Genetic independence between traits separated by metamorphosis is widespread but varies with biological function.

Why is metamorphosis so pervasive? Does it facilitate the independent (micro)evolution of quantitative traits in distinct life stages, similarly to how it enables some limbs and organs to develop at specific life stages? We tested this hypothesis by measuring the expression of 6400 genes in 41 Drosophila melanogaster inbred lines at larval and adult stages. Only 30% of the genes showed significant genetic correlations between larval and adult expression. By contrast, 46% of the traits showed some level of genetic independence between stages. Gene ontology terms enrichment revealed that across stages correlated traits were often involved in proteins synthesis, insecticide resistance and innate immunity, while a vast number of genes expression traits associated with energy metabolism were independent between life stages. We compared our results to a similar case: genetic constraints between males and females in gonochoric species (i.e. sexual antagonism). We expected selection for the separation between males and females to be higher than between juvenile and adult functions, as gonochorism is a more common strategy in the animal kingdom than metamorphosis. Surprisingly, we found that inter-stage constraints were lower than inter-sexual genetic constraints. Overall, our results show that metamorphosis enables a large part of the transcriptome to evolve independently at different life stages.

Microbiota acquisition and transmission in Drosophila flies.

Understanding the ecological and evolutionary dynamics of host-microbiota associations notably involves exploring how members of the microbiota assemble and whether they are transmitted along host generations. Here, we investigate the larval acquisition of facultative bacterial and yeast symbionts of Drosophila melanogaster and Drosophila suzukii in ecologically realistic setups. Fly mothers and fruit were major sources of symbionts. Microorganisms associated with adult males also contributed to larval microbiota, mostly in D. melanogaster. Yeasts acquired at the larval stage maintained through metamorphosis, adult life, and were transmitted to offspring. All these observations varied widely among microbial strains, suggesting they have different transmission strategies among fruits and insects. Our approach shows microbiota members of insects can be acquired from a diversity of sources and highlights the compound nature of microbiotas. Such microbial transmission events along generations should favor the evolution of mutualistic interactions and enable microbiota-mediated local adaptation of the insect host.

Microbiota-mediated competition between Drosophila species.

BACKGROUND: The influence of microbiota in ecological interactions, and in particular competition, is poorly known. We studied competition between two insect species, the invasive pest Drosophila suzukii and the model Drosophila melanogaster, whose larval ecological niches overlap in ripe, but not rotten, fruit. RESULTS: We discovered D. suzukii females prevent costly interspecific larval competition by avoiding oviposition on substrates previously visited by D. melanogaster. More precisely, D. melanogaster association with gut bacteria of the genus Lactobacillus triggered D. suzukii avoidance. However, D. suzukii avoidance behavior is condition-dependent, and D. suzukii females that themselves carry D. melanogaster bacteria stop avoiding sites visited by D. melanogaster. The adaptive significance of avoiding cues from the competitor's microbiota was revealed by experimentally reproducing in-fruit larval competition: reduced survival of D. suzukii larvae only occurred if the competitor had its normal microbiota. CONCLUSIONS: This study establishes microbiotas as potent mediators of interspecific competition and reveals a central role for context-dependent behaviors under bacterial influence. Video Abstract.

Influence of bacteria on the maintenance of a yeast during Drosophila melanogaster metamorphosis.

Interactions between microorganisms associated with metazoan hosts are emerging as key features of symbiotic systems. Little is known about the role of such interactions on the maintenance of host-microorganism association throughout the host's life cycle. We studied the influence of extracellular bacteria on the maintenance of a wild isolate of the yeast Saccharomyces cerevisiae through metamorphosis of the fly Drosophila melanogaster reared in fruit. Yeasts maintained through metamorphosis only when larvae were associated with extracellular bacteria isolated from D. melanogaster faeces. One of these isolates, an Enterobacteriaceae, favoured yeast maintenance during metamorphosis. Such bacterial influence on host-yeast association may have consequences for the ecology and evolution of insect-yeast-bacteria symbioses in the wild.

Yeast facilitates the multiplication of Drosophila bacterial symbionts but has no effect on the form or parameters of Taylor's law.

