Visualizing Molecular Functions and Cross-Species Activity of Sex-Peptide in Drosophila

The Drosophila melanogaster sex-peptide (melSP) is a seminal fluid component that induces postmating responses (PMR) of females via the sex-peptide receptor (SPR) . Although SP orthologs are found in many Drosophila species, their functions remain poorly characterized. It is unknown whether SP functions are conserved across species or rather specific to each species. Here, we developed a GFP-tagged melSP (G-SP) and used it to visualize cross-species binding activity to the female reproductive system of various species. First we demonstrated that ectopically expressed G-SP induced PMR in D. melanogaster females and bound to the female reproductive system, most notably to the common oviduct. No binding occurred in the females lacking SPR, indicating that G-SP binding was dependent on SPR. Next we tested whether G-SP binds to the common oviducts from 11 Drosophila species using dissected reproductive tracts. The binding was observed in six species belonging to the D. melanogaster species group, but not to those outside the group. Injection of melSP reduced the receptivity of females belonging to the D. melanogaster species group, but not of those outside the group, being consistent with the ability to bind G-SP. Thus the SP-mediated PMR appears to be limited to this species group. SPR was expressed in the oviducts at high levels in this group; therefore, we speculate that an enhanced expression of SPR in the oviduct was critical to establish the SP-mediated PMR during evolution.     

Identification of One Intron Loss and Phylogenetic Evolution of Dfak Gene in the Drosophila melanogaster Species Group

Intron loss and its evolutionary significance have been noted in Drosophila. The current study provides another example of intron loss within a single-copy Dfak gene in Drosophila. By using polymerase chain reaction (PCR), we amplified about 1.3 kb fragment spanning intron 5–10, located in the position of Tyr kinase (TyK) domain of Dfak gene from Drosophila melanogaster species group, and observed size difference among the amplified DNA fragments from different species. Further sequencing analysis revealed that D. melanogaster and D. simulans deleted an about 60 bp of DNA fragment relative to other 7 Drosophila species, such as D. elegans, D. ficusphila, D. biarmipes, D. takahashii, D. jambulina, D. prostipennis and D. pseudoobscura, and the deleted fragment located precisely in the position of one intron. The data suggested that intron loss might have occurred in the Dfak gene evolutionary process of D. melanogaster and D. simulans of Drosophila melanogaster species group. In addition, the constructed phylogenetic tree based on the Dfak TyK domains clearly revealed the evolutionary relationships between subgroups of Drosophila melanogaster species group, and the intron loss identified from D. melanogaster and D. simulans provides a unique diagnostic tool for taxonomic classification of the melanogaster subgroup from other group of genus Drosophila.     

CHEMICAL COMMUNICATION IN HAWAIIAN DROSOPHILA

We are interested in elucidating the extent to which lekking Hawaiian Drosophila species have diverged from their continental counterparts, which engage in sexual behavior at communal food sources, with regard to the chemical communication systems that the flies employ. Accordingly, we have analyzed flies from three closely related Hawaiian Drosophila species in the adiastola subgroup. These species are of interest because the males engage in a unique behavior: while courting, they raise their abdomens over their heads and emit anal droplets. Analysis of the flies' behavior, the hydrocarbons in males' anal droplets, and males' cuticular hydrocarbons suggest that females' responses to males may be mediated by cuticular pheromones and/or pheromones in males' extruded droplets that enable the females to distinguish conspecific from heterospeciflc males. Conversely, perception of cuticular hydrocarbons from conspecific females enables D. adiastola males to distinguish females from a closely related species from conspecific females. On the basis of these observations, we suggest that the adiastola subgroup species are unique among drosophilids in that they utilize an anal droplet-mediated pheromone communication system, some or all components of which are species specific. However, the lekking Hawaiian Drosophila species are similar to D. melanogaster and related continental species in that the Hawaiian flies employ a cuticular pheromone communication system, some components of which are sex and species-specific.     

