Development of ten microsatellite markers from the keystone mistletoe <Emphasis Type="Italic">Tristerix corymbosus</Emphasis> (Loranthaceae) using 454 next generation sequencing and their applicability to population genetic structure studies

Tristerix corymbosus (Loranthaceae) is a keystone mistletoe from the South American temperate rainforests. As most mistletoes, T. corymbosus relies on biotic pollination and seed dispersal, which may cause population structure. For a better understanding of its ecology, we isolated and characterized ten polymorphic microsatellite loci for this species. We used 454 Next Generation Sequencing to build a microsatellite library from a high quality DNA sample. We tested 90 sequences from which we obtained ten polymorphic markers. In order to assess their variability, the novel markers were tested in 48 individuals from three locations of the Valdivian Coastal Reserve in Chile. We also estimated genetic differences between pairs of populations using the F_ST statistic. The mean number of alleles per locus in the 48 individuals studied was 7.1 (2–17 alleles per locus). The observed and expected heterozygosity ranged from 0.298 to 0.634 and from 0.310 to 0.881, respectively. There were genetic differences among the three populations assessed, according to the F_ST values (ranging from 0.048 to 0.100, all significant) and, the number of alleles per locus ranged from 3.9 to 5.1. These are the first microsatellite markers developed for T. corymbosus, and they arise as a powerful tool for studying population structure, genetic diversity and gene flow at the landscape scale, along its distribution.     

Does the house sparrow Passer domesticus represent a global model species for egg rejection behavior?

Conspecific brood parasitism (CP) is a facultative breeding tactic whereby females lay their eggs in the nests of conspecifics. In some species, potential hosts have evolved the ability to identify and reject foreign eggs from their nest. Previous studies suggest that the ubiquitous house sparrow Passer domesticus in Spain and South Africa employs both CP and egg rejection, while a population in China does not. Given the species’ invasive range expansions, the house sparrow represents a potentially excellent global model system for parasitic egg rejection across variable ecological conditions. We examined the responses of house sparrows to experimental parasitism at three geographically distinct locations (in Israel, North America, and New Zealand) to provide a robust test of how general the findings of the previous studies are. In all three geographic regions egg rejection rates were negligible and not statistically different from background rates of disappearance of control eggs, suggesting that the house sparrow is not a suitable model species for egg rejection experiments on a global scale.     

PCR primers for microsatellite loci in the desert tortoise (Gopherus agassizii,Testudinidae)

The desert tortoise, Gopherus agassizii, is a threatened species native to the North American desert southwest and is recognized as having distinct Mojave and Sonoran populations. We identified six polymorphic microsatellite loci in the desert tortoise. All six loci were polymorphic in Sonoran samples. Five of the loci were variable in Mojave samples with varying degrees of amplification success. Two of the loci exhibited low allelic variation (2–3 alleles) while four were highly variable (8–27 alleles).     

Use of a Bacteriophage Lysin,PlyC, as an Enzyme Disinfectant against Streptococcus equi

