range of natural colors at our study site, cuttlefish closely matched the substrate spectra in a variety of body patterns. Theoretical calculations showed that this effect might be more pronounced at greater depths. We also showed that a non-biological method (“Spectral Angle Mapper”), commonly used for spectral shape similarity assessment in the field of remote sensing, shows moderate correlation to biological measures of color contrast. This performance is comparable to that of a traditional measure of spectral shape similarity, hue and chroma. This study is among the first to quantify color matching of camouflaged cuttlefish in the wild.     

Freezing behaviour facilitates bioelectric crypsis in cuttlefish faced with predation risk

Cephalopods, and in particular the cuttlefish Sepia officinalis, are common models for studies of camouflage and predator avoidance behaviour. Preventing detection by predators is especially important to this group of animals, most of which are soft-bodied, lack physical defences, and are subject to both visually and non-visually mediated detection. Here, we report a novel cryptic mechanism in S. officinalis in which bioelectric cues are reduced via a behavioural freeze response to a predator stimulus. The reduction of bioelectric fields created by the freeze-simulating stimulus resulted in a possible decrease in shark predation risk by reducing detectability. The freeze response may also facilitate other non-visual cryptic mechanisms to lower predation risk from a wide range of predator types.     

Temperature effects on hemocyanin oxygen binding in an antarctic cephalopod

The functional relevance of oxygen transport by hemocyanin of the Antarctic octopod Megaleledone senoi and of the eurythermal cuttlefish Sepia officinalis was analyzed by continuous and simultaneous recordings of changes in pH and hemocyanin oxygen saturation in whole blood at various temperatures. These data were compared to literature data on other temperate and cold-water cephalopods (octopods and giant squid). In S. officinalis, the oxygen affinity of hemocyanin changed at deltaP50/degrees C = 0.12 kPa (pH 7.4) with increasing temperatures; this is similar to observations in temperate octopods. In M. senoi, thermal sensitivity was much smaller (<0.01 kPa, pH 7.2). Furthermore, M. senoi hemocyanin displayed one of the highest levels of oxygen affinity (P50 < 1 kPa, pH 7.6, 0 degrees C) found so far in cephalopods and a rather low cooperativity (n50 = 1.4 at 0 degrees C). The pH sensitivity of oxygen binding (delta log P50/delta pH) increased with increasing temperature in both the cuttlefish and the Antarctic octopod. At low PO2 (1.0 kPa) and pH (7.2), the presence of a large venous oxygen reserve (43% saturation) insensitive to pH reflects reduced pH sensitivity and high oxygen affinity in M. senoi hemocyanin at 0 degrees C. In S. officinalis, this reserve was 19% at pH 7.4, 20 degrees C, and 1.7 kPa O2, a level still higher than in squid. These findings suggest that the lower metabolic rate of octopods and cuttlefish compared to squid is reflected in less pH-dependent oxygen transport. Results of the hemocyanin analysis for the Antarctic octopod were similar to those reported for Vampyroteuthis--an extremely high oxygen affinity supporting a very low metabolic rate. In contrast to findings in cold-adapted giant squid, the minimized thermal sensitivity of oxygen transport in Antarctic octopods will reduce metabolic scope and thereby contribute to their stenothermality.     

Contribution of pilA to Competitive Colonization of the Squid Euprymna scolopes by Vibrio fischeri

Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis. Hatchling squid lack these bacterial symbionts, and V. fischeri strains must compete to occupy this privileged niche. We cloned a V. fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins. Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly. Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene. In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization. In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing. The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V. fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used. ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain. V. fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V. fischeri isolates apparently possess pilA. The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.     

