Spatially explicit depiction of a floral epiphytic bacterial community reveals role for environmental filtering within petals.

The microbiome of flowers (anthosphere) is an understudied compartment of the plant microbiome. Within the flower, petals represent a heterogeneous environment for microbes in terms of resources and environmental stress. Yet, little is known of drivers of structure and function of the epiphytic microbial community at the within-petal scale. We characterized the petal microbiome in two co-flowering plants that differ in the pattern of ultraviolet (UV) absorption along their petals. Bacterial communities were similar between plant hosts, with only rare phylogenetically distant species contributing to differences. The epiphyte community was highly culturable (75% of families) lending confidence in the spatially explicit isolation and characterization of bacteria. In one host, petals were heterogeneous in UV absorption along their length, and in these, there was a negative relationship between growth rate and position on the petal, as well as lower UV tolerance in strains isolated from the UV-absorbing base than from UV reflecting tip. A similar pattern was not seen in microbes isolated from a second host whose petals had uniform patterning along their length. Across strains, the variation in carbon usage and chemical tolerance followed common phylogenetic patterns. This work highlights the value of petals for spatially explicit explorations of bacteria of the anthosphere.

A modifier screen identifies regulators of cytoskeletal architecture as mediators of Shroom-dependent changes in tissue morphology.

Regulation of cell architecture is critical in the formation of tissues during animal development. The mechanisms that control cell shape must be both dynamic and stable in order to establish and maintain the correct cellular organization. Previous work has identified Shroom family proteins as essential regulators of cell morphology during vertebrate development. Shroom proteins regulate cell architecture by directing the subcellular distribution and activation of Rho-kinase, which results in the localized activation of non-muscle myosin II. Because the Shroom-Rock-myosin II module is conserved in most animal model systems, we have utilized Drosophila melanogaster to further investigate the pathways and components that are required for Shroom to define cell shape and tissue architecture. Using a phenotype-based heterozygous F1 genetic screen for modifiers of Shroom activity, we identified several cytoskeletal and signaling protein that may cooperate with Shroom. We show that two of these proteins, Enabled and Short stop, are required for ShroomA-induced changes in tissue morphology and are apically enriched in response to Shroom expression. While the recruitment of Ena is necessary, it is not sufficient to redefine cell morphology. Additionally, this requirement for Ena appears to be context dependent, as a variant of Shroom that is apically localized, binds to Rock, but lacks the Ena binding site, is still capable of inducing changes in tissue architecture. These data point to important cellular pathways that may regulate contractility or facilitate Shroom-mediated changes in cell and tissue morphology.

Am I a Science Person? A Strong Science Identity Bolsters Minority Students' Sense of Belonging and Performance in College.

Identifying as a "science person" is predictive of science success, but the mechanisms involved are not well-understood. We hypothesized that science identity predicts success because it fosters a sense of belonging in science classrooms. Thus, science identity should be particularly important for first-generation and racial-minority students, who may harbor doubts about belonging in science. Two field studies in college Introductory Biology classes (Ns = 368, 639) supported these hypotheses. A strong science identity predicted higher grades, particularly for minority students. Also consistent with hypotheses, Study 2 found that self-reported belonging in college mediated the relationship between science identity and performance. Furthermore, a social belonging manipulation eliminated the relationship between science identity and performance among minority students. These results support the idea that a strong science identity is particularly beneficial for minority students because it bolsters belonging in science courses. Practical and theoretical implications are discussed.

Changing Social Contexts to Foster Equity in College Science Courses: An Ecological-Belonging Intervention.

