Heritable endosymbionts of Drosophila

Although heritable microorganisms are increasingly recognized as widespread in insects, no systematic screens for such symbionts have been conducted in Drosophila species (the primary insect genetic models for studies of evolution, development, and innate immunity). Previous efforts screened relatively few Drosophila lineages, mainly for Wolbachia. We conducted an extensive survey of potentially heritable endosymbionts from any bacterial lineage via PCR screens of mature ovaries in 181 recently collected fly strains representing 35 species from 11 species groups. Due to our fly sampling methods, however, we are likely to have missed fly strains infected with sex ratio-distorting endosymbionts. Only Wolbachia and Spiroplasma, both widespread in insects, were confirmed as symbionts. These findings indicate that in contrast to some other insect groups, other heritable symbionts are uncommon in Drosophila species, possibly reflecting a robust innate immune response that eliminates many bacteria. A more extensive survey targeted these two symbiont types through diagnostic PCR in 1225 strains representing 225 species from 32 species groups. Of these, 19 species were infected by Wolbachia while only 3 species had Spiroplasma. Several new strains of Wolbachia and Spiroplasma were discovered, including ones divergent from any reported to date. The phylogenetic distribution of Wolbachia and Spiroplasma in Drosophila is discussed.     

Conservation implications of hybridization in Hawaiian picture-winged Drosophila

In this review, we discuss the importance of hybridization among species for the conservation of Hawaiian picture-winged Drosophila. Hybridization can be a positive evolutionary process that creates new species and increases the adaptation of populations and species through the spread of adaptive alleles and traits. Conversely, hybridization can disrupt the genetic integrity of species or populations and this may be most detrimental among taxa that are recently hybridizing due to recent ecological changes. The loss of biodiversity in Hawaiian Drosophila through hybridization may be facilitated by habitat alteration and introduced species that reduce population sizes and alter geographic distributions of native species. We briefly review the evidence for hybridization in the genus Drosophila and then focus on hybridization in the Hawaiian picture-winged Drosophila. We examine three general approaches for identifying hybrids and for assessing the factors that appear to contribute to hybridization and the potential ecological and evolutionary outcomes of hybrids in natural populations. Overall, the potential for hybridization among species will likely increase the risk of extinction for Hawaiian picture-winged Drosophila species. Thus, it is important to consider the potential for hybridization among species when developing plans for the conservation of Hawaiian Drosophila.     

Tissue-Specific Expression Phenotypes of Hawaiian Drosophila Adh Genes in Drosophila Melanogaster Transformants

Interspecific differences in the tissue-specific patterns of expression displayed by the alcohol dehydrogenase (Adh) genes within the Hawaiian picture-winged Drosophila represent a rich source of evolutionary variation in gene regulation. Study of the cis-acting elements responsible for regulatory differences between Adh genes from various species is greatly facilitated by analyzing the behavior of the different Adh genes in a homogeneous background. Accordingly, the Adh gene from Drosophila grimshawi was introduced into the germ line of Drosophila melanogaster by means of P element-mediated transformation, and transformants carrying this gene were compared to transformants carrying the Adh genes from Drosophila affinidisjuncta and Drosophila hawaiiensis. The results indicate that the D. affinidisjuncta and D. grimshawi genes have relatively higher levels of expression and broader tissue distribution of expression than the D. hawaiiensis gene in larvae. All three genes are expressed at similar overall levels in adults, with differences in tissue distribution of enzyme activity corresponding to the pattern in the donor species. However, certain systematic differences between Adh gene expression in transformants and in the Hawaiian Drosophila are noted along with tissue-specific position effects in some cases. The implications of these findings for the understanding of evolved regulatory variation are discussed.     

New Drosophila P-like elements and reclassification of Drosophila P-elements subfamilies

Genomic searches for P-like transposable elements were performed (1) in silico in the 12 available Drosophila genomes and (2) by PCR using degenerate primers in 21 Neotropical Drosophila species. In silico searches revealed P-like sequences only in Drosophila persimilis and Drosophila willistoni. Sixteen new P-like elements were obtained by PCR. These sequences were added to sequences of previously described P-like elements, and a phylogenetic analysis was performed. The subfamilies of P-elements described in the literature (Canonical, M, O, T, and K) were included in the reconstructed tree, and all were monophyletic. However, we suggest that some subfamilies can be enlarged, other subdivided, and some new subfamilies may be proposed, totalizing eleven subfamilies, most of which contain new P-like sequences. Our analyses support the monophyly of P-like elements in Drosophilidae. We suggest that, once these elements need host-specific factors to be mobilizable, the horizontal transfer (HT) of P-like elements may be inhibited among more distant taxa. Nevertheless, HT among Drosophilidae species appears to be a common phenomenon.     

