Combining Motion Analysis and Microfluidics – A Novel Approach for Detecting Whole-Animal Responses to Test Substances

Small, early life stages, such as zebrafish embryos are increasingly used to assess the biological effects of chemical compounds in vivo. However, behavioural screens of such organisms are challenging in terms of both data collection (culture techniques, drug delivery and imaging) and data evaluation (very large data sets), restricting the use of high throughput systems compared to in vitro assays. Here, we combine the use of a microfluidic flow-through culture system, or BioWell plate, with a novel motion analysis technique, (sparse optic flow - SOF) followed by spectral analysis (discrete Fourier transformation - DFT), as a first step towards automating data extraction and analysis for such screenings. Replicate zebrafish embryos housed in a BioWell plate within a custom-built imaging system were subject to a chemical exposure (1.5% ethanol). Embryo movement was videoed before (30 min), during (60 min) and after (60 min) exposure and SOF was then used to extract data on movement (angles of rotation and angular changes to the centre of mass of embryos). DFT was subsequently used to quantify the movement patterns exhibited during these periods and Multidimensional Scaling and ANOSIM were used to test for differences. Motion analysis revealed that zebrafish had significantly altered movements during both the second half of the alcohol exposure period and also the second half of the recovery period compared to their pre-treatment movements. Manual quantification of tail flicking revealed the same differences between exposure-periods as detected using the automated approach. However, the automated approach also incorporates other movements visible in the organism such as blood flow and heart beat, and has greater power to discern environmentally-driven changes in the behaviour and physiology of organisms. We suggest that combining these technologies could provide a highly efficient, high throughput assay, for assessing whole embryo responses to various drugs and chemicals.     

Purification and partial characterization of a corticosteroid-binding globulin from hamster serum

Our objective was to characterize and purify the corticosteroid-binding proteins in hamster pregnancy serum. When [3H]cortisol-labeled pregnancy and proestrous serum were subjected to native polyacrylamide gel electrophoresis, a single peak of specific steroid-binding activity was detected in each, with identical electrophoretic mobility. The steroid-binding affinity (Ka = 1.07.10(8) M-1 for cortisol) is typical of corticosteroid-binding globulin from other species, but the steroid-binding specificity (cortisol greater than testosterone greater than progesterone) is not. An ultraviolet photoaffinity-labeling protocol was developed using 17 beta-hydroxy-4,6-[1,2-3H]androstadiene-3-one ([3H]androstadienolone), permitting analysis of ultraviolet photoaffinity-labeled proestrous and pregnancy serum by two-dimensional polyacrylamide gel electrophoresis and fluorography. Both sera contained the same labeled protein species. Corticosteroid-binding globulin was purified from pregnancy serum by DEAE-cellulose chromatography followed by steroid affinity chromatography on androstadienolone-17 beta-hemisuccinate-ethylenediamine-AffiGel 10. The purified protein (Mr = 62,250; pI = 3.95; n = 1; Stokes radius = 3.5; S = 4-5) was determined to be a glycoprotein. When analyzed by gel filtration and two-dimensional polyacrylamide gel electrophoresis, purified corticosteroid-binding globulin behaved the same as in unfractionated serum, and when ultraviolet photoaffinity-labeled with [3H]androstadienolone, purified corticosteroid-binding globulin produced the same fluorogram spot pattern seen in unfractionated serum. A specific corticosteroid-binding globulin antiserum was raised in rabbits, and this antiserum reacted with a single spot in Western blots of unfractionated serum. Thus, hamster pregnancy serum was determined to have one corticosteroid-binding protein. This protein is identical to the corticosteroid-binding globulin found in proestrous serum, with a higher titer in pregnancy serum. No other steroid-binding component is observed in hamster serum.     

Interactions between freshwater snails and tadpoles: competition and facilitation

Summary Freshwater snails and anuran tadpoles have been suggested to have their highest population densities in ponds of intermediate size where abiotic disturbance (e.g. desiccation) is low and large predators absent. Both snails and tadpoles feed on periphytic algae and, thus, there should be a large potential for competitive interactions to occur between these two distantly related taxa. In a field experiment we examined the relative strength of competition between two closely related snail species, Lymnaea stagnalis and L. peregra, and between L. stagnalis and tadpoles of the common frog, Rana temporaria. Snail growth and egg production and tadpole size at and time to metamorphosis were determined. Effects on the common food source, periphyton, were monitored with the aid of artificial substrates. Periphyton dry weight was dramatically reduced in the presence of snails and/or tadpoles. There were no competitive effects on growth or egg production of the two snail species when they were coexisting. Mortality of L. peregra was high (95%) after reproduction, but independent of treatment. Growth of L. stagnalis was reduced only at the highest tadpole densities, whereas egg production was reduced both by intraspecific competition and by competition with tadpoles. Differences in egg production were retained after tadpole metamorphosis. Tadpole larval period increased, weight of metamorphosing frogs decreased and growth rate was reduced as a function of increasing tadpole density. However, contrary to expectation, snails had a positive effect on tadpole larval period, weight and growth rate. Further, in experimental containers without snails there was a dense growth of the filamentous green alga Cladophora sp. We suggest that the facilitative effects of snails on tadpoles are due to an indirect mutualistic mechanism, involving competition between food sources of different quality (microalgae and Cladophora sp.) and tadpoles being competitively dominant over snails for the preferred food source (microalgae). In the presence of tadpoles snails will be forced to feed on low-quality Cladophora, increasing nutrient turnover rates, which results in enhanced productivity of microalgae, increasing tadpole food resources. Thus, tadpoles have a negative effect on snails through resource depression, while snails facilitate tadpole growth through an indirect enhancement of food availability.     

