Cortical area parcellation is a challenging problem that is often approached by combining structural imaging  with functional imaging . Diffusion MRI (dMRI) has been widely adopted to analyse white matter microstructure, but scarcely used to distinguish grey matter regions because of the reduced anisotropy there. Nevertheless, differences in the texture of the cortical 'fabric' have long been mapped by histologists to distinguish cortical areas. Reliable area-specific contrast in the dMRI signal has previously been demonstrated in selected occipital and sensorimotor areas. We expand upon these findings by testing several diffusion-based feature sets in a series of classification tasks. Using Human Connectome Project (HCP) 3T datasets and a supervised learning approach, we demonstrate that diffusion MRI is sensitive to architectonic differences between a large number of different cortical areas defined in the HCP parcellation. By employing a surface-based cortical imaging pipeline, which defines diffusion features relative to local cortical surface orientation, we show that we can differentiate areas from their neighbours with higher accuracy than when using only fractional anisotropy or mean diffusivity. The results suggest that grey matter diffusion may provide a new, independent source of information for dividing up the cortex. •Diffusion MRI provides architectonic contrast between a variety of cortical areas.•dMRI provides complementary information in areas of low myelin content.•High order rotational invariants provide more reliable parcellation results. Early studies of the microstructure of the human cerebral cortex revealed a laminar structure comprising of six layers of varying thickness, and cellular and axonal fibre composition . The hetIn vivo image-based methods for analysis of the grey matter have the potential to alleviate or eliminate some of these limitations. These methods are able to tackle inter-subject variability through the comparable ease of in vivo data collection. They can also be combined with additional multi-modal data from the same subject, to directly assess structure-function relationships, and lend themselves gracefully to observer-free algorithmic analyses. Some of these improvements have been applied to ex vivo data using observer independent intensity analysis  has become ubiquitous in the study of white matter (WM) microstructure . By measentation , fibre fentation , the volentation , and axoentation . Althougex vivo data .Initial grey matter (GM) applications of dMRI focused on the developing brain due to its increased anisotropy . Others ivo data . For exain vivo resolutions has recently become plausible through advancements such as simultaneous multi-slice acquisition, improved gradient systems, better motion/eddy current correction algorithms, and ultra-high field MRI . Diffusion datasets had 270 gradient directions across three b-shells, b=1000, 2000 and 3000 s/mm2, with twelve b=0 s/mm2 images interspersed. A spatial resolution of 1.25 mm3 was achieved using multiband accelerated imaging. Pre-processing steps conducted prior to data release included eddy current and motion correction (Data sets for 40 subjects were obtained from Human Connectome Project (HCP) Q3 500-Subjects release, specifically, subjects were selected from the unrelated participants subset. Data were from healthy participants and publicly available under ethics approval. In-depth descriptions of the acquisition parameters and pre-processing pipelines are provided in the HCP documentation . In summrrection .0.7 mm3) T1w and T2w structural scans which help reduce surface placement errors   provides2.iso), representing free-water content.The neurite orientation, dispersion and density imaging (NODDI)  is a pop3.In the third approach, we more finely characterised the shape of the local diffusion surface by first decomposing the HARDI data into a 6th order spherical harmonic (SH) series. A total of 9 features were generated for each b-shell. The first 4 features are fully rotationally invariant, whereas the last 5 are invariant in the local tangent plane to the cortical surface. Features 1–4 were the k = 1,2,3,4th moments of the apparent diffusion coefficients (ADCs). The 5th feature was the mean of the ADC in the direction of the surface normal, and the remaining 4 features were the k = 1,2,3,4th moments of the ADC in the plane that is parallel to the cortical surface. A related approach has already been tested on cortical tissue .4.A final approach used a 4th order tensor representation of the ADC and all its informatically  complete and irreducible invariants. These invariants fully describe the geometric characteristics of the ADC up to any orientation or pose in 3D. Higher order tensors were introduced as an alternative (and bijective) mathematical basis to spherical harmonics in Four different diffusion MRI analysis techniques were used to generate six feature sets see  that werThe latter two approaches better characterise the precise shape of the HARDI signal due to their sensitivity to higher-order details. Because GM regions have much lower anisotropy than WM, which often consists of coherently organised WM fascicles, we hypothesised that these higher order features might be better suited to capturing the complex and subtle variations that are known to distinguish different cortical areas.The S1 and M1 areas are very distinct from each other and consistently located across subjects. Therefore, classification between these two regions was chosen as a robust initial test-bed. Training labels were defined for each subject using the HCP multi-modal parcellation (HCP-MMP) atlas . Specififorest size=20, tree depth=7, other parameters at default values) to distinguish the two areas using a pool of 20 subjects. Data under the two regions were extracted for each feature set in each subject. The classifier was trained on a set of subjects for several training group sizes (TS), ranging from 1 to 19 subjects, and then tested on an unseen subject. The same training group was maintained until all feature sets were tested. A leave-one-out approach was implemented to ensure that all of the available subjects were tested in turn for each TS.Random forest classification (RFC)  was implWe hypothesised that the low-order features would dominate the classification because they have previously been shown to be very distinct between S1 and M1 .A hemisphere-wide parcellation pipeline was developed to test the DT6, DT9 and SH27 feature sets across a broader range of cortical areas. These three feature sets were prioritised because they encompass the differences we intended to test, i.e., DT9 includes surface specific features where DT6 does not, and SH27 is a higher-order method compared to DT9 and DT6. We also employed population averaging for these classification tests to increase the contrast-to-noise ratio between cortical areas.Of the 40 HCP subjects, 30 subjects were selected as the training pool and the remaining 10 were assigned as the test pool. 6 of the 30 training subjects were randomly selected at a time, to generate a total of 20 group average training sets. Surface-based averaging  was perfThe 180 areas of the HCP-MMP were utilized as a set of prospective hemisphere-wide, training labels. Given a test average, the cortical area marked by each of the training labels was tested in turn, using a multi-label RFC, against its neighbouring cortical areas. For example, when trying to predict the class of the data marked by the V1 label, the classifier was trained on data taken from 3 labels in the training averages: ProS (prostriata), V1 and V2 B. This nThe methods presented above were combined to produce a whole hemisphere parcellation on an individual subject to assess whether between-area contrasts can be detected without averaging.The neighbourhood multi-label RFC approach from the group average pipeline was used to classify the data marked by each of the 180 HCP-MMP areas for a single unseen subject. The training data was generated by concatenating the data for each of the neighbourhood labels from a group of training subjects, as in the binary M1/S1 tests. The training group was reduced to 10 subjects to reduce runtime and memory requirements of the classifier. During registration of the training labels, a small number of vertices (1–5%) were assigned to multiple classes. In each case, the vertex was assigned to its mode class label; in the absence of a mode class, the vertex assignment was selected randomly from its predicted classes.A quantitative vertex-wise comparison method was developed to evaluate the quality of different full hemisphere parcellation results. Here, quality was defined as the local spatial coherence of the parcellation. Given two corresponding parcellation results, A and B, the number of unique cluster IDs within a 90-nearest neighbour surface searchlight surrounding each vertex were counted, and the resulting cluster counts were subtracted from each other (A - B). A positive value , whereas the performance of SH27, 4T36 and DT6 were not significantly different from each other. ND3 and DT3 were also found to be significantly different from each other and all of the other feature sets (p < .001). Aside from the reasons given in the methods, we chose not to include DT3 and ND3 in subsequent experiments because they performed comparatively poorly here. 4T36 was also omitted because it performed so similarly to SH27.The DT9 feature set performs best in the classification between M1 and S1 for all values of TS larger than 2, achieving a mean classification accuracy of In The lateral and medial views of the parcellation result for the DT6, DT9, and SH27 feature sets are displayed in The overall trend comparing DT6, DT9, and SH27 (left to right) is an observable decrease in the granularity of the parcellation in some areas . This suggests that inclusion of a surface normal component (DT9 & SH27 compared to DT6) and the use of higher order features (SH27 compared