Streptococcus equi is the causative agent of the purulent infection equine strangles. This disease is transmitted through shedding of live bacteria from nasal secretions and abscess drainage or by contact with surfaces contaminated by the bacteria. Disinfectants are effective against S. equi, but inactivation by environmental factors, damage to equipment, and toxicity are of great concern. Bacteriophage-encoded lysins (cell wall hydrolases) have been investigated as therapeutic agents due to their ability to lyse susceptible gram-positive organisms. Here, we investigate the use of one lysin, PlyC, as a narrow-spectrum disinfectant against S. equi. This enzyme was active against >20 clinical isolates of S. equi, including both S. equi subsp. equi and S. equi subsp. zooepidemicus. Significantly, PlyC was 1,000 times more active on a per weight basis than Virkon-S, a common disinfecting agent, with 1 μg of enzyme able to sterilize a 10⁸ CFU/ml culture of S. equi in 30 min. PlyC was subjected to a standard battery of tests including the Use Dilution Method for Testing Disinfectants and the Germicidal Spray Products Test. Results indicate that aerosolized PlyC can eradicate or significantly reduce the S. equi load on a variety of materials found on common stable and horse-related equipment. Additionally, PlyC was shown to retain full activity under conditions that mimic a horse stable, i.e., in the presence of nonionic detergents, hard water, or organic materials. We propose PlyC as the first protein-based, narrow-spectrum disinfectant against S. equi, which may augment or supplement the use of broad-spectrum disinfectants in barns and stables where equine strangles is prevalent.Equine strangles is a highly contagious lymphadenitis of the head and neck that is uniquely caused by Streptococcus equi, predominantly S. equi subsp. equi, with some disease associated with S. equi subsp. zooepidemicus (24, 29). Progression of the purulent infection leads to acute swelling and subsequent abscess formation of the submaxillary, submandibular, and retropharyngeal lymph nodes causing a “strangling” of the pharynx, with severe cases necessitating a tracheotomy (24). Serious complications occur in approximately 20% of infected horses, and the overall mortality rate has been reported to be as high as 8% on farms where the infection is endemic (25). Even after recovery from infection, long-lasting, immune-mediated complications such as progressive muscle atrophy have been reported, which can adversely affect the career of a racing, show, or work horse (27).The route of S. equi transmission is through nasal secretions and drainage from abscesses. Infected horses can nasally shed bacteria for weeks, contaminating surfaces through which other horses can become infected. S. equi extract and attenuated-live vaccines exist, but they are often associated with abscess formation at the site of injection, short duration of immunity, poor efficacy, and the very real threat of a nascent infection from the vaccine (15, 24, 29). Thus, strangles continues to be a serious and widespread infectious disease of horses despite the presence of multiple commercially available vaccines.Strangles prevention strategies include good disinfection/hygiene practices, isolation of infected animals, and removal of equipment for sanitization where possible (9, 26; also R. E. Holland, D. G. Harris, and A. Monge, presented at the 52nd Annual Convention of the American Association of Equine Practitioners, San Antonio, TX, 2 to 6 December 2006). Current broad-spectrum disinfectants belong to one of several chemical categories including alcohols, aldehydes, biguanides, halogens, oxidizing agents, phenols, or quaternary ammonium compounds (6, 8). To various degrees, these compounds have been shown to be flammable, light sensitive, carcinogenic, corrosive to metals, irritating to mucous membranes, and/or toxic to livestock and humans (8, 10). Additionally, many factors that are often associated with cleaning stalls/barns (e.g., hard water, organic load, or detergents) can reduce or even ablate efficacy of chemical disinfectants (8). Importantly, studies have shown that these commonly used disinfectants can select for mutant bacteria with decreased susceptibility to biocides and antibiotics without compromising virulence (21).Recently, bacteriophage-encoded peptidoglycan hydrolases, collectively termed lysins and often referred to as “enzybiotics,” have been investigated as potential therapeutic agents against pathogens due to their ability to lyse the bacterial cell wall (12). These enzymes not only exert their lethal effects in the absence of bacteriophage (cause “lysis from without”) but also display specificity for a bacterial host, often for a particular genus, species, or even a subspecies depending on the lysin (11). For example, one lysin, PlyC, is known to lyse streptococcal species bearing a polyrhamnose epitope, which include group C streptococcus (i.e., S. equi subsp. equi) among other streptococci (19). As an adjunct to broad-spectrum disinfectants, we investigate here the use of the PlyC enzyme to help control the acquisition and spread of S. equi subsp. equi in horse stalls and barns.     

Clustering of Socioeconomic,Behavioural, and Neonatal Risk Factors for Infant Health in Pregnant Smokers

Background

Tobacco smoking is a major cause of morbidity and mortality, including during pregnancy. Although effective ways of promoting smoking cessation during pregnancy exist, the impact of these interventions has not been studied at a national level. We estimated the prevalence of smoking throughout pregnancy in the Netherlands and quantified associations of maternal smoking throughout pregnancy with socioeconomic, behavioural, and neonatal risk factors for infant health and development.