Variation in adult life history and stress resistance across five species of<Emphasis Type="Italic">Drosophila</Emphasis>

Dry weight at eclosion, adult lifespan, lifetime fecundity, lipid and carbohydrate content at eclosion, and starvation and desiccation resistance at eclosion were assayed on a long-term laboratory population ofDrosophila melanogaster, and one recently wild-caught population each of four other species ofDrosophila, two from themelanogaster and two from theimmigrans species group. The relationships among trait means across the five species did not conform to expectations based on correlations among these traits inferred from selection studies onD. melanogaster. In particular, the expected positive relationships between fecundity and size/lipid content, lipid content and starvation resistance, carbohydrate (glycogen) content and desiccation resistance, and the expected negative relationship between lifespan and fecundity were not observed. Most traits were strongly positively correlated between sexes across species, except for fractional lipid content and starvation resistance per microgram lipid. For most traits, there was evidence for significant sexual dimorphism but the degree of dimorphism did not vary across species except in the case of adult lifespan, starvation resistance per microgram lipid, and desiccation resistance per microgram carbohydrate. Overall,D. nasuta nasuta andD. sulfurigaster neonasuta (immigrans group) were heavier at eclosion than themelanogaster group species, and tended to have somewhat higher absolute lipid content and starvation resistance. Yet, these twoimmigrans group species were shorter-lived and had lower average daily fecundity than themelanogaster group species. The smallest species,D. malerkotliana (melanogaster group), had relatively high daily fecundity, intermediate lifespan and high fractional lipid content, especially in females.D. ananassae (melanogaster group) had the highest absolute and fractional carbohydrate content, but its desiccation resistance per microgram carbohydrate was the lowest among the five species. In terms of overall performance, the laboratory population ofD. melanogaster was clearly superior, under laboratory conditions, to the other four species if adult lifespan, lifetime fecundity, average daily fecundity, and absolute starvation and desiccation resistance are considered. This finding is contrary to several recent reports of substantially higher adult lifespan and stress resistance in recently wild-caught flies, relative to flies maintained for a long time in discretegeneration laboratory cultures. Possible explanations for these apparent anomalies are discussed in the context of the differing selection pressures likely to be experienced byDrosophila populations in laboratory versus wild environments. This paper is dedicated to the memory of our friend and former colleague Dr Hans Raj Negi, who tragically passed away at a very young age in a road accident in November 2003.     

INCOMPLETE BEHAVIORAL ISOLATION BETWEEN TWO DISTANTLY RELATED DROSOPHILA SPECIES

Two sympatric, distantly related Drosophila species, D. affinis and D. melanogaster, interact sexually in the laboratory and in the field. When mature males from one species are tested with females or sexually attractive males from the other species, the mature males perform courtship that, in most cases, is indistinguishable from the courtship that attractive conspecific flies elicit. Moreover, some of the D. affinis females that elicit courtship from D. melanogaster males copulate with them.     

Role of arylalkylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase in regulation of biogenic amines levels by gonadotropins in <Emphasis Type="Italic">Drosophila</Emphasis>

The effect of 20-hydroxyecdysone (20E) and the juvenile hormone (JH) on the activity of the arylalkylamine N-acetyltransferase (AANAT) was studied in young females of wild-type D. virilis and D. melanogaster. 20E feeding of the flies led to a decrease in AANAT activity in both species when dopamine (DA) was used as substrate, but did not affect the enzyme activity when octopamine (OA) was used as substrate. JH application increased AANAT activity with DA as substrate in both species, but did not change it with OA as substrate. AANAT activity was also measured in young females of a JH-deficient strain of D. melanogaster, apterous ^56f . A decrease in the enzyme activity was observed in the mutant females as compared to wild-type. Mechanisms of regulation of DA level by gonadotropins in Drosophila are discussed.     

