Separating two tightly linked species-defining phenotypes in Bactrocera with hybrid recombinant analysis.

BACKGROUND: Bactrocera tryoni and Bactrocera neohumeralis mate asynchronously; the former mates exclusively around dusk while the latter mates during the day. The two species also differ in the colour of the post-pronotal lobe (callus), which is predominantly yellow in B. tryoni and brown in B. neohumeralis. We have examined the genetic relationship between the two characters in hybrids, backcrosses and multigeneration hybrid progeny. RESULTS: Our analysis of the mating time of the parental species revealed that while B. tryoni mate exclusively at dusk, B. neohumeralis females pair with B. neohumeralis males during the day and with B. tryoni males at dusk. We found considerable variance in mating time and callus colour among hybrid backcross individuals of both sexes but there was a strong although not invariant trend for callus colour to co-segregate with mating time in both sexes. To genetically separate these two phenotypes we allowed the interspecific F1 hybrids to propagate for 25 generations (F25) without selection for mating time or callus colour, finding that the advanced hybrid population had moved towards B. tryoni phenotypes for both traits. Selection for day mating in replicate lines at F25 resulted in significant phenotypic shifts in both traits towards B. neohumeralis phenotypes in F26. However, we were unable to completely recover the mating time profile of B. neohumeralis and relaxation of selection for day mating led to a shift back towards dusk mating, but not yellow callus colour, by F35. CONCLUSION: We conclude that the inheritance of the two major species-defining traits is separable but tightly linked and involves more than one gene in each case. It also appears that laboratory conditions select for the B. tryoni phenotypes for mating time. We discuss our findings in relation to speciation theory and the likely effects of domestication during the generation of mass release strains for sterile insect control programmes.

Genome-wide patterns of differentiation over space and time in the Queensland fruit fly.

The Queensland fruit fly, Bactrocera tryoni, is a major pest of Australian horticulture which has expanded its range in association with the spread of horticulture over the last ~ 150 years. Its distribution in northern Australia overlaps that of another fruit fly pest to which some authors accord full species status, Bactrocera aquilonis. We have used reduced representation genome-wide sequencing to genotype 359 individuals taken from 35 populations from across the current range of the two taxa, plus a further 73 individuals from six of those populations collected 15-22 years earlier. We find significant population differentiation along an east-west transect across northern Australia which likely reflects limited but bidirectional gene flow between the two taxa. The southward expansion of B. tryoni has led to relatively little genetic differentiation, and most of it is associated with a move into previously marginal inland habitats. Two disjunct populations elsewhere in Australia and three on Melanesian islands are each clearly differentiated from all others, with data strongly supporting establishment from relatively few founders and significant isolation subsequently. Resequencing of historical samples from one of the disjunct Australian populations shows that its genetic profile has changed little over a 15-year period, while the Melanesian data suggest a succession of 'island hopping' events with progressive reductions in genetic diversity. We discuss our results in relation to the control of B. tryoni and as a model for understanding the genetics of invasion and hybridisation processes.

Extraordinary conservation of entire chromosomes in insects over long evolutionary periods.

Comparison of the genomes of different Drosophila species has shown that six different chromosomes, the so-called ''Muller elements," constitute the building blocks for all Drosophila species. Here, we confirm previous results suggesting that this conservation of the Muller elements extends far beyond Drosophila, to at least tephritid fruit flies, thought to have diverged from drosophilids 60-70 mYr ago. Less than 10 percent of genes differ in chromosome location between the two insect groups. Within chromosomes, however, the order is highly scrambled, as expected from the comparison between Drosophila species. The data also support the notion that the sex chromosomes of tephritid flies originated from an ancestor of the dot chromosome 4 of Drosophila. Overall, therefore, no new chromosome has been created for perhaps a billion generations over the two evolutionary lines. This stability at the chromosome level, which appears to extend to all Diptera including mosquitoes, is in stark contrast to other groups such as mammals, birds, fish and plants, in which chromosome numbers and organization vary enormously among species that have diverged over much fewer generations.

Wolbachia pseudogenes and low prevalence infections in tropical but not temperate Australian tephritid fruit flies: manifestations of lateral gene transfer and endosymbiont spillover?

