cAmphibians are suffering unprecedented global declines. A leading cause is the infectious disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis. Chytridiomycosis is a skin disease which disrupts transport of essential ions leading to death. Soluble factors produced by B. dendrobatidis impair amphibian and mammalian lymphocytes in vitro, but previous studies have not shown the effects of these inhibitory factors in vivo. To demonstrate in vivo inhibition of immunity by B. dendrobatidis, a modified delayed-type-hypersensitivity (DTH) protocol was developed to induce innate and adaptive inflammatory swelling in the feet of Xenopus laevis by injection of killed bacteria or phytohemagglutinin (PHA). Compared to previous protocols for PHA injection in amphibians, this method induced up to 20-fold greater inflammatory swelling. Using this new protocol, we measured DTH responses induced by killed bacteria or PHA in the presence of B. dendrobatidis supernatants. Swelling induced by single injection of PHA or killed bacteria was not significantly affected by B. dendrobatidis supernatants. However, swelling caused by a secondary injection of PHA, was significantly reduced by B. dendrobatidis supernatants. As previously described in vitro, factors from B. dendrobatidis appear to inhibit lymphocyte-mediated inflammatory swelling but not swelling caused by an inducer of innate leukocytes. This suggests that B. dendrobatidis is capable of inhibiting lymphocytes in a localized response to prevent adaptive immune responses in the skin. The modified protocol used to induce inflammatory swelling in the present study may be more effective than previous methods to investigate amphibian immune competence, particularly in nonmodel species.

28An important concern for conservation biologists is the continuing loss of global biodiversity. 29 This is especially true for amphibians, which are disappearing at an alarming rate. The emerging 30 fungal pathogen, Batrachochytrium dendrobatidis (Bd), causes the skin disease chytridiomycosis 31 and is a major contributor to amphibian population declines. Recent studies suggest that 32 symbiotic skin bacteria provide some protection against Bd. Possible mitigation strategies 33 include skin microbiome manipulation and bioaugmentation with potentially beneficial bacteria. 34Such approaches may employ a method to reduce resident bacteria on the skin to allow for new 35 colonization. Using the southern leopard frog, Rana (Lithobates) sphenocephala, we show here 36 that the skin of this species harbors a rich array of culturable bacterial species that inhibit Bd 37 growth in vitro. Use of a potent antibiotic cocktail dramatically reduced culturable skin bacteria 38 within 48 hours. Reduction of bacteria on post-metamorphic juvenile skin using this antibiotic 39 cocktail resulted in an increased Bd skin burden within two weeks of Bd exposure and a higher 40 Bd burden in those frogs that died. However, efforts to maintain a normal bacterial skin 41 community using a semi-natural mesocosm-derived source of microbes failed to provide long 42 term protection of these vulnerable frogs. Regardless of presence or absence of mesocosm-43 derived bacteria, Bd loads decreased over time in surviving frogs, suggesting the development of 44 an adaptive immune defense. In this species, development of an antimicrobial peptide defense is 45 © 2015. This manuscript version is made available under the Elsevier user license http://www.elsevier.com/open-access/userlicense/1.0/ 2 delayed, and thus, the microbial community and adaptive immune defenses may provide 46 protection at this critical period of development. 47 48 Keywords: amphibian skin microbiome; antibiotic depletion; antimicrobial peptides; 49 chytridiomycosis; southern leopard frogs; skin defenses 50 51 experiments to optimize bacterial reduction by antibiotics; and (3) as positive controls for AMP 87 expression at age 52 weeks post-metamorphosis. Juveniles raised in 2013 were used for: (1) 88 evaluation of AMP production following norepinephrine injection immediately after 89 metamorphosis and (2) a bacterial reduction and infection experiment described below.90 Norepinephrine-induced individuals were not used in the infection studies.91 3Mesocosm tanks were prepared in February each year as previously described (Holden et 92 al., 2015). Briefly, each tank was filled with ~613 L of well water. About 300 g of dry 93 deciduous leaf litter and one 500 ml aliquot of concentrated plankton from a nearby pond were 94 added. Fiberglass mesh screens (1-mm mesh) were used as lids to prevent colonization by Bd-95 infected adults or predators and to provide shading. A fiberglass composite ramp was placed in 96 each tank at a 30° angle to simulate the margin of a pond. Tanks were...

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation and decreased the need for antibiotics.

Auxotrophic strains of Agrobacterium tumefaciens can contribute to the development of more efficient transformation systems, especially for crops historically considered recalcitrant. Homologous recombination was used to derive methionine auxotrophs of two common A. tumefaciens strains, LBA4404 and EHA105. The EHA105 strains were more efficient for switchgrass transformation, while both the EHA105 and LBA4404 strains worked equally well for the rice control. Event quality, as measured by transgene copy number, was not affected by auxotrophy, but was higher for the LBA4404 strains than the EHA105 strains. Ultimately, the use of auxotrophs reduced bacterial overgrowth during co-cultivation, with a concomitant decrease in the need for antibiotics. In turn, reduced overgrowth allowed longer co-cultivation periods, with a trend towards higher transformation frequencies for switchgrass, but not for rice.