Heliconius Butterflies Host Characteristic and Phylogenetically Structured Adult-Stage Microbiomes

ABSTRACT Lepidoptera (butterflies and moths) are diverse and ecologically important, yet we know little about how they interact with microbes as adults. Due to metamorphosis, the form and function of their adult-stage microbiomes might be very different from those of microbiomes in the larval stage (caterpillars). We studied adult-stage microbiomes of Heliconius and closely related passion-vine butterflies (Heliconiini), which are an important model system in evolutionary biology. To characterize the structure and dynamics of heliconiine microbiomes, we used field collections of wild butterflies, 16S rRNA gene sequencing, quantitative PCR, and shotgun metagenomics. We found that Heliconius butterflies harbor simple and abundant bacterial communities that are moderately consistent among conspecific individuals and over time. Heliconiine microbiomes also exhibited a strong signal of the host phylogeny, with a major distinction between Heliconius and other butterflies. These patterns were largely driven by differing relative abundances of bacterial phylotypes shared among host species and genera, as opposed to the presence or absence of host-specific phylotypes. We suggest that the phylogenetic structure in heliconiine microbiomes arises from conserved host traits that differentially filter microbes from the environment. While the relative importance of different traits remains unclear, our data indicate that pollen feeding (unique to Heliconius) is not a primary driver. Using shotgun metagenomics, we also discovered trypanosomatids and microsporidia to be prevalent in butterfly guts, raising the possibility of antagonistic interactions between eukaryotic parasites and colocalized gut bacteria. Our discovery of characteristic and phylogenetically structured microbiomes provides a foundation for tests of adult-stage microbiome function, a poorly understood aspect of lepidopteran biology. IMPORTANCE Many insects host microbiomes with important ecological functions. However, the prevalence of this phenomenon is unclear because in many insect taxa, microbiomes have been studied in only part of the life cycle, if at all. A prominent example is butterflies and moths, in which the composition and functional role of adult-stage microbiomes are largely unknown. We comprehensively characterized microbiomes in adult passion-vine butterflies. Butterfly-associated bacterial communities are generally abundant in guts, consistent within populations, and composed of taxa widely shared among hosts. More closely related butterflies harbor more similar microbiomes, with the most dramatic shift in microbiome composition occurring in tandem with a suite of ecological and life history traits unique to the genus Heliconius. Butterflies are also frequently infected with previously undescribed eukaryotic parasites, which may interact with bacteria in important ways. These findings advance our understanding of butterfly biology and insect-microbe interactions generally.

Egg cannibalism by passion vine specialist Disonycha Chevrolat beetles (Coleoptera: Chrysomelidae: Galerucinae: Alticini)

Cannibalistic behavior is now recognized to be an important component of nutritional ecology in both carnivorous and herbivorous species, including many beetle families (Englert and Thomas 1970; Beaver 1974; Dickinson 1992; Bartlett 1987; Alabi et al. 2008). This habit was historically viewed by an incidental outcome of unnaturally crowded laboratory situations with little ecological importance (Fox 1975), but it is increasingly acknowledged that cannibalism represents a potentially advantageous behavior (Richardson et al. 2010). Here we report on multiple cases of egg cannibalism, or conspecific oophagy, by adults of two species of passion vine (Passiflora Linnaeus: Passifloraceae) specialist flea beetles in the genus Disonycha Chevrolat (Coleoptera: Chrysomelidae: Galerucinae: Alticini). This is the first report of egg cannibalism from the Galerucinae, and to our knowledge, only the fourth report of egg cannibalism by adults in the Chrysomelidae; the other three reports are of adult Chrysomelinae species eating conspecific eggs (Dickinson 1992; McCauley 1992; Schrod et al. 1996). We conclude this note with several questions raised by our observations, followed by a discussion that may contribute to explanations of this behavior.