BACKGROUND: Maternally inherited Wolbachia bacteria infect many insect species. They can also be transferred horizontally into uninfected host lineages. A Wolbachia spillover from an infected source population must occur prior to the establishment of heritable infections, but this spillover may be transient. In a previous study of tephritid fruit fly species of tropical Australia we detected a high incidence of identical Wolbachia strains in several species as well as Wolbachia pseudogenes. Here, we have investigated this further by analysing field specimens of 24 species collected along a 3,000 km climate gradient of eastern Australia. RESULTS: Wolbachia sequences were detected in individuals of nine of the 24 (37 %) species. Seven (29 %) species displayed four distinct Wolbachia strains based on characterisation of full multi locus sequencing (MLST) profiles; the strains occurred as single and double infections in a small number of individuals (2-17 %). For the two remaining species all individuals had incomplete MLST profiles and Wolbachia pseudogenes that may be indicative of lateral gene transfer into host genomes. The detection of Wolbachia was restricted to northern Australia, including in five species that only occur in the tropics. Within the more widely distributed Bactrocera tryoni and Bactrocera neohumeralis, Wolbachia also only occurred in the north, and was not linked to any particular mitochondrial haplotypes. CONCLUSIONS: The presence of Wolbachia pseudogenes at high prevalence in two species in absence of complete MLST profiles may represent footprints of historic infections that have been lost. The detection of identical low prevalence strains in a small number of individuals of seven species may question their role as reproductive manipulator and their vertical inheritance. Instead, the findings may be indicative of transient infections that result from spillover events from a yet unknown source. These spillover events appear to be restricted to northern Australia, without proliferation in host lineages further south. Our study highlights that tropical fruit fly communities contain Wolbachia pseudogenes and may be exposed to frequent horizontal Wolbachia transfer. It also emphasises that global estimates of Wolbachia frequencies may need to consider lateral gene transfer and Wolbachia spillover that may be regionally restricted, transient and not inherited.

The draft genome of the pest tephritid fruit fly Bactrocera tryoni: resources for the genomic analysis of hybridising species.

BACKGROUND: The tephritid fruit flies include a number of economically important pests of horticulture, with a large accumulated body of research on their biology and control. Amongst the Tephritidae, the genus Bactrocera, containing over 400 species, presents various species groups of potential utility for genetic studies of speciation, behaviour or pest control. In Australia, there exists a triad of closely-related, sympatric Bactrocera species which do not mate in the wild but which, despite distinct morphologies and behaviours, can be force-mated in the laboratory to produce fertile hybrid offspring. To exploit the opportunities offered by genomics, such as the efficient identification of genetic loci central to pest behaviour and to the earliest stages of speciation, investigators require genomic resources for future investigations. RESULTS: We produced a draft de novo genome assembly of Australia's major tephritid pest species, Bactrocera tryoni. The male genome (650-700 Mbp) includes approximately 150 Mb of interspersed repetitive DNA sequences and 60 Mb of satellite DNA. Assessment using conserved core eukaryotic sequences indicated 98% completeness. Over 16,000 MAKER-derived gene models showed a large degree of overlap with other Dipteran reference genomes. The sequence of the ribosomal RNA transcribed unit was also determined. Unscaffolded assemblies of B. neohumeralis and B. jarvisi were then produced; comparison with B. tryoni showed that the species are more closely related than any Drosophila species pair. The similarity of the genomes was exploited to identify 4924 potentially diagnostic indels between the species, all of which occur in non-coding regions. CONCLUSIONS: This first draft B. tryoni genome resembles other dipteran genomes in terms of size and putative coding sequences. For all three species included in this study, we have identified a comprehensive set of non-redundant repetitive sequences, including the ribosomal RNA unit, and have quantified the major satellite DNA families. These genetic resources will facilitate the further investigations of genetic mechanisms responsible for the behavioural and morphological differences between these three species and other tephritids. We have also shown how whole genome sequence data can be used to generate simple diagnostic tests between very closely-related species where only one of the species is scaffolded.

Australian endemic pest tephritids: genetic, molecular and microbial tools for improved Sterile Insect Technique.

