Physiological and biochemical investigations on egg stickiness in common carp

The properties and behaviour of common carp, Cyprinus carpio, eggs in water and in ovarian fluids were studied at different temperature, pH, and with divalent cation concentrations. The biochemical composition of zona radiata externa (ZRE) was analyzed qualitatively and quantitatively on amino acids, carbohydrates, uronic acid and sialic acids using chemical assays; on proteins using electrophoresis. Comparative biochemical studies were performed on the chub, Leuciscus cephalus, the vimba, Vimba vimba and the bleak, Chalcalburnus chalcoides. Eggs of common carp became sticky within seconds after mixing with water. Egg stickiness was not affected by water pH in a range of 6-9, by water temperatures between 4 and 30 degrees C, by divalent cations in concentrations < or =20 mmol/l, and by sodium chloride concentrations < or =50 mmol/l. Our investigations indicated that specific proteins of the cyprinid ovarian fluid are controlling (inhibiting) the initiation of egg stickiness: egg stickiness did not develop as long as the eggs were incubated in ovarian fluid. When however the ovarian fluid proteins were removed from the ovarian fluid by heat treatment, eggs developed stickiness within seconds, like they do in water. Biochemically, the ZRE consisted of nine types of proteins whereby four of them were glycoproteins. Glucose, fructose, galactose, and uronic acids were the major carbohydrates. Treatment of the egg membrane with invertase or amyloglucosidase did not affect the egg stickiness. Treatment with protease prevented stickiness. From these results and from additional histochemical results, we conclude that glycoproteins are likely to be the molecules responsible for stickiness.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins

Enzyme-linked immunosorbent assays (ELISAs) are applied for the quantification of a vast diversity of small molecules. However, ELISAs require that the antigen is present in a soluble form in the sample. Accordingly, the few ELISAs described so far targeting insoluble proteins such as integral membrane and scaffold proteins have been restricted by limited extraction efficiencies and the need to establish an individual solubilization protocol for each protein. Here we describe a sandwich ELISA that allows the quantification of a diverse array of synaptic membrane and scaffold proteins such as munc13-1, gephyrin, NMDA R1 (N-methyl-d-aspartate receptor subunit 1), synaptic vesicle membrane proteins, and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors). The assay is based on initial solubilization by the denaturing detergent sodium dodecyl sulfate (SDS), followed by partial SDS removal using the detergent Triton X-100, which restores antigenicity while keeping the proteins in solution. Using recombinant standard proteins, we determined assay sensitivities of 78 ng/ml to 77 pg/ml (or 74-0.1 fmol). Calibration of the assay using both immunoblotting and mass spectroscopy revealed that in some cases correction factors need to be included for absolute quantification. The assay is versatile, allows parallel processing and automation, and should be applicable to a wide range of hitherto inaccessible proteins.     

Influence of nest-floor slope on the nest choice of laying hens

Group nests in alternative housing systems for laying hens primarily fulfil the hen's needs for seclusion and protection. Commercial nests used in Switzerland are built according to the provisions of the Swiss Animal Welfare Legislation. However, nest types can differ in aspects, such as floor slope, that could have an impact on egg-laying behaviour. Floor slope has to be designed so that eggs roll away without breaking and so that hens feel comfortable laying their eggs. In commercial nests, the slope is usually between 12% and 18%. The aim of this study was to investigate the effect of floor slope on the hen's nest preference and laying behaviour. We predicted that hens would prefer nests with a lower sloped floor for evolutionary reasons and for reasons related to comfort.Eight pens, each with 17-18 white laying hens (LSL), were equipped with two roll-away nests (0.54 m²) having different floor slopes (12% and 18%). Eggs were collected each day (from approximately 20 weeks of age until 28 weeks of age); the number of eggs in each nest and on the floor of the pens was recorded. Behaviour inside the nest was filmed for two consecutive days during the main egg-laying time from the second hour to the fifth hour (4 h) after lights came on in week 27/28. The following data were recorded: number of hens in each nest, the nest visits/egg number ratio, the number of sitting events, the body alignment of hens sitting in the nest and the number and duration of nest visits. Data were analysed with a repeated-measures ANOVA. There was no difference between the numbers of eggs in the two nests, but more hens were counted in nests with a 12% slope (p = 0.027). The ratio between the number of nest visits and number of eggs did not differ significantly between the nests. However, we counted more sitting events in the nest with 12% slope (p = 0.007). The percentage of body alignment towards the back (p = 0.044) and towards the front (p = 0.028) of the nest differed between the nests. Furthermore, for nest visits lasting between 10 and 90 min, we found significant differences in the total number of nest visits (p = 0.039). For visits in this range of duration, we also found significant differences for nest visits with sitting (p = 0.025) and for the number of nest visits with egg laying (p = 0.049). All of these differences favoured the 12% nest.Both nests were generally accepted by the hens. However, because of the higher number of hens counted in the 12% nest and the higher amounts of nest visits and sitting events found in these nests, we recommend to use nests with a floor slope of 12% rather than 18%.     