Characterization and isolation of novel microsatellites from the Drosophila dunni subgroup

We have isolated and characterized 77 novel microsatellites from two species, Drosophila dunni and Drosophila nigrodunni, which are closely related Caribbean-island endemics from the Drosophila cardini species group. These species are very distantly related to all other Drosophila from which microsatellites have previously been characterized. We find that the average length of microsatellites isolated in these species is quite small, with an overall mean length of 9.8 repeat units for dinucleotide microsatellites in the two study species. The nucleotide composition of dinucleotides differs between the two species: D. nigrodunni has a predominance of (AC/GT)n repeats, whereas D. dunni has equal numbers of (AC/GT)n and (AG/CT)n repeats. Tri- and tetranucleotide repeats are not abundant in either species. We assayed the variability of eight microsatellites in a closely related third species, Drosophila arawakana, using wild-caught individuals from the island of Guadeloupe. We found the microsatellites to be extremely variable in this population, with observed heterozygosities ranging from 0.541 to 0.889. DNA amplification trials suggest that these eight microsatellites are widely conserved across the D. cardini group, with five of the eight producing amplification products in every species tested. However, the loci are very poorly conserved over greater phylogenetic distances. DNA amplification of the microsatellite loci was unreliable in members of the closely related Drosophila quinaria, Drosophila calloptera, Drosophila guarani and Drosophila tripunctata species groups. Furthermore, these microsatellites could not be detected in the genome of Drosophila melanogaster, despite the conservation of microsatellite flanking regions at some loci. These data indicate that Drosophila microsatellite loci are quite short lived over evolutionary timescales relative to many other taxa.     

Comparative morphology of nucleolar DNA in Drosophila. II. Drosophila fulvimaculoides and drosophila tumiditarsus

This paper reports the demonstration, using fluorescence microscopy, of nucleolar DNA in two species of Drosophila. In Drosophila fulvimaculoides, the nucleolar DNA presents a variable morphology, suggestive of puffing activity. This material, which sometimes shows a banded structure like that of the polytene chromosomes, is shown not to be coextensive with the Y chromosome. Nucleolar DNA is demonstrated in Drosophila tumiditarsus also, and previous reports of an association of the dot chromosome with the nucleolus in this species are confirmed. The special usefulness of these two species for various sorts of investigation in pointed out.     

Resource availability and population size in cactophilic Drosophila

1. Four species of Drosophila, Drosophila nigrospiracula ( Patterson & Wheeler 1942 ) , Drosophila mettleri ( Heed 1977 ) , Drosophila pachea ( Patterson & Wheeler 1942 ) , and Drosophila mojavensis ( Patterson & Crow 1940 ) , are endemic to the Sonoran Desert of North America and breed in different species of necrotic columnar cacti. Differences in resource availability have been suggested to explain the interspecific variability in fly population biology, but resource availability for these species has not been quantitatively assessed thoroughly in either spatial or temporal terms. The resource availability was quantified quarterly at three sites for 3 years and population sizes for each Drosophila species were estimated.
2. Spatial and temporal availability of resources differed significantly among species of host cacti, with organpipe cactus ( Stenocereus thurberi ) being the least abundant and senita ( Lophocereus schottii ) the most abundant spatially.
3. Drosophila species differed significantly in population size. The largest population sizes were found for D. nigrospiracula and D. mojavensis and smallest for D. pachea . Populations of D. mettleri were intermediate to these.
4. Population size was greatest for fly species utilizing host species having the largest and longest lasting necroses.
5. Resource availability does not explain the reduction of fly populations in the summer. Necroses were most abundant when flies were absent.     

