Adaptable, turn-on maturation (ATOM) fluorescent biosensors for multiplexed detection in cells.

A grand challenge in biosensor design is to develop a single-molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Here, we created a family of adaptable, turn-on maturation (ATOM) biosensors consisting of a monobody (circularly permuted at one of two positions) or a nanobody (circularly permuted at one of three positions) inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells coexpressing cyan, yellow and red ATOM sensors detected biosensor targets that were specifically localized to various subcellular compartments. Fluorescence activation involved ligand-dependent chromophore maturation with turn-on ratios of up to 62-fold in cells and 100-fold in vitro. Endoplasmic reticulum- and mitochondria-localized ATOM sensors detected ligands that were targeted to those organelles. The ATOM design was validated with three monobodies and one nanobody inserted into distinct fluorescent proteins, suggesting that customized ATOM sensors can be generated quickly.

Mimicking kidney flow shear efficiently induces aggregation of LECT2, a protein involved in renal amyloidosis.

Aggregation of leukocyte cell-derived chemotaxin 2 (LECT2) causes ALECT2, a systemic amyloidosis that affects the kidney and liver. Previous studies established that LECT2 fibrillogenesis is accelerated by the loss of its bound zinc ion and stirring/shaking. These forms of agitation create heterogeneous shear conditions, including air-liquid interfaces that denature proteins, that are not present in the body. Here, we determined the extent to which a more physiological form of mechanical stress-shear generated by fluid flow through a network of narrow channels-drives LECT2 fibrillogenesis. To mimic blood flow through the kidney, where LECT2 and other proteins form amyloid deposits, we developed a microfluidic device consisting of progressively branched channels narrowing from 5 mm to 20 µm in width. Shear was particularly pronounced at the branch points and in the smallest capillaries. Aggregation was induced within 24 h by shear levels that were in the physiological range and well below those required to unfold globular proteins such as LECT2. EM images suggested the resulting fibril ultrastructures were different when generated by laminar flow shear versus shaking/stirring. Importantly, results from the microfluidic device showed the first evidence that the I40V mutation accelerated fibril formation and increased both the size and the density of the aggregates. These findings suggest that kidney-like flow shear, in combination with zinc loss, acts in combination with the I40V mutation to trigger LECT2 amyloidogenesis. These microfluidic devices may be of general use for uncovering mechanisms by which blood flow induces misfolding and amyloidosis of circulating proteins.

Mechanistic insights into enhancement or inhibition of phase separation by different polyubiquitin chains.

Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin-binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.

Colorectal cancer and advanced adenoma incidence during post-polypectomy surveillance: a national cohort study in the English Bowel Cancer Screening Programme.

BACKGROUND: Improved colonoscopy quality has led to debate about whether all post-polypectomy surveillance is justified. We evaluated surveillance within the English Bowel Cancer Screening Programme (BCSP) to determine the yield of surveillance and identify predictive factors for surveillance outcome. METHODS: We performed a retrospective cohort study of individuals undergoing post-polypectomy surveillance between July 2006 and January 2017. BCSP records were linked to the National Cancer Registration Database to identify interval-type post-colonoscopy colorectal cancers (CRCs). Advanced adenoma and CRC at surveillance were documented. CRC incidence was compared with the general population using standardized incidence ratios (SIRs). Predictors of advanced adenomas at first surveillance (S1), and CRC during follow-up, were identified. RESULTS: 44 151 individuals (23 078 intermediate risk; 21 073 high risk) underwent 64 544 surveillance episodes. Advanced adenoma and CRC yields were, respectively, 10.0 % and 0.5 % at S1, 8.5 % and 0.4 % at S2, and 10.8 % and 0.4 % at S3. S1 yield was lowest in those with one index adenoma ≥ 10 mm (advanced adenoma 6.1 %; CRC 0.3 %). The SIR was 0.76 (95 %CI 0.66-0.88), accounted for by the intermediate risk group (intermediate risk SIR 0.61, 95 %CI 0.49-0.75; high risk SIR 0.95, 95 %CI 0.79-1.15). Adenoma multiplicity, presence of a large nonpedunculated adenoma, and greater villous component were associated with advanced adenoma at S1. Older age and multiplicity were significantly associated with CRC risk. CONCLUSION: This large, national analysis found low levels of CRC in those undergoing surveillance and low advanced adenoma yield in most subgroups. Less intensive surveillance in some subgroups is warranted, and surveillance may be avoided in those with a single large adenoma.