Proteomic Analysis of Chinook Salmon (Oncorhynchus tshawytscha) Ovarian Fluid

The ovarian, or coelomic, fluid that is released with the egg mass of many fishes is increasingly found to play an important role in several biological processes crucial for reproductive success. These include maintenance of oocyte fertility and developmental competence, prolonging of sperm motility, and enhancing sperm swimming speed. Here we examined if and how the proteome of chinook salmon (Oncorhynchus tshawytscha) ovarian fluid varied among females and then sought to examine the composition of this fluid. Ovarian fluid in chinook salmon was analyzed using 1D SDS PAGE and LC-MS/MS tryptic digest screened against Mascot and Sequest databases. We found marked differences in the number and concentrations of proteins in salmon ovarian fluid across different females. A total of 174 proteins were identified in ovarian fluid, 47 of which were represented by six or more peptides, belonging to one of six Gene Ontology pathways. The response to chemical stimulus and response to hypoxia pathways were best represented, accounting for 26 of the 174 proteins. The current data set provides a resource that furthers our understanding of those factors that influence successful egg production and fertilisation in salmonids and other species.     

Embryonic development in the Patagonian red snail Odontocymbiola magellanica (Neogastropoda: Volutidae): Morphology and biochemistry

Embryo morphology, feeding mechanism and changes in composition of the egg capsule content during development (intracapsular fluids and embryos) were studied in Odontocymbiola magellanica from newly spawned egg capsules to the pre-hatching juvenile stage. Changes in embryo morphology and behavior are presented, based on observations and micrographs of living specimens and scanning electron microscopy. The arrangement of velar cilia and athrocytes and shell gland location and development differed markedly from other studied caenogastropods. Embryo ingestion of intracapsular fluid was promoted by velar ciliary currents at least from the early veliger stage, while feeding by grazing on the inner membranous layer of the egg capsule was rarely observed until juveniles were about to hatch. The main growth of embryos occurred during the veliger stages. A significant nutritional investment in egg capsules, as compared with other South American volutids was observed. Nutrition from proteins seemed to predominate at the expense of a high molecular weight fraction (>220 kDa). Calcium concentration in the intracapsular fluid remained constant during development, but notably, the total intracapsular content (i.e., the amount contained in both fluid and embryos) increased 3-fold, which may be explained by extraction from the egg capsule magnesium-rich calcite cover, or alternatively, by uptake of calcium from the surrounding sea water. Ammonia, a major end-product of nitrogen metabolism in marine invertebrates, was present in both embryos and intracapsular fluid, from which it may easily diffuse to the surrounding sea water through the egg capsule wall. Our results on embryo morphology, development and biochemical changes provide useful comparative data for evolutionary and developmental studies in the Volutidae as well as in other caenogastropods.     

