Predicting the effects of multiple global change drivers on microbial communities remains challenging.

Microbial communities in many ecosystems are facing a broad range of global change drivers, such as nutrient enrichment, chemical pollution, and temperature change. These drivers can cause changes in the abundance of taxa, the composition of communities, and the properties of ecosystems. While the influence of single drivers is already described in numerous studies, the effect and predictability of multiple drivers changing simultaneously is still poorly understood. In this study, we used 240 highly replicable oxic/anoxic aquatic lab microcosms and four drivers (fertilizer, glyphosate, metal pollution, antibiotics) in all possible combinations at three different temperatures (20, 24, and 28°C) to shed light into consequences of multiple drivers on different levels of organization, ranging from species abundance to community and ecosystem parameters. We found (i) that at all levels of ecological organization, combinations of drivers can change the biological consequence and direction of effect compared to single drivers, (ii) that effects of combinations are further modified by temperature, (iii) that a larger number of drivers occurring simultaneously is often quite closely related to their effect size, and (iv) that there is little evidence that any of these effects are associated with the level of ecological organization of the state variable. These findings suggest that, at least in this experimental ecosystem approximating a stratified aquatic ecosystem, there may be relatively little scope for predicting the effects of combinations of drivers from the effects of individual drivers, or by accounting for the level of ecological organization in question, though there may be some scope for prediction based on the number of drivers that are occurring simultaneous. A priority, though also a considerable challenge, is to extend such research to consider continuous variation in the magnitude of multiple drivers acting together.

Contrasting resistance and resilience to light variation of the coupled oxic and anoxic components of an experimental microbial ecosystem.

Understanding how microbial communities of aquatic ecosystems respond to environmental change remains a critical challenge in microbial ecology. In this study, we used light-dependent oxic-anoxic micro-ecosystems to understand how the functioning and diversity of aerobic and anaerobic lake analog communities are affected by a pulse light deprivation. Continuous measurements of oxygen concentration were made and a time series of full-length 16S rRNA sequencing was used to quantify changes in alpha- and beta diversity. In the upper oxic layer, oxygen concentration decreased significantly under light reduction, but showed resilience in daily mean, minimum, and maximum after light conditions were restored to control level. Only the amplitude of diurnal fluctuations in oxygen concentrations did not recover fully, and instead tended to remain lower in treated ecosystems. Alpha diversity of the upper oxic layer communities showed a delayed increase after light conditions were restored, and was not resilient in the longer term. In contrast, alpha diversity of the anoxic lower layer communities increased during the light reduction, but was resilient in the longer term. Community composition changed significantly during light reduction, and showed resilience in the oxic layer and lack of resilience in the anoxic layer. Alpha diversity and the amplitude of daily oxygen fluctuations within and among treatments were strongly correlated, suggesting that higher diversity could lead to less variable oxygen concentrations, or vice versa. Our experiment showed that light deprivation induces multifaceted responses of community function (oxygen respiration) and structure, hence focusing on a single stability component could potentially be misleading.

Large and interacting effects of temperature and nutrient addition on stratified microbial ecosystems in a small, replicated, and liquid-dominated Winogradsky column approach.

Aquatic ecosystems are often stratified, with cyanobacteria in oxic layers and phototrophic sulfur bacteria in anoxic zones. Changes in stratification caused by the global environmental change are an ongoing concern. Increasing understanding of how such aerobic and anaerobic microbial communities, and associated abiotic conditions, respond to multifarious environmental changes is an important endeavor in microbial ecology. Insights can come from observational and experimental studies of naturally occurring stratified aquatic ecosystems, theoretical models of ecological processes, and experimental studies of replicated microbial communities in the laboratory. Here, we demonstrate a laboratory-based approach with small, replicated, and liquid-dominated Winogradsky columns, with distinct oxic/anoxic strata in a highly replicable manner. Our objective was to apply simultaneous global change scenarios (temperature, nutrient addition) on this micro-ecosystem to report how the microbial communities (full-length 16S rRNA gene seq.) and the abiotic conditions (O2 , H2 S, TOC) of the oxic/anoxic layer responded to these environmental changes. The composition of the strongly stratified microbial communities was greatly affected by temperature and by the interaction of temperature and nutrient addition, demonstrating the need of investigating global change treatments simultaneously. Especially phototrophic sulfur bacteria dominated the water column at higher temperatures and may indicate the presence of alternative stable states. We show that the establishment of such a micro-ecosystem has the potential to test global change scenarios in stratified eutrophic limnic systems.