Loss of bound zinc facilitates amyloid fibril formation of leukocyte-cell-derived chemotaxin 2 (LECT2).

Aggregation of the circulating protein leukocyte-cell-derived chemotaxin 2 (LECT2) causes amyloidosis of LECT2 (ALECT2), one of the most prevalent forms of systemic amyloidosis affecting the kidney and liver. The I40V mutation is thought to be necessary but not sufficient for ALECT2, with a second, as-yet undetermined condition being required for the disease. EM, X-ray diffraction, NMR, and fluorescence experiments demonstrate that LECT2 forms amyloid fibrils in vitro in the absence of other proteins. Removal of LECT2's single bound Zn2+ appears to be obligatory for fibril formation. Zinc-binding affinity is strongly dependent on pH: 9-13 % of LECT2 is calculated to exist in the zinc-free state over the normal pH range of blood, with this fraction rising to 80 % at pH 6.5. The I40V mutation does not alter zinc-binding affinity or kinetics but destabilizes the zinc-free conformation. These results suggest a mechanism in which loss of zinc together with the I40V mutation leads to ALECT2.

Tumor suppressor Tsc1 is a new Hsp90 co-chaperone that facilitates folding of kinase and non-kinase clients.

The tumor suppressors Tsc1 and Tsc2 form the tuberous sclerosis complex (TSC), a regulator of mTOR activity. Tsc1 stabilizes Tsc2; however, the precise mechanism involved remains elusive. The molecular chaperone heat-shock protein 90 (Hsp90) is an essential component of the cellular homeostatic machinery in eukaryotes. Here, we show that Tsc1 is a new co-chaperone for Hsp90 that inhibits its ATPase activity. The C-terminal domain of Tsc1 (998-1,164 aa) forms a homodimer and binds to both protomers of the Hsp90 middle domain. This ensures inhibition of both subunits of the Hsp90 dimer and prevents the activating co-chaperone Aha1 from binding the middle domain of Hsp90. Conversely, phosphorylation of Aha1-Y223 increases its affinity for Hsp90 and displaces Tsc1, thereby providing a mechanism for equilibrium between binding of these two co-chaperones to Hsp90. Our findings establish an active role for Tsc1 as a facilitator of Hsp90-mediated folding of kinase and non-kinase clients-including Tsc2-thereby preventing their ubiquitination and proteasomal degradation.

Clinical presentation, sonographic features and treatment options of segmental stiff skin syndrome.

Segmental stiff skin syndrome is a rare genetic connective tissue disease, which is often misdiagnosed. High-frequency ultrasonography can represent a useful clinical adjunct in the differential diagnosis of this condition, in conjunction with the clinical and histopathological findings. Treatment options are limited and evidence is scarce. We present the clinical, sonographic and histological features of five paediatric patients diagnosed at our institution and discuss their response to treatment.

Measurements of the radioactivity of the cloud from the accident at Windscale Works (Sellafield, England) in October 1957: data submitted to the International Geophysical Year (IGY; July 1957-December 1958).

A fire in a nuclear reactor at Windscale Works (Sellafield, England) in October 1957 led to an uncontrolled aerial release of radionuclides. At the time of the accident air was sampled at various locations in Europe to monitor atmospheric pollution, and the opportunity was taken to measure the sampling filters for activity concentrations of iodine-131, caesium-137 and polonium-210 at the Harwell research establishment (United Kingdom); when it was not possible to perform measurements at Harwell, original measurement data were supplied. This programme of activity measurements was performed in the context of work by the Advisory Committee on Nuclear Radiation of the International Geophysical Year (IGY; July 1957-December 1958). The International Geophysical Year was an international programme of research into a comprehensive range of geophysical phenomena. The results of this measurement programme were originally reported in Harwell Memorandum AERE-M857 (1961) and this Harwell report is reproduced in this paper because of its historical interest and because it is no longer readily accessible to researchers.

