Research on insect microbiota has greatly expanded over the past decade, along with a growing appreciation of the microbial contributions to insect ecology and evolution. Many of these studies use DNA sequencing to characterize the diversity and composition of insect-associated microbial communities. The choice of strategies used for specimen collection, storage, and handling could introduce biases in molecular assessments of insect microbiota, but such potential influences have not been systematically evaluated. Likewise, although it is common practice to surface sterilize insects prior to DNA extraction, it is not known if this time-consuming step has any effect on microbial community analyses. To resolve these methodological unknowns, we conducted an experiment wherein replicate individual insects of four species were stored intact for two months using five different methods—freezing, ethanol, dimethyl sulfoxide (DMSO), cetrimonium bromide (CTAB), and room-temperature storage without preservative—and then subjected to whole-specimen 16S rRNA gene sequencing to assess whether the structure of the insect-associated bacterial communities was impacted by these different storage strategies. Overall, different insect species harbored markedly distinct bacterial communities, a pattern that was highly robust to the method used to store samples. Storage method had little to no effect on assessments of microbiota composition, and the magnitude of the effect differed among the insect species examined. No single method emerged as “best,” i.e., one consistently having the highest similarity in community structure to control specimens, which were not stored prior to homogenization and DNA sequencing. We also found that surface sterilization did not change bacterial community structure as compared to unsterilized insects, presumably due to the vastly greater microbial biomass inside the insect body relative to its surface. We therefore recommend that researchers can use any of the methods tested here, and base their choice according to practical considerations such as prior use, cost, and availability in the field, although we still advise that all samples within a study be handled in an identical manner when possible. We also suggest that, in large-scale molecular studies of hundreds of insect specimens, surface sterilization may not be worth the time and effort involved. This information should help researchers design sampling strategies and will facilitate cross-comparisons and meta-analyses of microbial community data obtained from insect specimens preserved in different ways.

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. Importance Many 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.

The educational literature recognizes that people go through a number of stages in their intellectual development. During the first stage, called received knowledge or dualism, people expect knowledge to be handed to them by authority figures (thus "received") and think in terms of black and white (thus "dualism"). Our experience indicates that many computer science students are at this first stage of learning. To help students move beyond this stage, we describe a system and strategy, the PL-Detective, to be used in a Concepts of Programming Languages course. Assignments using this system directly confront students with the notion that they can create knowledge via interactions with the PL-Detective and that discussion with students (rather than asking the instructor) is an effective way of learning how to reason. We present experimental results that show that the PL-Detective is effective in helping students move beyond the stage of received knowledge.

Last year we described the PL-Detective, a system for building exercises and demonstrations in a programming languages course. One of the main goals of the PL-Detective was to provide an experimental environment with which students could interact in order to discover the information that they needed to complete the exercise. In this paper we evaluate the PL-Detective with respect to this goal. We present data from a class of 29 groups of two or three students that used the PL-Detective for 11 exercises. Our data shows that students are both effective and efficient at getting information from the PL-Detective.