Parental size and environmental conditions affect egg capsule production by Nassarius reticulatus (Linnaeus 1758) (Gastropoda: Nassariidae)

In shallow coastal habitats scavenging netted whelks Nassarius reticulatus attached egg capsules to the stipes of red algae Chondrus crispus and occasionally on Furcellaria lumbricalis and Plumaria plumose. In the laboratory egg capsules were laid on aquaria sides and lids by individuals ≥ 21 mm shell length. Larger size classes produced more egg capsules and spawned over a longer period and in doing so partitioned less energy into shell growth. Large netted whelks (25-28.9 mm) produced larger capsules which contained significantly more and larger eggs than those produced by smaller individuals (21-24.9 mm). Egg capsule production continued throughout the year by regularly fed N. reticulatus held at ambient seawater temperatures. Egg production increased in the spring and summer with peak production during June (15 °C), decreased between August and October and resumed again during the winter (November to February at ∼ 7 °C). During the summer (15-16 °C) egg capsules were smaller and contained smaller eggs than those deposited during the winter (7-10 °C), although the number of eggs · capsule⁻¹ was similar. Enforced food limitation reduced the number and size of the egg capsules, the number and size of eggs produced · female⁻¹ and the duration of the breeding period. Hatching success of N. reticulatus egg capsules was high (95%) even at winter seawater temperatures (11-8.5 °C) and the duration of embryonic development was fastest between 15 and 17.5 °C.     

Effect of essential dietary fatty acids on egg production and hatching success of the marine copepod Temora longicornis

The calanoid copepod Temora longicornis and its food (seston of size < 200 μm) was sampled during three successive seasons (2002-04) in the Trondheimsfjord, Central Norway. Egg production (24 h) and hatching success (72 h) was determined by incubation experiments, and the essential fatty acid (EFA) content of their in situ food was analysed. The dominant EFA in the seston were DHA (22:6n-3) and EPA (20:5n-3), whereas ARA (20:4n-6) were present in low quantities. Egg production and hatching success was relatively low during early spring and late autumn (~ 10 eggs female^- 1 day^- 1 and ~ 30%), and relatively high during summer. Spring phytoplankton, dominated by diatoms, contained low amounts of DHA. Dinoflagellates, small flagellates, and ciliates dominated during summer, when a high content of DHA was recorded.The rate of egg production of T. longicornis did not show any relationship with food concentration (r² = 0.003), but was positively correlated to temperature, although not statistically significantly (r² = 0.48, p = 0.05). The quantitative and percent DHA contents of the food was significantly related to the rate of egg production (r² = 0.96 and 0.95, respectively, p < 0.001), but no such relationship were observed for the quantitative or percent content of EPA and ARA in the seston. The egg production of T. longicornis during May-August was 43-47 eggs female^- 1 day^- 1, with dietary DHA contents higher than 7-8 mg DHA g^- 1 DW. Also the hatching success of T. longicornis was positively correlated to the quantitative content of DHA in the diet (r² = 0.88), but hatching was also inversely related to the percentage ARA (r² = 0.84). The maximum hatching success was found when the ARA content was < 0.15% of total fatty acids and the DHA:ARA ratio was > 50. The conclusion that DHA most strongly affected egg production whereas ARA affected hatching, fit well with earlier findings for fish. Our results do not exclude that toxic aldehydes interact with reproduction of calanoid copepods when diatoms are present, but we observed a consistent pattern where dietary DHA and ARA alone explained a majority of the variability in egg production and hatching of T. longicornis.     

Bemerkungen zur Biologie sowie Haltung und Vermehrung des Sokotra-Riesengeckos, Haemodracon riebeckii (Peters, 1882)

