The Neuro-Ecology of Drosophila Pupation Behavior

Many species of Drosophila form conspecific pupa aggregations across the breeding sites. These aggregations could result from species-specific larval odor recognition. To test this hypothesis we used larval odors of D. melanogaster and D. pavani, two species that coexist in the nature. When stimulated by those odors, wild type and vestigial (vg) third-instar larvae of D. melanogaster pupated on conspecific larval odors, but individuals deficient in the expression of the odor co-receptor Orco randomly pupated across the substrate, indicating that in this species, olfaction plays a role in pupation site selection. Larvae are unable to learn but can smell, the Syn^97CS and rut strains of D. melanogaster, did not respond to conspecific odors or D. pavani larval cues, and they randomly pupated across the substrate, suggesting that larval odor-based learning could influence the pupation site selection. Thus, Orco, Syn^97CS and rut loci participated in the pupation site selection. When stimulated by conspecific and D. melanogaster larval cues, D. pavani larvae also pupated on conspecific odors. The larvae of D. gaucha, a sibling species of D. pavani, did not respond to D. melanogaster larval cues, pupating randomly across the substrate. In nature, D. gaucha is isolated from D. melanogaster. Interspecific hybrids, which result from crossing pavani female with gaucha males clumped their pupae similarly to D. pavani, but the behavior of gaucha female x pavani male hybrids was similar to D. gaucha parent. The two sibling species show substantial evolutionary divergence in organization and functioning of larval nervous system. D. melanogaster and D. pavani larvae extracted information about odor identities and the spatial location of congener and alien larvae to select pupation sites. We hypothesize that larval recognition contributes to the cohabitation of species with similar ecologies, thus aiding the organization and persistence of Drosophila species guilds in the wild.     

Hybrid disadvantage in the larval foraging behaviour of the two neotropical species of <Emphasis Type="Italic">Drosophila pavani</Emphasis> and <Emphasis Type="Italic">Drosophila gaucha</Emphasis>

Flies from two populations of the Chilean endemic neotropical species Drosophila pavani and two populations of its sibling species Drosophila gaucha were crossed reciprocally to obtain intra- and interspecific hybrids. The developmental pathways of locomotor activity and feeding rate were analysed for eleven of twelve possible genotype groups. The hybrids showed reduced fitness indicated by a decrease in the measured traits. Hybrid disadvantage was strongest in interspecific hybrids, especially with respect to feeding behaviour. This evidence supports the contention that D. pavani and D. gaucha have evolved different coadapted gene pools controlling the developmental pathways for behavioural traits expressed during larval foraging; but genetic divergence affecting these behaviours has also taken place between locally adapted populations within each species.     

Identification of multiple odorant receptors essential for pyrethrum repellency in Drosophila melanogaster

Pyrethrum extract from dry flowers of Tanacetum cinerariifolium (formally Chrysanthemum cinerariifolium) has been used globally as a popular insect repellent against arthropod pests for thousands of years. However, the mechanistic basis of pyrethrum repellency remains unknown. In this study, we found that pyrethrum spatially repels and activates olfactory responses in Drosophila melanogaster, a genetically tractable model insect, and the closely-related D. suzukii which is a serious invasive fruit crop pest. The discovery of spatial pyrethrum repellency and olfactory response to pyrethrum in D. melanogaster facilitated our identification of four odorant receptors, Or7a, Or42b, Or59b and Or98a that are responsive to pyrethrum. Further analysis showed that the first three Ors are activated by pyrethrins, the major insecticidal components in pyrethrum, whereas Or98a is activated by (E)-β-farnesene (EBF), a sesquiterpene and a minor component in pyrethrum. Importantly, knockout of Or7a, Or59b or Or98a individually abolished fly avoidance to pyrethrum, while knockout of Or42b had no effect, demonstrating that simultaneous activation of Or7a, Or59b and Or98a is required for pyrethrum repellency in D. melanogaster. Our study provides insights into the molecular basis of repellency of one of the most ancient and globally used insect repellents. Identification of pyrethrum-responsive Ors opens the door to develop new synthetic insect repellent mixtures that are highly effective and broad-spectrum.     