Oviposition preference for spherical surfaces is shared among multiple Drosophila species except D. melanogaster (Diptera: Drosophilidae)

Oviposition preference for spherical substrates has been reported in some insects but not in Drosophila species until the recent finding that Drosophila suzukii preferentially lays eggs on spherical surfaces with a smaller radius, whereas D. melanogaster does not. This finding raised two questions: (i) Was this trait specifically acquired in D. suzukii or lost in D. melanogaster? (ii) In the latter case, is it due to the long-term laboratory culture using oviposition substrates with flat surfaces? To answer these questions, we examined the oviposition preference of three Drosophila species using the stocks recently established from wild individuals. As with D. suzukii, D. simulans and D. takahashii showed significant preference for spherical surfaces with a smaller radius, suggesting that this trait is shared by multiple Drosophila species. In contrast, D. melanogaster did not show any preference for either smaller or larger radii, showing that the preference already has been lost in the natural population of D. melanogaster. It may be possible that the loss of oviposition preference for spherical surfaces is involved in the evolutionary process of D. melanogaster becoming a human commensal.     

Geographic variation in reproductive success of the parasitoid Asobara tabida in larvae of several Drosophila species

Abstract.

• 1 Asobara tabida is a parasitoid of Drosophila larvae in fermenting substrates. Because it is a widespread species, it may encounter different biotic and abiotic circumstances in various parts of its range.
• 2 The species composition of the host population varies over the parasitoid's range: D.obscura-group species (especially D.subobscura) are the main hosts for northwestern and central European parasitoids; D.melanogaster is the main host for southern European parasitoids.
• 3 D.melanogaster larvae can defend themselves against A.tabida by encapsulating the parasitoid egg, and survival in D.melanogaster is always lower than in D.subobscura.
• 4 Parasitoids from southern European populations are much better able to survive in D.melanogaster than their northwestern and central European conspecifics; parasitoids from different populations are equally well able to survive in D.subobscura.
• 5 The lower survival in D.melanogaster may be partly compensated for by the larger size of parasitoids emerging from this host species compared to parasitoids emerging from D.subobscura.
• 6 Within population groups, larger A.tabida females have more eggs in their ovarioles. Additionally, southern European females have more eggs and less fat than northern and western/central European females. The relationship between size and longevity is ambiguous.
• 7 It is concluded that parasitoids from different populations are adapted to region-specific circumstances.

     

Divergence of the Regulation of alpha-Amylase Activity in Drosophila melanogaster, Drosophila funebris, and Drosophila saltans

The regulation of amylase activity in threeDrosophila species, D. melanogaster,D. funebris and D. saltans, wasanalyzed by measuring the specific activity levels infour dietary environments, cornmeal, glucose, 5% starch, and 10% starch, at threedevelopmental stages, i.e., the third-instar larval,pupal, and 2-day-old adult stages. The developmentalprofiles of amylase activity for the threeDrosophila species showed that the level of activity washigh at the larval and adult stages but substantiallylow at the pupal stage, suggesting thatDrosophila does not utilize starch at the pupalstage. Divergence in the regulation of amylase was observed amongthe three Drosophila species on the followingpoints. (1) The order of amylase specific activity wasD. melanogaster > D. funebris >D. saltans. (2) The response pattern to the dietary environment varied amongthe species and changed during development. (3) Thetiming of the switch in the response pattern to thedietary environment during development was before pupation in D. funebris and D.saltans but after pupation in D.melanogaster. The significance of the divergence inthe regulation of amylase activity for adaptation to astarch environment in Drosophila is discussed.     

