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

Positive Darwinian selection results in resistance to cardioactive toxins in true toads (Anura: Bufonidae)

Members of the Family Bufonidae, true toads, are famous for their endogenously synthesized cardioactive steroids that serve as defensive toxins. Evolution of resistance to these toxins is not understood. We sequenced a key region of the toxin's binding site in the Na+/K+ ATPase for relevant taxa representing Hyloidea (including bufonids), Ranoidea and Archaeobatrachia and tested for positive selection in a phylogenetic context. Bufonidae were distinct from other Hyloidea at 4-6 of 12 sites and, with one exception, had a homologous amino acid sequence. Melanophryniscus stelzneri had a distinct sequence, consistent with other independent evidence for a differentiated toxin. Tests within Bufonidae detected positive selection within the binding region, providing, to our knowledge, the first evidence of this type for positive selection within Amphibia. There was no evidence for positive selection on Bufonidae or M. stelzneri lineages. Sequence change in Leptodactylus ocellatus, a leptodactylid predator of Bufonidae, provides a molecular basis for predator resistance possibly associated with gene duplication

Biological control of the cane toad in Australia: A review

The marine toad Bufo marinus is native to northern South America, parts of Central America and Southern Texas. It was deliberately introduced into Australia's tropical north-east in 1935 in an unsuccessful attempt to control the cane beetle, a damaging insect pest of sugarcane crops. The toads quickly established in the new environment and began to spread. Today, they inhabit most of the Australian tropics and sub-tropics and have reached Western Australia. Models predict that global warming will enable the toads to extend their range further south. They cause severe environmental impacts, as all life stages of B. marinus contain bufadienolides, alkaloid substances toxic to vertebrates, resulting in death of the predators ingesting it. The continental scale of this biological invasion in combination with the remoteness of the areas affected, poses a specific set of challenges to potential control approaches for cane toads. This review covers different biocontrol strategies pursued over the past 8 years, with particular focus on an immunological approach aiming at the disruption of toad metamorphosis. So far, research efforts have failed to produce a tool for large-scale reduction of toad populations. Considerations of future research priorities and efforts are also discussed

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

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

Physical measures of <i>B. marinus</i> development across metamorphosis unchanged by recombinant adult globin injection.

<p>a: Wet weights (mg), lengths (mm) and speed of development (days) for animals at six different developmental stages injected with rAdglob (□) or FCA control (▴, dotted line), as well as normal uninjected animals (◊) were recorded. n = 10 animals for each stage and error bars show standard deviation. b: Swimming performance of rAdglob treated and FCA control tadpoles at approximately stage 36 (or 10 days post injection) is shown. Burst swim speed represents the absolute swim speed normalised to body length, and the full data range (vertical line), standard deviation (box) and mean (horizontal line) are indicated. c: Jumping performance of rAdglob treated and FCA control amimals at stage 46 is shown. Longest jump distance was normalised to body lengths.</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

Sampling schedule for tadpoles infected with recombinant BIV.

<p>n = 5. Stage 20, infection; Stage 28, 6 days post infection; stage 33, just prior to the onset of adult globin production; stage 42, just after the onset of adult globin production; post TR, 1–2 weeks post tail resorption.</p