The Socotra Giant Gecko, Haemodracon riebeckii, is the largest species of lizard on Socotra Island. The nocturnal, arboreal and rupiculous living geckos are omnivorous. Two pairs were kept in terrariums and were fed with various insects (crickets, locusts, cockroaches), sweet fruits and other feeding stuff (such as meat, fish). Temporarily H. riebeckii was kept together with other lizards (Eublepharis macularius, Trachylepis socotrana), without any signs of aggressive behaviour. Juveniles and adults of both sexes are able to produce a sound. These acoustic signals seem to be related to predators, because never any intraspecific function could be observed. Within seven years of captive breeding two females produced 253 eggs. Usually two white and sticky soft-shelled eggs were laid within one clutch, more rarely a single egg was laid. The two eggs of a clutch were always laid on the same day. H. riebeckii belongs to the geckos that bury their eggs and practice some brood care, but no special parental care. The female is able to proof with her hind legs the deep and shape of a hollow in the substrate to bury the eggs, which were buried in a sticky and soft-shelled condition. They are oval in shape (egg length 16.4-19.8 mm, egg width 12.4-17.8 mm, quotient EL:EW 1.22±0.05) and have in the beginning a weight ranging from 1.7100 to 2.5201 g. As typical for geckos with hard-shelled eggs the egg weight decreases during the incubation period. The loss can be between 5.59 to 30.29%. The development of eggs up to hatching of young depends upon temperature and the germinal stage in the laid egg. The time difference between the hatching of the young within one clutch of two eggs was usually 1 to 5 days. In some cases there were, however, longer differences (up to 61 days), which are probably caused by different developmental stages of the embryos during the time of egg laying. The shortest incubation period recorded during our investigations was 83 days for eggs incubated at constant temperature of 28 to 29.5 °C and the longest 359 days at 26 to 26.5 °C. Constant high incubation temperatures caused a premature hatching of young. In normal hatched young were the yolk sac retracted and the navel closed. In premature hatched young were the yolk not resorbed and the mortality within the first three month comparatively high. The snout-vent length (SVL) of newly hatched young is from 27 to 39 mm and the tail length (TL) from 25 to 38 mm (SVL:TL index 0.90-1.27), the weight is from 0.7688 to 1.5366 g. Young specimens are distinguished from adults by the brown/white striped lower jaw and the white-banded tail. Young which hatched in the terrarium were eaten by the adults. A loss of young can be avoided if they are raised individually.     

Development of cryopreservation protocols for early stage zebrafish (Danio rerio) ovarian follicles using controlled slow cooling

Cryopreservation of germplasm of aquatic species offers many benefits to the fields of aquaculture, conservation and biomedicine. Although successful fish sperm cryopreservation has been achieved with many species, there has been no report of successful cryopreservation of fish embryos and late stage oocytes which are large, chilling sensitive and have low membrane permeability. In the present study, cryopreservation of early stage zebrafish ovarian follicles was studied for the first time using controlled slow freezing. The effect of cryoprotectant, freezing medium, cooling rate, method for cryoprotectant removal, post-thaw incubation time and ovarian follicle developmental stage were investigated. Stages I and II ovarian follicles were frozen in 4 M methanol and 3 M DMSO in either L-15 medium or KCl buffer. Ovarian follicle viability was assessed using trypan blue, FDA + PI staining and ADP/ATP assay. The results showed that KCl buffer was more beneficial than L-15 medium, methanol was more effective than DMSO, optimum cooling rates were 2-4 °C/min, stepwise removal of cryoprotectant improved ovarian follicle viability significantly and stage I ovarian follicles were more sensitive to freezing. The results also showed that FDA + PI staining and ADP/ATP assay were more sensitive than TB staining. The highest follicle viabilities after post-thaw incubation for 2 h obtained with FDA + PI staining were 50.7 ± 4.0% although ADP/ATP ratios of the cryopreserved follicles were significantly increased indicating increased cell damage. Studies are currently being carried out on in vitro maturation of these cryopreserved ovarian follicles.     

Regulation of the proliferative activity of ovarian surface epithelial cells by follicular fluid

Despite critical roles of the ovarian surface epithelium (OSE) in ovulation and post-ovulatory wound repair, little is known about the physiological mechanism regulating OSE proliferation. A role of follicles and corpora lutea in locally regulating the proliferative activity of OSE has been suggested. In this study, the effects of follicular and luteal products on proliferation of cultured OSE cells were tested using cells obtained from seasonally anoestrous ewes. Follicular fluid but not luteal extracts induced OSE cell proliferation (2.5-fold relative to untreated controls; P < 0.0001). The response of OSE cells was not affected by follicle size or previous charcoal-extraction of follicular fluid (P > 0.1). Treatment with IGF-1 (2.2-fold; P < 0.01), EGF (1.9-fold; P < 0.01) and, to a lesser extent, FSH (P < 0.05) also induced OSE cell proliferation. In contrast, oestradiol or progesterone did not induce cell proliferation or enhance the effects of FSH on proliferation (P > 0.1). It was concluded that follicular fluid can directly stimulate ovine OSE cell proliferation and that this effect is attributable to non-steroidal mitogens.     