Interactions between microbial symbionts influence their demography and that of their hosts. Taylor's power law (TL)-a well-established relationship between population size mean and variance across space and time-may help to unveil the factors and processes that determine symbiont multiplications. Recent studies suggest pervasive interactions between symbionts in Drosophila melanogaster. We used this system to investigate theoretical predictions regarding the effects of interspecific interactions on TL parameters. We assayed twenty natural strains of bacteria in the presence and absence of a strain of yeast using an ecologically realistic set-up with D. melanogaster larvae reared in natural fruit. Yeast presence led to a small increase in bacterial cell numbers; bacterial strain identity largely affected yeast multiplication. The spatial version of TL held among bacterial and yeast populations with slopes of 2. However, contrary to theoretical prediction, the facilitation of bacterial symbionts by yeast had no detectable effect on TL's parameters. These results shed new light on the nature of D. melanogaster's symbiosis with yeast and bacteria. They further reveal the complexity of investigating TL with microorganisms.

Do traits separated by metamorphosis evolve independently? Concepts and methods.

Despite the ubiquity of complex life cycles, we know little of the evolutionary constraints exerted by metamorphosis. Here, we present pitfalls and methods to answer whether animals with a complex life cycle can independently adapt to the environments encountered at each life stage, with a specific focus on the microevolution of quantitative characters. We first discuss challenges associated with study traits and populations. We further emphasize the benefits of using a combination of approaches. We then develop how multivariate methods can limit several issues by revealing genetic patterns that are invisible when only considering trait-by-trait genetic correlations. Finally, we detail how Lande's work on sexual dimorphism can be applied in measuring G matrices across life stages. The methods and tools described here will contribute towards building a predictive framework for trait evolution across life stages.

Performance of Metarhizium anisopliae-treated foam in combination with Phytoseiulus longipes Evans against Tetranychus evansi Baker & Pritchard (Acari: Tetranychidae).

BACKGROUND: Tetranychus evansi (Te) is an exotic pest of solanaceous crops in Africa. The predatory mite Phytoseiulus longipes (Pl) and the fungus Metarhizium anisopliae (Ma) are potential biocontrol agents of Te. The present study investigated the efficacy of fungus-treated foam placed above or below the third Te-infested tomato leaf. The persistence of fungus-treated foam and the performance of Pl with and without fungus-treated foam were evaluated. RESULTS: The fungus-treated foam was effective when Te infestation was below the third tomato leaf as no damage was recorded on any of the upper tomato leaves up to 30 days post-treatment. However, in the control treatments, the infestation increased considerably from 9 ± 0.3% to 100 ± 0% (mean ± standard error) at 15 days post-treatment. The reuse of the fungus-treated foam at 15, 30 and 45 days post-treatment resulted in 19 ± 1.4%, 25 ± 1.2% and 54 ± 2.1%, respectively, infestation by Te. The fungus-treated foam and Pl alone were efficient, but there was no benefit to combining them for use against Te. CONCLUSION: The fungus-treated foam is an effective method to optimise the use of Ma in screenhouse conditions. These two control agents could be integrated in an integrated pest management strategy for crop protection. However, these results need to be confirmed in large field trials. © 2018 Society of Chemical Industry.

The virome of Drosophila suzukii, an invasive pest of soft fruit.

Drosophila suzukii (Matsumura) is one of the most damaging and costly pests to invade temperate horticultural regions in recent history. Conventional control of this pest is challenging, and an environmentally benign microbial biopesticide is highly desirable. A thorough exploration of the pathogens infecting this pest is not only the first step on the road to the development of an effective biopesticide, but also provides a valuable comparative dataset for the study of viruses in the model family Drosophilidae. Here we use a metatransciptomic approach to identify viruses infecting this fly in both its native (Japanese) and invasive (British and French) ranges. We describe eighteen new RNA viruses, including members of the Picornavirales, Mononegavirales, Bunyavirales, Chuviruses, Nodaviridae, Tombusviridae, Reoviridae, and Nidovirales, and discuss their phylogenetic relationships with previously known viruses. We also detect 18 previously described viruses of other Drosophila species that appear to be associated with D. suzukii in the wild.

Comparison of cauliflower-insect-fungus interactions and pesticides for cabbage root fly control.