to DT6 & DT9) both provide useful additional information to the training and classification process.Qualitative assessment of the lateral surface indicates that area 3b (part of S1) has the most distinct HARDI signal profile compared to its neighbouring areas, as all of the feature sets achieve a reasonable correspondence to the training label for this area. Some regions show markedly different classification outcomes between the different feature sets (dotted arrows). For example, the MT region is subdivided by SH27, but recovered more completely by DT6 and DT9. The SH27 subdivision may reflect inter-subject variability in the location of MT proper .Inspection of the medial surface surprisingly reveals that none of the feature vectors strongly differentiated the primary (V1) and secondary (V2) visual areas (black arrows). Areas close to the medial surface interface with the corpus callosum, e.g., retrosplenial complex and hippocampus, can be accurately classified by all feature sets and are known to be architectonically distinct from most other medial and lateral cortical areas.The searchlight comparison of the results is shown in The bar plots in The quality of each parcellation was compared more rigorously using the searchlight cluster counting method B. Again,This work provides evidence that diffusion MRI is a sensitive and anatomically meaningful contrast for identifying differences between cortical areas at 3T resolutions. We demonstrated that the M1 and S1 cortices can be reliably distinguished from each other using a simple white matter model, the diffusion tensor, as previously suggested . The traAreas of high reproducibility are not limited to myelin rich areas; for example, 78% of the vertices in the inferior frontal sulcus area, IFJa, were correctly assigned using the SH27 feature set despite this area having six neighbours. Such examples indicate that dMRI provides useful contrast in regions where myelin density is not as informative. However, as the fidelity of the training labels is questionable in these areas, further investigation will be required to illuminate what is driving classification outcomes in these regions. Nevertheless, the results suggests that dMRI could be a useful modality to incorporate in future studies that aim to non-invasively fingerprint the differing microstructure in cortical units. This hypothesis was further supported by the analysis of the single subject, whole hemisphere parcellation in which area-like clusters were demonstrated in similar regions to the group average results.3 is required at 3T to consistently image Stria of Gennari  contain internal architectonic boundaries that are just as striking as any between-area boundary see  on MT. Tin vivo, cyto- and myeloarchitectonic reference map of the entire cortical surface. However, the method adequately demonstrated that diffusion MRI represents a complementary modality for future studies of cortical microstructure.It should also be considered that we used only one of the many competing atlases. We remain some distance from being able to generate a definitive The classification efficacy of several diffusion-based feature sets were assessed. In particular, we wanted to determine whether either (a) the explicit inclusion of radial and tangential diffusion properties  or (b) the use of higher-order feature sets, improves between-area contrast compared to commonly used scalar metrics, such as FA.Initial tests on S1/M1 classification found relatively poor performance in lower order feature sets that only used a single b-shell or combined b-shells before classification . The consistently poor performance of DT3 compared to DT6 and DT9 supports the notion that different b-values can probe different aspects of cortical microstructure . The relThe DT-based feature sets perform well despite diffusion at higher b-values (b > 1000) not respecting the Gaussian assumptions of this model . One posThe full hemisphere, group average, and single subject results provide evidence in favour of using the more generalized, higher-order spherical harmonic feature sets. In contrast to the trend observed in the binary experiment, we found that in this multi-class problem SH27, not DT9, obtains the most accurate definition of 3b. It is possible that the DT-based features are not sensitive enough to describe differences between the larger set of classes. S1 has 3 neighbours in this 4-way classification meaning the three times as many features that SH27 provides are beneficial.The more generalized features of SH27 provide a more coherent clustering result across the cortex, particularly for the single subject result. This again suggests that these features describe cortical tissue domains more effectively. Perhaps the higher order feature sets are less influenced by confounding effects, such as changes in global diffusivity between subjects, than the DT feature set as a result of their increased dimensionality. Support for the higher order feature sets is again evidenced in the distribution of areas in which SH27 out performs DT6 and DT9. It achieves better results for areas of high myelination, primary areas, or areas for which the training labels can be considered more reliable. On the other hand, it is possible that the SH27 feature set is more susceptible to overfitting in regions where the diffusion signatures of neighbouring areas are less distinct from each other. The combination of DT9 and SH27 can also be used as a feature set to maximise performance across the cortex see . A few rThe inclusion of features that take the orientation of the local cortical surface into account , consistently offered an advantage over the scalar DT6. This is clear when comparing DT6 to DT9 in both the binary classification and group average experiments. However, we cannot conclusively say that such features are always necessary. Comparing the results of SH27 to 4T36 indicates that explicit reference to the local tissue orientation might not be required if the feature set provides a functionally complete description of the ADC. Further testing of 4T36 across a broader set of areas is required to confirm this.Ultimately, deciding which feature set to use requires a nuanced approach that considers the specific aims of future studies. If attempting to delineate architectonic domains, the above results indicate that high order decomposition approaches might be more appropriate when describing the texture of the 'fabric' of the neuropil at an intermediate scale. On the other hand, an advantage of biophysical models is that they provide features that are more readily interpreted. For example, they are more useful if one wishes to understand the specific microstructural changes at the level of single fibres that can affect abnormal brain function.The above works omit a set of recently emerging techniques, which aim to separate the contribution of microstructural tissue composition from the mesoscopic orientation distribution within the dMRI signal . These mOne of the limiting factors of the above work was the relatively coarse resolution of the diffusion data compared to the thickness of cortical laminae. This only allowed data to be sampled at a single cortical depth, which may have led to a failure to sufficiently capture variations in laminar structure, particularly for thicker areas of the cortex i.e., gyral crowns or area 4 (M1). It was also insufficient for reliably characterising the properties of V1. The relatively large voxels may have introduced noise by differential mixing of signals from different laminae in different locations. A finer sampling of different depths in each cortical column has the potential to provide a closer approach to the classical histological analysis of the cortex. The low resolution is also likely to incur partial volume effects, which we aimed to minimise by sampling at the middle cortical depth. However, further analysis in needed to determine the extent to which partial voluming from WM and CSF impact the results. The higher spatial resolutions that can be obtained at 7T have great potential for resolving these issues and validating the above findings.There are several avenues that could be explored in more depth in future studies. For example, the labels were back-projected from a reference brain, but it would be interesting to investigate whether generating labels on individual HCP subjects can improve accuracy. Furthermore, we found that the feature ranking information provided by the RFC was highly variable between different regions – additional analysis could shed light on which features are more discriminative for which areas.The results so far have concentrated on one hemisphere, but there are many interesting questions that will be able to be addressed in future studies where homologous regions are compared across hemispheres, given the well-documented differences in function between them.Our results provide support for including surface-based HARDI data analysis as an additional, independent measure of cortical microstructure to aid e.g. quantitative T1, which has been widely used as a proxy for myelin density. We demonstrated that higher-order decomposition methods provide a more consistent characterisation of grey matter microenvironments in regions for which the classification method can be considered most reliable. However, even simple lower order models such as the diffusion tensor provide contrast between cortical areas. In particular, combining the traditional diffusion tensor metrics of FA and MD with the surface specific radiality index is very powerful in binary classification between M1 and S1. Further work at higher resolutions and improved SNR will likely enhance the performance of these methods. With expected advances in data acquisition methods, it is likely that surface-based analysis of grey matter diffusion will become a new standard tool for probing microstructural variations among the complex mosaic of distinct cortical areas that make up the human neocortex.