Relatively low upper threshold temperature in lizards from cool habitats

We used the slender forest skink (Scincella modesta) as a model animal to test for the hypothesis that the upper threshold of incubation temperature is relatively low in lizards using shaded (and thus, cool) habitats. Eight gravid females were collected in early May 2005 from a population in Hangzhou, Zhejiang (eastern China). All females laid a single clutch of 7–13 eggs between mid-May and early June. Eggs were incubated at 24, 28 and 30 (±0.2) °C. None of eggs incubated at 30 °C hatched. Eggs incubated at 24 and 28 °C differed in incubation length but not in hatching success. The incubation length at 24 and 28 °C averaged 22.3 and 20.3 days, respectively. Hatchlings from eggs incubated at 24 and 28 °C did not differ in all examined morphological traits, but hatchlings from eggs incubated at 28 °C performed apparently worse in the racetrack than did their counterparts from eggs incubated at 24 °C. The temperature of 28 °C is close to the upper thermal threshold for successful embryonic development in S. modesta. Compared to other oviparous lizards using open (and thus, warm) habitats, the upper thermal threshold and the range of optimal temperatures for embryonic development are both lower in S. modesta. Our study supports the previous conclusion that species living in thermally different habitats may differ in the upper thermal threshold and the range of optimal temperatures for embryonic development.     

Ovarian follicular dynamics and plasma steroid concentrations are not significantly different in ewes given intravaginal sponges containing either 20 or 40 mg of fluorogestone acetate

Although various progestagens are often used to induce and synchronize estrus and ovulation in ruminants, concerns regarding residues are the impetus to develop alternative approaches, including reduced doses of progestagens. Therefore, the objective was to determine whether ovarian function was affected by halving the dose of fluorogestone acetate in intravaginal sponges for synchronizing ovulation in sheep during the physiologic breeding season. Twenty Manchega ewes, 4-6-year-old, were randomly allocated to receive an intravaginal sponge containing either 20 mg (P20, n = 10) or 40 mg of fluorogestone acetate (P40, n = 10). Cloprostenol (125 μg) was given at sponge insertion, and all sponges were removed after 6 d. Ovarian follicular dynamics (monitored by daily ultrasonography) and other aspects of ovarian function did not differ significantly between the two groups. Ovulatory follicles (OF) grew at a similar growth rate (r = 0.62; P < 0.001), with comparable initial and maximum diameters (4.2 ± 0.4 to 6.0 ± 0.3 mm in P20 vs. 4.6 ± 0.6 to 5.7 ± 0.2 mm in P40, mean ± S.E.M.). Plasma estradiol concentrations (determined once daily) increased linearly during the 72 h interval after sponge removal (1.3 ± 0.1 to 3.3 ± 0.1 pg/mL for P20, P < 0.005 and 1.4 ± 0.1 to 3.1 ± 0.2 pg/mL for P40, P < 0.005). Ten days after sponge removal, ovulation rates (1.2 ± 0.2 for P20 and 1.4 ± 0.3 for P40), and plasma progesterone concentrations (3.8 ± 0.35 ng/mL for P20 and 3.9 ± 0.38 ng/mL for P40) were similar. In conclusion, reducing the dose of fluorogestone acetate from 40 to 20 mg did not affect significantly ovarian follicular dynamics or other aspects of ovarian function.     

Using S-adenosyl-l-homocysteine capture compounds to characterize S-adenosyl-l-methionine and S-adenosyl-l-homocysteine binding proteins

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K_d values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established K_d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.     

Developing an in vitro method for Eimeria tenella attachment to its preferred and non-preferred intestinal sites

A frozen section method utilising chicken intestinal tissue was developed to study the Eimeria tenella attachment ex vivo. In order to examine Eimeria-epithelial cell attachment, 10⁵E. tenella sporozoites were incubated with each caecal frozen section (6, 10 and 14 μm) for 1 h in 5% CO₂ incubator at 41 °C. E. tenella sporozoites attached successfully to enterocytes in 14 μm thick of caecal sections. Sporozoite attachment to caecal sections was shown to be dependent on the number of parasites added. To evaluate the method, E. tenella sporozoites were incubated to its preferred (caecum) and non-preferred (duodenum and jejunum) intestinal sites. The number of sporozoites attached to the caecal enterocytes was significantly greater (P < 0.0001) in comparison with the limited number of sporozoites attached to enterocytes of non-preferred intestinal sites. This method was shown to be able to reveal differences in binding capability and allows for comparison of intestinal site attachment.     