Molecular evolution of Drosophila odorant receptor genes

A total of 752 odorant receptor (Or) genes, including pseudogenes, were identified in 11 Drosophila species and named after their orthologs in Drosophila melanogaster. The 813 Or genes, including 61 from D. melanogaster, were classified into 59 orthologous groups that are well supported by gene phylogeny. By reconciling with the gene family phylogeny, we estimated the number of gene duplication/loss events and intron gain/loss events in the species phylogeny. We found that these events are particularly frequent in Drosophila grimshawi, Drosophila willistoni, and obscura group. More than half of the duplicated genes stay as tandem arrays, whose size range from 2 to 8. These genes vary in sequence and some likely underwent positive selection, indicating that the gene duplication was important for flies to acquire new olfactory functions. We hypothesize that Or genes conferred the basic olfactory repertoire to ancestral flies before the speciation of the Drosophila and Sophophora subgenera about 40 Mya. This repertoire has been largely maintained in the current species, whereas lineage-specific gene duplication seems to have led to additional specialization in some species in response to specific ecological conditions.     

X chromosome DNA variation in Drosophila virilis.

Comparisons of polymorphism patterns between distantly related species are essential in order to determine their generality. However, most work on the genus Drosophila has been done only with species of the subgenus Sophophora. In the present work, we have sequenced one intron and surrounding coding sequences of 6 X-linked genes (chorion protein s36, elav, fused, runt, suppressor of sable and zeste) from 21 strains of wild-type Drosophila virilis (subgenus Drosophila). From these data, we have estimated the average level of DNA polymorphism, inferred the effective population size and population structure of this species, and compared the results with those obtained for other Drosophila species. There is no reduction in variation at two loci close to the centromeric heterochromatin, in contrast to Drosophila melanogaster.     

Characterization of a species-specific satellite DNA of Drosophila buzzatii

The pBuM189 satellite DNA family was found to be species specific for Drosophila buzzatii . It consists of slightly AT-rich tandemly arranged repeats with a high copy number in the genome and shows a very high level of intraspecific sequence similarity. pBuM189 repeats cannot be detected in the genomes of closely related species such as Drosophila serido , Drosophila borborema and Drosophila koepferae . The data support the marginal systematic position of D. buzzatii within the buzzatii cluster of the Drosophila repleta group.     

Preadult competition between Drosophila subobscura and Drosophila pseudoobscura

The Palaearctic species Drosophila subobscura has recently colonized a large area of North America where it coexists with Drosophila pseudoobscura. The viability and developmental rate of these species were studied at 13 d?C, 18d?C and 23 d?C and at densities of 10, 50, 100 and 200 eggs per vial. The two species were differently affected by density and temperature in the ranges studied. Both intra- and interspecific cultures showed that D. pseudoobscura was best adapted to 23 d?C, where it was clearly the dominant species. On the other hand, at 18 d?C and especially at 13 d?C, although D. subobscura was less viable than D. pseudoobscura, its developmental time was shorter, which may give advantage to this species. Results reported here agree with the observed distribution of these species in North America.     

An intron loss of Dfak gene in species of the Drosophila melanogaster subgroup and phylogenetic analysis

Drosophila focal adhesion kinase (Dfak) gene is a single-copy nuclear gene. Previous study revealed that Drosophila melanogaster and Drosophila simulans had lost an intron precisely within the tyrosine kinase (TyK) domain of this gene. However, this did not happen in several other Drosophila species, including Drosophila elegans, Drosophila ficusphila, Drosophila biarmipes, Drosophila jambulina, Drosophila prostipennis, Drosophila takahashii, and Drosophila pseudoobscura. In the current study, homologous sequences of Drosophila sechellia, Drosophila mauritiana, Drosophila yakuba, Drosophila teissieri, Drosophila santomea, and Drosophila erecta were amplified by polymerase chain reaction, and further sequencing analysis indicated that these species were missing a TyK domain intron, indicating they were closely related. The relationship of the D. melanogaster species group was reconstructed using TyK domain nucleotide sequences. The resulting phylogenetic tree revealed that these 8 species were the most related species in the melanogaster group. These results strongly support previously proposed classifications based on morphological and molecular data.     