Methodology/Principal Findings

Data of five national surveys, containing records of 14,553 Dutch mothers and their offspring were analyzed. From 2001 to 2007, the overall rate of smoking throughout pregnancy fell by 42% (from 13.2% to 7.6%) mainly as a result of a decrease among highly educated women. In the lowest-educated group, the overall rate of smoking throughout pregnancy was six times as high as in the highest-educated group (18.7% versus 3.2%). Prenatal tobacco smoke exposure was associated with increased risk of extremely preterm (≤28 completed weeks) (OR 7.25; 95% CI 3.40 to 15.38) and small-for-gestational age (SGA) infants (OR 3.08; 95% CI 2.66 to 3.57). Smoking-attributable risk percents in the population (based on adjusted risk ratios) were estimated at 29% for extremely preterm births and at 17% for SGA outcomes. Infants of smokers were more likely to experience significant alcohol exposure in utero (OR 2.08; 95%CI 1.25 to 3.45) and formula feeding in early life (OR 1.91; 95% CI 1.69 to 2.16).

Conclusions

The rates of maternal smoking throughout pregnancy decreased significantly in the Netherlands from 2001 to 2007. If pregnant women were to cease tobacco use completely, an estimated 29% of extremely preterm births and 17% of SGA infants may be avoided annually.     

Redox‐dependent lipoylation of mitochondrial proteins in Plasmodium falciparum

Lipoate scavenging from the human host is essential for malaria parasite survival. Scavenged lipoate is covalently attached to three parasite proteins: the H‐protein and the E2 subunits of branched chain amino acid dehydrogenase (BCDH) and α‐ketoglutarate dehydrogenase (KDH). We show mitochondrial localization for the E2 subunits of BCDH and KDH, similar to previously localized H‐protein, demonstrating that all three lipoylated proteins reside in the parasite mitochondrion. The lipoate ligase 1, LipL1, has been shown to reside in the mitochondrion and it catalyses the lipoylation of the H‐protein; however, we show that LipL1 alone cannot lipoylate BCDH or KDH. A second mitochondrial protein with homology to lipoate ligases, LipL2, does not show ligase activity and is not capable of lipoylating any of the mitochondrial substrates. Instead, BCDH and KDH are lipoylated through a novel mechanism requiring both LipL1 and LipL2. This mechanism is sensitive to redox conditions where BCDH and KDH are exclusively lipoylated under strong reducing conditions in contrast to the H‐protein which is preferentially lipoylated under less reducing conditions. Thus, malaria parasites contain two different routes of mitochondrial lipoylation, an arrangement that has not been described for any other organism.     

Evolutionally guided enzyme design

A combination of "rational" and "irrational" strategies for the creation of enzymes with novel properties is proving to be a powerful concept in the field of enzyme engineering. Guided by principles of physical organic chemistry, rational design strategies are used to identify suitable target enzymes and to choose appropriate molecular biological methods for engineering purposes. In contrast, irrational (or random) strategies are centered around the biological paradigm of stochastic molecular evolution. As illustrated in this review, such a hybrid approach is particularly useful for the design of new modular enzymes. (c) 1996 John Wiley & Sons, Inc.     

Stable isotopes of Lithosiini and lichens in Hong Kong show the biodindicator potential of lichenivorous moths

Urban landscapes provide unique environments for a wide variety of plants and animals, but their suitability may be limited by anthropogenic impacts such as pollution. We examined the potential utility of lichen and lichen-feeding moths as biodindicators of air pollution in Hong Kong by comparing carbon (C) and nitrogen (N) stable isotope values in lichens, lichenivorous and non-lichenivorous moths (Lepidoptera: Erebidae) and a moth outgroup (Lepidoptera: Geometridae). Our results show that stable isotope values for C and N were similar for lichens and lichen feeding moths, while non-lichen feeding moths formed a distinct group. In addition, we found consistent δ¹³C and δ¹⁵N values across moth body parts, indicating that any portion of the specimen is suitable for isotopic fingerprinting. Our results highlight that lichen feeding moths may be useful for integrating signals of atmospheric nitrogen pollution and could therefore have utility in monitoring and quantifying air quality over time and space.