Dispersal and prepupation behavior of Chilean sympatric Drosophila species that breed in the same site in nature

We investigated dispersal patterns of Drosophila larvae searchingfor pupation sites over three substrates to determine the roleof spatial heterogeneity and presence of other species on prepupationbehavior. We used D. melanogaster, D. hydei, and D. pavani whoseparents emerged from apples collected in one orchard. Each speciesshowed different preferences for substrates on which to pupate,particularly in the presence of another Drosophila species.Larval locomotion rate and turning behavior in D. melanogaster,D. hydei, and D. pavani were modified depending this upon thetype of substrate (agar and sand) on which the larvae crawled.These two behaviors are involved in dispersal and aggregationof pupae. Distance between pupae of the same species decreaseswhen larvae of another species pupate on the same substrate.Aggregated distributions over the substrates lead to patcheswith few or no individuals. These could serve as pupation sitesfor other Drosophila species that, in nature, also emerge fromsmall breeding sites.     

Meiotic,genomic and evolutionary properties of crossover distribution in Drosophila yakuba

The number and location of crossovers across genomes are highly regulated during meiosis, yet the key components controlling them are fast evolving, hindering our understanding of the mechanistic causes and evolutionary consequences of changes in crossover rates. Drosophila melanogaster has been a model species to study meiosis for more than a century, with an available high-resolution crossover map that is, nonetheless, missing for closely related species, thus preventing evolutionary context. Here, we applied a novel and highly efficient approach to generate whole-genome high-resolution crossover maps in D. yakuba to tackle multiple questions that benefit from being addressed collectively within an appropriate phylogenetic framework, in our case the D. melanogaster species subgroup. The genotyping of more than 1,600 individual meiotic events allowed us to identify several key distinct properties relative to D. melanogaster. We show that D. yakuba, in addition to higher crossover rates than D. melanogaster, has a stronger centromere effect and crossover assurance than any Drosophila species analyzed to date. We also report the presence of an active crossover-associated meiotic drive mechanism for the X chromosome that results in the preferential inclusion in oocytes of chromatids with crossovers. Our evolutionary and genomic analyses suggest that the genome-wide landscape of crossover rates in D. yakuba has been fairly stable and captures a significant signal of the ancestral crossover landscape for the whole D. melanogaster subgroup, even informative for the D. melanogaster lineage. Contemporary crossover rates in D. melanogaster, on the other hand, do not recapitulate ancestral crossovers landscapes. As a result, the temporal stability of crossover landscapes observed in D. yakuba makes this species an ideal system for applying population genetic models of selection and linkage, given that these models assume temporal constancy in linkage effects. Our studies emphasize the importance of generating multiple high-resolution crossover rate maps within a coherent phylogenetic context to broaden our understanding of crossover control during meiosis and to improve studies on the evolutionary consequences of variable crossover rates across genomes and time.     

Dosage Compensation of X-Linked Muller Element F Genes but Not X-Linked Transgenes in the Australian Sheep Blowfly

In most animals that have X and Y sex chromosomes, chromosome-wide mechanisms are used to balance X-linked gene expression in males and females. In the fly Drosophila melanogaster, the dosage compensation mechanism also generally extends to X-linked transgenes. Over 70 transgenic lines of the Australian sheep blowfly Lucilia cuprina have been made as part of an effort to develop male-only strains for a genetic control program of this major pest of sheep. All lines carry a constitutively expressed fluorescent protein marker gene. In all 12 X-linked lines, female larvae show brighter fluorescence than male larvae, suggesting the marker gene is not dosage compensated. This has been confirmed by quantitative RT-PCR for selected lines. To determine if endogenous X-linked genes are dosage compensated, we isolated 8 genes that are orthologs of genes that are on the fourth chromosome in D. melanogaster. Recent evidence suggests that the D. melanogaster fourth chromosome, or Muller element F, is the ancestral X chromosome in Diptera that has reverted to an autosome in Drosophila species. We show by quantitative PCR of male and female DNA that 6 of the 8 linkage group F genes reside on the X chromosome in L. cuprina. The other two Muller element F genes were found to be autosomal in L. cuprina, whereas two Muller element B genes were found on the same region of the X chromosome as the L. cuprina orthologs of the D. melanogaster Ephrin and gawky genes. We find that the L. cuprina X chromosome genes are equally expressed in males and females (i.e., fully dosage compensated). Thus, unlike in Drosophila, it appears that the Lucilia dosage compensation system is specific for genes endogenous to the X chromosome and cannot be co-opted by recently arrived transgenes.     

Pupation Site Selection in Four Drosophilid Species: Aggregation and Contact

This study of pupation site selection was undertaken to assess the frequency and orientation of pupae making physical contact in four sympatric species in the family Drosophilidae: Drosophila melanogaster, D. simulans, D. funebris, and Zaprionus tuberculatus. Pupation behavior was assayed in a vial containing a small food cup. Using nearest-neighbor analysis, we found that pupae were aggregated. Furthermore, the frequency of contact between pupae was far greater than could be explained by chance, in all four species. In the three species of Drosophila, about a third of the contacts were between intimately paired pupae whose long axes were parallel; we call this arrangement synapsis. In Z. tuberculatus, most pupal contacts were unoriented. When D. melanogaster larvae were reared with each of the other species, heterospecific pupal contact, including synapsis, occurred. Our discovery of pupal contact in several drosophilids expands the known repertory of social behavior in this family.     

