Osmotic and ionic regulation in response to salinity variations and cold resistance in the Arctic under-ice amphipod <Emphasis Type="Italic">Apherusa glacialis</Emphasis>

Amphipods living at the underside of Arctic sea ice are exposed to varying salinities due to freezing and melting, and have to cope with the resulting osmotic stress. Extracellular osmotic and ionic regulation at different salinities, thermal hysteresis, and supercooling points (SCPs) were studied in the under-ice amphipod Apherusa glacialis. The species is euryhaline, capable to regulate hyperosmotically at salinities S _R < 30 g/kg, and osmoconforms at salinities S _R ≥ 30 g/kg. Hyperosmotic regulation is an adaptation to thrive in low-salinity meltwater below the ice. Conforming to the ambient salinity during freezing reduces the risk of internal ice formation. Thermal hysteresis was not observed in the haemolymph of A. glacialis. The SCP of the species was −7.8 ± 1.9°C. Several ions were specifically downregulated ([Mg²⁺], [SO₄ ²⁻]), or upregulated ([K⁺], [Ca²⁺]) in comparison to the medium. Strong downregulation of [Mg²⁺], is probably necessary to avoid an anaesthetic effect at low temperatures.     

Sympagic amphipods in the Arctic pack ice: redescriptions of <Emphasis Type="Italic">Eusirus holmii</Emphasis> Hansen, 1887 and <Emphasis Type="Italic">Pleusymtes karstensi</Emphasis> (Barnard, 1959)

During an expedition into the Arctic Ocean, in September 2004, six different species of amphipods were collected in the ice above 82°N. All six species (Apherusa glacialis, Gammarus wilkitzkii, Onisimus nanseni, O. glacialis, Pleusymtes karstensi and Eusirus holmii) were observed to be living adjacent to the sea ice or partly within its brine channels. The nature of the association with the ice for the last two species is uncertain, but the finding raises important questions regarding our knowledge of the sympagic fauna. Based on the obtained material, the two species E. holmii and P. karstensi are redescribed, and their association with the sea ice is discussed.     

Consumers of sea urchins,<Emphasis Type="Italic"> Paracentrotus lividus</Emphasis> and<Emphasis Type="Italic"> Arbacia lixula</Emphasis>, in shallow Mediterranean rocky reefs

Underwater observations on fish and asteroid consumers (i.e. predators and scavengers) of sea urchins, Paracentrotus lividus and Arbacia lixula, were carried out at several locations in shallow Mediterranean rocky reefs. Observations conducted in the marine reserve of Torre Guaceto (Adriatic Sea) revealed that sparid fishes, Diplodus sargus and D. vulgaris, are the main fish predators of small (<1 cm in test diameter) and medium (1–4 cm) sea urchins, whereas the labrids Coris julis and Thalassoma pavo preyed only upon small sea urchins. Large D. sargus were able to prey upon small and medium, and occasionally large (>4 cm) sea urchins, whereas medium and small Diplodus preyed mainly upon small sea urchins. The number of sea urchins preyed upon by fishes was negatively related to sea urchin size for both species. P. lividus appeared to be subject to higher predation levels than A. lixula. The scavenger guild comprised 11 fish species, with D. sargus, D. vulgaris, Coris julis and Chromis chromis accounting for about 80% of scavenger fishes. Observations performed at several locations in the Mediterranean on the predatory asteroid Marthasterias glacialis revealed that only 3% of the detected individuals were preying upon sea urchins. Due to the importance of sea urchins for assemblage structure and functioning of Mediterranean rocky reef ecosystems, these results may have also important implications for management of fishing activities.Communicated by H.-D. Franke     

Salinity sensitivity of early embryos of the Antarctic sea urchin, <Emphasis Type="Italic">Sterechinus neumayeri</Emphasis>

Embryos and larvae of the Antarctic sea urchin, Sterechinus neumayeri, have received considerable experimental attention assessing impacts of low temperature on development; however, salinity effects are not well documented because heretofore, the Antarctic coastal marine environment has been remarkably stenohaline. In this study, subtle decreases of 2 and 4 parts per 1,000 in standard salinity were tested to see if the developmental rate of S. neumayeri embryos would be impacted by a potential hyposmotic stress. At 30 psu, significantly fewer embryos (2 individuals out of 198 tested) reached morula stage by 36 h post-fertilization in comparison embryos in control treatments at 34 psu. Antarctic sea urchins are an important component of marine environments due to their grazing activities. Reductions in larval recruitment success due to the influx of freshwater from melting ice shelves resulting from global climate change could have far-reaching impacts on benthic ecosystem structure in Antarctica.     

