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.     

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.     

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.     

Distribution and dietary regulation of an associated facultative Rhizobiales-related bacterium in the omnivorous giant tropical ant,Paraponera clavata

We document a facultative Bartonella-like Rhizobiales bacterium in the giant tropical ant, Paraponera clavata. In a lowland tropical rainforest in Costa Rica, 59 colonies were assayed for the prevalence of the Bartonella-like bacterium (BLB), 14 of which were positive. We addressed three questions: First, how does the prevalence of BLB within colonies vary with environmental conditions? Second, how does diet affect the prevalence of BLB in P. clavata? Third, how does the distribution of BLB among colonies reflect ambient differences in food resources and foraging habits? A variety of environmental variables that may be predictive of the presence of BLB were measured, and diet manipulations were conducted to test whether the prevalence of BLB responded to supplemental carbohydrate or prey. The ambient frequency of BLB is much higher in young secondary forests, but is nearly absent from older secondary forests. The prevalence of BLB inside field colonies increased over the duration of a 2-week carbohydrate supplementation; however, water and prey supplementation did not alter the prevalence of BLB. The diets of the colonies located in young secondary forest, compared to other habitats, have a diet richer in carbohydrates and lower in prey. The abundance of carbohydrate, or the relative lack of N, in a colony’s diet influences the occurrence of the BLB microbe in P. clavata. As experimental diet manipulations can affect the facultative presence of an N-cycling microbe, a consistent diet shift in diet may facilitate the emergence of tighter symbioses.     

Insights into the role of age and social interactions on the sexual attractiveness of queens in an eusocial bee, <Emphasis Type="Italic">Melipona flavolineata</Emphasis> (Apidae,Meliponini)

The attraction of sexual partners is a vital necessity among insects, and it involves conflict of interests and complex communication systems among male and female. In this study, we investigated the developing of sexual attractiveness in virgin queens (i.e., gynes) of Melipona flavolineata, an eusocial stingless bee. We followed the development of sexual attractiveness in 64 gynes, belonging to seven age classes (0, 3, 6, 9, 15, 18 days post-emergence), and we also evaluated the effect of different social interactions (such as competition between queens and interactions with workers) on the development of attractiveness in other 60 gynes. We used the number of males that tried to mate with a focal gyne as a representative variable of its sexual attractiveness. During the essays, each gyne was individually presented to 10 sexually mature males, and during 3 min, we counted the number of males that everted their genitalia in response to the presence of a gyne. Here, we show that M. flavolineata gynes are capable to (i) maintain their sexual attractiveness for long periods through adult life, (ii) they need a minimum social interaction to trigger the development of sexual attractiveness, and (iii) that gynes express this trait only within a social context. We conclude that the effective occurrence of matings is conditional on potential social interactions that gynes experienced before taking the nuptial flight, when they are still in the nest. These findings bring insights into the factors determining reproductive success in social insects.     

Bone histology sheds light on the nature of the “dermal armor” of the enigmatic sauropod dinosaur <Emphasis Type="Italic">Agustinia ligabuei</Emphasis> Bonaparte, 1999

Agustinia ligabuei is an Early Cretaceous sauropod dinosaur from the northwest of Patagonia that is currently the topic of debate with respect to its phylogenetic position and atypical dermal armor. The presence of four morphotypes of laminar and transversely elongated putative osteoderms was used to consider Agustinia as an armored sauropod. Regarding the different hypotheses about the identity of the bony structures of Agustinia (e.g., osteoderms, cervical or dorsal ribs, hypertrophied elements), a comparative histological analysis has been carried out. Histological evidence is presented herein and reveals that none of the morphotypes of Agustinia shows a primary bone tissue formed by structural fiber bundles as in other sauropod dinosaur osteoderms. Furthermore, on the basis of their gross morphology and microstructure, the bony structures originally classified as types 1 + 4 and 3 are more comparable respectively with dorsal and cervical ribs than any other kind of dermal or bony element. Due to poor preservation, the nature of the type 2 cannot be assessed but is here tentatively assigned to a pelvic girdle element. Although a phylogenetic reassessment of Agustinia is not the purpose of this paper, our paleohistological analyses have broader implications: by not supporting the dermal armor hypothesis for Agustinia, its inclusion in Lithostrotia is not justified in the absence of other diagnostic features.