Drosophila Avoids Parasitoids by Sensing Their Semiochemicals via a Dedicated Olfactory Circuit

Detecting danger is one of the foremost tasks for a neural system. Larval parasitoids constitute clear danger to Drosophila, as up to 80% of fly larvae become parasitized in nature. We show that Drosophila melanogaster larvae and adults avoid sites smelling of the main parasitoid enemies, Leptopilina wasps. This avoidance is mediated via a highly specific olfactory sensory neuron (OSN) type. While the larval OSN expresses the olfactory receptor Or49a and is tuned to the Leptopilina odor iridomyrmecin, the adult expresses both Or49a and Or85f and in addition detects the wasp odors actinidine and nepetalactol. The information is transferred via projection neurons to a specific part of the lateral horn known to be involved in mediating avoidance. Drosophila has thus developed a dedicated circuit to detect a life-threatening enemy based on the smell of its semiochemicals. Such an enemy-detecting olfactory circuit has earlier only been characterized in mice and nematodes.     

An omnivorous arthropod, <Emphasis Type="Italic">Nesidiocoris tenuis</Emphasis>, induces gender-specific plant volatiles to which conspecific males and females respond differently

Nesidiocoris tenuis Reuter (Heteroptera: Miridae) is an omnivorous mirid bug that preys on diverse generalist herbivorous arthropods. N. tenuis adults are attracted to volatiles from plants induced by their prey, such as tobacco cutworms (CCW) (Spodoptera litura larvae) and two-spotted spider mites (Tetranychus urticae). N. tenuis adults also induce volatiles when they infest plants. In this study, we focused on olfactory responses of N. tenuis males and females to volatiles from eggplants and sesame plants induced by conspecifics of the same or different gender by using a Y-tube olfactometer. Males were attracted to volatiles from plants of both species induced by either males or females. The male preference was biased to volatiles from plants of both species induced by females, probably because the biased response would facilitate their mate-finding. Females were attracted only to volatiles from plants of both species induced by females. Mating occurs multiple times in this species. Thus, the responses would indirectly affect mating of males and females. Slight but significant qualitative and quantitative differences were detected between the volatiles of plants of both species induced by N. tenuis females and those of the plants induced by conspecific males. N. tenuis might use such differences in their gender-specific responses.     

Alien and native plant life‐forms respond differently to human and climate pressures

Aim To investigate whether differences in the elevational trend in native and alien species richness were dependent on climate or human pressures. Specifically we tested whether life‐form and/or alien/native status modifies the response of plant species richness to human population and temperature along: (1) a complete elevational gradient, and (2) within separate elevational bands that, by keeping temperature within a narrow range, elucidate the effects of human pressures more clearly. Location Two provinces (c. 7507 km²) on the southern border of the European Alps (Italy), subdivided into 240 contiguous sampling cells (c. 35.7 km²). Methods We used an extensive dataset on alien and native species richness across an elevation gradient (20–2900 m a.s.l.). Richness of natives and naturalized aliens were separately related to temperature, human population and Raunkiaer life‐form using general linear mixed models. Life‐form describes different plant strategies for survival during seasons with adverse cold/arid conditions. Results The relationship between species richness and temperature for natives was strongly dependent on life‐form, while aliens showed a consistent positive trend. Similar trends across alien and native life‐forms were found for the relationship between species richness and human population along the whole gradient and within separate elevational bands. Main conclusions The absence of life‐form‐dependent responses amongst aliens supports the hypothesis that the distribution of alien plant species richness was more related to propagule pressure and availability of novel niches created by human activities than to climatic filtering. While climate change will potentially contribute to relaxing species thermal constraints, the response of alien species to future warming will also be contingent on changes in anthropogenic pressures.     

Does sexual experience affect the strength of male mate choice for high‐quality females in Drosophila melanogaster?