A novel family of mitochondrial proteins is represented by the <Emphasis Type="Italic">Drosophila</Emphasis> genes <Emphasis Type="Italic">slmo</Emphasis>, <Emphasis Type="Italic">preli-like</Emphasis> and <Emphasis Type="Italic">real-time</Emphasis>

Mitochondria play essential roles in development and disease. The characterisation of mitochondrial proteins is therefore of particular importance. The slowmo (slmo) gene of Drosophila melanogaster has been shown to encode a novel type of mitochondrial protein, and is essential in the developing central nervous system. The Slmo protein contains a conserved PRELI/MSF1p domain, found in proteins from a wide variety of eukaryotic organisms. However, the function of the proteins of this family is currently unknown. In this study, the evolutionary relationships between members of the PRELI/MSF1p family are described, and we present the first analysis of two novel Drosophila genes predicted to encode proteins of this type. The first of these, preli-like (prel), is expressed ubiquitously during embryonic development, whilst the second, real-time (retm), is expressed dynamically in the developing gut and central nervous system. retm encodes a member of a novel conserved subclass of larger PRELI/MSF1p domain proteins, which also contain the CRAL-TRIO motif thought to mediate the transport of small hydrophobic ligands. Here we provide evidence that, like Slmo, both the Prel and Retm proteins are localised to the mitochondria, indicating that the function of the PRELI/MSF1p domain is specific to this organelle.Edited by P. Simpson     

Structure,regulation, and function of <Emphasis Type="Italic">micro1</Emphasis> in the sea urchin <Emphasis Type="Italic">Hemicentrotus pulcherrimus</Emphasis>

The animal-vegetal axis of sea urchin embryos is morphologically apparent at the 16-cell stage, when the mesomeres, macromeres, and micromeres align along it. At this stage, the micromere is the only autonomously specified blastomere that functions as a signaling center. We used a subtraction PCR survey to identify the homeobox gene micro1 as a micromere-specific gene. The micro1 gene is a representative of a novel family of paired-like class homeobox genes, along with PlHbox12 from Paracentrotus lividus and pmar1 from Strongylocentrotus purpuratus. In the present study, we showed that micro1 is a multicopy gene with six or more polymorphic loci, at least three of which are clustered in a 30-kb region of the genome. The micro1 gene is transiently expressed during early cleavage stages in the micromere. Recently, nuclear -catenin was shown to be essential for the specification of vegetal cell fates, including micromeres, and the temporal and spatial coincidence of micro1 expression with the nuclear entry of -catenin is highly suggestive. We demonstrated that micro1 is a direct target of -catenin. In addition, we showed that micro1 is necessary and sufficient for micromere specification. These observations on the structure, regulation, and function of micro1 lead to the conclusion that micro1 and pmar1 (and potentially PlHbox12) are orthologous.     

Trade-offs between microhabitat selection and physiological plasticity in the Antarctic springtail, <Emphasis Type="Italic">Cryptopygus antarcticus</Emphasis> (Willem)

In the maritime Antarctic, terrestrial arthropods have recourse to two strategies to mitigate low summer temperatures: (1) physiological plasticity and (2) avoidance via microhabitat insulation. This study investigated the interaction between these strategies in the springtail, Cryptopygus antarcticus, established in situ within contrasting microcosms (buffered vs. exposed) and within two sets of habitat simulations (wet vs. dry) over diurnal scales through the Antarctic summer. Significant differences were found in the cold hardiness of springtails sampled simultaneously from each microcosm. Exposed animals showed greater plasticity in the “true” austral summer, but as field temperatures declined preceding the onset of winter, buffered animals showed greater resilience. Overall, water was found to inhibit the buffering effect of moss and there was a significantly greater discrimination between buffered and exposed microcosms in the dry treatment. Analysis of microhabitat temperatures indicate that it is thermal variability not lower temperature that is responsible for the greater plasticity of exposed animals.     