Molecular phylogeny of the subgenus Sophophora of Drosophila derived from large subunit of ribosomal RNA sequences

RNA sequencing has been used to assess the relationships among species of the subgenus Sophophora of the genus Drosophila. Two divergent domains, D1 and D2, of the large ribosomal RNA (28S), totalling 550 nucleotides have been sequenced using the rRNA direct sequencing method. A tree has been reconstructed from the neighbor-joining algorithm and the confidence intervals were evaluated by the bootstrap procedure. Results have shown that the branching of the willistoni and saltans groups of the subgenus Sophophora is very ancient and probably predates that of the subgenus Drosophila. The other groups and subgroups of Sophophora are clustered in three main lineages: 1) the melanogaster and oriental subgroups; 2) the montium subgroup; 3) the ananassae subgroup of the melanogaster group clustered with the fima and obscura groups. Thus, in comparison with our results, several taxa of various ranks appear paraphyletic (the genus Drosophila, the subgenus Sophophora and the melanogaster group). Our biochemical phylogeny is only in partial agreement with the pattern of Throckmorton's radiations as well as with classical taxonomy, both based on morphological data.     

Molecular evolution of sex-biased genes in the Drosophila ananassae subgroup

Background  

Genes with sex-biased expression often show rapid molecular evolution between species. Previous population genetic and comparative genomic studies of Drosophila melanogaster and D. simulans revealed that male-biased genes have especially high rates of adaptive evolution. To test if this is also the case for other lineages within the melanogaster group, we investigated gene expression in D. ananassae, a species that occurs in structured populations in tropical and subtropical regions. We used custom-made microarrays and published microarray data to characterize the sex-biased expression of 129 D. ananassae genes whose D. melanogaster orthologs had been classified previously as male-biased, female-biased, or unbiased in their expression and had been studied extensively at the population-genetic level. For 43 of these genes we surveyed DNA sequence polymorphism in a natural population of D. ananassae and determined divergence to the sister species D. atripex and D. phaeopleura.     

Molecular evolution of the sex peptide network in Drosophila

Successful reproduction depends on interactions between numerous proteins beyond those involved directly in gamete fusion. Although such reproductive proteins evolve in response to sexual selection pressures, how networks of interacting proteins arise and evolve as reproductive phenotypes change remains an open question. Here, we investigated the molecular evolution of the ‘sex peptide network’ of Drosophila melanogaster, a functionally well‐characterized reproductive protein network. In this species, the peptide hormone sex peptide (SP) and its interacting proteins cause major changes in female physiology and behaviour after mating. In contrast, females of more distantly related Drosophila species do not respond to SP. In spite of these phenotypic differences, we detected orthologs of all network proteins across 22 diverse Drosophila species and found evidence that most orthologs likely function in reproduction throughout the genus. Within SP‐responsive species, we detected the recurrent, adaptive evolution of several network proteins, consistent with sexual selection acting to continually refine network function. We also found some evidence for adaptive evolution of several proteins along two specific phylogenetic lineages that correspond with increased expression of the SP receptor in female reproductive tracts or increased sperm length, respectively. Finally, we used gene expression profiling to examine the likely degree of functional conservation of the paralogs of an SP network protein that arose via gene duplication. Our results suggest a dynamic history for the SP network in which network members arose before the onset of robust SP‐mediated responses and then were shaped by both purifying and positive selection.     

MITOCHONDRIAL–NUCLEAR EPISTASIS AFFECTS FITNESS WITHIN SPECIES BUT DOES NOT CONTRIBUTE TO FIXED INCOMPATIBILITIES BETWEEN SPECIES OF DROSOPHILA

Efficient mitochondrial function requires physical interactions between the proteins encoded by the mitochondrial and nuclear genomes. Coevolution between these genomes may result in the accumulation of incompatibilities between divergent lineages. We test whether mitochondrial–nuclear incompatibilities have accumulated within the Drosophila melanogaster species subgroup by combining divergent mitochondrial and nuclear lineages and quantifying the effects on relative fitness. Precise placement of nine mtDNAs from D. melanogaster, D. simulans, and D. mauritiana into two D. melanogaster nuclear genetic backgrounds reveals significant mitochondrial–nuclear epistasis affecting fitness in females. Combining the mitochondrial genomes with three different D. melanogaster X chromosomes reveals significant epistasis for male fitness between X‐linked and mitochondrial variation. However, we find no evidence that the more than 500 fixed differences between the mitochondrial genomes of D. melanogaster and the D. simulans species complex are incompatible with the D. melanogaster nuclear genome. Rather, the interactions of largest effect occur between mitochondrial and nuclear polymorphisms that segregate within species of the D. melanogaster species subgroup. We propose that a low mitochondrial substitution rate, resulting from a low mutation rate and/or efficient purifying selection, precludes the accumulation of mitochondrial–nuclear incompatibilities among these Drosophila species.     