Cabbage root fly (Delia radicum L.) control represents a major challenge in brassica production, therefore different management strategies for its control were tested in conventionally managed open field cauliflower production. Strategies included treatments with low-risk methods such as nitrogen lime, the insecticide spinosad and the Beauveria bassiana ATCC 74040-based biopesticide Naturalis. Their effects were compared with treatments based on nonformulated fungal species Metarhizium brunneum, B. bassiana, Clonostachys solani, Trichoderma atroviride, T. koningiopsis, and T. gamsii and commercial insecticides λ-cyhalothrin and thiamethoxam. Spinosad and thiamethoxam were pipetted to individual plants before transplanting; λ-cyhalothrin was sprayed after transplanting; nitrogen lime was applied at first hoeing. Nonformulated fungi were delivered onto cauliflower plantlets' roots as a single pretransplantation inoculation. The cabbage root fly population dynamics exhibited a strong spatiotemporal variation. The lowest number of cabbage root fly pupae recovered from cauliflower roots in the field experiments was recorded in plants treated with spinosad (significant reduction), followed by Naturalis and one of the tested M. brunneum strains (nonsignificant reduction). Significantly more pupae were counted in the nitrogen lime treatment. The field experiments showed that a single drench of cauliflower plantlets with spinosad offered consistent and enduring cabbage root fly control. Naturalis and nonformulated fungal isolates did not decrease cabbage root fly pressure significantly, apparently due to lack of statistical power. The implications of the substantial intra- and inter-annual pest pressure variation and the benefits of using single plant treatments are discussed, and recommendations for improvement of rhizosphere-competence utilizing biological control strategies provided.

Stable Polycomb-dependent transgenerational inheritance of chromatin states in Drosophila.

Transgenerational epigenetic inheritance (TEI) describes the transmission of alternative functional states through multiple generations in the presence of the same genomic DNA sequence. Very little is known about the principles and the molecular mechanisms governing this type of inheritance. Here, by transiently enhancing 3D chromatin interactions, we established stable and isogenic Drosophila epilines that carry alternative epialleles, as defined by differential levels of Polycomb-dependent trimethylation of histone H3 Lys27 (forming H3K27me3). After being established, epialleles can be dominantly transmitted to naive flies and can induce paramutation. Importantly, epilines can be reset to a naive state by disruption of chromatin interactions. Finally, we found that environmental changes modulate the expressivity of the epialleles, and we extended our paradigm to naturally occurring phenotypes. Our work sheds light on how nuclear organization and Polycomb group (PcG) proteins contribute to epigenetically inheritable phenotypic variability.

Deciphering the Routes of invasion of Drosophila suzukii by Means of ABC Random Forest.

Deciphering invasion routes from molecular data is crucial to understanding biological invasions, including identifying bottlenecks in population size and admixture among distinct populations. Here, we unravel the invasion routes of the invasive pest Drosophila suzukii using a multi-locus microsatellite dataset (25 loci on 23 worldwide sampling locations). To do this, we use approximate Bayesian computation (ABC), which has improved the reconstruction of invasion routes, but can be computationally expensive. We use our study to illustrate the use of a new, more efficient, ABC method, ABC random forest (ABC-RF) and compare it to a standard ABC method (ABC-LDA). We find that Japan emerges as the most probable source of the earliest recorded invasion into Hawaii. Southeast China and Hawaii together are the most probable sources of populations in western North America, which then in turn served as sources for those in eastern North America. European populations are genetically more homogeneous than North American populations, and their most probable source is northeast China, with evidence of limited gene flow from the eastern US as well. All introduced populations passed through bottlenecks, and analyses reveal five distinct admixture events. These findings can inform hypotheses concerning how this species evolved between different and independent source and invasive populations. Methodological comparisons indicate that ABC-RF and ABC-LDA show concordant results if ABC-LDA is based on a large number of simulated datasets but that ABC-RF out-performs ABC-LDA when using a comparable and more manageable number of simulated datasets, especially when analyzing complex introduction scenarios.

Isolation, characterization and PCR multiplexing of microsatellite loci for a mite crop pest, Tetranychus urticae (Acari: Tetranychidae).