A Single Protein Disruption Site Results in Efficient Reassembly by Multiple Engineering Methods.

Disrupting a protein's sequence by cleavage or insertion of a hinge domain forms the basis for protein engineering tools, including fragment complementation, circular permutation, and domain swapping. Despite the utility of these designs, their widespread implementation has been limited by the difficulty in choosing where to interrupt the protein sequence: the resulting fragments often aggregate or fail to reassemble. Here, we show that an optimal site exists within ribose binding protein (RBP) that, when disrupted, results in the most efficient formation of fragment-complemented and domain-swapped species. Cleaving RBP at this site also produces a highly stable, cooperatively folded circular permutant. This hot-spot site was identified by an experimental approach involving selection among competing folds. We find that efficiency in the case of RBP is determined by kinetic factors (survival of the first) rather than thermodynamics (survival of the fittest). Together with emerging computational tools, this limited data set defines a pathway for designing robust platforms for molecular switches and biosensors based on the aforementioned protein modifications.

Evidence of degradation of hair corticosterone in museum specimens.

Researchers increasingly rely on non-invasive physiological indices, such as glucocorticoid (GC) levels, to interpret how vertebrates respond to changes in their environment. Recently, hair GCs have been of particular interest, because they are presumed stable over long periods of storage, which may facilitate the study of large-scale spatial and temporal patterns of stress in mammals. In the current study, we evaluated the stability of hair corticosterone levels in museum specimens, and the potential effects of different museum curation treatments. Using deer mouse (Peromyscus maniculatus) specimens collected from Vancouver Island (11 sites, 82 individuals) over 76 years, we found that specimens collected earlier in the 20th century had lower hair corticosterone than more recently collected specimens. These results suggest that hair hormone levels may not be stable over decades of storage time. We then subjected hair samples collected from white-footed mouse (Peromyscus leucopus, n = 36) to 3 different museum curation treatments, and found that borax lowered hair corticosterone levels relative to control samples, but air drying samples, or treating them with turpentine had no effect. Our results present a source of concern for the use of museum specimens for hair hormone analysis, and for studying long term trends in glucocorticoid levels.

Thiosemicarbazones Functioning as Zinc Metallochaperones to Reactivate Mutant p53.

Small-molecule restoration of wild-type structure and function to mutant p53 (so-called mutant reactivation) is a highly sought-after goal in cancer drug development. We previously discovered that small-molecule zinc chelators called zinc metallochaperones (ZMCs) reactivate mutant p53 by restoring zinc binding to zinc-deficient p53 mutants. The lead compound identified from the NCI-60 human tumor cell lines screen, NSC319726 (ZMC1), belongs to the thiosemicarbazone (TSC) class of metal ion chelators that bind iron, copper, magnesium, zinc, and other transition metals. Here, we have investigated the other TSCs, NSC319725 and NSC328784, identified in the same screen, as well as the more well studied TSC, 3-AP (Triapine), to determine whether they function as ZMCs. We measured the zinc Kd zinc ionophore activity, ability to restore zinc to purified p53 DNA binding domain (DBD), and ability to restore site-specific DNA binding to purified R175H-DBD in vitro. We tested all four TSCs in a number of cell-based assays to examine mutant p53 reactivation and the generation of reactive oxygen species (ROS). We found that NSC319725 and NSC328784 behave similarly to ZMC1 in both biophysical and cell-based assays and are heretofore named ZMC2 (NSC319725) and ZMC3 (NSC328784). 3-AP generates a ROS signal similar to ZMC1-3, but it fails to function as a ZMC both in vitro and in cells and ultimately does not reactivate p53. These findings indicate that not all TSCs function as ZMCs, and much of their activity can be predicted by their affinity for zinc.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy.

Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

A longitudinal cohort study of HIV 'treatment as prevention' in gay, bisexual and other men who have sex with men: the Treatment with Antiretrovirals and their Impact on Positive And Negative men (TAIPAN) study protocol.

BACKGROUND: Australia has increased coverage of antiretroviral treatment (ART) over the past decade, reaching 73% uptake in 2014. While ART reduces AIDS-related deaths, accumulating evidence suggests that it could also bolster prevention efforts by reducing the risk of HIV transmission ('treatment as prevention'). While promising, evidence of community-level impact of treatment as prevention on reducing HIV incidence among gay and bisexual men is limited. We describe a study protocol that aims to determine if scale up of testing and treatment for HIV leads to a reduction in community viraemia and, in turn, if this reduction is temporally associated with a reduction in HIV incidence among gay and bisexual men in Australia's two most populous states. METHODS: Over the period 2009 to 2017, we will establish two cohorts making use of clinical and laboratory data electronically extracted retrospectively and prospectively from 73 health services and laboratories in the states of New South Wales and Victoria. The 'positive cohort' will consist of approximately 13,000 gay and bisexual men (>90% of all people living with HIV). The 'negative cohort' will consist of at least 40,000 HIV-negative gay and bisexual men (approximately half of the total population). Within the negative cohort we will use standard repeat-testing methods to calculate annual HIV incidence. Community prevalence of viraemia will be defined as the proportion of men with a viral load ≥200RNA copies/mm3, which will combine viral load data from the positive cohort and viraemia estimates among those with an undiagnosed HIV infection. Using regression analyses and adjusting for behavioural and demographic factors associated with infection, we will assess the temporal association between the community prevalence of viraemia and the incidence of HIV infection. Further analyses will make use of these cohorts to assess incidence and predictors of treatment initiation, repeat HIV testing, and viral suppression. DISCUSSION: This study will provide important information on whether 'treatment as prevention' is associated with a reduction in HIV incidence at a community level among gay and bisexual men.

Synthetic metallochaperone ZMC1 rescues mutant p53 conformation by transporting zinc into cells as an ionophore.

p53 is a Zn(2+)-dependent tumor suppressor inactivated in >50% of human cancers. The most common mutation, R175H, inactivates p53 by reducing its affinity for the essential zinc ion, leaving the mutant protein unable to bind the metal in the low [Zn(2+)]free environment of the cell. The exploratory cancer drug zinc metallochaperone-1 (ZMC1) was previously demonstrated to reactivate this and other Zn(2+)-binding mutants by binding Zn(2+) and buffering it to a level such that Zn(2+) can repopulate the defective binding site, but how it accomplishes this in the context of living cells and organisms is unclear. In this study, we demonstrated that ZMC1 increases intracellular [Zn(2+)]free by functioning as a Zn(2+) ionophore, binding Zn(2+) in the extracellular environment, diffusing across the plasma membrane, and releasing it intracellularly. It raises intracellular [Zn(2+)]free in cancer (TOV112D) and noncancer human embryonic kidney cell line 293 to 15.8 and 18.1 nM, respectively, with half-times of 2-3 minutes. These [Zn(2+)]free levels are predicted to result in ∼90% saturation of p53-R175H, thus accounting for its observed reactivation. This mechanism is supported by the X-ray crystal structure of the [Zn(ZMC1)2] complex, which demonstrates structural and chemical features consistent with those of known metal ionophores. These findings provide a physical mechanism linking zinc metallochaperone-1 in both in vitro and in vivo activities and define the remaining critical parameter necessary for developing synthetic metallochaperones for clinical use.

Modulating effects of WT1 on interferon-ß-vitamin D association in MS.

