Assessment of Virally Vectored Autoimmunity as a Biocontrol Strategy for Cane Toads

BACKGROUND: The cane toad, Bufo (Chaunus) marinus, is one of the most notorious vertebrate pests introduced into Australia over the last 200 years and, so far, efforts to identify a naturally occurring B. marinus-specific pathogen for use as a biological control agent have been unsuccessful. We explored an alternative approach that entailed genetically modifying a pathogen with broad host specificity so that it no longer caused disease, but carried a gene to disrupt the cane toad life cycle in a species specific manner. METHODOLOGY/PRINCIPAL FINDINGS: The adult beta globin gene was selected as the model gene for proof of concept of autoimmunity as a biocontrol method for cane toads. A previous report showed injection of bullfrog tadpoles with adult beta globin resulted in an alteration in the form of beta globin expressed in metamorphs as well as reduced survival. In B. marinus we established for the first time that the switch from tadpole to adult globin exists. The effect of injecting B. marinus tadpoles with purified recombinant adult globin protein was then assessed using behavioural (swim speed in tadpoles and jump length in metamorphs), developmental (time to metamorphosis, weight and length at various developmental stages, protein profile of adult globin) and genetic (adult globin mRNA levels) measures. However, we were unable to detect any differences between treated and control animals. Further, globin delivery using Bohle iridovirus, an Australian ranavirus isolate belonging to the Iridovirus family, did not reduce the survival of metamorphs or alter the form of beta globin expressed in metamorphs. CONCLUSIONS/SIGNIFICANCE: While we were able to show for the first time that the switch from tadpole to adult globin does occur in B. marinus, we were not able to induce autoimmunity and disrupt metamorphosis. The short development time of B. marinus tadpoles may preclude this approach

Old habits die hard? The fragility of eco-driving mental models and why green driving behaviour is difficult to sustain

Tangible incentives, training and feedback systems have been shown to reduce drivers’ fuel consumption in several studies. However, the effects of such tools are often short-lived or dependent on continuous cues. Several studies found that many drivers already possess eco-driving mental models, and are able to activate them, for instance when an experimenter asks them to “drive fuel-efficiently”. However, it is unclear how sustainable mental models are. The aim of the current study was to investigate the resilience of drivers’ eco-driving mental models following engagement with a workload task, implemented as a simplified version of the Twenty Questions Task (TQT). Would drivers revert to ‘everyday’ driving behaviours following exposure to heightened workload? A driving simulator experiment was conducted whereby 15 participants first performed a baseline drive, and then in a second session were prompted to drive fuel-efficiently. In each drive, the participants drove with and without completing the TQT. The results of two-way ANOVAs and Wilcoxon signed-rank tests support that they drive more slowly and keep a more stable speed when asked to eco-drive. However, it appears that drivers fell back into ‘everyday’ habits over time, and after the workload task, but these effects cannot be clearly isolated from each other. Driving and the workload task possibly invoked unrelated thoughts, causing eco-driving mental models to be deactivated. Future research is needed to explore ways to activate existing knowledge and skills and to use reminders at regular intervals, so new driver behaviours can be proceduralised and automatised and thus changed sustainably

No effect on globin protein profile following infection of tadpoles with virus carrying rAdglob.

<p>200 ng protein per well from lysed red blood cell samples taken from metamorphs then stained with silver stain. a: Lanes 1 and 12, See Blue Plus2 Pre Stained Standard (Invitrogen); Lanes 2–4, blood from 3 control animals bathed in 10² TCID₅₀/ml of rBIV/neo^r; Lanes 5–10, blood from 6 test animals bathed in 10² TCID₅₀/ml of rBIV/neo^r/adglo. Lane 11, positive control, globin from a 2 month old toadlet. b: Panel shows silver stain and Western blot antibody detection of adult globin; lane i See Blue Plus2 Pre Stained Standard (Invitrogen); lane ii, positive control, globin from a 2 month old toadlet; lanes iii, iv and v, 500, 1000 and 5000 ng of protein from fluid of untreated stage 42 tadpoles.</p

Time course detection of rHb within tadpoles after injection.

<p>Western blot using rabbit antibody to adult globin to detect persistence of rHb emulsion. n = 3 animals pooled per time point. Actin indicates loading per protein sample (mAb mouse anti-actin used at 1∶5000).</p

Tadpole to adult globin switch detected in normal cane toad development.

<p>a: mRNA data expressed as number of copies of adult or tadpole globin mRNA detected by real time PCR across various tadpole and metamorphic stages. Mean copy numbers were normalised using a toad actin housekeeping gene. Animals were staged according to Limbaugh and Volpe, 1957. Toadlet (*) development was approximately one month post-metamorphosis. b: Detection of globin proteins as determined by western blot analysis using specific antibodies to tadpole and adult globins. Coomassie staining indicates the loading level for each lane. Recombinant proteins for adult and tadpole globin (rAdglob and rTadglob, respectively), as well as native adult globin (Adult) were included as positive controls.</p

IgG antibody response to globin antigen not detected in metamorphs by ELISA.

<p>IgG levels as measured by ELISA in metamorphs (stage 46) injected with adult globin (rAdglob treated) compared with untreated metamorphs (FCA control). Positive controls show IgG levels in metamorphs and an adult injected with ovalbumin. Sera from rAdglob treated and FCA control animals used at 1∶80; ovalbumin treated metamorph sera used at 1∶320; ovalbumin treated adult sera used at 1∶1000.</p