Biodiversity increases and decreases ecosystem stability.

Losses and gains in species diversity affect ecological stability1-7 and the sustainability of ecosystem functions and services8-13. Experiments and models have revealed positive, negative and no effects of diversity on individual components of stability, such as temporal variability, resistance and resilience2,3,6,11,12,14. How these stability components covary remains poorly understood15. Similarly, the effects of diversity on overall ecosystem stability16, which is conceptually akin to ecosystem multifunctionality17,18, remain unknown. Here we studied communities of aquatic ciliates to understand how temporal variability, resistance and overall ecosystem stability responded to diversity (that is, species richness) in a large experiment involving 690 micro-ecosystems sampled 19 times over 40 days, resulting in 12,939 samplings. Species richness increased temporal stability but decreased resistance to warming. Thus, two stability components covaried negatively along the diversity gradient. Previous biodiversity manipulation studies rarely reported such negative covariation despite general predictions of the negative effects of diversity on individual stability components3. Integrating our findings with the ecosystem multifunctionality concept revealed hump- and U-shaped effects of diversity on overall ecosystem stability. That is, biodiversity can increase overall ecosystem stability when biodiversity is low, and decrease it when biodiversity is high, or the opposite with a U-shaped relationship. The effects of diversity on ecosystem multifunctionality would also be hump- or U-shaped if diversity had positive effects on some functions and negative effects on others. Linking the ecosystem multifunctionality concept and ecosystem stability can transform the perceived effects of diversity on ecological stability and may help to translate this science into policy-relevant information.

The effect of mating on immunity can be masked by experimental piercing in female Drosophila melanogaster.

Mating and immunity are two major components of fitness and links between them have been demonstrated in a number of recent investigations. In Drosophila melanogaster, a seminal fluid protein, sex-peptide (SP), up-regulates a number of antimicrobial peptide (AMP) genes in females after mating but the resulting effect on pathogen resistance is unclear. In this study, we tested (1) whether SP-induced changes in gene expression affect the ability of females to kill injected non-pathogenic bacteria and (2) how the injection process per se affects the expression of AMP genes relative to SP. The ability of virgin females and females mated to SP lacking or control males to clear bacteria was assayed using an established technique in which Escherichia coli are injected directly into the fly body and the rate of clearance of the injected bacteria is determined. We found no repeatable differences in clearance rates between virgin females and females mated to SP producing or SP lacking males. However, we found that the piercing of the integument, as occurs during injection, up-regulates AMP gene expression much more strongly than SP. Thus, assays that involve piercing, which are commonly used in immunity studies, can mask more subtle and biologically relevant changes in immunity, such as those induced by mating.

Sex-peptide-regulated female sexual behavior requires a subset of ascending ventral nerve cord neurons.

BACKGROUND: Male-derived Sex-peptide (SP) elicits egg laying and rejection of courting males in mated Drosophila females. Little is known about the genes that specify the underlying neuronal circuits and mediate this switch in female sexual behavior. RESULTS: Here we show that the egghead gene involved in glycosphingolipid biosynthesis provides an essential component to the SP response. We have isolated viable alleles of the vital egghead gene that abolish egghead expression from a distal promoter resulting in the absence of the largest transcript of this complex transcription unit. Temporally and spatially restricted expression of egghead revealed a requirement for egghead early in the development of apterous-expressing ventral nerve cord neurons to rescue the SP response. In viable egghead alleles, these ascending interneurons, three per abdominal and seven per thoracic hemisegment, fail to innervate the central brain. egghead expression in apterous neurons rescues neuronal targeting and the response to SP. Furthermore, neurotransmission in apterous neurons is required to elicit the SP response. CONCLUSION: Together with the former finding of SP binding to afferent nerves , these results suggest that SP-mediated modification of sensory input switches female sexual behavior from the virgin to the mated state.