Physiological constraints on the life cycle and distribution of the Antarctic fairy shrimp <Emphasis Type="Italic">Branchinecta gaini</Emphasis>

Maturation to adulthood and successful reproduction in the Antarctic fairy shrimp, Branchinecta gaini, must be completed within a physiologically challenging temporal window of ca. 2.5 months in the southern Antarctic Peninsula. Although adults show considerable metabolic opportunism at positive temperatures, little is known of their tolerance of two physiological insults potentially typical to pool life in the maritime Antarctic: sub-zero temperatures and salinity. B. gaini are freeze-avoiding crustaceans with temperatures of crystallisation (T _cs) of −5°C. No antifreeze proteins were detected in the haemolymph. Adults osmoregulate in relation to temperature, but rapid mortality in saline solutions of even low concentration, indicate they cannot osmoregulate in relation to salinity. Survival of ice encasement at temperatures above their T _c was found to be pressure but not time dependent: at severe inoculative ice pressures, there was little immediate survival and none survived after 48 h below −2°C; at mild inoculative ice pressures, immediate survival was ca. 100% at −3°C, but <20% after 48 h. There was no significant difference in survival after 1 and 6 h encasement at −3°C. Observations of ventilation suggest that it is not low temperature per se, but ice that represents the primary cryo-stress, with ventilatory appendages physically handcuffed below the freezing point of pool water. Both sub-zero temperatures and salinity represent real physiological constraints on adult fairy shrimp.     

Fitness and life history parameters of <Emphasis Type="Italic">Leptomastix epona</Emphasis> and <Emphasis Type="Italic">Pseudaphycus flavidulus</Emphasis>, two parasitoids of the obscure mealybug <Emphasis Type="Italic">Pseudococcus viburni</Emphasis>

Fitness and life table parameters of two endoparasitoids of the obscure mealybug Pseudococcus viburni (Signoret), the solitary Leptomastix epona (Walker) and the gregarious Pseudaphycus flavidulus (Brèthes), were examined in relation to temperature and host size with a view to determine the efficacy of the parasitoids as biocontrol agents of the pest. Three temperature levels (21°C, 26°C and 31°C) and two host sizes classes (small, which mostly comprised third instar nymphs and large, which consisted of female adults) were studied. The lower developmental threshold and thermal constant of the host and the parasitoids were found similar so the coincidence of pest and parasitoids is likely. The rate of development of the parasitoids increased with a linear trend as the temperature increased from 21°C to 31°C. Temperature had a significant effect on mummification in both parasitoid species and on successful parasitism by P. flavidulus. Host size had a significant effect on the mummification caused by L. epona and on the proportion of the male offspring which emerged as well as on the successful parasitism by P. flavidulus. Life table parameters of the parasitoids were estimated in small and large hosts at 26°C in the laboratory. Both parasitoids achieved a greater intrinsic rate of natural increase and gross reproductive rate in addition to a shorter generation and doubling time in large mealybugs compared with small ones. Consequently, large hosts are expected to have a higher impact on the rise of the parasitoids population and the potential of the parasitoids to control the mealybug population improves with the increase of host size. Handling Editor: Torsen Meiners.     

Adaptations to terrestrial overwintering of hatchling northern map turtles,<Emphasis Type="Italic"> Graptemys geographica</Emphasis>

We conducted a 3-year field and laboratory study of winter biology in hatchlings of the northern map turtle (Graptemys geographica). At our study area in northern Indiana, hatchlings routinely overwintered in their natal nests, emerging after the weather warmed in spring. Winter survival was excellent despite the fact that hatchlings were exposed frequently to subfreezing temperatures (to –5.4 °C). In the laboratory, cold-acclimated hatchlings exhibited low rates of evaporative water loss (mean=2.0 mg g^–1 day^–1), which would enable them to conserve body water during winter. Laboratory-reared hatchlings were intolerant of freezing at –2.5 °C for 24 h, conditions that are readily survived by freeze-tolerant species of turtles. Winter survival of hatchling G. geographica probably depended on their extensive capacity for supercooling (to –14.8 °C) and their well-developed resistance to inoculative freezing, which may occur when hatchlings contact ice and ice-nucleating agents present in nesting soil. Supercooled hatchlings survived a brief exposure to –8 °C. Others, held at –6 °C for 5 days, maintained ATP concentrations at control levels, although they did accumulate lactate and glucose, probably in response to tissue hypoxia. Therefore, anoxia tolerance, as evidenced by the viability of hatchlings exposed to N₂ gas for 8 days, may promote survival during exposure to subfreezing temperatures.Abbreviations EWL evaporative water loss - FP_eq equilibrium freezing point - INA ice-nucleating agents - T_c temperature of crystallizationCommunicated by L.C.-H. Wang     