Identification and comparisons of the yolk polypeptides and the genes which code for them in D. melanogaster sibling species

Summary The yolk proteins stored in Drosophila, oocytes for utilisation during embryogenesis are an ideal system for studying the regulation of gene expression during development. The 3 major polypeptides found in yolk in D. melanogaster are synthesised in the fat body and ovarian follicle cells and selectively accumulated by the oocyte during vitellogenesis. In order to understand more about their regulation and the mechanism of uptake, studies on other species are necessary.Three yolk polypeptides have previously been identified in the D. melanogaster sibling species (D. melanogaster, D. simulans, D. mauritiana, D. erecta, D. teissieri, D. orena and D. yakuba). In D. melanogaster three genes located on the X chromosome are known to code for these yolk polypeptides. in this study genomic Southern transfers and in situ hybridisation experiments were carried out on the sibling species. Using the three cloned yolk protein genes from D. melanogaster, homologous sequences could be detected in the sibling species. It is suggested that three yolk protein genes occur in each of these species, all being located on the X chromosome, and that two of the genes are very closely linked in these same species. Yolk protein gene-homologous DNA sequences have also been identified in two more distantly related species D. funebris and D. virilis.     

Evolution of the LINE-like I element in the Drosophila melanogaster species subgroup

LINE-like retrotransposons, the so-called I elements, control the system of I-R (inducer-reactive) hybrid dysgenesis in Drosophila melanogaster. I elements are present in many Drosophila species. It has been suggested that active, complete I elements, located at different sites on the chromosomes, invaded natural populations of D. melanogaster recently (1920–1970). But old strains lacking active I elements have only defective I elements located in the chromocenter. We have cloned I elements from D. melanogaster and the melanogaster subgroup. In D. melanogaster, the nucleotide sequences of chromocentral I elements differed from those on chromosome arms by as much as 7%. All the I elements of D. mauritiana and D. sechellia are more closely related to the chromosomal I elements of D. melanogaster than to the chromocentral I elements in any species. No sequence difference was observed in the surveyed region between two chromosomal I elements isolated from D. melanogaster and one from D. simulans. These findings strongly support the idea that the defective chromocentral I elements of D. melanogaster originated before the species diverged and the chromosomal I elements were eliminated. The chromosomal I elements reinvaded natural populations of D. melanogaster recently, and were possibly introduced from D. simulans by horizontal transmission.     

Comparative mapping of cosmids and gene clones from a 1.6 Mb chromosomal region of Drosophila melanogaster in three species of the distantly related subgenus Drosophila

The successful hybridization of cosmid clones from Drosophila melanogaster (Sophophora subgenus) to the salivary gland chromosomes of other species as distantly related as those in the Drosophila subgenus attests their great potential for unravelling genome evolution. We have carried out, using 28 cosmids and 13 gene clones, a study of the organization of the D. melanogaster 95A-96A chromosomal region in three Drosophila subgenus species: D. repleta, D. buzzattii and D. virilis. These clones were first used to built an accurate map of this 1.6 Mb region of D. melanogaster chromosome 3R (Muller’s element E). Then, they were hybridized and mapped to the homologous chromosome 2 of the other three distantly related species. The studied region is disseminated over 13 different sites of chromosome 2 in the Drosophila subgenus species, which implies a minimum of 12 inversion breakpoints fixed between the two subgenera. Extrapolation to the entire chromosome gives 90 fixed inversions. The D. melanogaster Pp1-96A-Acr96Aa segment conserved in D. repleta and D. buzzatii is longer than previously thought and is also conserved in D. virilis. In addition, three other D. melanogaster segments conserved in the three Drosophila subgenus species were found. Finally, our data indicate significant statistical differences in the evolution rate of Muller’s element E among lineages, a result that agrees well with the previous cytogenetic data. Received: 22 July 1998; in revised form: 11 November 1998 / Accepted: 12 November 1998     

