Patterns of Variation in the Usage of Fatty Acid Chains among Classes of Ester and Ether Neutral Lipids and Phospholipids in the Queensland Fruit Fly.

Modern lipidomics has the power and sensitivity to elucidate the role of insects' lipidomes in their adaptations to the environment at a mechanistic molecular level. However, few lipidomic studies have yet been conducted on insects beyond model species such as Drosophila melanogaster. Here, we present the lipidome of adult males of another higher dipteran frugivore, Bactrocera tryoni. We describe 421 lipids across 15 classes of ester neutral lipids and phospholipids and ether neutral lipids and phospholipids. Most of the lipids are specified in terms of the carbon and double bond contents of each constituent hydrocarbon chain, and more ether lipids are specified to this degree than in any previous insect lipidomic analyses. Class-specific profiles of chain length and (un)saturation are broadly similar to those reported in D. melanogaster, although we found fewer medium-length chains in ether lipids. The high level of chain specification in our dataset also revealed widespread non-random combinations of different chain types in several ester lipid classes, including deficits of combinations involving chains of the same carbon and double bond contents among four phospholipid classes and excesses of combinations of dissimilar chains in several classes. Large differences were also found in the length and double bond profiles of the acyl vs. alkyl or alkenyl chains of the ether lipids. Work on other organisms suggests some of the differences observed will be functionally consequential and mediated, at least in part, by differences in substrate specificity among enzymes in lipid synthesis and remodelling pathways. Interrogation of the B. tryoni genome showed it has comparable levels of diversity overall in these enzymes but with some gene gain/loss differences and considerable sequence divergence from D. melanogaster.

Genetic variation for rectal gland volatiles among recently collected isofemale lines and a domesticated strain of Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae).

Divergence between populations in mating behaviour can function as a potent premating isolating mechanism and promote speciation. However, very few cases of inherited intraspecific variation in sexual signalling have been reported in tephritid fruit flies, despite them being a highly speciose family. We tested for such variation in one tephritid, the Queensland fruit fly, Bactrocera tryoni (Qfly). Qfly mating behaviour depends on volatiles secreted from male rectal glands but no role for the volatiles from female rectal glands has yet been reported. We previously detected over 100 volatile compounds in male rectal glands and identified over 30 of them. Similar numbers were recorded in females. However, many compounds showed presence/absence differences between the sexes and many others showed quantitative differences between them. Here we report inherited variation among 24 Qfly lines (23 isofemale lines established from recent field collections and one domesticated line) in the abundance of three esters, two alcohols, two amides, an aldehyde and 18 unidentified volatiles in male rectal glands. We did not find any compounds in female rectal glands that varied significantly among the lines, although this may at least partly reflect lower female sample numbers. Most of the 26 male compounds that differed between lines were more abundant in the domesticated line than any of the recently established isofemale lines, which concurs with other evidence for changes in mating behaviour during domestication of this species. There were also large differences in several of the 26 compounds among the isofemale lines, and some of these differences were associated with the regions from which the lines were collected. While some of the variation in different compounds was correlated across lines, much of it was not, implicating involvement of multiple genes. Our findings parallel reports of geographic variation in other Qfly traits and point to inherited differences in reproductive physiology that could provide a basis for evolution of premating isolation between ecotypes.

Multi-locus genotyping of stored sperm reveals female remating rates in wild populations of the Queensland fruit fly.

Female insects commonly have more than one mate during a breeding period ('polyandry'), storing and using sperm from multiple males. In addition to its evolutionary significance, insect polyandry has practical implications for pest management that relies on the sterile insect technique (SIT). The Queensland fruit fly, Bactrocera tryoni (Froggatt), is a major horticultural pest in Australia, and outbreaks are managed by SIT in some regions. The present study provides the first evidence for polyandry in female B. tryoni from field populations from New South Wales (NSW) and Queensland (QLD) through multi-locus genotyping (ten microsatellite markers in four fluorescent multiplexes) of the stored sperm in ovipositing females. Polyandry level was significantly higher in the NSW collection (80.0 %) than the QLD collection (26.1 %), suggesting substantial regional and/or temporal variation. These findings have important implications for the use of SIT to suppress B. tryoni populations and to eradicate outbreaks.

Diversity and sex differences in rectal gland volatiles of Queensland fruit fly, Bactrocera tryoni (Diptera: Tephritidae).