In diverse classrooms, stereotypes are often "in the air," which can interfere with learning and performance among stigmatized students. Two studies designed to foster equity in college science classrooms (Ns = 1,215 and 607) tested an intervention to establish social norms that make stereotypes irrelevant in the classroom. At the beginning of the term, classrooms assigned to an ecological-belonging intervention engaged in discussion with peers around the message that social and academic adversity is normative and that students generally overcome such adversity. Compared with business-as-usual controls, intervention students had higher attendance, course grades, and 1-year college persistence. The intervention was especially impactful among historically underperforming students, as it improved course grades for ethnic minorities in introductory biology and for women in introductory physics. Regardless of demographics, attendance in the intervention classroom predicted higher cumulative grade point averages 2 to 4 years later. The results illustrate the viability of an ecological approach to fostering equity and unlocking student potential.

Exposure to cuticular bacteria can alter host behavior in a funnel-weaving spider.

Contact with environmental microbes are arguably the most common species interaction in which any animal participates. Studies have noted diverse relationships between hosts and resident microbes, which can have strong consequences for host development, physiology, and behavior. Many of these studies focus specifically on pathogens or beneficial microbes, while the benign microbes, of which the majority of bacteria could be described, are often ignored. Here, we explore the nature of the relationships between the grass spider Agelenopsis pennsylvanica and bacteria collected from their cuticles in situ. First, using culture-based methods, we identified a portion of the cuticular bacterial communities that are naturally associated with these spiders. Then, we topically exposed spiders to a subset of these bacterial monocultures to estimate how bacterial exposure may alter 3 host behavioral traits: boldness, aggressiveness, and activity level. We conducted these behavioral assays 3 times before and 3 times after topical application, and compared the changes observed in each trait with spiders that were exposed to a sterile control treatment. We identified 9 species of bacteria from the cuticles of 36 spiders and exposed groups of 20 spiders to 1 of 4 species of cuticular bacteria. We found that exposure to Dermacoccus nishinomiyaensis and Staphylococcus saprophyticus was associated with a 10-fold decrease in the foraging aggressiveness of spiders toward prey in their web. Since bacterial exposure did not have survival consequences for hosts, these data suggest that interactions with cuticular bacteria, even non-pathogenic bacteria, could alter host behavior.

Erratum: Exposure to cuticular bacteria can alter host behavior in a funnel-weaving spider.

[This corrects the article DOI: 10.1093/cz/zox064.]

An inclusive Research Education Community (iREC): Impact of the SEA-PHAGES program on research outcomes and student learning.

Engaging undergraduate students in scientific research promises substantial benefits, but it is not accessible to all students and is rarely implemented early in college education, when it will have the greatest impact. An inclusive Research Education Community (iREC) provides a centralized scientific and administrative infrastructure enabling engagement of large numbers of students at different types of institutions. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) is an iREC that promotes engagement and continued involvement in science among beginning undergraduate students. The SEA-PHAGES students show strong gains correlated with persistence relative to those in traditional laboratory courses regardless of academic, ethnic, gender, and socioeconomic profiles. This persistent involvement in science is reflected in key measures, including project ownership, scientific community values, science identity, and scientific networking.

Function and immuno-localization of aquaporins in the Antarctic midge Belgica antarctica.

Aquaporin (AQP) water channel proteins play key roles in water movement across cell membranes. Extending previous reports of cryoprotective functions in insects, this study examines roles of AQPs in response to dehydration, rehydration, and freezing, and their distribution in specific tissues of the Antarctic midge, Belgica antarctica (Diptera, Chironomidae). When AQPs were blocked using mercuric chloride, tissue dehydration tolerance increased in response to hypertonic challenge, and susceptibility to overhydration decreased in a hypotonic solution. Blocking AQPs decreased the ability of tissues from the midgut and Malpighian tubules to tolerate freezing, but only minimal changes were noted in cellular viability of the fat body. Immuno-localization revealed that a DRIP-like protein (a Drosophila aquaporin), AQP2- and AQP3 (aquaglyceroporin)-like proteins were present in most larval tissues. DRIP- and AQP2-like proteins were also present in the gut of adult midges, but AQP4-like protein was not detectable in any tissues we examined. Western blotting indicated that larval AQP2-like protein levels were increased in response to dehydration, rehydration and freezing, whereas, in adults DRIP-, AQP2-, and AQP3-like proteins were elevated by dehydration. These results imply a vital role for aquaporin/aquaglyceroporins in water relations and freezing tolerance in B. antarctica.