Evolutionary relationships of Drosophila mojavensis geographic host races and their sister species Drosophila arizonae

The cactophilic Drosophila mojavensis species group living in the deserts and dry tropical forests of the southwestern United States and Mexico provides a valuable system for studies in diversification and speciation. Rigorous studies of the relationships between host races of D. mojavensis and the relationships among the members of the species group (D. mojavensis, Drosophila arizona, and Drosophila navojoa) are lacking. We used mitochondrial CO1 sequence data to address the phylogenetics and population genetics of this species group. In this study we have found that the sister species D. mojavensis and D. arizonae share no mitochondrial haplotypes and thus show no evidence for recent introgression. We estimate the divergence time between D. mojavensis and D. arizonae to be between 1.91 and 2.97 million years ago. D. arizonae shows little structure in our population genetic analyses but there is phylogenetic differentiation between southeastern and northern populations of D. arizonae. Drosophila mojavensis shows significant population and phylogenetic structure across the four geographic regions of its distribution. The mitochondrial data support an origin of D. mojavensis on the mainland with early differentiation into the populations now found in the Mojave Desert and the Mainland Sonoran Desert and later colonization of the Baja Peninsula, in contrast to previous models. Also, the sister clade to D. mojavensis/D. arizonae includes D. navojoa and Drosophila huaylasi. By defining the genetic relationships among these populations, we provide a foundation for more sophisticated hypothesis testing regarding the timing of early speciation events and host switches in this species group.     

Phylogenetic taxonomy in Drosophila: Problems and prospects

The genus Drosophila is one of the best-studied model systems in modern biology, with twelve fully sequenced genomes available. In spite of the large number of genetic and genomic resources, little is known concerning the phylogenetic relationships, ecology, and evolutionary history of all but a few species. Recent molecular systematic studies have shown that this genus is comprised of at least three independent lineages and that several other genera are actually embedded within Drosophila. This genus accounts for over 2000 described, and many more undescribed, species. While some Drosophila researchers are advocating dividing this genus into three or more separate genera, others favor maintaining Drosophila as a single large genus. With the recent sequencing of the genomes of multiple Drosophila species and their expanding use in comparative biology, it is critical that the Drosophila research community understands the taxonomic framework underlying the naming and relationships of these species. The subdivision of this genus has significant biological implications, ranging from the accurate annotation of single genes to understanding how ecological adaptations have occurred over the history of the group.     

Evolutionary dynamics of wAu-like Wolbachia variants in neotropical Drosophila spp

Wolbachia bacteria are common intracellular symbionts of arthropods and have been extensively studied in Drosophila. Most research focuses on two Old Word hosts, Drosophila melanogaster and Drosophila simulans, and does not take into account that some of the Wolbachia associations in these species may have evolved only after their fast global expansion and after the exposure to Wolbachia of previously isolated habitats. Here we looked at Wolbachia of Neotropical Drosophila species. Seventy-one lines of 16 Neotropical Drosophila species sampled in different regions and at different time points were analyzed. Wolbachia is absent in lines of Drosophila willistoni collected before the 1970s, but more recent samples are infected with a strain designated wWil. Wolbachia is absent in all other species of the willistoni group. Polymorphic wWil-related strains were detected in some saltans group species, with D. septentriosaltans being coinfected with at least four variants. Based on wsp and ftsZ sequence data, wWil of D. willistoni is identical to wAu, a strain isolated from D. simulans, but can be discriminated when using a polymorphic minisatellite marker. In contrast to wAu, which infects both germ line and somatic tissues of D. simulans, wWil is found exclusively in the primordial germ line cells of D. willistoni embryos. We report on a pool of closely related Wolbachia strains in Neotropical Drosophila species as a potential source for the wAu strain in D. simulans. Possible evolutionary scenarios reconstructing the infection history of wAu-like Wolbachia in Neotropical Drosophila species and the Old World species D. simulans are discussed.     

The Spiroplasma heritable bacterial endosymbiont of Drosophila

Since the discovery of the small, gram-positive bacterium, Spiroplasma, as a sex-ratio distorting agent in Drosophila over 50 years ago, substantial progress has been made in understanding the relationship of this bacteria with its insect host. Thus far, spiroplasmas have been found as heritable endosymbionts in sixteen different species of Drosophila. In some species these bacteria cause a male-killing phenotype, where the males die during embryogenesis. In other species, however, Spiroplasma does not cause male-killing, and its fitness effects are unclear. Though recent research has identified multiple factors that affect the prevalence and transmission of spiroplasmas in Drosophila populations, much work remains to fully characterize this symbiosis. Spiroplasma is the only identified heritable bacterial endosymbiont of Drosophila, other than Wolbachia, and can serve as a useful as model for elucidating the nature of insect/bacterial interactions.     