Although females are traditionally thought of as the choosy sex, there is increasing evidence in many species that males will preferentially court or mate with certain females over others when given a choice. In the fruit fly, Drosophila melanogaster, males discriminate between potential mating partners based on a number of female traits, including species, mating history, age, and condition. Interestingly, many of these male preferences are affected by the male''s previous sexual experiences, such that males increase courtship toward types of females that they have previously mated with and decrease courtship toward types of females that have previously rejected them. D. melanogaster males also show courtship and mating preferences for larger females over smaller females, likely because larger females have higher fecundity. It is unknown, however, whether this preference shows behavioral plasticity based on the male''s sexual history as we see for other male preferences. Here, we manipulate the sexual experience of D. melanogaster males and test whether this manipulation has any effect on the strength of male mate choice for large females. We find that sexually inexperienced males have a robust courtship preference for large females that is unaffected by previous experience mating with, or being rejected by, females of differing sizes. Given that female body size is one of the most common targets of male mate choice across insect species, our experiments with D. melanogaster may provide insight into how these preferences develop and evolve.     

CRISPR/Cas9 in locusts: Successful establishment of an olfactory deficiency line by targeting the mutagenesis of an odorant receptor co-receptor (Orco)

Locusts are important agricultural pests worldwide and regarded as study models for entomology. However, the absence of targeted gene manipulation systems for locusts has restricted their applications for research. Herein, we report the successful use of the CRISPR/Cas9 system to induce a targeted heritable mutagenesis of the migratory locust, Locusta migratoria. The target sequence of gRNA was designed to disrupt the gene encoding the odorant receptor co-receptor (Orco) and examine the roles of the odorant receptor pathway in the locust. Microinjection of the mixture of Cas9-mRNA and Orco-gRNA into the locust eggs resulted in efficient target-gene editing at a rate of 71.7% in G0 animals and achieved a germline efficiency of up to 88.1% in G1 animals. By a crossing strategy, we successfully established stable Orco mutant lines. EAGs and SSRs indicated that the fourth-instar nymphs of the Orco mutants showed severely impaired electrophysiological responses to multiple odors. The Orco mutant locusts lost an attraction response to aggregation pheromones under the crowding conditions. The locomotor activity and body coloration of the Orco mutant locusts did not significantly differ from those of the two other genotypes. This study provides an easy and effective approach by using the CRISPR/Cas9 system for generating loss-of-function mutants for functional genetic studies of locusts and for managing insect pests.     

The Discovery,Distribution, and Evolution of Viruses Associated with Drosophila melanogaster

Drosophila melanogaster is a valuable invertebrate model for viral infection and antiviral immunity, and is a focus for studies of insect-virus coevolution. Here we use a metagenomic approach to identify more than 20 previously undetected RNA viruses and a DNA virus associated with wild D. melanogaster. These viruses not only include distant relatives of known insect pathogens but also novel groups of insect-infecting viruses. By sequencing virus-derived small RNAs, we show that the viruses represent active infections of Drosophila. We find that the RNA viruses differ in the number and properties of their small RNAs, and we detect both siRNAs and a novel miRNA from the DNA virus. Analysis of small RNAs also allows us to identify putative viral sequences that lack detectable sequence similarity to known viruses. By surveying >2,000 individually collected wild adult Drosophila we show that more than 30% of D. melanogaster carry a detectable virus, and more than 6% carry multiple viruses. However, despite a high prevalence of the Wolbachia endosymbiont—which is known to be protective against virus infections in Drosophila—we were unable to detect any relationship between the presence of Wolbachia and the presence of any virus. Using publicly available RNA-seq datasets, we show that the community of viruses in Drosophila laboratories is very different from that seen in the wild, but that some of the newly discovered viruses are nevertheless widespread in laboratory lines and are ubiquitous in cell culture. By sequencing viruses from individual wild-collected flies we show that some viruses are shared between D. melanogaster and D. simulans. Our results provide an essential evolutionary and ecological context for host–virus interaction in Drosophila, and the newly reported viral sequences will help develop D. melanogaster further as a model for molecular and evolutionary virus research.     