Among Australian endemic tephritid fruit flies, the sibling species Bactrocera tryoni and Bactrocera neohumeralis have been serious horticultural pests since the introduction of horticulture in the nineteenth century. More recently, Bactrocera jarvisi has also been declared a pest in northern Australia. After several decades of genetic research there is now a range of classical and molecular genetic tools that can be used to develop improved Sterile Insect Technique (SIT) strains for control of these pests. Four-way crossing strategies have the potential to overcome the problem of inbreeding in mass-reared strains of B. tryoni. The ability to produce hybrids between B. tryoni and the other two species in the laboratory has proved useful for the development of genetically marked strains. The identification of Y-chromosome markers in B. jarvisi means that male and female embryos can be distinguished in any strain that carries a B. jarvisi Y chromosome. This has enabled the study of homologues of the sex-determination genes during development of B jarvisi and B. tryoni, which is necessary for the generation of genetic-sexing strains. Germ-line transformation has been established and a draft genome sequence for B. tryoni released. Transcriptomes from various species, tissues and developmental stages, to aid in identification of manipulation targets for improving SIT, have been assembled and are in the pipeline. Broad analyses of the microbiome have revealed a metagenome that is highly variable within and across species and defined by the environment. More specific analyses detected Wolbachia at low prevalence in the tropics but absent in temperate regions, suggesting a possible role for this endosymbiont in future control strategies.

Comprehensive transcriptome analysis of early male and female Bactrocera jarvisi embryos.

BACKGROUND: Developing embryos are provided with maternal RNA transcripts and proteins, but transcription from the zygotic nuclei must be activated to control continuing embryonic development. Transcripts are generated at different stages of early development, and those involved in sex determination and cellularisation are some of the earliest to be activated. The male sex in tephritid fruit flies is determined by the presence of a Y chromosome, and it is believed that a transcript from the Y-chromosome sets in motion a cascade that determines male development, as part of the greater maternal to zygotic transition (MTZ). Here we investigate the poly(A+) transcriptome in early male and female embryos of the horticultural pest Bactrocera jarvisi (Diptera: Tephritidae). RESULTS: Bactrocera jarvisi embryos were collected over two pre-blastoderm time periods, 2-3h and 3-5h after egg laying. Embryos were individually sexed using a Y-chromosome marker, allowing the sex-specific poly(A+) transcriptome of single-sex embryo pools to be deep-sequenced and assembled de novo. Transcripts for sixteen sex-determination and two cellularisation gene homologues of Drosophila melanogaster (Diptera: Drosophilidae) were identified in early embryos of B. jarvisi, including transcripts highly upregulated prior to cellularisation. No strong candidates for transcripts derived solely from the Y chromosome were recovered from the poly(A+) fraction. CONCLUSIONS: Bactrocera jarvisi provides an excellent model for embryonic studies due to available Y-chromosome markers and the compact time frame for zygotic transcription and the sex-determined state. Our data contribute fundamental information to sex-determination research, and provide candidates for the sourcing of gene promoters for transgenic pest-management strategies of tephritid fruit flies.

DNA methylation: bisulphite modification and analysis.

DNA methylation is an important epigenetic modification of DNA in mammalian genomes. DNA methylation patterns are established early in development, modulated during tissue-specific differentiation and disrupted in many disease states, including cancer. To understand further the biological functions of these changes, accurate and reproducible methods are required to fully analyze the DNA methylation sequence. Here, we describe the 'gold-standard' bisulphite conversion protocol that can be used to re-sequence DNA from mammalian cells in order to determine and quantify the methylation state of a gene or genomic region at single-nucleotide resolution. The process of bisulphite treatment exploits the different sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulphite under acidic conditions--in which cytosine undergoes conversion to uracil, whereas 5-MeC remains unreactive. Bisulphite conversion of DNA, in either single tubes or in a 96-well format, can be performed in a minimum of 8 h and a maximum of 18 h, depending on the amount and quality of starting DNA.

The cryptochrome (cry) gene and a mating isolation mechanism in tephritid fruit flies.

