Butterfly eyespots represent novel complex traits that display substantial diversity in number and size within and across species. Correlative gene expression studies have implicated a large suite of transcription factors, including Distal-less (Dll), Engrailed (En), and Spalt (Sal), in eyespot development in butterflies, but direct evidence testing the function of any of these proteins is still missing. Here we show that the characteristic two-eyespot pattern of wildtype Bicyclus anynana forewings is correlated with dynamic progression of Dll, En, and Sal expression in larval wings from four spots to two spots, whereas no such decline in gene expression ensues in a four-eyespot mutant. We then conduct transgenic experiments testing whether overexpression of any of these genes in a wild-type genetic background is sufficient to induce eyespot differentiation in these pre-patterned wing compartments. We also produce a Dll-RNAi transgenic line to test how Dll down-regulation affects eyespot development. Finally we test how ectopic expression of these genes during the pupal stages of development alters adults color patters. We show that over-expressing Dll in larvae is sufficient to induce the differentiation of additional eyespots and increase the size of eyespots, whereas down-regulating Dll leads to a decrease in eyespot size. Furthermore, ectopic expression of Dll in the early pupal wing led to the appearance of ectopic patches of black scales. We conclude that Dll is a positive regulator of focal differentiation and eyespot signaling and that this gene is also a possible selector gene for scale melanization in butterflies.

Bacteriophage shape the composition and function of microbial communities. Yet it remains difficult to predict the effect of phage on microbial interactions. Specifically, little is known about how phage influence mutualisms in networks of crossfeeding bacteria. We mathematically modeled the impacts of phage in a synthetic microbial community in which Escherichia coli and Salmonella enterica exchange essential metabolites. In this model, independent phage attack of either species was sufficient to temporarily inhibit both members of the mutualism; however, the evolution of phage resistance facilitated yields similar to those observed in the absence of phage. In laboratory experiments, attack of S. enterica with P22vir phage followed these modeling expectations of delayed community growth with little change in the final yield of bacteria. In contrast, when E. coli was attacked with T7 phage, S. enterica, the nonhost species, reached higher yields compared with no-phage controls. T7 infection increased nonhost yield by releasing consumable cell debris, and by driving evolution of partially resistant E. coli that secreted more carbon. Our results demonstrate that phage can have extensive indirect effects in microbial communities, that the nature of these indirect effects depends on metabolic and evolutionary mechanisms, and that knowing the degree of evolved resistance leads to qualitatively different predictions of bacterial community dynamics in response to phage attack.

Bacteriophage shape the composition and function of microbial communities. Yet, it remains difficult to predict the effect of phage on microbial interactions. Specifically, little is known about how phage influence mutualisms in networks of cross-feeding bacteria. We modeled the impacts of phage in a synthetic microbial community in which Escherichia coli and Salmonella enterica exchange essential metabolites. In this model, phage attack of either species was sufficient to inhibit both members of the mutualism; however, the evolution of phage resistance ultimately allowed both species to attain yields similar to those observed in the absence of phage. In laboratory experiments, attack of S. enterica with P22vir phage followed these modeling expectations of delayed community growth with little change in the final yield of bacteria. In contrast, when E. coli was attacked with T7 phage, S. enterica, the non-host species, reached higher yields compared to no-phage controls. T7 increased non-host yield by releasing consumable cell debris and by driving evolution of phage resistant E. coli that secreted more carbon. Additionally, E. coli evolved only partial resistance, increasing the total amount of lysed cells available for S. enterica to consume. Our results demonstrate that phage can have extensive indirect effects in microbial communities, and that the nature of these indirect effects depends on metabolic and evolutionary mechanisms.

Borrelia burgdorferi, a causative agent of Lyme borreliosis, is a zoonotic pathogen that survives in nutrient-limited environments within a tick, prior to transmission to its mammalian host. Survival under these prolonged nutrient-limited conditions is thought to be similar to survival during stationary phase, which is characterized by growth cessation and decreased protein production. Multiple ribosome-associated proteins are implicated in stationary-phase survival of Escherichia coli. These proteins include hibernation-promoting factor (HPF), which dimerizes ribosomes and prevents translation of mRNA. Bioinformatic analyses indicate that B. burgdorferi harbors an hpf homolog, the bb0449 gene. BB0449 protein secondary structure modeling also predicted HPF-like structure and function. However, BB0449 protein was not localized in the ribosome-associated protein fraction of in vitro-grown B. burgdorferi. In wild-type B. burgdorferi, bb0449 transcript and BB0449 protein levels are low during various growth phases. These results are inconsistent with patterns of synthesis of HPF-like proteins in other bacterial species. In addition, two independently derived bb0449 mutants successfully completed the mouse-tick infectious cycle, indicating that bb0449 is not required for prolonged survival in the nutrient-limited environment in the unfed tick or any other stage of infection by B. burgdorferi. We suggest either that BB0449 is associated with ribosomes under specific conditions not yet identified or that BB0449 of B. burgdorferi has a function other than ribosome conformation modulation. L yme disease, the most common tick-transmitted disease in the United States (1), is caused by infection with the spirochete Borrelia burgdorferi (2, 3). This pathogen's complex infectious cycle includes an Ixodes tick vector and multiple vertebrate hosts. The Ixodes tick vector, like all hard ticks, has three developmental stages: larva, nymph, and adult. Typically, larval ticks become infected with B. burgdorferi by feeding on an infected vertebrate host, often a small mammal. B. burgdorferi survives within the tick midgut (2) as larval ticks molt into nymphs (transstadial passage) and the nymphs wait to feed again, which can include overwintering (4, 5). Following the larval molt, infected nymphs can transmit B. burgdorferi to naive vertebrate hosts, which, in turn, serve as a reservoir of spirochetes for larval ticks. After fed nymphs molt, infected adult ticks can also transmit B. burgdorferi, but adults typically feed on larger animals upon which larvae do not feed. Moreover, little or no transovarial transmission of spirochetes occurs (6, 7). Therefore, adult ticks do not significantly contribute to the maintenance of B. burgdorferi populations (8), and neither do humans, which are incidental hosts of B. burgdorferi. Hence, the larval and nymphal tick life stages are relevant to the zoonotic life cycle of B. burgdorferi.It has been generally accepted that B. burgdorferi populations are sustained in nature by the long-term infect...