Heliconius butterflies host characteristic and phylogenetically structured adult-stage microbiomes

Lepidoptera (butterflies and moths) are diverse and ecologically important, yet we know little about how they interact with microbes as adults. Due to metamorphosis, the form and function of their adult-stage microbiomes might be very different from microbiomes in the larval stage (caterpillars). We studied adult-stage microbiomes of Heliconius and closely related passion-vine butterflies (Heliconiini), which are an important model system in evolutionary biology. To characterize the structure and dynamics of heliconiine microbiomes, we used field collections of wild butterflies, 16S rRNA gene sequencing, quantitative PCR, and shotgun metagenomics. We found that Heliconius harbor simple and abundant bacterial communities that are moderately consistent among conspecific individuals and over time. Heliconiine microbiomes also exhibited a strong signal of host phylogeny, with a major distinction between Heliconius and other butterflies. These patterns were largely driven by differing relative abundances of bacterial phylotypes shared among host species and genera, as opposed to the presence or absence of host-specific phylotypes. We suggest that phylogenetic structure in heliconiine microbiomes arises from conserved host traits that differentially filter microbes from the environment. While the relative importance of different traits remains unclear, our data indicate that pollen-feeding (unique to Heliconius) is not a primary driver. Using shotgun metagenomics, we also discovered trypanosomatids and microsporidia to be prevalent in butterfly guts, raising the possibility of antagonistic interactions between eukaryotic parasites and co-localized gut bacteria. Our discovery of characteristic and phylogenetically structured microbiomes provides a foundation for tests of adult-stage microbiome function, a poorly understood aspect of lepidopteran biology.ImportanceMany insects host microbiomes with important ecological functions. However, the prevalence of this phenomenon is unclear, because in many insect taxa microbiomes have only been studied in part of the life cycle, if at all. A prominent example is the butterflies and moths, in which the composition and functional role of adult-stage microbiomes are largely unknown. We comprehensively characterized microbiomes in adult passion-vine butterflies. Butterfly-associated bacterial communities are generally abundant in guts, consistent within populations, and composed of taxa widely shared among hosts. More closely related butterflies harbor more similar microbiomes, with the most dramatic shift in microbiome composition occurring in tandem with a suite of ecological and life history traits unique to the genus Heliconius. Butterflies are also frequently infected with previously undescribed eukaryotic parasites, which may interact with bacteria in important ways. These findings advance our understanding of butterfly biology and of insect-microbe interactions generally.

Performance consequences of food mixing in two passion vine leaf-footed bugs, Holymenia clavigera (Herbst, 1784) and Anisoscelis foliacea marginella (Dallas, 1852) (Hemiptera; Coreidae)

Holymenia clavigera (Herbst) and Anisoscelis foliacea marginella (Dallas) (Hemiptera: Coreidae: Anisoscelini) are distributed in southern Brazil and use various passion vine species (Passifloraceae) as host-plants. Preliminary observations indicate a high coexistence of these species in terms of host-plant use; in addition, there is a strong similarity regarding egg and nymph morphology. In this study, the most suitable feeding sites for nymph performance on wild (Passiflora suberosa Linnaeus and Passiflora misera Humbold, Bonpland et Kunth) and cultivated (Passiflora edulis Sims) hosts were determined by rearing them on each host and on the combination of hosts. Performance was determined by evaluating nymph development and survivorship, and adult size at emergence. Plant parts used were also recorded. For both species, P. suberosa was the most suitable host plant. First instar nymphs of both species fed on terminal buds more frequently when compared to other plant parts. Second instar nymphs switched to green fruits, whose behavior was more pronounced for H. clavigera. Thus, H. clavigera and A. foliacea marginella immatures are extremely similar in terms of host-plant use and consequences for performance, in addition to their morphological similarity. We suggest that these coreids may have evolved through several processes, including parsimony between the immature stages after speciation, evolutionary convergence, mimicry or genetic drift.

Selection of wild hosts for feeding by passion vine hopper Scolypopa australis (Walker) (Hemiptera Ricaniidae) in the Bay of Plenty

Passion vine hopper Scolypopa australis (Walker) (Hemiptera Ricaniidae) a pest of kiwifruit occurs on a wide range of native and introduced plants that are probably not equally preferred as hosts First instar nymphs and adults were observed on 69 and 53 respectively of 42 plant species found at varying frequency in a survey of gullies adjacent to kiwifruit orchards in the Bay of Plenty In choice tests with four plant species common in gullies firstinstar nymphs and adults preferred the native species mahoe (Melicytus ramiflorus) Under nochoice conditions survival from first to late instar was also best on mahoe Of 10 plant species in a nochoice test adults survived best and laid most eggs on the native species wineberry (Aristotelia serrata)