The objective was to examine whether attendance in the mass cervical screening programme has implications for the prognosis when cervical cancer is diagnosed. We performed a retrospective analysis of all cases of cervical cancer between 1st of January 2012 and 31st of December 2014 in the Region of Southern Denmark. The cases were retrieved from the Danish National Pathology Registry, PatoBank. Odds ratios (OR) with confidence intervals (95% CI) were calculated for attendees versus non-attendees of the screening programme by using χ2-test. 216 patients were included in the study. 61.6% of the study population had not attended the screening programme. Patients who had attended the programme were characterised by disease in low stage , treatment with surgery alone  and a lower risk of death . Adenocarcinomas were more often detected among attendees of the programme compared to squamous cell carcinomas . Statistically significant results regarding relapse of cancer  and lymph node metastases  were not found. Cervical cancer detected in women who had attended the mass cervical screening programme prior to the diagnosis, was shown to have a statistically significant lower FIGO stage (p = 0.0004) and was therefore linked to less extensive treatment options. Continued focus on increasing the participation rate of the programme is of importance, as the nonattendance rate continues to be high. However, the incidence of cervical cancer still remains slightly higher in Denmark  compared to the other Scandinavian countries3 and the vision of an attendance rate at minimum 75 % countrywide is not yet reached4.The incidence of cervical cancer has decreased markedly since the onset of mass cervical screening in the 1960s in Denmark. The programme is well-known to detect easily treatable precursors to cancer and thereby reducing the incidence of cervical cancer2. Participation in the screening programme, follow-up and treatment is free of charge.In Denmark, women aged 23–49 years are invited for cervical screening every third year by a personally addressed invitation letter and thereafter every fifth year until the age of 65. For women aged 23–59 years, the screening test is a Papanicolaou cytological sample from the uterine cervix, usually taken by the general practitioner. For women aged 60–64 years a high-risk human papillomavirus (hr-HPV) DNA-test is performed on the collected sample. If the hr-HPV DNA-test is negative, the woman is withdrawn from the screening programme, but if the test is positive for hr-HPV 16/18 the woman is referred for colposcopy, and if the test is positive for other hr-HPV types the cytological specimen will be evaluated by microscopy5. To improve attendance in the cervical screening programme the Danish National Board of Health recommends that women receive two recalls with an interval of 3 months if they do not respond to the first invitation. The general practitioner receives a reminder when follow-up of an abnormal or inadequate cervical sample has failed2. Liquid-based cytology has also been introduced in order to reduce the number of inadequate cytological samples and allow for both cytological and HPV test on the same sample7. Although several implementations have been made to enhance the efficiency of mass cervical screening, approximately 400 Danish women are still diagnosed with cervical cancer every year3. Three challenges remain predominant: non-attendance to the screening programme, false negative screening samples and lack of follow-up of abnormal or inadequate screening samples. To elucidate how much each of these three reasons contribute to reduce the effectiveness of the programme, the Danish National Board of Health recommended in 2007 and 2012 that new cases of cervical cancer should be reassessed by audit, including re-evaluation of “normal” cervical samples  up to 3.5 or 5.5 years prior to the cancer diagnosis for the younger and older target populations respectively8.The screening programme has continuously been reassessed in several ways to optimise the outcome by reducing the incidence of cervical cancer, but especially in the last 1–2 decades there has been a stagnation of the cervical cancer incidence in DenmarkThe aim of this study was to investigate whether attendance to the mass cervical screening programme is associated with a better prognosis for women diagnosed with cervical cancer focusing on the following prognostic factors: stage of disease, histological diagnosis, lymph node metastases, relapse of cancer and death. It was hypothesised that women attending the screening programme have a better prognosis than non-attendees.st of January 2012 to 31st of December 2014 in the Region of Southern Denmark, which has a population of 1.2 million9. In that period, 69% of invited women attended the programme10.This register-based study was conducted at Department of Pathology and Department of Gynecology and Obstetrics, at Odense University Hospital, Odense, Denmark. Data were collected retrospectively. The study describes audit results for patients diagnosed with cervical cancer in a three-year study period from 1Medical records concerning information about cancer diagnosis, stage of disease, previous screening history and treatment were examined.All patients with a histologically verified primary diagnosis of cervical cancer in the study period, living in the Region of Southern Denmark and treated at Odense University Hospital were retrieved from the Danish Pathology Register, PatoBank. The patients were traced in the PatoBank by searching for relevant Systematised Nomenclature of Medicine (SNOMED) codes; T83 for location in the uterine cervix and relevant M-codes for histological cancer diagnoses. The PatoBank is a nationwide database that contains information about all specimens, including cervical samples, examined at departments of pathology in Denmark. The information in the PatoBank was linked to the patients’ electronically medical records by the CPR-number, which is a ten-digit personal identification number assigned to all Danish citizens. The treatment of cervical cancer is centralized to Odense University Hospital in the Region of Southern Denmark.Information about age and stage of disease  at the time of diagnosis as well as information about lymph node metastases , treatment  and relapse of cervical cancer or death was retrieved from patients’ records. Cytological and histological diagnoses prior to the cervical cancer diagnosis as well as histological diagnosis of the cancer and results from the audit were retrieved from the PatoBank. Follow up was completed in May 2016.Patients or the public were not involved in any way in our work as it was a register based retrospective study.1. In this study we allocated the patients to the same seven groups as listed below. The groups of audit are further elaborated in local guidelines provided for the Department of Pathology at Odense University Hospital, which is also accounted for in the following:The Danish National Board of Health provides guidelines for the categorization of the patients into non-attendees (group 1) and attendees (groups 2–7)Deficient screening history (not attended the screening programme) – no cervical samples  3.5 years (23–49 years of age) or 5.5 years (50–64 years of age) prior to the diagnosis of cancer.2.Sufficient screening history  – at least one “normal” cervical sample test 3.5 or 5.5 years prior to the cancer diagnosis re-evaluated as true negative.3.False negative cytology – a cytological sample defined as “normal” 3.5 or 5.5 years prior to the cancer diagnosis, but diagnosed as High-grade Squamous Intraepithelial Lesion or worse at re-evaluation.4.False negative histology - a histological sample defined as “normal” 3.5 or 5.5 years prior to the cancer diagnosis, but diagnosed as Cervical Intraepithelial Neoplasia grade 2 or worse at re-evaluation.5.Lack of follow-up of abnormal or inadequate cytological samples.6.Lack of follow-up of abnormal or inadequate histological samples.7.Other – includes cases where audit was not feasible due to untraceable samples.In the Region of Southern Denmark, all the departments of pathology use liquid-based cytology technique (Thinprep® or Surepath®), and three or four departments were using semi-automatic computer assisted screening in the study period.2-test was performed on the groups 2–7 versus group 1 using SPSS version 23. Only results with p-values <0.05 were considered statistically significant.A χA total of 216 patients diagnosed with cervical cancer were found to fulfil the inclusion criteria for this study. The mean age of the patients was 48.5 ± 16.8 (range 23–95). The study results are shown in Table We found that 133 (61.6%) of 216 patients had a deficient screening history, of whom 36 (27.1%) were outside (older than) the recommended screening age and 97 (72.9%) within the recommended screening age. Thus, 61.6% of the study population were non-attendees of the screening programme, Fig. Re-evaluation of cytological samples revealed false negative cervical samples in 13 (6.0%) patients. In six (2.8%) cases, follow-up of cervical cytological samples had failed and in four cases (1.9%) audit was not feasible due to untraceable cytological glass slides.The distribution of histological subtypes in the seven audit groups is shown in Fig. 11 compared to 73 (54.9%) of the 133 patients who had not attended the programme, meaning that 19.3% of the attendees and 45.1% of the non-attendees were diagnosed with FIGO stage II and higher. For one patient the stage of disease was unknown as