Two sibling species of tephritid fruit fly, Bactrocera tryoni and Bactrocera neohumeralis, are differentiated by their time of mating, which is genetically determined and requires interactions between the endogenous circadian clock and light intensity. The cryptochrome (cry) gene, a light-sensitive component of the circadian clock, was isolated in the two Bactrocera species. The putative amino acid sequence is identical in the two species. In the brain, in situ hybridization showed that cry is expressed in the lateral and dorsal regions of the central brain where PER immunostaining was also observed and in a peripheral cell cluster of the antennal lobes. Levels of cry mRNA were analyzed in whole head, brain, and antennae. In whole head, cry is abundantly and constantly expressed. However, in brain and antennae the transcript cycles in abundance, with higher levels during the day than at night, and cry transcripts are more abundant in the brain and antennae of B. neohumeralis than in that of B. tryoni. Strikingly, these results are duplicated in hybrid lines, generated by rare mating between B. tryoni and B. neohumeralis and then selected on the basis of mating time, suggesting a role for the cry gene in the mating isolation mechanism that differentiates the species.

Expression of an Otx gene in the adult rudiment and the developing central nervous system in the vestibula larva of the sea urchin Holopneustes purpurescens.

Expression of the Otx gene, HprOtx, from the sea urchin Holopneustes purpurescens, is described during the development of the adult echinoid rudiment in the vestibula larva of this species. The adult rudiment forms directly after gastrulation in the vestibula larva since, unlike the pluteus larva of most other sea urchin species, it is not a feeding larva. The expression is described during the period from hatching to a late vestibula larva. At hatching, HprOtx is expressed throughout the ectoderm of the gastrula. A short time later, expression is absent from the ectoderm on the oral side of the gastrula where the vestibule will form. In an early vestibula larva, HprOtx is not expressed in the ectodermal floor of the vestibule but is expressed in an asymmetric pattern in the aboral ectoderm. As the vestibule invaginates, HprOtx is newly expressed in the ectodermal floor of the vestibule as it develops into the neuroectoderm that is the anlage of the circum-oral central nervous system. The expression is at first in the central part of the floor, then it extends outwards to the ectoderm around the five primary podia and to the epineural folds between the podia. The epineural folds later close to form the radial nerves and the circum-oral nerve ring. In a late vestibula larva, HprOtx is expressed in the radial nerves and the nerve ring. The expression of an Otx gene in the developing echinoid central nervous system is interpreted as an instance of conserved gene expression in echinoderm development.

Bactrocera tryoni and closely related pest tephritids--molecular analysis and prospects for transgenic control strategies.

Bactrocera tryoni is a serious pest of horticulture in eastern Australia. Here we review molecular data relevant to pest status and development of a transformation system for this species. The development of transformation vectors for non-drosophilid insects has opened the door to the possibility of improving the sterile insect technique (SIT), by genetically engineering factory strains of pest insects to produce male-only broods. Transposition assays indicate that all five of the vectors currently used for transformation in non-drosophilid species have the potential to be useful as transformation vectors in B. tryoni. Evidence of cross mobilization of hobo by an endogenous Homer element emphasises the necessity to understand the endogenous transposons within a species. The sex-specific doublesex and yolk protein genes have been characterized with a view to engineering a female-specific lethal gene or modifying gene expression through RNA interference (RNAi). Data are presented which indicate the potential of RNAi to modify the sex ratio of resultant broods. An understanding of how pest status is determined and maintained is being addressed through the characterization of genes of the circadian clock that enable the fly to adapt to environmental cues. Such an understanding will be useful in the future to the effective delivery of sophisticated pest control measures.

cDNA hox sequences 3' of the homeobox isolated from the sea urchin Holopneustes purpurescens are definitive for sea urchin Hox orthologues.

In an investigation into Hox genes in sea urchin development, cDNA nucleotide sequences were isolated from seven Hox genes of Holopneustes purpurescens by 3' RACE. Each sequence extended from the homeobox through the coding region 3' of the homeobox to the 3'untranslated region and, in the majority of sequences, to a poly(A) signal and a poly(A) tail. The cDNA sequences 3' of the homeoboxes were very different between the seven Hox genes. In contrast, the sequences within each Hox gene 3' of the homeobox were very similar, especially at the amino acid level, between the sea urchin species for which sequences are available. The coding regions 3' of the homeodomains are thus a means of unambiguously identifying homologies between sea urchin Hox genes. A phylogenetic analysis where amino acid motifs in these 3' coding regions were aligned with similar motifs in chordate species led us to classify two posterior sea urchin Hox genes, of uncertain affinity, as orthologues of Hox9 and Hox10 genes. Putative cytoplasmic polyadenylation elements were mapped in the 3' untranslated region of sea urchin Hox genes.