Gene structure and transcriptional regulation of <Emphasis Type="Italic">dnaK</Emphasis> and <Emphasis Type="Italic">dnaJ</Emphasis> genes from a psychrophilic bacterium, <Emphasis Type="Italic">Colwellia maris</Emphasis>

The dnaK and dnaJ genes, encoding heat shock proteins, were cloned from a psychrophilic bacterium, Colwellia maris. Significant homology was evident comparing DnaK and DnaJ of the psychrophilile with the counterparts of mesophilic and thermophilic bacteria. In the DnaJ protein, three conserved regions of the Hsp40 family were observed. A putative promoter similar to the sigma32 consensus sequence was found upstream of the dnaK gene. The G+C content in the 5'-untranslated region of the dnaK gene was much lower than that in the corresponding region of mesophilic bacteria. Northern-blot analysis and primer-extension analysis showed that both genes were transcribed separately as monocistronic mRNAs. Following several temperature upshifts from 10 to 26 degrees C, maximum induction of the dnaK and dnaJ mRNAs was detected at 20 degrees C, suggesting that this temperature induces the heat shock response in this bacterium. In addition, the level of the induction of the dnaJ gene was much lower than that of the dnaK gene. These findings together revealed several specific features of the heat shock response at a relatively low temperature in psychrophiles.     

The hepatocytes of the brown trout (<Emphasis Type="Italic">Salmo trutta</Emphasis> f. <Emphasis Type="Italic">fario</Emphasis>): a stereological study of their number and size during the breeding cycle

A firm knowledge of the normal structure is crucial for evaluating pathological processes and morphofunctional correlations. Stereological liver structure characterization had its debut for mammals in the 1960s, but only in the 1980s did it start to be used in fishes. Using stereology, our aim was to verify the hypothesis that in parallel with the well-known annual seasonal changes in the liver–body ratio of brown trout, hepatocytes would vary their number and/or size, and that gender differences likely exist. Three-year-old specimens were used. Five animals per gender were examined in May (endogenous vitellogenesis), September (exogenous vitellogenesis), and February (spawning season end). The liver was fixed by perfusion, and its total volume estimated. Systematically sampled material was embedded in epoxy or in metachrylate resins. Stereology was executed on light and electron microscopy images. Unbiased design-based techniques were applied, using physical disectors and differential point counting. Target parameters were the relative (per unit volume) and total number of hepatocytes, the mean cell and nuclear volumes, and the total volumes of hepatocytes and their nuclei. Data support that in both genders the number of hepatocytes and the volume of its nucleus change along the breeding cycle. The cell number increased from endogenous to exogenous vitellogenesis (accompanying relative liver size gains), later followed by a decline in the cell number, still detectable after the spawning season. The total liver volumes of the cell and nucleus also increased from May to September in females, despite that the mean hepatocyte nuclear volume showed a minimum in September. No statistical changes in the mean cell volume were detected, regardless of the tendency for lower mean values in September. Changes were more marked in females and showed a higher correlation with the gonad weight. It was firstly suggested that numerical (rather than cell size) changes govern the shifts of the relative liver weight seen during the brown trout annual breeding cycle, and eventually of other fishes. We hypothesized that there are seasonal cycles of hepatocyte mitosis (from after spawning to exogenous vitellogenesis) and of apoptosis (at spawning). These cycles would be regulated by sex steroids, being more striking in females.     