Illuminating cell signaling: Using Vibrio harveyi in an introductory biology laboratory.

Cell signaling is an essential cellular process that is performed by all living organisms. Bacteria communicate with each other using a chemical language in a signaling pathway that allows bacteria to evaluate the size of their population, determine when they have reached a critical mass (quorum sensing), and then change their behavior in unison to carry out processes that require many cells acting together to be effective. Here, we describe a laboratory exercise in which the students observe the induction of bioluminescence or light production as an output of the quorum sensing pathway in Vibrio harveyi. Using both wildtype and mutant bacterial strains they explore the induction of community behavior via cell-cell communication by determining whether there is a correlation between the density of the bacterial population and the production of light by the bacterial culture. Using data from a cross-feeding assay the students make predictions about the identity of their strains and directly test these predictions using conditioned media from various liquid cultures. This two part exercise is designed for an introductory biology course to begin familiarizing students with collecting data, making predictions based upon the data and directly testing their hypotheses using a model organism with a cell signaling pathway that has a simple visual output: light production.

Localization of a Drosophila DRIP-like aquaporin in the Malpighian tubules of the house cricket, Acheta domesticus.

Malpighian tubules (Mt) are the primary excretory and osmoregulatory organs of insects, capable of rapidly transporting extraordinary volumes of fluid when stimulated by diuretic factors. In the house cricket, Acheta domesticus, the Mt are composed of three morphologically distinct regions (proximal, mid, and distal). Unlike the dipteran Mt, which have both primary and stellate cells, each region of the Acheta Mt consists of a morphologically uniform cell type. The mid and distal regions are both secretory in function and increase secretion rate in response to dibutyryl cAMP (cAMP). Achetakinin-2, while acting synergistically with cAMP on the mid-Mt, inhibits secretion by the distal Mt, and the effects can be reversed by cAMP. Using an antibody to the water-specific Drosophila aquaporin (DRIP), we demonstrated that DRIP-like immunoreactivity was found in both the distal and mid-Mt. The distribution of the aquaporin altered in response to stimulation and was consistent with the secretory data. The regulation of secretion in Acheta Mt is quite different from that of Drosophila, with both cation and anion/water transport occurring in the same cells. This is the first demonstration of the presence of an insect aquaporin, namely DRIP, in the Mt of an order other than the Diptera.

Developmental expression and biophysical characterization of a Drosophila melanogaster aquaporin.

Aquaporins (AQPs) accelerate the movement of water and other solutes across biological membranes, yet the molecular mechanisms of each AQP's transport function and the diverse physiological roles played by AQP family members are still being defined. We therefore have characterized an AQP in a model organism, Drosophila melanogaster, which is amenable to genetic manipulation and developmental analysis. To study the mechanism of Drosophila Malpighian tubule (MT)-facilitated water transport, we identified seven putative AQPs in the Drosophila genome and found that one of these, previously named DRIP, has the greatest sequence similarity to those vertebrate AQPs that exhibit the highest rates of water transport. In situ mRNA analyses showed that DRIP is expressed in both embryonic and adult MTs, as well as in other tissues in which fluid transport is essential. In addition, the pattern of DRIP expression was dynamic. To define DRIP-mediated water transport, the protein was expressed in Xenopus oocytes and in yeast secretory vesicles, and we found that significantly elevated rates of water transport correlated with DRIP expression. Moreover, the activation energy required for water transport in DRIP-expressing secretory vesicles was 4.9 kcal/mol. This low value is characteristic of AQP-mediated water transport, whereas the value in control vesicles was 16.4 kcal/mol. In contrast, glycerol, urea, ammonia, and proton transport were unaffected by DRIP expression, suggesting that DRIP is a highly selective water-specific channel. This result is consistent with the homology between DRIP and mammalian water-specific AQPs. Together, these data establish Drosophila as a new model system with which to investigate AQP function.