Life history adaptations and stress tolerance of four domestic species of Drosophila

Life history traits and stress tolerance were studied in four domestic species of Drosophila–D. melanogaster, D. simulans, D. auraria and D. immigrans– to understand how they adapt to their environments. In all species, larval weight approximately doubled in 1 day. The relative egg weight (egg weight : pupal weight) was smaller and the larval period was longer in D. immigrans than in the other three species. The pupal period was the longest in D. auraria. However, the adaptive significance of these differences in larval and pupal periods was not clear. The pupal case was generally thicker in the larger species, probably to support the larger pupal body. The start of oviposition was earliest and reproductive effort was greatest in female D. simulans, followed by female D. melanogaster. In contrast, starvation tolerance and the increase in bodyweight after eclosion was greater in D. immigrans and D. auraria than in the other two species. Pupal desiccation tolerance was greatest in D. melanogaster and lowest in D. auraria, and the less tolerant species seemed to select more humid sites for pupation. Adult tolerance to desiccation was greatest in D. melanogaster and lowest in D. simulans. In contrast, adult cold tolerance was greater in D. auraria and adult heat tolerance was lower in D. immigrans than in the other species. These differences in life history traits and stress tolerance represent the Drosophila species differential adaptations, and are assumed to allow coexistence of the species.     

Relationships within the Melanogaster subgroup species of the genus Drosophila (Sophophora)

Five members of the melanogaster species subgroup of the subgenus Sophophora have been studied cytologically, their mitotic chromosomes analysed after Giemsa, C-banding and quinacrine staining. In all five species (D. yakuba, D. teissieri, D. erecta, D. orena and D. mauritiana) n=4 and all of the species except D. orena have a typical melanogaster like mitotic karyotype though there are clear differences between species in the distribution of both C⁺ and Q⁺ material. D. orena has large metacentric X and Y chromosomes due to the accumulation of intensively fluorescing material on these elements with respect to their homologues in melanogaster. This extra heterochromatin of D. orena correlates with a very high proportion of satellite DNA in its nuclear genome (S. Barnes, unpublished). The polytene chromosomes of these species were studied after quinacrine staining and Q⁺ material found to be restricted to the polytene fourth chromosomes, with the exception of D. orena which possesses considerable Q⁺ material in its chromocentre. These findings are discussed in the light of other studies of karyotype evolution in the genus Drosophila.     

Ethanol Tolerance and Alcohol Dehydrogenase (ADH; EC1.1.1.1) Activity in Species of the cardini Group of Drosophila

Ethanol tolerance, alcohol dehydrogenase (ADH;EC1.1.1.1) activity, and tissue-specific expression wereexamined in species of the cardini group ofDrosophila using D. melanogaster as astandard of comparison. In contrast to most fruit-breeding species, allcardini species examined, two from the cardini subgroupand five from the dunni subgroup, were ethanol sensitive(LC₅₀ 2.05%) and the mean ADH activityof males ranges from only 8 to 16% that of D.melanogaster Adh^FF. Among all sevencardini species, there were small but significantdifferences in ethanol tolerance and ADH activity.Differences in enzyme mobility were in accordance with the proposedphylogeny for the dunni-subgroup species. ADH isexpressed in the fat body and midgut. Males of D.acutilabella and of D. belladunni havesignificantly less ethanol tolerance and express less ADH activitythan females in zymograms and histologicalpreparations.