Characterization of a cDNA encoding the 55 kDa B regulatory subunit of Arabidopsis protein phosphatase 2A

Type 2A serine/threonine protein phosphatases (PP2A) are key components in the regulation of signal transduction and control of cell metabolism. The activity of these protein phosphatases is modulated by regulatory subunits. While PP2A activity has been characterized in plants, little is known about its regulation. We used the polymerase chain reaction to amplify a segment of a cDNA encoding the B regulatory subunit of PP2A from Arabidopsis. The amplified DNA fragment of 372 nucleotides was used as a probe to screen an Arabidopsis cDNA library and a full-length clone (AtB) of 2.1 kbp was isolated. The predicted protein encoded by AtB is 43 to 46% identical and 53 to 56% similar to its yeast and mammalian counterparts, and contains three unique regions of amino acid insertions not present in the animal B regulatory subunit. Genomic Southern blots indicate the Arabidopsis genome contains at least two genes encoding the B regulatory subunit. In addition, other plant species also contain DNA sequences homologous to the B regulatory subunit, indicating that regulation of PP2A activity by the 55 kDa B regulatory subunit is probably ubiquitous in plants. Northern blots indicate the AtB mRNA accumulates in all Arabidopsis tissues examined, suggesting the protein product of the AtB gene performs a basic housekeeping function in plant cells.     

Developmental expression of the murine Prl-1 protein tyrosine phosphatase gene

The expression of the murine Prl-1 protein tyrosine phosphatase gene was examined in normal embryos from E10.5 through E18.5. Prl-1 mRNA was detected in the brain, neural tube, and dorsal root ganglia, and in several non-neuronal tissues, including the skeletal system. Heart and skeletal muscle were consistently negative. At E13.5, Prl-1 was expressed in the condensing prechondrogenic cells of the vertebrae, whereas at E18.5, Prl-1 mRNA was localized to the hypertrophic chondrocytes. The dynamic expression of Prl-1 during cartilage differentiation may suggest a functional role in skeletal development.     

Divergent environments and population bottlenecks fail to generate premating isolation in Drosophila pseudoobscura

While the feasibility of bottleneck-induced speciation is in doubt, population bottlenecks may still affect the speciation process by interacting with divergent selection. To explore this possibility, I conducted a laboratory speciation experiment using Drosophila pseudoobscura involving 78 replicate populations assigned in a two-way factorial design to both bottleneck (present vs. absent) and environment (ancestral vs. novel) treatments. Populations independently evolved under these treatments and were then tested for assortative mating and male mating success against their common ancestor. Bottlenecks alone did not generate any premating isolation, despite an experimental design that was conducive to bottleneck-induced speciation. Premating isolation also did not evolve in the novel environment treatment, neither in the presence nor absence of bottlenecks. However, male mating success was significantly reduced in the novel environment treatment, both as a plastic response to this environment and as a result of environment-dependent inbreeding effects in the bottlenecked populations. Reduced mating success of derived males will hamper speciation by enhancing the mating success of immigrant, ancestral males. Novel environments are generally thought to promote ecological speciation by generating divergent natural selection. In the current experiment, however, the novel environment did not cause the evolution of any premating isolation and it reduced the likelihood of speciation through its effects on male mating success.     

Global and regional patterns in lotic meiofauna

• 1 Parsimony analysis of endemicity (PAE) was used to assess patterns in the distribution of harpacticoid copepods (all freshwater species and stream species only) at global and regional scales. These analyses provided a focus for reviewing large scale patterns and processes in freshwater meiofauna.
• 2 On a global scale, PAE suggested that large‐scale biogeographical events have been most important in shaping present‐day distributions in the Canthocamptidae. A small proportion (4%) of canthocamptid species were widespread (i.e. occurred in more than one biogegraphical region), suggesting that dispersal may also play a role in determining distribution at the species level. Global distribution patterns for other meiofauna suggest varying roles for dispersal and vicariant events. No consistent latitudinal trends in species diversity were evident, although a lack of distributional data for many regions, and uncertainty over the status of many cosmopolitan species, precludes more robust analyses. Molecular techniques should prove useful in identifying truly cosmopolitan taxa.
• 3 On a regional scale, a PAE within Western Europe demonstrated a clear link between the distribution of canthocamptid species and the extent of the Last (Wiechselian) glaciation. Northern and southern areas of Europe contain distinctive harpacticoid faunas and the recolonisation of northern Europe appears to have been from the Balkans rather than other Mediterranean peninsulae. The high harpacticoid diversity in southern Europe, may reflect a lack of glacial disruption of groundwater habitats.
• 4 A PAE of lotic data for harpacticoid copepods within the Holarctic reflected the global PAE for freshwater harpacticoids as a whole, but not the regional PAE. A high proportion of stream‐dwelling harpacticoids are widespread species, but only one (Bryocamptus zschokkei) was found in streams across the Holarctic. Other cosmopolites were restricted to streams in Europe or North America, suggesting that species‘ niche requirements might differ among regions. There appeared to be some convergence in the composition of lotic copepod communities in terms of the number of species within genera.
• 5 We conclude that large‐scale processes inevitably have a major influence on the local composition of lotic meiofaunal communities, but that the relative importance of small scale vs. large scale processes is unclear at present, largely due to a paucity of suitable data.