OBJECTIVE: To investigate whether those genes involved in the vitamin D pathway modulate the relationship between 25-hydroxyvitamin D (25(OH)D) and IFN-ß, the relationship between IFN-ß and sun in predicting 25(OH)D, and the interaction between IFN-ß and 25(OH)D in modulating relapse risk in patients with MS. METHODS: Prospective cohort study of 169 participants with MS and genotype data followed 2002-2005. Gene-IFN-ß and gene-IFN-ß-sun interactions predicting 25(OH)D evaluated by multilevel mixed-effects linear regression. Gene-IFN-ß interactions with 25(OH)D in modulating in relapse risk assessed using survival analysis. RESULTS: The cohort was 71.6% female and of mean age 47.8. Two-independent intronic genotyped SNPs (rs10767935 and rs5030244) in WT1 significantly modified the IFN-ß-25(OH)D association after adjustment (P(interaction) = 0.001, 0.0002; P(adj) = 0.003, 0.006, respectively). There was a marked difference in the interaction between self-reported sun exposure and IFN-ß in predicting 25(OH)D by level of rs10767935, although this did not reach statistical significance. No SNPs modified the interaction between IFN-ß and 25(OH)D in predicting relapse. CONCLUSIONS: We have demonstrated that two-independent SNPs (rs10767935 and rs5030244) in WT1 modified the IFN-ß-25(OH)D association in patients with MS. Some evidence was shown for a difference in the sun-IFN-ß-25(OH)D association by level of rs10767935. These findings indicate that WT1 variants may play a role in altering the effects of IFN-ß on vitamin D in MS.

Converting a binding protein into a biosensing conformational switch using protein fragment exchange.

Biosensors can be used in applications ranging from identifying disease biomarkers to detecting spatial and temporal distributions of specific molecules in living cells. A major challenge facing biosensor development is how to functionally couple a biological recognition domain to an output module so that the binding event can be transduced to a visible and quantifiable signal [e.g., Förster resonance energy transfer (FRET)]. Most designs achieve coupling by means of a binding protein that changes conformation upon interacting with its target. This approach is limited by the fact that few proteins possess such natural allosteric mechanisms, and for those that do, the conformational change is frequently not extensive enough to produce a large change in distance between FRET donor and acceptor groups. Here, we introduce protein fragment exchange (FREX) to address both problems. FREX employs two components: a folded binding protein and a fragment duplicated from it, the latter of which can be chosen from many possible fragments. The system is rationally tuned so that addition of ligand induces a conformational change in which the fragment exchanges positions with the corresponding segment of the binding protein. Placing fluorescent donor and acceptor groups on the binding protein and fragment reduces the background level of FRET of the unbound sensor, resulting in a ratiometric FRET response that is expected to be strong and reproducible from protein to protein. FREX is demonstrated using fibronectin III, a monobody binding scaffold that has been tailored to recognize multiple targets. Sensors labeled with Alexa FRET pairs exhibit ratiometric FRET changes of up to 8.6-fold and perform equally well in buffer and serum. A genetically encoded variant of this sensor is shown to be functional in cell lysates and in mammalian cell cultures.

Lung ventilation volumetry with same-breath acquisition of hyperpolarized gas and proton MRI.

The purpose of this work was to assess the reproducibility of percentage of ventilated lung volume (PV) measured from hyperpolarized (HP) (3)He and (1)H anatomical images acquired in the same breath-hold when compared with PV measured from (3)He and (1)H images from separate breath-holds. Volumetric (3)He ventilation and (1)H anatomical images of the same resolution were acquired during the same breath-hold. To assess reproducibility, this procedure was performed twice with a short gap between acquisitions. In addition, (1)H images were also acquired in a separate breath for comparison. PV ((3)He ventilated volume divided by (1)H total lung volume) was calculated using the single-breath-hold images (PV(single)) and the separate-breath-hold images (PV(separate)). Short-term reproducibility of PV measurement was assessed for both single- and separate-breath acquisitions. Dice similarity coefficients (DSCs) were calculated to quantify spatial overlap between (3)He and (1)H segmentations for the single- and separate-breath-hold acquisitions. The efficacy of using the separate-breath method combined with image registration was also assessed. The mean magnitude difference between the two sets of PV values (±standard deviation) was 1.49 ± 1.32% for PV(single) and 4.19 ± 4.10% for PV(separate), with a significant difference (p < 0.01). The mean magnitude difference between the two PV values for the registered separate-breath technique (PV(sep-registered)) was 2.27 ± 2.23%. Bland-Altman analysis showed that PV measured with single-breath acquisitions was more repeatable than PV measured with separate-breath acquisitions, regardless of image registration. DSC values were significantly greater (p < 0.01) for single-breath acquisition than for separate-breath acquisition. Acquisition of HP gas ventilation and (1)H anatomical images in a single breath-hold provides a more reproducible means of percentage lung ventilation volume measurement than the previously used separate-breath-hold scan approach, and reduces errors.