she abstained from further investigation and was consequently excluded from the statistical analysis regarding FIGO stage. Attendance to the screening programme was associated with higher odds for being diagnosed with less advanced stage of disease (FIGO stage I) compared to non-attendance .Among the 83 patients who had attended the screening programme, a total of 67 (80.7%) were diagnosed with FIGO stage IA total of 106 (49.1%) patients received surgical treatment alone, while 109 (50.5%) patients received other treatment, of whom 14 (6.5%) received palliative oncological therapy alone because of FIGO stage IV and/or comorbidity; all 14 patients were non-attendees, Table Of the 133 non-attendees of the screening programme, 39 (29.3%) patients had lymph node metastases at the time of diagnosis, while 17 (20.5%) of the 83 attendees of the programme had lymph node metastases. Additionally, 13 (9.8%) patients among the non-attendees and six (7.2%) among the attendees had relapse of cancer. Statistically significant results regarding lymph node metastases  and relapse of cancer  were not achieved in this study.In our study, a total of 34 (15.7%) patients were registered dead (unknown cause of death); 27 (20.3%) among non-attendees and 7 (8.4%) among attendees. Attendees of the screening programme had a lower risk of death  compared to non-attendees.5 affects the prognosis of cervical cancer. The odds for being diagnosed with a less advanced disease stage (FIGO stage I) were 3.14 times higher given that attendance to the screening programme had been adequate. This is consistent with previous studies, which emphasize a higher risk of advanced stage of disease when not attending a screening programme13. Similarly, attendees of the screening programme had 2.63 times higher odds for being sufficiently treated by surgery alone and not requiring more extensive treatment options. This was an expected observation, as FIGO stage I (80.7%) dominated among attendees of the screening programme. Furthermore, the risk of death was found to be lower among attendees of the screening programme. However, cause of death was unknown and follow-up of the patients varied. Among the 133 non-attendees, 36 women were outside the recommended screening age (older), and consequently their risk of death of any cause is higher. In the study of Kristensson et al.14, non-attendees were found to be socio-economically disadvantaged compared to attendees and several other studies outlines socioeconomic factors as important variables for screening behaviour and also stage of cervical cancer16. Thus, higher age and poor socioeconomic factors among the non-attendees could also have influenced the results regarding death, but this was not further investigated.We investigated whether attending a well-organized screening programmeWe found a trend towards a lower risk of lymph node metastases and relapse for women who attended the screening program, but data were not statistically significant (p = 0.19 and p = 0.47 respectively). Furthermore, it was difficult to extract data regarding lymph node metastases, as this information relied on histology for some patients, while PET-CT was the determinant for other patients. For the latter, information about lymph node metastases was not always consistently reported. It is also important to underline that follow-up of the study population regarding relapse of cancer has varied as a result of a divergent time of cancer diagnosis. Although attending the screening programme seems to improve the prognosis of cervical cancer, a larger sample is required to investigate the same positive effect of attendance on lymph node metastases and relapse of cancer.18, which is consistent with the rate found in this study. A recent Danish study from the Capital Region of Denmark reports a somewhat lower non-attendance rate of 45.5%19. This difference may be explained by different demographics of the two study populations, as Kirschner et al19. describes an urban population of women, while our study describes a population from both urban and rural areas. This may reflect diversity in the screening behaviour between the two study populations. However, when reporting a non-attendance rate, women outside the recommended screening age should not be taken into account. The non-attendance rate reflects the efficiency of the screening programme and should thus be preserved women within the screening population but with absence of a cervical cytology available for re-evaluation. It seems of continued relevance to place emphasis on increasing the participation in the cervical screening programme as attendees of the screening programme in this study trended to have less advanced stage of disease at time of diagnosis. HPV self-testing for women who do not respond to the two recalls could be one option to detect this major known risk factor for cervical cancer development22. In the recent guidelines from The Danish National Board of Health from 2018, it is recommended to offer the possibility of self-sampling in the up-coming years, to enhance the participation in the screening programme23. This option is partially implemented in some regions in Denmark.61.6% of the women with cervical cancer had not attended the screening programme, i.e. the women either did not respond to the invitation letter or to the two recalls and for some cases or the women did not fulfil the criteria of being an attendee . Non-attendance rates between 54–64% have previously been reportedet al.19, which reflects an overall proper reading of cytological glass slides. However false negative cytology results from interpretation or location bias and continued improvement of both collection and interpretation techniques is required.Of the patients included, 6.0% had false negative cytology, which is comparable to the 9.8% reported by Kirschner 19. Interval cancers reflect incompleteness in the screening programme. Thus, the programme cannot be an isolated preventive approach against cervical cancer of which the HPV vaccine has been an additional approach since 200624. In 2009, the HPV vaccine became a part of the Danish childhood vaccination programme recommending HPV vaccination of girls at the age of 1225. Furthermore the 9-valent HPV vaccine (Gardasil 9) is now also offered to boys turning 12 years in summer of 2019, thereby enhancing heard immunity26. It is expected that the dual impact of HPV vaccination for both girls and boys, and the cervical screening programme will further reduce the incidence of cervical cancer.In this study, 27.8% of the patients with cervical cancer had attended the screening programme and were diagnosed with malignant disease despite all previous samples were judged normal; in other words they had developed interval cancers. It has been debated whether interval cancers are due to sampling errors or are an expression of rapidly developing cancers30. This is partly explained by the risk of sampling errors, as endocervical components are more difficult to collect and can be difficult to identify and diagnose by cytological examination32. Adenocarcinomas were more often found among attendees compared to non-attendees in this study, which may reflect deficiency in the screening process, as pre-cursors to adenocarcinomas were not detected prior to the cancer diagnosis. In addition, adenocarcinomas were detected in 22 (36.7%) of the 60 patients with true negative cytology, i.e. group 2, and in five (38.5%) of the 13 patients with false negative cytology, i.e. group 3. These findings emphasize the importance of sampling techniques and the need for continued attention on the interpretation of cytomorphological changes in cells of glandular origin. Implementation of HPV-DNA tests as the primary screening method might lead to improved detection of glandular lesions. The Danish National Board of Health has recently requested the regions to conduct a controlled, differentiated implementation of HPV screening for the age-group 30–59 years of age which would allow for an evaluation of the efficiency of HPV screening versus cytological sampling23.Several studies highlight that it is more difficult to detect adenocarcinoma compared to squamous cell carcinoma through the screening programmeet al. proposes to extend the screening population to include women above 65 years of age, as the incidence of cervical cancer among elderly women remains high33. However, according to Lynge et al. the current high incidence of cervical cancer among elderly women may represent residuals of unscreened or underscreened birth cohorts34. Further decrease in the incidence of cervical cancer among the elderly women is expected, as the present recommendations offer women aged 60–64 years a hr-HPV DNA-test exiting the screening programme with follow-up of the hr-HPV positive women. Thus, it is debatable whether an expansion of the screening population will be more beneficial than the present actions. Future results upon the incidence of cervical cancer among the elderly women, taking the current guidelines into account, are required to determine the efficiency of the hr-HPV DNA-test and decide whether further initiatives are necessary.Of the 216 patients who had developed cervical cancer, 41 (19.0%) were above 65 years of age and thus outside the recommended screening age. Only one of these patients had a history with conization because of cervical dysplasia prior to the diagnosis of cervical cancer. Hammer This study was strengthened by the use of PatoBank, which allowed access to all available cervical samples for all