Power matters in closing the phenotyping gap

Much of our understanding of physiology and metabolism is derived from investigating mouse mutants and transgenic mice, and open-access platforms for standardized mouse phenotyping such as the German Mouse Clinic (GMC) are currently viewed as one powerful tool for identifying novel gene-function relationships. Phenotyping or phenotypic screening involves the comparison of wild-type control mice with their mutant or transgenic littermates. In our study, we explored the extent to which standardized phenotyping will succeed in detecting biologically relevant phenotypic differences in mice generated and provided by different collaborators. We analyzed quantitative metabolic data (body mass, energy intake, and energy metabolized) collected at the GMC under the current workflow, and used them for statistical power considerations. Our results demonstrate that there is substantial variability in these parameters among lines of wild-type C57BL/6 (B6) mice from different sources. Given this variable background noise in mice that serve as controls, subtle phenotypes in mutant or transgenic littermates may be overlooked. Furthermore, a phenotype observed in one cohort of a mutant line may not be reproducible (to the same extent) in mice coming from a different environment or supplier. In the light of these constraints, we encourage researchers to incorporate information on intrastrain variability into future study planning, or to perform advanced hierarchical analyses. Both will ultimately improve the detectability of novel phenotypes by phenotypic screening. Carola W. Meyer and Ralf Elvert contributed equally to this work.     

A male gift to its partner? Cyanogenic glycosides in the spermatophore of longwing butterflies (<Emphasis Type="Italic">Heliconius</Emphasis>)

Males of several insect species transfer nuptial gifts to females during mating, typically in the form of a protein-rich spermatophore. In chemically defended species, males could potentially enhance such a gift with chemicals that help protect the female, her eggs, or both. This was shown for lepidopteran species that accumulate pyrrolizidine alkaloids. Most Heliconius butterflies are presumably protected from predators by virtue of de novo synthesized and/or sequestered cyanogenic glycosides. Males of Heliconius species are known to transfer nutritional gifts to the females but whether defensive chemicals could also be transferred is not known. To ascertain whether transfer of cyanogens occurs, we dissected freshly mated females from nine different Heliconius species and analyzed spermatophores for cyanogenic glycosides. We found cyanogens in the spermatophores of all nine species. This is the first time cyanogenic glycosides are reported in the spermatophores of arthropods. We discuss the implications of these findings for Heliconius biology and for other cyanogenic insects as well. We suggest that chemically defended species commonly lace their nuptial gifts with defensive chemicals to improve gift quality.     

Prenatal cannibalism in an insect

Host selection and infection strategies of parasitoids often correlate with high parental investment and low numbers of progeny. In this study, we investigate how additional internal mechanisms might shape brood size and fitness of the offspring. Emblemasoma auditrix is a parasitoid fly in which about 38 larvae hatch simultaneously in utero. After host location, a single larva is deposited into the host, where it rapidly develops and pupates after about 5 days. The search for hosts can take several weeks, and during that time, the larvae arrest their development and remain in the first larval instar. Nevertheless, the larvae increase in weight within the uterus, and this growth correlates to a decrease in the number of larvae, although no larvae are deposited. Thus, our data indicate a first case of prenatal cannibalism in an invertebrate with larvae feeding on each other within the uterus of the adult.     

Experience,but not distance,influences the recruitment precision in the stingless bee <Emphasis Type="Italic">Scaptotrigona mexicana</Emphasis>

Recruitment precision, i.e. the proportion of recruits that reach an advertised food source, is a crucial adaptation of social bees to their environment. Studies with honeybees showed that recruitment precision is not a fixed feature, but it may be enhanced by factors like experience and distance. However, little is known regarding the recruitment precision of stingless bees. Hence, in this study, we examined the effects of experience and spatial distance on the precision of the food communication system of the stingless bee Scaptotrigona mexicana. We conducted the experiments by training bees to a three-dimensional artificial patch at several distances from the colony. We recorded the choices of individual recruited foragers, either being newcomers (foragers without experience with the advertised food source) or experienced (foragers that had previously visited the feeder). We found that the average precision of newcomers (95.6 ± 2.61%) was significantly higher than that of experienced bees (80.2 ± 1.12%). While this might seem counter-intuitive on first sight, this “loss” of precision can be explained by the tendency of experienced recruits to explore nearby areas to find new rewarding food sources after they had initially learned the exact location of the food source. Increasing the distance from the colony had no significant effect on the precision of the foraging bees. Thus, our data show that experience, but not the distance of the food source, affected the patch precision of S. mexicana foragers.     

