Contrasting responses of black fly species (Diptera: Simuliidae) to experimental whole-stream warming

1. As global temperatures continue to rise, assessment of how species within ecological communities respond to shifts in temperature has become increasingly important. However, such assessments require detailed long-term observations or ecosystem-level manipulations that allow for interactions among species and the potential for species dispersal and exchange with the regional species pool.
2. We examined the effects of experimental whole-stream warming on a larval black fly assemblage in southwest Iceland. We used a paired-catchment design, in which we studied the warmed stream and a nearby reference stream for 1 year prior to warming and 2 years during warming and estimated population abundance, biomass, secondary production, and growth rates for larvae of three black fly species.
3. Experimental warming by 3.8°C had contrasting effects on the three black fly species in the assemblage. The abundance, biomass, growth, and production of Prosimulium ursinum decreased in the experimental stream during the warming manipulation. Despite increasing in the reference stream, the abundance, biomass, and production of another species, Simulium vernum, decreased in the experimental stream during warming.
4. In contrast, warming had an overall positive effect on Simulium vittatum. While warming had little effect on the growth of overwintering cohorts of S. vittatum, warming led to an additional cohort during the summer months and increased its abundance, biomass, and production. Overall, family-level production was enhanced by warming, despite variation in species-level responses.
5. Our study illustrates that the effects of climate warming are likely to differ even among closely related species. Moreover, our study highlights the need for further investigation into the uneven effects of warming on individual species and how those variable effects influence food web dynamics and ecosystem function.

     

Rapid GC mol% screening of primary culture lysates using horizontal slab gel electrophoresis

An electrophoretic technique for the rapid screening of GC mol% of bacterial DNA was modified and evaluated. Modifications of the technique included its adaptation to horizontal slab electrophoresis. Primary culture lysates (one per gel) of bacterial strains with unknown ratios of G+C/A+T+G+C (GC mol%), and reference strains whose GC mol% had been determined by thermal denaturation, were simultaneously electrophoresed for 2 h in polyacrylamide-agarose gels and mobilites of the chromosomal DNA bands were compared with the GC mol% values obtained from thermal denaturation curves. Results indicated a positive correlation (r = 0.80, df = 23) between GC mol% and electrophoretic mobility. The procedure, as modified, requires a minimum of equipment and resources and allows for the determination of GC mol% values with sufficient accuracy to serve as a means for inexpensive and routine screening of bacterial isolates.     

Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy

Chinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time-consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day-to-day changes in the cellular microenvironment need further in-depth characterization. In this study, two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom-built multimodal microscope with two-photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale-up. To our knowledge, this is the first study to use FLIM as a label-free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.     

Interaction between an ammonium-oxidizer,Nitrosomonas sp., and two heterotrophic bacteria,Nocardia atlantica andPseudomonas sp.: A note

Closed culture experiments showed that the ability of an estuarine chemolithotrophic ammonium-oxidizer,Nitrosomonas sp., to convert ammonium to nitrite was substantially increased by as much as 150% and 50% when grown in the presence of two heterotrophs isolated from the same environment. The heterotrophs were identified asNocardia atlantica andPseudomonas sp. The growth of the heterotrophs was stimulated by as much as one order of magnitude in the presence of the ammonium-oxidizer.     

Porcine big gastrin: Sequence,synthesis, and immunochemical studies

The synthesis of the tetracontapeptide corresponding to a revised sequence for porcine big gastrin is described. Both the synthetic and natural material were identical by radioimmunoassay using L33 antiserum, which is specific for the N-terminal region of the sequence.