relevant cervical cancer cases in Denmark. Furthermore, the unique CPR-system allowed for accurate linkage of information between the PatoBank and medical records. All cases of cervical cancer detected in 2012–2014 in the Region of Southern Denmark were included and the diagnoses of cervical cancer were further confirmed, as Systematised Nomenclature of Medicine codes were compared to information in medical records. A limitation in this study was the limited time of follow-up and the lack of information about the cause of death. Furthermore, baseline characteristic linked to screening behaviour as socioeconomic factors was not feasible and thus not adjusted for in the analysis.2. Conclusively, the patients are either classified as attendees or non-attendees of the screening programme. Hence, the audit process can be regarded as a quality assurance of the screening programme, which is an opinion appearing explicitly in the audit guidelines in the United Kingdom35. Thus, in the audit process, it only seems sensible to re-evaluate true screening tests, i.e. cervical samples taken upon an invitation to the screening programme. However, all cervical samples coded as “normal” in the Danish PatoBank up to 5.5 years prior to the cancer diagnosis are being re-evaluated, without distinguishing between true screening tests and tests taken outside the screening programme, e.g. tests taken on medical indication or opportunistic tests. The reason for that is not further explained, but it can be assumed that it is being done for legal reasons (compensation claims). All women with “normal” cervical samples up to 5.5 years from the cancer diagnosis are, in the audit process, considered as attendees of the screening programme and the word “attendee” is consequently misused. Ideally, it should be preserved to women actually attending the screening programme upon an invitation, but the current coding practice does not make this possible. It is difficult to conclude from the information in PatoBank whether the cervical cancer actually was detected upon screening  or on symptoms, as symptoms may have occurred in a screening interval period. A woman could possibly attend her general practitioner with contact-bleeding (symptom) and be an “attendee of the mass cervical screening program” if a specimen was found in her PatoBank history up to 5.5 years prior to cancer diagnosis. If the purpose of an audit is to assure the quality and efficiency of the screening programme, it could be considered to re-organize the present audit process, so only true screening tests influence the audit conclusions. Re-organization of the audit process could involve attachment of more precise information to the samples in the PatoBank, which would make it easier to distinguish between true screening tests and tests taken outside the screening programme. Inspiration can be drawn from the United Kingdom, where the audit process is able to differentiate between women who had attended the screening programme and women who had not when developing cervical cancer, especially with focus on the reasons for non-attendance in spite of an invitation36.During this study, we gained insight into the performance of the audit process in the Region of Southern Denmark, and as we experienced several challenges connected to this procedure, it is of relevance to comment on these. The audit process intends to ensure the quality of the detection and treatment of cervical cancerA trend of a lower stage of disease at diagnosis, less extensive treatment of cervical cancer and lower risk of death was found among attendees of the screening programme. Besides lowering the incidence, attendance was shown to improve prognosis for women who are diagnosed with cancer despite regular cervical cytology tests. More focus on the screening programme and its ability to detect adenocarcinomas is also desirable, as the proportion of adenocarcinomas among the attendees of the screening programme with true negative (36.7%) and false negative cytology (38.5%) appears rather high.Our study results thus provide a mapping of screening behaviour and prognostic factors among women diagnosed with cervical cancer and thereby add a useful baseline for additional studies with nationwide data reviewing the efficiency of the screening programme. Further clinical information following cervical samples could add to the audit process’ ability to differentiate between women who had attended the screening programme and women who had not when developing cervical cancer. An optimization of the audit process as well as national data covering the screening behaviour in Denmark are needed to clarify the complete efficiency of the screening programme.The study was approved by the Danish Data Protection Agency  and the Danish Patient Safety Authority . These authorities determined that informed consent was not required because the study used de-identified data from approved databases.


Drosophila Genetic Reference Panel (DGRP) to identify the mutations involved in resistance to two widely used classes of insecticides: organophosphates  and pyrethroids (deltamethrin). Most variation in parathion resistance was associated with mutations in the target gene Ace, while most variation in deltamethrin resistance was associated with mutations in Cyp6a23, a gene encoding a detoxification enzyme never previously associated with resistance. A “nested GWAS” further revealed the contribution of other loci: Dscam1 and trpl were implicated in resistance to parathion, but only in lines lacking Wolbachia. Cyp6a17, the paralogous gene of Cyp6a23, and CG7627, an ATP-binding cassette transporter, were implicated in deltamethrin resistance. We observed signatures of recent selective sweeps at all of these resistance loci and confirmed that the soft sweep at Ace is indeed driven by the identified resistance mutations. Analysis of allele frequencies in additional population samples revealed that most resistance mutations are segregating across the globe, but that frequencies can vary substantially among populations. Altogether, our data reveal that the widely used OP and pyrethroid insecticides imposed a strong selection pressure on natural insect populations. However, it remains unclear why, in Drosophila, resistance evolved due to changes in the target site for OPs, but due to a detoxification enzyme for pyrethroids.Resistance to insecticides has evolved in multiple insect species, leading to increased application rates and even control failures. Understanding the genetic basis of insecticide resistance is fundamental for mitigating its impact on crop production and disease control. We performed a GWAS approach with the  Insecticides are widely used for control of agricultural and structural pests, and to control insect vectors of disease. It is difficult, or perhaps impossible, to exactly calculate the economic and human health benefits associated with insecticide use, but they are significant. For example, depending on the crop and level of insect pressure present in a given year, insecticides can boost yields by 6–79% . In justFor more than twenty years, the availability of molecular tools has facilitated the identification of mutations responsible for changes in protein structure and also in gene expression causing insecticide resistance. Out of necessity these studies were usually carried out on strains that had been selected in the laboratory, in an effort to make the resistance allele(s) homozygous. Identification of the mutations responsible for resistance allowed for the frequency of these mutations to be examined in field populations. In the postgenomic era, Genome-Wide Association Studies (GWAS) offer the potential to examine how the evolution of insecticide resistance occurs at a whole genome level, without having to select a resistant strain in the laboratory. GWAS studies have been recently used to look at the pattern of resistance to a banned insecticide, , an organophosphate ) and a neonicotinoid insecticide (imidacloprid)  and increased detoxification by cytochrome P450s [CYPs] and esterases/hydrolases are the major mechanisms of resistance .OP and pyrethroid insecticides are widely used today. OPs were developed in the late 1940s and were the most widely used class of insecticides for more than three decades. Pyrethroid insecticides were commercialized in the 1980s and rapidly replaced OPs as the most widely used class of insecticides for about 20 years. A great deal has been learned about the basis of resistance to these two classes of insecticides. Mutations in the target site  in the whole genome. To this purpose, we performed GWAS using the Drosophila Genetic Reference Panel (DGRP), a panel of 205 lines of D. melanogaster mostly homozygous and fully sequenced and derived from a wild caught population . The use of inbred fly lines allowed us to assess the impact of pesticides on distinct, but constant genetic backgrounds to tease out the effect of the genotype from environmental effects. The association of a particular allele at a particular locus with the degree of resistance of each line to an insecticide allowed us to identify candidate genes belonging to the quantitative trait loci (QTL) underlying the resistance to those insecticides. Using an approach that first performed a GWAS with all the Drosophila lines of the panel followed by another GWAS including only the lines that did not carry the major effect allele (nested GWAS), we were able to identify and validate a set of genes of major and minor effect on resistance to OPs (parathion) and to pyrethroids (deltamethrin). These classes of insecticides were selected because they have been widely used for decades and are representatives of the 3rd and 2nd most widely used classes of insecticides today . We thus expected these pesticides to have exerted significant selection pressure on D. melanogaster. Using other Drosophila genetic panels, we investigated the presence of our detected mutations in other natural populations and evaluated the signal of selection on our detected mutations.The aim of this study was to investigate the variation in resistance of individuals collected from a field population toward two classes of currently used insecticides in a natural population of Drosophila stocks were raised at 22° on standard Cornmeal agar medium, with a relative humidity of 60–70%, and a photoperiod of 12L:12D, unless specified. For the Genome Wide Association Study (GWAS), most of the isogenic lines of the Drosophila Genetic Reference Panel were used (194 lines were exposed to parathion and 195 to deltamethrin)  or the da-Gal4; TSubi-Gal80 conditional driver . F1 progeny was obtained by crossing virgin females (25 isolated within 8 h of emergence) of the driver strain with males (∼15) of the UAS-transgene line. The F1 progenies  were raised at 18° until three days after emergence, and then switched to 29° for a week to trigger maximum transgene expression before being assayed for resistance to deltamethrin at 29°. The F1 progenies  were raised and assayed for resistance at 25°. As a control, the driver virgin females were crossed to the appropriate background lines Attp2, Attp40 or 1118w (see Table S1).All Drosophila lines and the appropriate background lines were obtained from the Bloomington Drosophila Stock Center  and the Vienna Drosophila Ressource Center (VDRC) (Table S1). Three mutant lines, four transgenic UAS-RNAi lines and one overexpression line from the parathion candidate gene list were available for knockout, knockdown or overexpress of trpl, olf413, fru or Dscam1 genes. One mutant line and nine transgenic UAS-RNAi lines from the deltamethrin candidate gene list were used for knockout of Cyp6a17 or knockdown of Cyp6a9, Cyp6a17, Cyp6a19, Cyp6a20, Cyp6a22, Cyp6a23, CG7627 and tou, respectively.Thirty-three transgenic 2 scintillation vial , which was coated evenly on the inside surface using a hotdog roller machine  for 20 min under a fume hood until all the acetone had evaporated. Treated vials were incubated at 23° for 20 hr before flies were transferred inside. Approximately 20 5-8 days old adult males for each line were assayed per vial for each insecticide. We do not exclude the possibility that the ranking of susceptibility among lines would be affected by the sex used. However, there is no reason to think that major effect genes would be sex dependent and adult males provide the technical advantage that they do not strongly alter the vial conditions in which they are kept, unlike the females that produce larvae. Therefore, using males, allowed us to control better the environmental conditions and have a more robust evaluation of the genetic effect on the phenotypes. Vials were stoppered with a piece of cotton covered with a square of nylon tulle fabric and secured with a staple. The stopper was injected with 2 ml of 20% sugar water after addition of the flies, and assays were held at 25° with a photoperiod 12L:12D. 1 ml of distilled H2O was added to the stoppers after 24 h. For GWAS, mortality was assessed at 2.5 h, 5 h, 11 h, 24 h, and 48 h after flies were added to each vial for parathion and at 48 h for deltamethrin. Ataxic flies were counted as dead and five separate experiments were conducted over five continuous weeks. For validation experiments, mortality was assessed 24 h after insecticide treatment. F1 males (3-7-day-old) from each of the crosses were tested using single dose assays for parathion or deltamethrin. Preliminary experiments were done with both insecticides to determine the optimal concentration to use (i.e. one that would fully resolve the DGRP lines). Five DGRP lines were randomly selected and tested at multiple concentrations. After we felt the optimal concentration had been determined we validated this across 18 randomly selected lines.The residual contact application method was used to examine the relative susceptibility of DGRP lines for the insecticides, parathion and deltamethrin. Parathion  and deltamethrin  were each dissolved in acetone to final concentrations of 1.5 µg/ml and 0.7 µg/ml respectively. 0.5 ml insecticide solution was added to a 38.6 cme.g., some loci have information for all lines, other do not), thus, sample sizes used in each association tested changes from a locus to another. Not all SNPs are therefore suitable for testing the association between the genetic variation at one locus and the resistance to insecticide. We selected SNPs for our association study based on 2 criteria: 1- avoid a complete collinearity (possibly confounding) between alleles and Wolbachia status ; 2- we had enough lines per treatment to run the model. Prior to each test, we therefore calculated a two-by-two matrix with Wolbachia status and allele identity  summarizing the sum of lines for each category. We further included in our association only the SNPs where at least three of the categories had five lines. All the analyses were performed with custom made script.The genetic diversity of the DGRP lines comprises about 4 millions SNPs. However, the genotypic information for each line differs between loci  + frailty . The variable “Experiment” and the identity of the lines were accounted for as random effect following a Gaussian distribution. For the second insecticide, deltamethrin, we tested with a linear regression based on a binomial distribution of the error (function GLMER from the R package “lme4”), the survival at 48h post-exposure of the individuals carrying each allele. We could not use a survival analysis because between 2.5h and 48h some ataxic individuals could recover  before eventually dying. Therefore, the model was as following: cbind  ∼ Wolbachia + SNP +(1|DGRP_lines). The identity of the lines was accounted for as a random effect following a Gaussian distribution. We compared this analysis to the analysis accounting for the variable “Experiment” as a random effect. The results were not strongly different but the approach including a random effect required much more computer time . Therefore, we performed our analyses without this term. To identify other genes responsible for the resistance in absence of major effect alleles, we performed a “Nested-GWAS” which consists in running the same analysis on the lines that are not 100% survival. In other words, we attempted to find the alleles responsible for the remaining variation.We next estimated the significance of the alleles at each selected SNP for the survival of each line to parathion and deltamethrin. For parathion, we used a parametric survival analysis with a log-normal distribution of the error . The model was as following: Surv  ∼ D. melanogaster genome annotation in version 6 with the convert tool from Flybase. The effect and the characterization of the mutation’s effect at each candidate SNP were provided using VEP from the website Ensembl (http://www.ensembl.org/info/docs/tools/vep/index.html). Candidates to be validated were chosen based on the shape of the peak in the Manhattan plot and the function provided by VEP (likelihood to be involved in the resistance). Then, those with a non-synonymous mutation were favored.Candidate SNPs were among the alleles where the p-value was below 0.0001. We then converted the positions provided for the version 5 of the multcomp in R).Validation of selected candidates were tested by exposing the genotypes and their control to the same conditions as in the GWAS. Differences of proportion of surviving individuals 48 hr post exposure were statistically tested with a generalized linear model with a quasibinomial distribution of the error. We used a general linear hypothesis test (glht) with Tukey post Hoc pairwise comparisons , to ascertain differences between pairs of treatments . Only the lines that were included in the GWAS analysis were used. We further filtered out any site with more than 18% missing data. Indels were removed and the data were subset to biallelic sites. Missing data were imputed and remaining heterozygous sites were phased with Beagle 4.1, using windows of 50,000 sites and 15 iterations per window  or absence (“0”) of individual insecticide resistance mutations . Trees were estimated and drawn using the R package ape (For the H12 selection scans and haplotype trees presented in r window . Each aung trees . At the http://www.johnpool.net/genomes.html). The same 18% missing data filter was applied prior to imputation, and the remaining sites were again phased using Beagle 4.1, using windows of 50,000 sites and 15 iterations per window . Most variation in deltamethrin resistance is associated with Cyp6a23, a probable detoxification enzyme. Both major effect genes were found under selection and we identified traces of soft sweep around their loci. Importantly, the alleles of the major effect genes we identified were not a particularity of our sampled population but were found in two other wild-caught D. melanogaster populations present in the Global Diversity panel lines (Down syndrome cell adhesion molecule 1 (Dscam1) and transient receptor potential-like (trpl) in the resistance to parathion, and of Cyp6a17 and CG7627, an ATP-binding cassette transporter in the resistance to