Evidence of rich microbial communities in the subsoil of a boreal acid sulphate soil conducive to greenhouse gas emissions

The largest areas of acid sulphate (AS) soils in Europe are located in Finland, where 67,000–130,000 ha of AS soils are in agricultural use. In addition to their acidifying effects on waters, AS soils might be a significant source of greenhouse gases. In this pilot research, carbon and nitrogen content and microbial activity were studied in an AS and a non-AS soil. Large carbon and nitrogen stocks (110 Mg C_org ha^?1 and 15 Mg N_tot ha^?1) as well as high substrate induced respiration (33 μg CO₂–C g^?1h^?1) were found in the C horizons of the AS soil but not in the non-AS soil. High microbial activity in these horizons of the AS soil was further confirmed by the measurement of dehydrogenase activity, basal respiration, the numbers of culturable bacterial cells, and the ratio of culturable to total numbers of cells. Still, the denitrifying enzyme activity was very low in the anaerobic horizons of the AS soil, indicating the prevalence of microbes other than denitrifiers. We suspect that the microbial community originated with the genesis of AS soil and has been supported by the large stocks of accumulated carbon and mineral nitrogen in the C horizons. If these permanently water-saturated subsoils are exposed to oxygen and their microbial activity consequently increases, large carbon and nitrogen stocks are likely to be mobilised, resulting in increased emission of greenhouse gases. Additional studies of boreal AS soils are needed to assess their potential contribution to increases in greenhouse gas fluxes at the local, regional, and global scales.     

An in vitro/in vivo screening assay as a sensitive tool to assess endocrine disruptive activity in surface water

Adult male fathead minnow were exposed for 14 or 28-days under flow-through conditions to undiluted filtered water samples from the rivers Meuse and Rhine in the Netherlands. The experiment included two vessels per treatment each containing 10 fish and samples of five fish were taken after 14 and 28 days. Additional groups were exposed to 17alpha-ethinylestradiol (EE2) as a reference and untreated drinking water as a negative control. Major endpoints examined included induction of vitellogenin (VTG) synthesis, VTG mRNA activity, hepato- and gonadosomatic indices (HSI and GSI) and gonadal histology. No significant difference was recorded in body weight or mean GSI values between the various treatments. Only exposure to Meuse water resulted in significantly higher HSI means after 14 days. Histological examination showed no apparent effects on gonadal tissue except for eosinophilic blood plasma in fish exposed to Meuse water or EE2. After 14 and 28 days, elevated VTG and VTG mRNA levels were measured in most livers of the fish exposed to Meuse water, but not in the fish exposed to Rhine water. This was confirmed by measuring estrogenic responses in the in vitro ER CALUX assay. Induction of VTG synthesis proved to be the most sensitive endpoint in the Non Spawning Male Fish Assay for in vivo detection of bio-available estrogenic activity supplementary to a sensitive in vitro assay. The other endpoints examined varied too much and required a higher number of fish or replicates to achieve sufficient power for statistical testing making them less animal friendly.     

Presence, biotransformation and effects of sulfophenylcarboxylic acids in the benthic fish Solea senegalensis

The presence of linear alkylbenzene sulfonates (LAS) and their degradation intermediates, sulfophenylcarboxylic acids (SPCs), with concentrations up to 100 ppb has been found in surface waters taken from the estuary of the river Guadalete (Cádiz, SW of Spain). Higher concentrations were found at the sampling site located adjacent to the discharge outlet of a wastewater treatment plant (WWTP). The concentrations decreased downstream to a few ppb as a result of dilution, sorption, and degradation processes, which were taking place along the estuary. Once the presence of both xenobiotics was confirmed in the environment, an in vivo assay was conducted to study their biotransformation and effects in the benthic fish Solea senegalensis. A flow-through system was employed, consisting of an exposure phase (120 h) with environmental levels of the surfactant (200, 500 and 800 microg/L of 2?C(10)LAS), followed by a depuration stage (72 h). The generation of SPCs has been quantified during these phases in both water and fish, with LAS biotransformation shown in all cases. The antioxidant enzymes catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), the phase II enzyme glutathione S-transferase (GST), and the phase III acid and alkaline phosphatases (AcP, ALP) were also estimated and utilized as biomarkers.     