Characterization of <Emphasis Type="Italic">Francisella</Emphasis> sp., GM2212, the first <Emphasis Type="Italic">Francisella</Emphasis> isolate from marine fish,Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella sp., isolate GM2212^T, previously isolated from diseased farmed Atlantic cod Gadus morhua in Norway is characterized. The complete 16S rDNA, 16S–23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and a hypothetical lipoprotein (LpnB) is sequenced and compared with Francisella tularensis and Francisella philomiragia. All these sequences support a close relationship between GM2212^T and F. philomiragia. The bacterium grows at 10–25°C with an optimum at about 20°C, a temperature range clearly different from F. tularensis and F. philomiragia. GM2212^T is catalase-positive, indole positive, oxidase-negative, do not produce H₂S in Triple Sugar Iron agar, and does not hydrolyze gelatin, is resistant to erythromycin and susceptible to ceftazidime, the latter five characteristics separating it from F. philomiragia. Cysteine enhances growth. Acid is produced from d-glucose, maltose, sucrose (weak) but not from lactose or glycerol. GM2212^T grows on both MacConkey agar and in nutrient broth (6% NaCl). The bacterium is resistant to trimethoprim-sulfamethoxazole, penicillines, cefuroxime and erythromycin; but is susceptible to ceftazidime, tetracycline, gentamicin, ciprofloxacin. Based on the molecular and phenotypical characteristics, we suggest that this GM2212 isolate, may represent a new species of Francisella. Isolate GM2212^T (=CNCM I-3481^T = CNCM I-3511^T = DSM 18777^T).     

Duplication of <Emphasis Type="Italic">AP1</Emphasis> within the <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L. <Emphasis Type="Italic">AP1/FUL</Emphasis> clade is followed by rapid amino acid and regulatory evolution

The AP1/FUL clade of MADS box genes have undergone multiple duplication events among angiosperm species. While initially identified as having floral meristem identity and floral organ identity function in Arabidopsis, the role of AP1 homologs does not appear to be universally conserved even among eudicots. In comparison, the role of FRUITFULL has not been extensively explored in non-model species. We report on the isolation of three AP1/FUL genes from cultivated spinach, Spinacia oleracea L. Two genes, designated SpAPETALA1-1 (SpAP1-1) and SpAPETALA1-2 (SpAP1-2), cluster as paralogous genes within the Caryophyllales AP1 clade. They are highly differentiated in the 3′, carboxyl-end encoding region of the gene following the third amphipathic alpha-helix region, while still retaining some elements of a signature AP1 carboxyl motifs. In situ hybridization studies also demonstrate that the two paralogs have evolved different temporal and spatial expression patterns, and that neither gene is expressed in the developing sepal whorl, suggesting that the AP1 floral organ identity function is not conserved in spinach. The spinach FRUITFULL homolog, SpFRUITFULL (SpFUL), has retained the conserved motif and groups with Caryophyllales FRUITFULL homologs. SpFUL is expressed in leaf as well as in floral tissue, and shows strong expression late in flower development, particularly in the tapetal layer in males, and in the endothecium layer and stigma, in the females. The combined evidence of high rates of non-synonymous substitutions and differential expression patterns supports a scenario in which the AP1 homologs in the spinach AP1/FUL gene family have experienced rapid evolution following duplication. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Annual reproductive cycle and rate of the spermatogenic process in male mosquitofish <Emphasis Type="Italic">Gambusia affinis</Emphasis>

The present study investigates the relationship between the annual cycle of testicular development and external environment and the rate of spermatogenesis in the mosquitofish Gambusia affinis based on histological observations of testes. The annual reproductive cycle of the mosquitofish was divided into two periods, i.e., the spermatogenic period (May–October) and resting period (October–April). In the spermatogenic period, the transition from spermatogonia to spermatocytes begins and meiosis actively progresses. In the resting period, the transition from spermatogonia to spermatocytes ceases, meiosis of spermatocytes that already shifted by this period gradually progresses, and a considerable number of sperm balls are produced. Onset of spermatogenesis seems to be related to both a rise in water temperature and a prolonged photoperiod. 5-bromo-2-deoxyuridine (BrdU) was a useful in vivo marker of DNA synthesizing spermatogenic cells. The results of immunohistochemical detection of injected BrdU indicated that 5 days are needed for the conversion of spermatocytes to spermatids, 5 days for spermatids to spermatozoa, and 10 days for spermatozoa to sperm balls.