Direct observation demonstrates that Liprin-alpha is required for trafficking of synaptic vesicles.

Axonal transport is required for the elaboration and maintenance of synaptic morphology and function. Liprin-alphas are scaffolding proteins important for synapse structure and electrophysiology. A reported interaction with Kinesin-3 (Kif1a) suggested Liprin-alpha may also be involved in axonal transport. Here, at the light and ultrastructural levels, we discover aberrant accumulations of synaptic vesicle markers (Synaptotagmin and Synaptobrevin-GFP) and clear-core vesicles along Drosophila Liprin-alpha mutant axons. Analysis of presynaptic markers reveals reduced levels at Liprin-alpha synapses. Direct visualization of Synaptobrevin-GFP transport in living animals demonstrates a decrease in anterograde processivity in Liprin-alpha mutants but also an increase in retrograde transport initiation. Pull-down assays reveal that Liprin-alpha interacts with Drosophila Kinesin-1 (Khc) but not dynein. Together, these findings suggest that Liprin-alpha promotes the delivery of synaptic material by a direct increase in kinesin processivity and an indirect suppression of dynein activation. This work is the first to use live observation in Drosophila mutants to demonstrate the role of a scaffolding protein in the regulation of bidirectional transport. It suggests the synaptic strength and morphology defects linked to Liprin-alpha may in part be due to a failure in the delivery of synaptic-vesicle precursors.

Functions of the ectodomain and cytoplasmic tyrosine phosphatase domains of receptor protein tyrosine phosphatase Dlar in vivo.

The receptor protein tyrosine phosphatase (PTPase) Dlar has an ectodomain consisting of three immunoglobulin (Ig)-like domains and nine fibronectin type III (FnIII) repeats and a cytoplasmic domain consisting of two PTPase domains, membrane-proximal PTP-D1 and C-terminal PTP-D2. A series of mutant Dlar transgenes were introduced into the Drosophila genome via P-element transformation and were then assayed for their capacity to rescue phenotypes caused by homozygous loss-of-function genotypes. The Ig-like domains, but not the FnIII domains, are essential for survival. Conversely, the FnIII domains, but not the Ig-like domains, are required during oogenesis, suggesting that different domains of the Dlar ectodomain are involved in distinct functions during Drosophila development. All detectable PTPase activity maps to PTP-D1 in vitro. The catalytically inactive mutants of Dlar were able to rescue Dlar(-/-) lethality nearly as efficiently as wild-type Dlar transgenes, while this ability was impaired in the PTP-D2 deletion mutants DlarDeltaPTP-D2 and Dlar(bypass). Dlar-C1929S, in which PTP-D2 has been inactivated, increases the frequency of bypass phenotype observed in Dlar(-/-) genotypes, but only if PTP-D1 is catalytically active in the transgene. These results indicate multiple roles for PTP-D2, perhaps by acting as a docking domain for downstream elements and as a regulator of PTP-D1.

Drosophila liprin-alpha and the receptor phosphatase Dlar control synapse morphogenesis.

Here, we examine the synaptic function of the receptor protein tyrosine phosphatase (RPTP), Dlar, and an associated intracellular protein, Dliprin-alpha, at the Drosophila larval neuromuscular junction. We show that Dliprin-alpha and Dlar are required for normal synaptic morphology. We also find that synapse complexity is proportional to the amount of Dlar gene product, suggesting that Dlar activity determines synapse size. Ultrastructural analysis reveals that Dliprin-alpha and Dlar are required to define the size and shape of the presynaptic active zone. Accordingly, there is a concomitant decrease in synaptic transmission in both mutants. Finally, epistasis analysis indicates that Dliprin-alpha is required for Dlar's action at the synapse. These data suggest a model where Dliprin-alpha and Dlar cooperate to regulate the formation and/or maintenance of a network of presynaptic proteins.