Exponential Megapriming PCR (EMP) Cloning—Seamless DNA Insertion into Any Target Plasmid without Sequence Constraints

We present a fast, reliable and inexpensive restriction-free cloning method for seamless DNA insertion into any plasmid without sequence limitation. Exponential megapriming PCR (EMP) cloning requires two consecutive PCR steps and can be carried out in one day. We show that EMP cloning has a higher efficiency than restriction-free (RF) cloning, especially for long inserts above 2.5 kb. EMP further enables simultaneous cloning of multiple inserts.     

Neuronal Basis of Innate Olfactory Attraction to Ethanol in Drosophila

The decision to move towards a mating partner or a food source is essential for life. The mechanisms underlying these behaviors are not well understood. Here, we investigated the role of octopamine – the invertebrate analogue of noradrenaline – in innate olfactory attraction to ethanol. We confirmed that preference is caused via an olfactory stimulus by dissecting the function of the olfactory co-receptor Orco (formally known as OR83b). Orco function is not required for ethanol recognition per se, however it plays a role in context dependent recognition of ethanol. Odor-evoked ethanol preference requires the function of Tbh (Tyramine β hydroxalyse), the rate-limiting enzyme of octopamine synthesis. In addition, neuronal activity in a subset of octopaminergic neurons is necessary for olfactory ethanol preference. Notably, a specific neuronal activation pattern of tyraminergic/octopaminergic neurons elicit preference and is therefore sufficient to induce preference. In contrast, dopamine dependent increase in locomotor activity is not sufficient for olfactory ethanol preference. Consistent with the role of noradrenaline in mammalian drug induced rewards, we provide evidence that in adult Drosophila the octopaminergic neurotransmitter functions as a reinforcer and that the molecular dissection of the innate attraction to ethanol uncovers the basic properties of a response selection system.     

Analysis and visualization of H7 influenza using genomic,evolutionary and geographic information in a modular web service

We have reported previously on use of a web‐based application, Supramap ( http://supramap.org ) for the study of biogeographic, genotypic, and phenotypic evolution. Using Supramap we have developed maps of the spread of drug‐resistant influenza and host shifts in H1N1 and H5N1 influenza and coronaviruses such as SARS. Here we report on another zoonotic pathogen, H7 influenza, and provide an update on the implementation of Supramap as a web service. We find that the emergence of pathogenic strains of H7 is labile with many transitions from high to low pathogenicity, and from low to high pathogenicity. We use Supramap to put these events in a temporal and geospatial context. We identify several lineages of H7 influenza with biomarkers of high pathogenicity in regions that have not been reported in the scientific literature. The original implementation of Supramap was built with tightly coupled client and server software. Now we have decoupled the components to provide a modular web service for POY ( http://poyws.org ) that can be consumed by a data provider to create a novel application. To demonstrate the web service, we have produced an application, Geogenes ( http://geogenes.org ). Unlike in Supramap, in which the user is required to create and upload data files, in Geogenes the user works from a graphical interface to query an underlying dataset. Geogenes demonstrates how the web service can provide underlying processing for any sequence and metadata database. © The Willi Hennig Society 2012.     

Using a Lethality Index to Assess Susceptibility of Tribolium confusum and Oryzaephilus surinamensis to Insecticides

We evaluated knockdown caused by four insecticides: alpha-cypermethrin, chlorfenapyr, pirimiphos-methyl and fipronil against adults of Tribolium confusum Jacquelin Duval, the confused flour beetle and Oryzaephilus surinamensis (L.), the sawtoothed grain beetle. Bioassays were conducted on concrete and metal surfaces. Adults of the tested species were exposed on both surfaces treated with the above insecticides at two doses (low and high). Knockdown assessment was done after 15, 30 and 60 min of adult exposure in the treated surfaces. Also, after 1, 3, 5, 7 and 14 d of exposure, a lethality index was calculated with an equation resulting to values from 0 to 100, where 100 indicated complete mortality and 0 complete survival. We also developed a lethality index by ranking each adult on each surface from 0 to 4, 0: adults moved normally, 1: adults were knocked down, but were able to walk for short intervals, 2: adults were knocked down and unable to walk, but with visible movement of antennae etc., 3: adults were knocked down, with very minimal movement of the tarsi and the antennae and 4: adults were dead (no movement). Knockdown of adults immediately after exposure (15–60 min) was higher for pirimiphos-methyl followed by alpha-cypermethrin, for both dose rates tested and species, but only on the metal surface. The lethality index was nearly 100 for all insecticides after 5d of exposure for O. surinamensis, while for T. confusum the adult lethality index was considerably lower for alpha-cypermethrin, suggesting that that recovery from knockdown occurred. Chlorfenapyr was the only insecticide that was more effective on concrete than on metal, while the reverse was noted for the other three insecticides. These results show that knockdown has different levels, which can be used as indicators of insect mortality or recovery.     

Modeling the effects of El Niño, density-dependence, and disturbance on harbor seal (Phoca vitulina) counts in Drakes Estero, California: 1997–2007

Harbor seal ( Phoca vitulina ) haul-out site use may be affected by natural or anthropogenic factors. Here, we use an 11-yr (1997–2007) study of a seal colony located near a mariculture operation in Drakes Estero, California, to test for natural (El Niño-Southern Oscillation (ENSO), density-dependence, long-term trends) and anthropogenic (disturbance or displacement related to oyster production activities) factors that may influence the use of haul-out subsites. Annual mariculture related seal disturbance rates increased significantly with increases in oyster harvest ( r _s= 0.55). Using generalized linear models (GLMs) ranked by best fit and Akaike's Information Criteria, ENSO and oyster production (as a proxy for disturbance/displacement) best explained the patterns of seal use at all three subsites near the mariculture operations, with effects being stronger at the two subsites closest to operations. Conversely, density-dependence and linear trend effects poorly explained the counts at these subsites. We conclude that a combination of ENSO and mariculture activities best explain the patterns of seal haul-out use during the breeding/pupping season at the seal haul-out sites closest to oyster activities.