Brood‐parasite‐induced female‐biased mortality affects songbird demography: negative implications for conservation

Parasites, of all sorts, can profoundly affect host population dynamics. Parasites commonly cause sex‐biased mortality and this can add to their impact. Female‐biased mortality in particular can destabilize dynamics and promote population collapse. We previously reported in a correlative study that brown‐headed cowbird Molothrus ater brood parasitism of song sparrows Melospiza melodia appears to cause female‐biased host nestling mortality. Here, we report results from ‘infestation’ and ‘de‐infestation’ experiments designed to test whether brood parasitism causes female‐biased mortality, and we document the resulting demographic impact using a simulation model. Experimental cowbird infestation of song sparrow nests halved the proportion of female host nestlings (0.31±0.07 vs 0.59±0.06; infested vs unparasitized nests at day 6) replicating the halving reported in naturally cowbird‐parasitized nests (0.28±0.01 vs 0.57±0.05; parasitized vs unparasitized). De‐infestation of naturally cowbird‐parasitized nests in turn wholly eliminated any effect on the proportion of female host nestlings (0.53±0.13 vs 0.54±0.06; de‐infested vs unparasitized) confirming that brood parasitism is the cause. This halving of the proportion of females fledging is likely to be as significant as nest predation in affecting population dynamics, based on the elasticities derived from our demographic model (–0.50 vs –0.59). Experimental infestation reduced the testosterone levels, begging behaviour, and body mass of six day old female host nestlings, whereas males were largely unaffected, suggesting that it is the exacerbation of intra‐brood competition that may be primarily responsible for the resulting female‐biased mortality. The brown‐headed cowbird is invasive in most of North America and has been implicated in regional population declines of many native species. We suggest that female‐biased host offspring mortality is likely to be commonplace among the 144 host species the cowbird successfully parasitizes, and we discuss the negative implications for songbird conservation, given the projected demographic impact.     

Influence of baking enzymes on antimicrobial activity of five bacteriocin‐like inhibitory substances produced by lactic acid bacteria isolated from Lithuanian sourdoughs

Aim: To evaluate the effect of four different baking enzymes on the inhibitory activity of five bacteriocin‐like inhibitory substances (BLIS) produced by lactic acid bacteria (LAB) isolated from Lithuanian sourdoughs. Methods and Results: The overlay assay and the Bioscreen methods revealed that the five BLIS exhibited an inhibitory effect against spore germination and vegetative outgrowth of Bacillus subtilis, the predominant species causing ropiness in bread. The possibility that the observed antibacterial activity of BLIS might be lost after treatment with enzymes used for baking purposes was also examined. Conclusions: The enzymes tested; hemicellulase, lipase, amyloglucosidase and amylase had little or no effect on the majority of the antimicrobial activities associated with the five BLIS studied. Significance and Impact of the Study: This study suggests a potential application in the sourdough baking industry for these antimicrobial producing LAB strains in the control of B. subtilis spore germination and vegetative outgrowth.     

A dual expression platform to optimize the soluble production of heterologous proteins in the periplasm of <Emphasis Type="Italic">Escherichia coli</Emphasis>

The functional analysis of individual proteins or of multiprotein complexes—since the completion of several genome sequencing projects—is in focus of current scientific work. Many heterologous proteins contain disulfide-bonds, required for their correct folding and activity, and therefore, need to be transported to the periplasm. The production of soluble and functional protein in the periplasm often needs target-specific regulatory genetic elements, leader peptides, and folding regimes. Usually, the optimization of periplasmic expression is a step-wise and time-consuming procedure. To overcome this problem we developed a dual expression system, containing a degP-promoter-based reporter system and a highly versatile plasmid set. This combines the differential protein expression with the selection of a target-specific expression plasmid. For the validation of this expression tool, two different molecular formats of a recombinant antibody directed to the human epidermal growth factor receptor and human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) were used. By application of this expression system we demonstrated that the amount of functional protein is inversely proportional to the on-line luciferase signal. We showed that this technology offers a simple tool to evaluate and improve the yield of functionally expressed proteins in the periplasm, which depends on the used regulatory elements and folding strategies.     

A cation-pi interaction discriminates among sodium channels that are either sensitive or resistant to tetrodotoxin block

Voltage-gated sodium channels control the upstroke of the action potential in excitable cells of nerve and muscle tissue, making them ideal targets for exogenous toxins that aim to squelch electrical excitability. One such toxin, tetrodotoxin (TTX), blocks sodium channels with nanomolar affinity only when an aromatic Phe or Tyr residue is present at a specific location in the external vestibule of the ion-conducting pore. To test whether TTX is attracted to Tyr401 of NaV1.4 through a cation-pi interaction, this aromatic residue was replaced with fluorinated derivatives of Phe using in vivo nonsense suppression. Consistent with a cation-pi interaction, increased fluorination of Phe401, which reduces the negative electrostatic potential on the aromatic face, caused a monotonic increase in the inhibitory constant for block. Trifluorination of the aromatic ring decreased TTX affinity by approximately 50-fold, a reduction similar to that caused by replacement with the comparably hydrophobic residue Leu. Furthermore, we show that an energetically equivalent cation-pi interaction underlies both use-dependent and tonic block by TTX. Our results are supported by high level ab initio quantum mechanical calculations applied to a model of TTX binding to benzene. Our analysis suggests that the aromatic side chain faces the permeation pathway where it orients TTX optimally and interacts with permeant ions. These results are the first of their kind to show the incorporation of unnatural amino acids into a voltage-gated sodium channel and demonstrate that a cation-pi interaction is responsible for the obligate nature of an aromatic at this position in TTX-sensitive sodium channels.