Unified theory of 1f and conductance noise in nerve membrane

A theory of $\text{1}\text{f}$ and conductance noise is given for ionic channels in nerve membrane. The theory is based on the assumption that the channels are in constant, stochastically independent, rotational motion within a fluid bilayer membrane. The resulting expression for the current noise power density S contains a conduction noise term consistent withStevens (1972) and Hill & Chen (1972) and a $\text{1}\text{f}$ noise term consistent with Lundstrom & McQueen (1974) and Clay & Shlesinger (1976). The expression for S also contains a third term which is the spectrum of the product of the single channel conduction noise and $\text{1}\text{f}$ noise correlation functions. This term is independent of the number of channels in the membrane, R. Consequently, the expression for S effectively reduces to a sum of $\text{1}\text{f}$ and conduction noise for R 10–100 which is in agreement with noise measurements on squid axon. The theory is applied in detail to potassium squid noise measurements of Conti, DeFelice & Wanke (1975) using the stochastic analysis of single file ion motion developed in our previous paper (Clay & Shlesinger (1976)).     

Exposure to road traffic noise and cognitive development in schoolchildren in Barcelona,Spain: A population-based cohort study

BackgroundRoad traffic noise is a prevalent and known health hazard. However, little is known yet about its effect on children’s cognition. We aimed to study the association between exposure to road traffic noise and the development of working memory and attention in primary school children, considering school-outdoor and school-indoor annual average noise levels and noise fluctuation characteristics, as well as home-outdoor noise exposure.Methods and findingsWe followed up a population-based sample of 2,680 children aged 7 to 10 years from 38 schools in Barcelona (Catalonia, Spain) between January 2012 to March 2013. Children underwent computerised cognitive tests 4 times (n = 10,112), for working memory (2-back task, detectability), complex working memory (3-back task, detectability), and inattentiveness (Attention Network Task, hit reaction time standard error, in milliseconds). Road traffic noise was measured indoors and outdoors at schools, at the start of the school year, using standard protocols to obtain A-weighted equivalent sound pressure levels, i.e., annual average levels scaled to human hearing, for the daytime (daytime LAeq, in dB). We also derived fluctuation indicators out of the measurements (noise intermittency ratio, %; and number of noise events) and obtained individual estimated indoor noise levels (LAeq) correcting for classroom orientation and classroom change between years. Home-outdoor noise exposure at home (Lden, i.e., EU indicator for the 24-hour annual average levels) was estimated using Barcelona’s noise map for year 2012, according to the European Noise Directive (2002). We used linear mixed models to evaluate the association between exposure to noise and cognitive development adjusting for age, sex, maternal education, socioeconomical vulnerability index at home, indoor or outdoor traffic-related air pollution (TRAP) for corresponding school models or outdoor nitrogen dioxide (NO₂) for home models. Child and school were included as nested random effects.The median age (percentile 25, percentile 75) of children in visit 1 was 8.5 (7.8; 9.3) years, 49.9% were girls, and 50% of the schools were public. School-outdoor exposure to road traffic noise was associated with a slower development in working memory (2-back and 3-back) and greater inattentiveness over 1 year in children, both for the average noise level (e.g., ‒4.83 points [95% CI: ‒7.21, ‒2.45], p-value < 0.001, in 2-back detectability per 5 dB in street levels) and noise fluctuation (e.g., ‒4.38 [‒7.08, ‒1.67], p-value = 0.002, per 50 noise events at street level). Individual exposure to the road traffic average noise level in classrooms was only associated with inattentiveness (2.49 ms [0, 4.81], p-value = 0.050, per 5 dB), whereas indoor noise fluctuation was consistently associated with all outcomes. Home-outdoor noise exposure was not associated with the outcomes. Study limitations include a potential lack of generalizability (58% of mothers with university degree in our study versus 50% in the region) and the lack of past noise exposure assessment.ConclusionsWe observed that exposure to road traffic noise at school, but not at home, was associated with slower development of working memory, complex working memory, and attention in schoolchildren over 1 year. Associations with noise fluctuation indicators were more evident than with average noise levels in classrooms.

In a cohort study, Maria Foraster and colleagues study associations between exposure to road traffic noise at schools, and trajectories of working memory and inattentiveness among schoolchildren aged 7 to 10 years in Barcelona, Spain.