Stepwise conversion of a binding protein to a fluorescent switch: application to Thermoanaerobacter tengcongensis ribose binding protein.

Alternate frame folding (AFF) is a protein engineering methodology the purpose of which is to convert an ordinary binding protein into a molecular switch. The AFF modification entails duplicating an amino- or carboxy-terminal segment of the protein and appending it to the opposite end of the molecule. This duplication allows the protein to interconvert, in a ligand-dependent fashion, between two mutually exclusive native folds: the wild-type structure and a circularly permuted form. The fold shift can be detected by placement of extrinsic fluorophores at sites sensitive to the engineered conformational change. Here, we apply the AFF mechanism to create several ribose-sensing proteins derived from Thermoanaerobacter tengcongensis ribose binding protein. Our purpose is to systematically explore the parameters of the AFF design. These considerations include the site of circular permutation, the length and location of the duplicated segment, thermodynamic and kinetic optimization of the switching mechanism, and placement of extrinsic fluorophores. Three of the four AFF variants created here undergo the expected conformational shift and exhibit a ribose-dependent fluorescence change. The fourth construct fails to switch folds upon addition of ribose, likely because the circularly permuted form folds much more slowly than the nonpermuted form. This disparity apparently introduces a kinetic barrier that partitions the refolding molecules to the nonpermuted structure. The results of this study serve as a guideline for applying the AFF modification to other proteins of biomedical, diagnostic, and industrial interest.

Niche differentiation and colonization of a novel environment by an asexual parasitic wasp.

How do asexual taxa become adapted to a diversity of environments, and how do they persist despite changing environmental conditions? These questions are linked by their mutual focus on the relationship between genetic variation, which is often limited in asexuals, and the ability to respond to environmental variation. Asexual taxa originating from a single ancestor present a unique opportunity to assess rates of phenotypic and genetic change when access to new genetic variation is limited to mutation. Diachasma muliebre is an asexual Hymenopteran wasp that is geographically and genetically isolated from all sexual relatives. D. muliebre attack larvae of the western cherry fruit fly (Rhagoletis indifferens), which in turn feed inside bitter cherry fruit (Prunus emarginata) in August and September. R. indifferens has recently colonized a new host plant with an earlier fruiting phenology (June/July), domesticated sweet cherries (P. avium), and D. muliebre has followed its host into this temporally earlier niche. We tested three hypotheses: 1) that all D. muliebre lineages originate from a single asexual ancestor; 2) that different D. muliebre lineages (as defined by unique mtDNA haplotypes) have differentiated on their ancestral host in an important life-history trait, eclosion timing; and 3) that early-eclosing lineages have preferentially colonized the new sweet cherry niche. We find that mitochondrial COI and microsatellite data provide strong support for a single ancestral origin for all lineages. Furthermore, COI sequencing revealed five mitochondrial haplotypes among D. muliebre, and individual wasps possessing one distinctive mitochondrial haplotype (haplotype II) eclosed as reproductive adults significantly earlier than wasps with all other haplotypes. In addition, this early-eclosing lineage of D. muliebre is one of two lineages that have colonized the P. avium habitat, consistent with the preferential colonization hypothesis. These data suggest that D. muliebre has evolved adaptive phenotypic variation despite limited genetic variation, and that this variation has subsequently allowed an expansion of some wasps into a novel habitat. The D. muliebre system may allow for in-depth study of adaptation and long-term persistence of asexual taxa.