Rocks and Walls: Natural Versus Secondary Habitats

Walls are often considered secondary habitats for vegetation of natural rock surfaces. Compared with rocks, walls differ in many features, for example the presence of a binding material (mainly calcareous mortar), location in settlements and exposure to human impact. A data set of 1,205 phytosociological relevés recorded on horizontal wall tops, wall verticals and rock verticals in the Czech Republic was used to compare their vegetation with regard to i) species composition (frequent species, species diversity, endangered and alien species) and ii) the ecological requirements of the respective species. Gamma diversity of vascular plant species was comparable in all habitat types (242 species on wall tops, 212 species on wall verticals and 197 species on rock verticals). Wall verticals had higher beta diversity, but lower alpha diversity than rocks. Remarkable differences were found comparing the diversity of alien species. Whereas alpha and gamma diversities of aliens were higher on both wall habitats, beta diversity of aliens was the highest on rocks. The high floristic heterogeneity of walls is mainly attributable to the large pool of species from the surrounding urbanized landscape (e.g., cultivated ornamental species and synanthropic weeds) that are favoured by high nutrient inputs. In contrast, species characteristic of rocks are mainly substrate specialists. Walls and rocks share the frequent occurrence of ferns, grasses and herbs typical of forest understorey and clearings. Compared with rocks, walls are generally colonized by species requiring higher nutrient content, soil reaction, temperature and moisture. Secondary wall habitats might be suitable for some rare and endangered species, but contrary to rocks their occurrences are only accidental and temporary. The representation of aliens was considerably higher on walls (approximately 35%) than on rocks (9%).     

Evolutionary pattern of the gtwin retrotransposon in the Drosophila melanogaster subgroup

The gtwin retrotransposon was recently discovered in the Drosophila melanogaster genome and it is evolutionarily closer to gypsy endogenous retrovirus. This study has identified gtwin homologous sequences in the genome of D. simulans, D. sechellia, D. erecta and D. yakuba by performing homology searches against the public genome database of Drosophila species. The phylogenetic analyses of the gtwin env gene sequences of these species have shown some incongruities with the host species phylogeny, suggesting some horizontal transfer events for this retroelement. Moreover, we reported the existence of DNA sequences putatively encoding full-length Env proteins in the genomes of Drosophila species other than D. melanogaster. The results suggest that the gtwin element may be an infectious retrovirus able to invade the genome of new species, supporting the gtwin evolutionary picture shown in this work.     

Feeding damage by larvae of the mustard leaf beetle deters conspecific females from oviposition and feeding

Herbivorous insects may be informed about the presence of competitors on the same host plant by a variety of cues. These cues can derive from either the competitor itself or the damaged plant. In the mustard leaf beetle Phaedon cochleariae (Coleoptera, Chrysomelidae), adults are known to be deterred from feeding and oviposition by the exocrine glandular secretion of conspecific co-occurring larvae. We hypothesised that the exocrine larval secretion released by feeding larvae may adsorb to the surface of Chinese cabbage leaves, and thus, convey the information about their former or actual presence. Further experiments tested the influence of leaves damaged by conspecific larvae, mechanically damaged leaves, larval frass and regurgitant on the oviposition and feeding behaviour of P. cochleariae. Finally, the effect of previous conspecific herbivory on larval development and larval host selection was assessed. Our results show that (epi)chrysomelidial, the major component of the exocrine secretion from P. cochleariae larvae, was detectable by GC-MS in surface extracts from leaves upon which larvae had fed. However, leaves exposed to volatiles of the larval secretion were not avoided by female P. cochleariae for feeding or oviposition. Thus, we conclude that secretion volatiles did not adsorb in sufficient amounts on the leaf surface to display deterrent activity towards adults. By contrast, gravid females avoided to feed and lay their eggs on leaves damaged by second-instar larvae for three days when compared to undamaged leaves. Mechanical damage of leaves and treatment of artificially damaged leaves with larval frass or regurgitant did not affect oviposition and feeding of P. cochleariae. Since no adverse effects of previous herbivory on larval development were detected, we suggest that female P. cochleariae avoid Chinese cabbage leaves damaged by feeding larvae for other reasons than escape from competition or avoidance of direct negative effects that result from consuming induced plant material.     

Temporal shifts in interactions between alien trees and the alien Argentine ant on native ants

Synergistic effects between various stressors on native biodiversity are poorly understood, especially adverse synergisms between different invasive organisms. While it is known that alien trees and the invasive Argentine ant Linepithema humile individually impact indigenous ant assemblages, little is known about how the impacts of these two types of aliens have a joint effect, and even less about the temporal dynamics of this interaction. We found that the Argentine ant benefited from invasion by alien trees through creation of less extreme environmental conditions at the hottest and coolest times of the year. We also found that the impacts of the two types of aliens were synergistic on the native ant assemblage. However, this synergistic impact also varied in intensity throughout the year, especially so in the more open natural sites. The alien tree canopy, when dense, created more constant environmental conditions throughout the year, leading to more constant Argentine ant levels and consequently more even impacts on the native ants. The various native ants varied considerably in their response to the impacts of both types of aliens, with many formicine ants being particularly sensitive. Our results show that the synergistic impacts, both negative and positive, of the two alien types are particular to each native ant species. Furthermore, the intensity of the adverse synergism is highly variable across the year.