The presence of Drosophila melanogaster sex peptide-like immunoreactivity in the accessory glands of male Helicoverpa armigera.

In this study a highly specific polyclonal antibody to DrmSP was produced and used to develop and standardize a sensitive direct ELISA. Structure-activity studies revealed that the antiserum is specific to the N-terminal of DrmSP. This ELISA was used for the detection of DrmSP-like immunoreactivity in the reproductive tissues of male Helicoverpa armigera moths at femtomole levels. Two positive immunoreactive peaks were found in HPLC purified extracts of male accessory glands. The immunoreactive peak, which contained a higher amount of immunoreactivity, was also found to be pheromonostatic in PBAN-injected decapitated females as well as in intact female moths during their peak pheromone production. Lower levels of DrmSP-like immunoreactivity were found in younger males (1-2 day-old) when compared to older males (3-7 day-old).

The sex-peptide DUP99B is expressed in the male ejaculatory duct and in the cardia of both sexes.

Mating elicits two postmating responses in many insect females: the egg laying rate increases and sexual receptivity is reduced. In Drosophila melanogaster, two peptides of the male genital tract, sex-peptide and DUP99B, elicit these postmating responses when injected into virgin females. Here we show that the gene encoding DUP99B is expressed in the male ejaculatory duct and in the cardia of both sexes. The DUP99B that is synthesized in the ejaculatory duct is transferred, during mating, into the female genital tract. Expression of the gene is first seen in a late pupal stage. Males containing an intact ejaculatory duct, but lacking accessory glands, initiate the two postmating responses in their female partners [Xue, L. & Noll, M. (2000) Proc. Natl Acad. Sci. USA97, 3272-3275]. Although such males synthesize DUP99B in wild-type quantities, they elicit only weak postmating responses in their mating partners. Males lacking the Dup99B gene elicit the two postmating responses to the same extent as wild-type males. These results suggest that both sex-peptide and DUP99B can elicit both responses in vivo. However, sex-peptide seems to play the major role in eliciting the postmating responses, while DUP99B may have specialized for other, as yet unknown, functions.

Ductus ejaculatorius peptide 99B (DUP99B), a novel Drosophila melanogaster sex-peptide pheromone.

We have characterized a glycosylated, 31 amino-acid peptide of 4932 Da isolated from Drosophila melanogaster males. The mature peptide contains a sugar moiety of 1184 Da at a NDT consensus glycosylation site and a disulfide bond. It is synthesized in the male ejaculatory duct via a 54 amino-acid precursor containing an N-terminal signal peptide and Arg-Lys at the C-terminus which is cleaved off during maturation. The gene contains an intron of 53 bp and is localized in the cytological region 99B of the D. melanogaster genome. The peptide is therefore named DUP99B (for ductus ejaculatorius peptide, cytological localization 99B). The C-terminal parts of mature DUP99B and D. melanogaster sex-peptide (ACP70A) are highly homologous. Injected into virgin females, DUP99B elicits the same postmating responses as sex-peptide (increased oviposition, reduced receptivity). These effects are also induced by de-glycosylated native peptide or synthetic DUP99B lacking the sugar moiety. Presence of the glycosyl group, however, decreases the amount needed to elicit the postmating responses. Homologies in the coding regions of the two exons of DUP99B and sex-peptide, respectively, suggest that the two genes have evolved by gene duplication. Thus, we consider these two genes to be members of the new sex-peptide gene family.