BACKGROUND: Tetranychus urticae is a highly polyphagous species with a cosmopolitan distribution that has the status of pest in more than 100 economically significant crops all over the world. Despite a number of previous efforts to isolate genetic markers, only a reduced set of microsatellite loci has been published. Taking advantage of the whole genome sequence of T. urticae that recently became available; we isolated and characterized a new set of microsatellite loci and tested the level of polymorphism across populations originating from a wide geographical area. RESULTS: A total of 42 microsatellite sequences widespread in the T. urticae genome were identified, the exact position in the genome recorded, and PCR amplification of microsatellite loci tested with primers defined here. Fourteen loci showed unambiguous genotype patterns and were further characterized. Three multiplex polymerase chain reaction sets were optimized in order to genotype a total of 24 polymorphic loci, including 10 previously published Tetranychus-specific loci. The microsatellite kits successfully amplified 686 individuals from 60 field populations for which we assessed the level of genetic diversity. The number of alleles per locus ranged from 3 to 16 and the expected heterozygosity values ranged from 0.12 to 0.81. Most of the loci displayed a significant excess of homozygous and did not model the Hardy-Weinberg equilibrium. This can be explained by the arrhenotokous mode of reproduction of T. urticae. CONCLUSIONS: These primers represent a valuable resource for robust studies on the genetic structure, dispersal and population biology of T. urticae, that can be used in managing this destructive agricultural pest.

Dispersal behavior of Tetranychus evansi and T. urticae on tomato at several spatial scales and densities: implications for integrated pest management.

Studying distribution is necessary to understand and manage the dynamics of species with spatially structured populations. Here we studied the distribution in Tetranychus evansi and T. urticae, two mite pests of tomato, in the scope of evaluating factors that can influence the effectiveness of Integrated Pest Management strategies. We found greater positive density-dependent distribution with T. evansi than T. urticae when assayed on single, detached tomato leaves. Indeed, T. evansi distribution among leaflets increased with initial population density while it was high even at low T. urticae densities. Intensity and rate of damage to whole plants was higher with T. evansi than T. urticae. We further studied the circadian migration of T. evansi within plant. When T. evansi density was high the distribution behavior peaked between 8 am and 3 pm and between 8 pm and 3 am local time of Kenya. Over 24 h the total number of mites ascending and descending was always similar and close to the total population size. The gregarious behavior of T. evansi combined with its rapid population growth rate, may explain why few tomato plants can be severely damaged by T. evansi and how suddenly all the crop can be highly infested. However the localisation and elimination of the first infested plants damaged by T. evansi could reduce the risk of outbreaks in the entire crop. These findings suggest also that an acaricide treated net placed on the first infested plants could be very effective to control T. evansi. Moreover circadian migration would therefore accentuate the efficiency of an acaricide treated net covering the infested plants.

Phenotypic plasticity in evolutionary rescue experiments.

Population persistence in a new and stressful environment can be influenced by the plastic phenotypic responses of individuals to this environment, and by the genetic evolution of plasticity itself. This process has recently been investigated theoretically, but testing the quantitative predictions in the wild is challenging because (i) there are usually not enough population replicates to deal with the stochasticity of the evolutionary process, (ii) environmental conditions are not controlled, and (iii) measuring selection and the inheritance of traits affecting fitness is difficult in natural populations. As an alternative, predictions from theory can be tested in the laboratory with controlled experiments. To illustrate the feasibility of this approach, we briefly review the literature on the experimental evolution of plasticity, and on evolutionary rescue in the laboratory, paying particular attention to differences and similarities between microbes and multicellular eukaryotes. We then highlight a set of questions that could be addressed using this framework, which would enable testing the robustness of theoretical predictions, and provide new insights into areas that have received little theoretical attention to date.

Quorum sensing and density-dependent dispersal in an aquatic model system.

Many organisms use cues to decide whether to disperse or not, especially those related to the composition of their environment. Dispersal hence sometimes depends on population density, which can be important for the dynamics and evolution of sub-divided populations. But very little is known about the factors that organisms use to inform their dispersal decision. We investigated the cues underlying density-dependent dispersal in inter-connected microcosms of the freshwater protozoan Paramecium caudatum. In two experiments, we manipulated (i) the number of cells per microcosm and (ii) the origin of their culture medium (supernatant from high- or low-density populations). We found a negative relationship between population density and rates of dispersal, suggesting the use of physical cues. There was no significant effect of culture medium origin on dispersal and thus no support for chemical cues usage. These results suggest that the perception of density - and as a result, the decision to disperse - in this organism can be based on physical factors. This type of quorum sensing may be an adaptation optimizing small scale monitoring of the environment and swarm formation in open water.