Effects of extreme temperatures on phenotypic variation and developmental stability in Drosophila melanogaster and Drosophila buzzatii

The effect of stressful and nonstressful rearing temperatures on phenotypic variation of four quantitative characters (thorax length, wing length, number of sternopleural chaetae, number of arista branches) and on developmental stability (fluctuating asymmetry) of the three latter characters was estimated in two Drosophila species: Drosophila melanogaster and Drosophila buzzatii . In both species, a general trend for increasing of phenotypic variation and fluctuating asymmetry at stress temperatures was observed; in fluctuating asymmetry, this effect was more pronounced. An increase of phenotypic variation under stress was shown for all characters examined except sternopleural chaeta number in D. buzzatii . Comparison of species responses suggests that the increase of variation in D. melanogaster was somewhat higher than in D. buzzatii .     

Alcohol dehydrogenase from Drosophila funebris and Drosophila immigrans: Molecular and evolutionary aspects

Alcohol dehydrogenase from Drosophila funebris and D. immigrans is evident at all developmental stages. The highest activity level appears in third-instar larvae and declines to a lower level at all later stages of development. Both species are monomorphic. The enzyme is a dimer consisting of two identical subunits with molecular weight 27,600. The pI values are 8.6 for D. funebris and 9.02 for D. immigrans. The optimum pH is 8.6 and 8.7 for D. funebris and D. immigrans, respectively. The Km values for NAD+, propan-2-ol, and butan-2-ol are 0.15, 2.90, and 2.08 mM, respectively, for D. funebris and 0.16, 1.53, and 1.49 mM, respectively, for D. immigrans. The half-life for the purified enzyme is 45 days for D. funebris and 18 days for D. immigrans at 4 degrees C. Data on the amino acid composition of both enzymes and peptide maps of alcohol dehydrogenase of D. immigrans reveal that they have marked homologies between them and also with alcohol dehydrogenases of other species. D. funebris shows reduced levels of alcohol dehydrogenase synthesis but has the highest specific activity reported to date for a Drosophila species. D. immigrans synthesises six times more enzyme but the specific activity is comparable to that of other species of Drosophila. This evidence could explain their different alcohol tolerance. The molecular properties of these alcohol dehydrogenases together with other species of Drosophila suggest that the alcohol dehydrogenase of Drosophila has arisen by divergent evolution from a common ancestral gene.     

Horizontal transmission of Wolbachia in a Drosophila community

Abstract.  1.  Wolbachia bacteria are reproductive parasites of arthropods and infect an estimated 20% of all insect species worldwide. In order to understand patterns of Wolbachia infection, it is necessary to determine how infections are gained or lost. Wolbachia transmission is mainly vertical, but horizontal transmission between different host species can result in new infections, although its ecological context is poorly understood. Horizontal transmission is often inferred from molecular phylogenies, but could be confounded by recombination between different Wolbachia strains.
2. This study addressed these issues by using three genes: wsp , ftsZ , and groE , to study Wolbachia infections in fruit- and fungus-feeding Drosophila communities in Berkshire, U.K.
3. Identical sequences were found for all three genes in Drosophila ambigua and Drosophila tristis. This suggests horizontal transmission of Wolbachia between these two previously unstudied Drosophila species, which may be the result of the two host species sharing the same food substrates or parasites.
4.  Wolbachia infections might be lost from species due to curing by naturally occurring antibiotics and the presence of these is likely to vary between larval food substrates.
5. It was investigated whether Wolbachia incidence was lower in fungus-feeding than in fruit-feeding Drosophila species, but no significant difference based on food substrate was found.     

Evolution of arginine kinase within the genus Drosophila

The rate of evolution and extant levels of polymorphism for arginine kinase were examined in species of the genus Drosophila. Surveys of 11 species for electrophoretic variation revealed an average heterozygosity of 0.003. Using antisera prepared against arginine kinase purified from a representative of each of three major subdivisions of the genus, immunological distances were measured by microcomplement fixation (MC'F) assay for 20 species of Drosophila. These data are consistent with the broad outline of the phylogenetic relationships within the genus suggested by other kinds of data. The unit evolutionary period for Drosophila arginine kinase was estimated to be 59 million years (MY). This contrasts with an estimate of 30 MY as the unit evolutionary period for vertebrate creatine kinase. It is suggested that this difference in evolutionary rate arises because a single gene encodes Drosophila arginine kinase, whereas at least three different genes encode the various forms of vertebrate creatine kinase.