The use of a metallothionein-like-proteins (MTLP) kinetic approach for metal bioavailability monitoring in dredged material

Ecotoxicological effects associated with contaminants present in dredged material from Spain were determined using a marine biotest based on the determination of metallothionein-like-protein concentrations (MTLPs) in the shore crab Carcinus maenas. Intermoult female C. maenas were exposed in the laboratory to sediments from three Spanish ports, Ría de Huelva (SW, Spain), La Coru?a (NW, Spain) and Bahía de Cádiz (SW, Spain) per replicate during 21 days. Hepatopancreas samples from crabs were taken for metallothioneins analysis on days 0, 7 and 21. Furthermore, chemical analysis was performed in the stations to determine the degree and nature of sediment contamination (Cr, Ni, Cu, Zn, Cd, Pb, Hg, As, PAHs and PCBs). A significant increase (p<0.05) in metallothionein concentration was observed over time in individuals exposed to sediment from the port Ría de Huelva characterized by high concentrations of metals. A toxicokinetic approach is proposed in this study related to the use of this biomarker in C. maenas to evaluate bioavailability associated with metals present in dredged material. As a first step, this toxicokinetic approach might reveal as a sensitive tool for evaluating bioavailability of contaminants present in dredged material.     

A new Dasypodini armadillo (Xenarthra: Cingulata) from San Gregorio Formation,Pliocene of Venezuela: affinities and biogeographic interpretations

We describe Pliodasypus vergelianus gen. et sp. nov., a Dasypodini armadillo from the middle Pliocene of Venezuela (Vergel Member, San Gregorio Formation). Although scarce, the remains are remarkable because of their geochronologic proximity to the main phase of Great American Biotic Interchange (GABI). The cladistic analysis conducted reveals that Pliodasypus groups with Dasypus and both are sister taxa of Propraopus, whereas Anadasypus is at a basal position. With respect to the records of tribe Dasypodini, after its oldest representative (Anadasypus, middle and late Miocene), the chronologically subsequent form is Pl. vergelianus (middle Pliocene), followed by Dasypus bellus in higher northern latitudes (late Pliocene), and then by widespread occurrences in the Pleistocene of North America (D. bellus) and South America (Propraopus, Dasypus punctatus, and Dasypus novemcinctus). Thus, we infer that Dasypus differentiated in the late Pliocene at low latitudes in the northern South America. It leads to two alternative hypotheses of dispersal: (a) some early Dasypus remained cryptically in South America until the Pleistocene, whereas others dispersed to North America between 2.2 and 2.7 Ma, or (b) they dispersed to North America subsequently to the emersion of the Panamanian isthmus and D. bellus differentiated there; later, during the Pleistocene, D. bellus entered South America and experienced speciation. The same process of re-ingression has been proposed to other xenarthrans, breaking with the traditional assumption that the GABI was unidirectional.     

A time-compressed simulated geomagnetic storm influences the nest-exiting flight angles of the stingless bee Tetragonisca angustula

Insects have been used as models for understanding animal orientation. It is well accepted that social insects such as honeybees and ants use different natural cues in their orientation mechanism. A magnetic sensitivity was suggested for the stingless bee Schwarziana quadripunctata, based on the observation of a surprising effect of a geomagnetic storm on the nest-exiting flight angles. Stimulated by this result, in this paper, the effects of a time-compressed simulated geomagnetic storm (TC-SGS) on the nest-exiting flight angles of another stingless bee, Tetragonisca angustula, are presented. Under an applied SGS, either on the horizontal or vertical component of the geomagnetic field, both nest-exiting flight angles, dip and azimuth, are statistically different from those under geomagnetic conditions. The angular dependence of ferromagnetic resonance (FMR) spectra of whole stingless bees shows the presence of organized magnetic nanoparticles in their bodies, which indicates this material as a possible magnetic detector.