Rectal gland volatiles are key mediators of sexual interactions in tephritid fruit flies. We used solid-phase microextraction (SPME) plus gas chromatography-mass spectrometry (GC-MS) and gas chromatography-flame ionization detection (GC-FID) to substantially expand rectal gland chemical characterisation of the Queensland fruit fly (Bactrocera tryoni (Diptera: Tephritidae); Qfly). The SPME GC-MS analysis identified 24 of the 30 compounds previously recorded from Qfly rectal glands, plus another 21 compounds that had not previously been reported. A few amides and fatty acid esters dominated the chromatograms of males and females respectively, but we also found other esters, alcohols and aldehydes and a ketone. The GC-FID analyses also revealed over 150 others, as yet unidentified, volatiles, generally in lesser amounts. The GC-FID analyses also showed 49 and 12 compounds were male- and female-specific, respectively, both in single sex (virgin) and mixed sex (mostly mated) groups. Another ten compounds were male-specific among virgins but undetected in mixed sex groups, and 29 were undetected in virgins but male-specific in mixed sex groups. The corresponding figures for females were four and zero, respectively. Most short retention time peaks (including a ketone and an ester) were male-specific, whereas most female-biased peaks (including five fatty acid esters) had long retention times. Our results indicate previously unsuspected diversity of rectal gland volatiles that might have pheromone functions in males, but far fewer in females.

Population differences and domestication effects on mating and remating frequencies in Queensland fruit fly.

Females of many insect species are unreceptive to remating for a period following their first mating. This inhibitory effect may be mediated by either the female or her first mate, or both, and often reflects the complex interplay of reproductive strategies between the sexes. Natural variation in remating inhibition and how this phenotype responds to captive breeding are largely unexplored in insects, including many pest species. We investigated genetic variation in remating propensity in the Queensland fruit fly, Bactrocera tryoni, using strains differing in source locality and degree of domestication. We found up to threefold inherited variation between strains from different localities in the level of intra-strain remating inhibition. The level of inhibition also declined significantly during domestication, which implied the existence of genetic variation for this trait within the starting populations as well. Inter-strain mating and remating trials showed that the strain differences were mainly due to the genotypes of the female and, to a lesser extent, the second male, with little effect of the initial male genotype. Implications for our understanding of fruit fly reproductive biology and population genetics and the design of Sterile Insect Technique pest management programs are discussed.

A wAlbB Wolbachia Transinfection Displays Stable Phenotypic Effects across Divergent Aedes aegypti Mosquito Backgrounds.

Aedes mosquitoes harboring intracellular Wolbachia bacteria are being released in arbovirus and mosquito control programs. With releases taking place around the world, understanding the contribution of host variation to Wolbachia phenotype is crucial. We generated a Wolbachia transinfection (wAlbBQ) in Aedes aegypti and performed backcrossing to introduce the infection into Australian or Malaysian nuclear backgrounds. Whole Wolbachia genome sequencing shows that the wAlbBQ transinfection is nearly identical to the reference wAlbB genome, suggesting few changes since the infection was first introduced to A. aegypti over 15 years ago. However, these sequences were distinct from other available wAlbB genome sequences, highlighting the potential diversity of wAlbB in natural Aedes albopictus populations. Phenotypic comparisons demonstrate the effects of wAlbB infection on egg hatching and nuclear background on fecundity and body size but no interactions between wAlbB infection and nuclear background for any trait. The wAlbB infection was stable at high temperatures and showed perfect maternal transmission and cytoplasmic incompatibility regardless of the host background. Our results demonstrate the stability of wAlbB across host backgrounds and point to its long-term effectiveness for controlling arbovirus transmission and mosquito populations. IMPORTANCEWolbachia bacteria are being used to control the transmission of dengue virus and other arboviruses by mosquitoes. For Wolbachia release programs to be effective globally, Wolbachia infections must be stable across mosquito populations from different locations. In this study, we transferred Wolbachia (strain wAlbB) to Aedes aegypti mosquitoes with an Australian genotype and introduced the infection to Malaysian mosquitoes through backcrossing. We found that the phenotypic effects of Wolbachia are stable across both mosquito backgrounds. We sequenced the genome of wAlbB and found very few genetic changes despite spending over 15 years in a novel mosquito host. Our results suggest that the effects of Wolbachia infections are likely to remain stable across time and host genotype.