deltamethrin.Our results indicate that the resistance to an OP and pyrethroid in the DGRP lines is largely due to a single major locus, that additional loci provide minor effects, and that these loci differ between parathion and deltamethrin. Most variation in parathion resistance is associated with mutations in i.e., when ataxia was not a confounding effect anymore). The proportions of survival 48 h post-exposure were compared between lines for deltamethrin. We found striking and reproducible variation in the DGRP lines’ survival to both insecticides . Survival to parathion was monitored at 2.5 h, 5 h, 11 h, 24 h and 48 h post-exposure and the susceptibility of each line was estimated by comparing the time death took to happen among lines. For deltamethrin we could not use the time death took to happen because flies were ataxic early in the process but could sometimes recover before dying. We analyzed survival at 2.5 h post-exposure to deltamethrin as we had recorded the information, but the results did not differ strongly from those at 48 h post exposure. Thus, because in vials housing several flies we could not separate ataxia from death at that time point, we decide to only consider the proportion of dead individuals 48 h post-exposure . Infection with Wolbachia did not correlate with resistance to parathion  nor to resistance to deltamethrin . Because resistance to different abiotic stresses could have shared mechanisms, we tested the correlations between resistance to parathion or deltamethrin and these stresses; namely the resistance to paraquat, starvation and ethanol that were measured in other studies . We did not detect any correlations with resistance to parathion. However, resistance to deltamethrin in our study correlated positively with both resistance to paraquat  and resistance to starvation . Further studies would be needed to investigate these correlations, particularly because they were performed in different laboratories at different times. We next asked whether the variation we observed was due to genetic or environmental differences. The variation in insecticide resistance in our population was explained more by genetic variance than by environmental variance, with 88% heritability for sensitivity to parathion and 61% for deltamethrin  suggested multiple loci could be involved in resistance  > 8) with resistance to parathion  > 5 and 787 had -log10 > 4). The presumptive genetic alterations and consequences for the genes close to these SNPs/indels can be found in Table S2. Based on both the significance of the association . To identify these secondary loci associated with parathion resistance, we next performed a nested GWAS. For that purpose, we ran a new GWAS using only a subset of lines (n= 124) that did not carry the resistance allele for the most significant SNP . This association was tested over 1,212,116 remaining SNPs/indels. Among those, we identified a list of candidates with the same criterion as above . From this list, we selected four candidate genes based on the annotated function of the protein and the availability in stock centers of genetic tools to perform functional validation: trpl , Survival with lognormal distribution: interaction SNP and Wolbachia: deviance= 455.39, P < 0.0001; trpl), Survival with lognormal distribution: interaction SNP and Wolbachia: deviance= 735.69, P < 0.0001]. This result suggests strongly that Wolbachia could have a direct role in the resistance to insecticides, but this effect depends on host genotype. Alternatively, it is possible that Wolbachia’s presence alters the activity of other unidentified genes involved in resistance. We next analyzed the impact of loss of function (null) alleles or RNAi knockdown of these candidate genes on the susceptibility to parathion. RNAi-mediated knock-down of olf413 or fru expression did not result in any changes in survivorship, suggesting they are not involved in resistance to parathion or that the changes in protein structure rather than in expression level are involved . Altogether, our data demonstrate that the genetic basis for the variation in resistance to parathion is multigenic, with a major effect due to non-synonymous mutations in Ace and secondary roles due to mutations in Dscam1 and trpl that can be buffered by the presence of Wolbachia.Overall, our results strongly suggest that i.e., -log10 > 8) to resistance to deltamethrin at the 48h time point but other, less strongly associated, SNPs/indels could be considered as potential candidates  > 5 and 1066 had -log10 > 4) . We therefore decided to test all six Cyps  using all the available RNAi lines against these Cyp genes and using the one null line (Cyp6a17) available. Knocking down Cyp6a23 and Cyp6a17 increased susceptibility of flies to deltamethrin  and Cyp6a17 (secondary effect), although we do not know whether there are any mutations in Cyp6a17 that could provide resistance. Remarkably, these two neighboring genes are paralogous  .Using the same strategy outlined above, we analyzed the association of 2,171,433 SNPs/indels with deltamethrin survival. In total, 6 loci were strongly significantly associated . The association was tested over 1,872,071 SNPs and we identified 11 SNPs/indels significantly associated  > 8), 142 with a -log10 > 5 and 766 with a -log10 > 4 with resistance against deltamethrin (Table S5). Among the significant SNPs/indels, an isolated indel with a high p-value  = 6.44, Figure S8B) was close and upstream from the gene CG7627, which appears to have ATPase activity and be involved in transmembrane movement of substances. Flies in which we downregulated the expression of CG7627 by RNAi had a lower probability to die from the exposure to deltamethrin when compared to their control (toutatis (tou) which interestingly was associated with resistance to deltamethrin in both the GWAS and nested GWAS  and is supposedly involved in nervous system development  that did not carry the resistance allele for the most significant SNP  . Many of these signals rank among the most pronounced sweep signals detected genome-wide, suggesting that the evolution of pesticide resistance constitutes one of the strongest adaptive response experienced by D. melanogaster in its recent evolutionary history.We found that a small number of individual loci explain most of the variation in resistance across the DGRP lines for both parathion and deltamethrin, suggesting that these loci could have undergone recent positive selection. To test this hypothesis, we performed a genome-wide scan of the DGRP lines using the H12 statistic . One of these clusters is located close to a cluster of haplotypes carrying only the third resistance mutation (001), suggesting a short evolutionary distance between these haplotypes. All haplotypes we observed in the DGRP that carried resistance mutations at two of the three sites (011 & 110) also fell in this group. This is consistent with a scenario in which these two-mutation haplotypes represent transition haplotypes to three-mutation haplotypes, or back-mutations. We observed several low-frequency haplotypes with only one resistance mutation  that did not appear to cluster with any of the other resistance haplotypes, suggesting that these haplotypes arose independently from wildtype alleles, as has been proposed previously . Ace.To provide further evidence that the sweep signal at Cyp6a23/Cyp6a17 loci we also detected sweep signatures in our H12 scan, although these signals were much weaker than at the Ace locus. One possible explanation for this is that the Cyp6a locus has undergone a very soft sweep from standing variation, which is consistent with the fact that the haplotype tree at this locus does not show any noticeable clustering of resistance alleles  constitute only ∼22% of the strains from Tasmania, but ∼74% of the DGRP strains. By contrast, fully resistant strains (11) constitute ∼75% of the strains from Tasmania, yet only ∼17% in the DGRP. These patterns could suggest that more intense pyrethroid selection has occurred in Tasmania compared to the rest of the world. Allele frequency differences are even more pronounced at Ace. Here, haplotypes with none of the three resistance mutations (000) comprise ∼96% of the strains from Zimbabwe, but only ∼37% of strains from Beijing, suggesting that the least intense organophosphate selection has occurred in the Zimbabwe population. Among the resistant haplotypes at Ace, there is also surprising variation in terms of the frequencies of individual resistance allele combinations. For instance, the most common combination of resistance alleles in the DGRP is 111 at ∼32%. Most of the other possible configurations with one or two resistance mutations also occur, yet at much lower frequencies. In the Beijing sample, however, the most frequency resistant configuration is 010 at ∼47%, with the three-mutation configuration (111) present in only ∼3% of strains. This extensive diversity in resistant haplotypes is consistent with a non-mutation-limited scenario in which individual resistance mutations can evolve rapidly and repeatedly at individual loci, such that even complex, multi-step adaptations can arise quickly with intermediate configurations not necessarily reaching high population frequency  comprising fly strains from five different continents  is known for its involvement in self-avoidance mechanisms that are key during neurogenesis. It is not entirely surprising that it plays a role in the resistance against an insecticide that disrupts the nervous system. The later, CG7627, is known to be involved in membrane