Genetic influence on disease spread following arrival of infected carriers.

Epidemiology in host meta-populations depends on parasite ability to disperse between, establish and persist in distinct sub-populations of hosts. We studied the genetic factors determining the short-term establishment, and long-term maintenance, of pathogens introduced by infected hosts (i.e. carriers) into recipient populations. We used experimental populations of the freshwater ciliate Paramecium caudatum and its bacterial parasite Holospora undulata. Parasite short-term spread (approximately one horizontal transmission cycle) was affected mainly by carrier genotype, and its interactions with parasite and recipient genotypes. By contrast, parasite longer term spread (2-3 horizontal transmission cycles) was mostly determined by parasite isolate. Importantly, measures of parasite short-term success (reproductive number, R) were not good predictors for longer term prevalence, probably because of the specific interactions between host and parasite genotypes. Analogous to variation in vectorial capacity and super-spreader occurrence, two crucial components of epidemiology, we show that carrier genotype can also affect disease spread within meta-populations.

Taylor's Law holds in experimental bacterial populations but competition does not influence the slope.

Populations vary in time and in space, and temporal variation may differ from spatial variation. Yet, in the past half century, field data have confirmed both the temporal and spatial forms of Taylor's power Law, a linear relationship between log(variance) and log(mean) of population size. Recent theory predicted that competitive species interactions should reduce the slope of the temporal version of Taylor's Law. We tested whether this prediction applied to the spatial version of Taylor's Law using simple, well-controlled laboratory populations of two species of bacteria that were cultured either separately or together for 24 h in media of widely varying nutrient richness. Experimentally, the spatial form of Taylor's Law with a slope of 2 held for these simple bacterial communities, but competitive interactions between the two species did not reduce the spatial Taylor's Law slope. These results contribute to the widespread usefulness of Taylor's Law in population ecology, epidemiology and pest control.

Reverse evolution: selection against costly resistance in disease-free microcosm populations of Paramecium caudatum.

Evolutionary costs of parasite resistance arise if genes conferring resistance reduce fitness in the absence of parasites. Thus, parasite-mediated selection may lead to increased resistance and a correlated decrease in fitness, whereas relaxed parasite-mediated selection may lead to reverse evolution of increased fitness and a correlated decrease in resistance. We tested this idea in experimental populations of the protozoan Paramecium caudatum and the parasitic bacterium Holospora undulata. After eight years, resistance to infection and asexual reproduction were compared among paramecia from (1) "infected" populations, (2) uninfected "naive" populations, and (3) previously infected, parasite-free "recovered" populations. Paramecia from "infected" populations were more resistant (+12%), but had lower reproduction (-15%) than "naive" paramecia, indicating an evolutionary trade-off between resistance and fitness. Recovered populations showed similar reproduction to naive populations; however, resistance of recently (<3 years) recovered populations was similar to paramecia from infected populations, whereas longer (>3 years) recovered populations were as susceptible as naive populations. This suggests a weak, convex trade-off between resistance and fitness, allowing recovery of fitness, without complete loss of resistance, favoring the maintenance of a generalist strategy of intermediate fitness and resistance. Our results indicate that (co)evolution with parasites can leave a genetic signature in disease-free populations.

Adaptation of Paramecium caudatum to variable conditions of temperature stress.

The environment is rarely constant and organisms are exposed to spatial and temporal variation that will impact life-histories. It is important to understand how such variation affects the adaptation of organisms to their local environment. We compare the adaptation of populations of the ciliate Paramecium caudatum exposed to constant (23 °C or 35 °C) and temporally variable temperature environments (random daily fluctuations between 23 °C or 35 °C). Consistent with theory, our experiment shows the evolution of specialists when evolution proceeds in constant environments and generalists when the environment is temporally variable. In addition, we demonstrate costs for specialists of being locally adapted through reduced fitness in novel environments. Conversely, we do not find any costs for generalists, as all populations from variable environments had equal or superior performance to specialists in their own environment. The lack of a cost for generalists is emphasised by the presence of a super generalist that has the highest performance at both assay temperatures.