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.

Climate stress resistance in male Queensland fruit fly varies among populations of diverse geographic origins and changes during domestication.

BACKGROUND: The highly polyphagous Queensland fruit fly (Bactrocera tryoni Froggatt) expanded its range substantially during the twentieth century and is now the most economically important insect pest of Australian horticulture, prompting intensive efforts to develop a Sterile Insect Technique (SIT) control program. Using a "common garden" approach, we have screened for natural genetic variation in key environmental fitness traits among populations from across the geographic range of this species and monitored changes in those traits induced during domestication. RESULTS: Significant variation was detected between the populations for heat, desiccation and starvation resistance and wing length (as a measure of body size). Desiccation resistance was correlated with both starvation resistance and wing length. Bioassay data for three resampled populations indicate that much of the variation in desiccation resistance reflects persistent, inherited differences among the populations. No latitudinal cline was detected for any of the traits and only weak correlations were found with climatic variables for heat resistance and wing length. All three stress resistance phenotypes and wing length changed significantly in certain populations with ongoing domestication but there was also a strong population by domestication interaction effect for each trait. CONCLUSIONS: Ecotypic variation in heat, starvation and desiccation resistance was detected in Australian Qfly populations, and these stress resistances diminished rapidly during domestication. Our results indicate a need to select source populations for SIT strains which have relatively high climatic stress resistance and to minimise loss of that resistance during domestication.

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.

The Effect of Nonrandom Mating on Wolbachia Dynamics: Implications for Population Replacement and Sterile Releases in Aedes Mosquitoes.

Wolbachia bacteria are known to cause deviations from random mating and affect sperm competition (SC) in some of their arthropod hosts. Because these effects could influence the effectiveness of Wolbachia in mosquito population replacement and suppression programs, we developed a theoretical framework to investigate them and we collected relevant data for the wMel infection in Aedes aegypti. Using incompatibility patterns as a measure of mating success of infected versus uninfected mosquitoes, we found some evidence that uninfected males sire more offspring than infected males. However, our theoretical framework suggests that this effect is unlikely to hamper Wolbachia invasion and has only minor effects on population suppression programs. Nevertheless, we suggest that mating effects and SC need to be monitored in an ongoing manner in release programs, given the possibility of ongoing selection for altered mating patterns.

Fitness of wAlbB Wolbachia Infection in Aedes aegypti: Parameter Estimates in an Outcrossed Background and Potential for Population Invasion.

Wolbachia endosymbionts are potentially useful tools for suppressing disease transmission by Aedes aegypti mosquitoes because Wolbachia can interfere with the transmission of dengue and other viruses as well as causing deleterious effects on their mosquito hosts. Most recent research has focused on the wMel infection, but other infections also influence viral transmission and may spread in natural populations. Here, we focus on the wAlbB infection in an Australian outbred background and show that this infection has many features that facilitate its invasion into natural populations including strong cytoplasmic incompatibility, a lack of effect on larval development, an equivalent mating success to uninfected males and perfect maternal transmission fidelity. On the other hand, the infection has deleterious effects when eggs are held in a dried state, falling between wMel and the more virulent wMelPop Wolbachia strains. The impact of this infection on lifespan also appears to be intermediate, consistent with the observation that this infection has a titer in adults between wMel and wMelPop. Population cage experiments indicate that the wAlbB infection establishes in cages when introduced at a frequency of 22%, suggesting that this strain could be successfully introduced into populations and subsequently persist and spread.

Larval competition extends developmental time and decreases adult size of wMelPop Wolbachia-infected Aedes aegypti.

The intracellular endosymbiont Wolbachia has been artificially transinfected into the dengue vector Aedes aegypti, where it is being investigated as a potential dengue biological control agent. Invasion of Wolbachia in natural populations depends upon the fitness of Wolbachia-infected Ae. aegypti relative to uninfected competitors. Although Wolbachia infections impose fitness costs on the adult host, effects at the immature stages are less clear, particularly in competitive situations. We look for effects of two Wolbachia infections, wMel and wMelPop, on intra-strain and inter-strain larval competition in Ae. aegypti. Development of Wolbachia-infected larvae is delayed in mixed cohorts with uninfected larvae under crowded-rearing conditions. Slow developing wMelPop-infected larvae have reduced adult size compared with uninfected larvae, and larvae with the wMel infection are somewhat larger and have greater viability relative to uninfected larvae when in mixed cohorts. Implications for successful invasion by these Wolbachia infections under field conditions are considered.