Thermal dependence of reproductive allocation in a tropical lizard

In ectotherms, environmental temperature is the most prominent abiotic factor that modulates life-history traits. We explored the influence of environmental temperature on reproduction in the Madagascar ground gecko (Paroedura picta) by measuring reproductive traits of females at constant temperatures (24, 27, 30 °C). Females of this species lay clutches of one or two eggs within short intervals. For each female, we measured egg mass for the first five clutches. For one clutch, we also measured the energetic content of eggs via bomb calorimetry. Temperature positively influenced the rate of egg production, but females at 30 °C laid smaller eggs than did females at either 24 or 27 °C. Dry mass of eggs scaled allometrically with wet mass, but this relationship was similar among thermal treatments. Females at all temperatures produced eggs with similar energy densities. Females at 24 °C allocated less energy per time unit (≈8 mW) to reproduction than did females from higher temperatures (≈12 mW). However, females at either 24 or 27 °C allocated significantly more energy per egg than did females at 30 °C. Our results demonstrate that a complex thermal sensitivity of reproductive rate can emerge from distinct thermal sensitivities of egg size, egg composition and clutch frequency.     

Penicillin-binding protein 2x of Streptococcus pneumoniae: three new mutational pathways for remodelling an essential enzyme into a resistance determinant

Mutations in the transpeptidase domain of penicillin-binding protein 2x (PBP2x) of Streptococcus pneumoniae that reduce the affinity to beta-lactams are important determinants of resistance to these antibiotics. We have now analyzed in vitro and in vivo properties of PBP2x variants from cefotaxime-resistant laboratory mutants and a clinical isolate. The patterns of two to four resistance-specific mutations present in each of the proteins, all of which are placed between 6.6 and 24 Å around the active site, fall into three categories according to their positions in the three-dimensional structure. The first PBP2x group is characterized by mutations at the end of helix α11 and carries the well-known T550A change and/or one mutation on the surface of the penicillin-binding domain in close contact with the C-terminal domain. All group I proteins display very low acylation efficiencies, ≤ 1700 M^− 1 s^− 1, for cefotaxime. The second class represented by PBP2x of the mutant C505 shows acylation efficiencies below 100 M^− 1 s^− 1 for both cefotaxime and benzylpenicillin and contains the mutation L403F at a critical site close to the active serine. PBP2x of the clinical isolate 669 reveals a third mutational pathway where at least the two mutations Q552E and S389L are important for resistance, and acylation efficiency is reduced for both beta-lactams to around 10,000 M^− 1 s^− 1. In each group, at least one mutation is located in close vicinity to the active site and mediates a resistance phenotype in vivo alone, whereas other mutations might exhibit secondary effects only in context with other alterations.     

Bioenergetics of early life-history stages of the brachyuran crab Cancer setosus in response to changes in temperature

In many marine invertebrates, a latitudinal cline in egg size is considered an adaptive response to a decrease in temperature, and enhances the energetic fitness of their larvae at hatching. However, the amount of energy carried over from the egg to the larval stage depends on the metabolic efficiency of egg development. In the present study, eggs of the brachyuran crab Cancer setosus were sampled for their dry mass (DM), carbon (C), nitrogen (N), and fatty acid (FA) content throughout development from blastula stage until hatching of zoea 1-larvae at Antofagasta (23°S) and Puerto Montt 41°S (Chile) under different temperature treatments (12, 16 and 19 °C). Hatching zoea 1 larvae contained 60 ± 3% of the initial blastula egg C content, regardless of site or temperature. However, the ontogenetic decrease in egg C content was to a significantly higher extend based on the utilization of energy-rich FA at 12 °C (− 1.16 µg/egg) compared to the 19 °C treatments in Antofagasta and Puerto Montt (− 0.63 to − 0.73 µg FA per egg). At 19 °C egg-metabolism was based to a substantial extend on protein, which allowed for the saving of energy-richer lipids. We conclude that the production of larger eggs with high FA content appears to be adaptive not only to fuel the larval development, but is also a response to the prolonged egg developmental times at lower temperatures.