Assessing quality of life-shortening Wolbachia-infected Aedes aegypti mosquitoes in the field based on capture rates and morphometric assessments.

BACKGROUND: Recent releases have been carried out with Aedes aegypti mosquitoes infected with the wMelPop mosquito cell-line adapted (wMelPop-CLA) strain of Wolbachia. This infection introduced from Drosophila provides strong blockage of dengue and other arboviruses but also has large fitness costs in laboratory tests. The releases were used to evaluate the fitness of released infected mosquitoes, and (following termination of releases) to test for any effects of wMelPop-CLA on wing size and shape when mosquitoes were reared under field conditions. METHODS: We monitored gravid females via double sticky traps to assess the reproductive success of wMelPop-CLA-infected females and also sampled the overall mosquito population post-release using Biogent Sentinel traps. Morphometric analyses were used to evaluate infection effects on wing shape as well as size. RESULTS: Oviposition success as assessed through double sticky traps was unrelated to size of released mosquitoes. However, released mosquitoes with lower wing loading were more successful. Furthermore, wMelPop-CLA-infected mosquitoes had 38.3% of the oviposition success of uninfected mosquitoes based on the predicted infection frequency after release. Environmental conditions affected wing shape and particularly size across time in uninfected mosquitoes, but not in naturally-reared wMelPop-CLA-infected mosquitoes. Although the overall size and shape do not differ between naturally-reared wMelPop-CLA-infected and uninfected mosquitoes, the infected mosquitoes tended to have smaller wings than uninfected mosquitoes during the cooler November in comparison to December. CONCLUSION: These results confirm the lower fitness of wMelPop-CLA infection under field conditions, helping to explain challenges associated with a successful invasion by this strain. In the long run, invasion may depend on releasing strains carrying insecticide resistance or egg desiccation resistance, combined with an active pre-release population suppression program.

Body size and wing shape measurements as quality indicators of Aedes aegypti mosquitoes destined for field release.

There is increasing interest in rearing modified mosquitoes for mass release to control vector-borne diseases, particularly Wolbachia-infected Aedes aegypti for suppression of dengue. Successful introductions require release of high quality mosquitoes into natural populations. Potential indicators of quality are body size and shape. We tested to determine if size, wing/thorax ratio, and wing shape are associated with field fitness of Wolbachia-infected Ae. aegypti. Compared with field-collected mosquitoes, released mosquitoes were larger in size, with lower size variance and different wing shape but similar in wing-thorax ratio and its associated variance. These differences were largely attributed to nutrition and to a minor extent to wMel Wolbachia infection. Survival potential of released female mosquitoes was similar to those from the field. Females at oviposition sites tended to be larger than those randomly collected from BG-Sentinel traps. Rearing conditions should thus aim for large size without affecting wing/thorax ratios.

A novel association between a SNP in CYBRD1 and serum ferritin levels in a cohort study of HFE hereditary haemochromatosis.

There is emerging evidence that there are genetic modifiers of iron indices for HFE gene mutation carriers at risk of hereditary hemochromatosis. A random sample, stratified by HFE genotype, of 863 from a cohort of 31 192 people of northern European descent provided blood samples for genotyping of 476 single nucleotide polymorphisms (SNPs) in 44 genes involved in iron metabolism. Single SNP association testing, using linear regression models adjusted for sex, menopause and HFE genotype, was conducted for four continuously distributed outcomes: serum ferritin (log transformed), transferrin saturation, serum transferrin, and serum iron. The SNP rs884409 in CYBRD1 is a novel modifier specific to HFE C282Y homozygotes. Median unadjusted serum ferritin concentration decreased from 1194 microg/l (N = 27) to 387 microg/l (N = 16) for male C282Y homozygotes and from 357 microg/l (N = 42) to 69 microg/l (N = 12) for females, comparing those with no copies to those with one copy of rs884409. Functional testing of this CYBRD1 promoter polymorphism using a heterologous expression assay resulted in a 30% decrease in basal promoter activity relative to the common genotype (P = 0.004). This putative genetic modifier of iron overload expression accounts for 11% (95% CI 0.4%, 22.6%) of the variance in serum ferritin levels of C282Y homozygotes.