transport. We do not know much about this gene, but other proteins that are capable of transporting xenobiotics can alter the toxicity of insecticides  and pyrethroids. This is important simply to prevent over-interpretation of our results. For example, the mutations in Ace that resulted in parathion resistance in the DGRP lines are likely the result of cumulative selection with multiple OPs (and carbamates), not necessarily the result of selection only with parathion. Conversely, Cyp6a23 is not involved in resistance to DDT, nitenpyram, dicyclanil nor diazinon , but not for the other (Vssc for deltamethrin). This is not limited to the DGRP lines as evaluation of the Global Diversity Lines also showed that mutation in Vssc was not present. This makes D. melanogaster quite unusual as Vssc mutations are very common in pest species and have been found in at least one strain from virtually every pyrethroid/DDT resistant species examined  (D. melanogaster at this position is CTT (same as house fly). Thus, a single nucleotide change could produce known resistance mutations at this position. Similarly, the T929I mutation can also confer pyrethroid resistance (D. melanogaster could accommodate this change with a single nucleotide mutation (from ACA to ATA). However ethyl methanesulfonate (EMS) mutagenesis led to the recovery of para (the D. melanogaster Vssc) mutants that were up to 22-fold resistant to DDT, and up to 10-fold resistant to deltamethrin , although cyclodiene selection of the same populations was highly successful (para mutations can be made that result in insensitivity to pyrethoids (and DDT), but such mutations do not appear to underlie resistance in field populations of D. melanogaster (based on the DGRP and GDL lines and laboratory selections of field populations). It is difficult to reconcile why selection favored changes in a target site for OPs and yet favored changes in a detoxification gene for pyrethroids.While it is remarkable that the GWAS analysis for both insecticides identified a single locus, it is curious that in one case variation in toxicity was linked to mutations in the target site gene (Cyp6g1 with a secondary effect seen for CHKov1 (Ace with secondary effects seen for Dscam1 and trpl. Although mutations in Ace are a common mechanism of resistance to OPs (and carbamates), it has long been recognized that mutations in Ace that give insensitivity to one insecticide may provide little or no resistance to other OPs (or carbamates)  . HoweverCyp6g1 was detected for DDT, azinphos-methyl and imidacloprid resistance. Thus, the GWAS analysis for DDT may not represent what evolved in the population due to DDT use, but rather what evolved in the population over the last 40 years that conferred cross-resistance to DDT.DDT was widely used from 1946 until resistance problems became wide spread (about 1960) and other more effective insecticides were introduced. DDT was banned by EPA in 1972. Organophosphates were introduced in the mid-1940s and became the most widely used class of insecticides from about 1955 – 1987. Pyrethroids were introduced about 1980 and rapidly rose to become the most widely used class of insecticides from about 1989-2000. Neonicotinoids  was registered for use in fruit about 1994 and have been the most widely used class of insecticides since about 2000. The DGRP lines were collected in 2003 (D. melanogaster, that are not the targeted pest and highlight that pesticide management should take into account the effect on the whole insect community. Furthermore, the fact that resistance can be buffered by the presence of the common endosymbiont Wolbachia and can evolve through changes in target site or in detoxification enzyme depending on the insecticides and on the insect species make evolution of resistance in those communities fairly unpredictable. However, resistance alleles were present in populations sampled throughout the world showing that even if unpredictable, evolution of resistance to insecticide is repeatable.Altogether our study confirms that insecticides apply a strong selection pressure even on insects, like


Ambystoma mexicanum) is an important model organism in biomedical research. Much current attention is focused on the axolotl's amazing ability to regenerate tissues and whole organs after injury. However, not forgotten is the axolotl's equally amazing ability to thwart aspects of tissue maturation and retain juvenile morphology into the adult phase of life. Unlike close tiger salamander relatives that undergo a thyroid hormone regulated metamorphosis, the axolotl does not typically undergo a metamorphosis. Instead, the axolotl exhibits a paedomorphic mode of development that enables a completely aquatic life cycle. The evolution of paedomorphosis allowed axolotls to exploit relatively permanent habitats in Mexico, and preadapted axolotls for domestication and laboratory study. In this perspective, we first introduce the axolotl and the various meanings of paedomorphosis, and then stress the need to move beyond endocrinology-guided approaches to understand the axolotl's hypothyroid state. With the recent completion of the axolotl genome assembly and established methods to manipulate gene functions, the axolotl is poised to provide new insights about paedomorphosis and the role of thyroid hormone in development and evolution.The Mexican axolotl ( Ambystoma mexicanum) have been studied in laboratories throughout the world for over two-hundred years  relatives of the axolotl are known to invariably undergo a metamorphosis  to regulate thyroid activity . In contA. mexicanum/A. andersoni paedomorphic hybrids were created and administered 50 mM T4 at the time metamorphosis occurs in metamorphic tiger salamanders. Siblings exhibited tremendous variation in metamorphic timing (160-day range) and some individuals remained paedomorphic after 150 days of continuous T4 treatment. Genetic linkage mapping was then used to identify three moderate effect QTL (met1-3) that additively explained variation in metamorphic timing, including met1 first identified in A. mexicanum/A. tigrinum hybrids. This study showed that metamorphic timing is associated with QTL that segregate allelic variation for responsiveness to T4.As was introduced above, genetic studies are beginning to resolve the location of genetic factors within the axolotl genome that regulate metamorphic timing and expression of paedomorphosis. Primarily, axolotls have been crossed to metamorphic tiger salamanders to segregate alleles that affect paedomorph expression and metamorphic timing, however we highlight a study  that cromet1-3. The expressed sequence tag (EST) that was used to initially map met1 showed similarity to nerve growth factor receptor (ngfr). As more genes were mapped, it became clear that this ngfr-like gene corresponded to nradd and met1 located to a genomic region that was uniquely structured during vertebrate evolution. The genes in this region are found on separate chromosomes in all other vertebrates, including the newt  which is member of a different salamander family  had not been sequenced and thus it was difficult to develop molecular probes and investigate gene functions. However, in just the past couple of years, the axolotl genome has been incrementally sequenced and recently a chromosome-level genome assembly was completed . Now it met1 genomic region (met1 (met1 genomic region were purposely chosen for sequencing to more finely resolve the boundaries of recombination that differentiate early (metamorphic) vs. late (paedomorphic) metamorphosing individuals. Inspection of these recombination boundaries against the background of the physically ordered loci yielded a high confidence genomic region of relatively few candidate genes , ccn1-like (LOC102363594), and c16orf89 (LOC102943813). While camp is known to be syntenic with setd2, kif9, and khlh18 in the human genome, ccn1-like and c16orf89 are not expected to map to the met1 region, which only contains loci from human chromosomes 3 and 17. ccn1 is a member of the CCN gene family, which contains six different members (CCN1-6). CCN family members typically have an N-terminal secretory signal peptide and four structural domains: an insulin-like growth factor binding protein-like domain, a von Willebrand factor type C repeat (VWC) domain, a thrombospondin-homology type 1 repeat (TSP1) domain, and a C-terminal cysteine-knot-containing (CT) domain. Interestingly, the met1 ccn1-like gene shows sequence similarity to ccn1 but has a 30 bp deletion in the hinge region between the VWC and TSP1 domains  that regulate global patterns of transcription during early neural development in mammals maybe interesting candidates to pursue because brain transcriptional activity is generally lower in larval axolotls than larval tiger salamanders (nradd) mediate early neuronal development in mammals, and based upon transcriptional data, c16orf89 is predicted to play a role in thyroid gland development and function. Given the multifunctional roles of CCN family members, ccn1-like presents an attractive candidate. However, nothing is known about this genes function and none of the genes discussed above seem to standout above the rest. As a path forward, we believe that all of the genes in the met1 region can be quickly tested for function using CRISPR-Cas9, which is known to efficiently knock-out genes in the axolotl. For example, CRISPR-Cas9 was used recently to create insertion/deletion polymorphisms in fat3 to generate offspring that presented limb and kidney defect phenotypes seen in the short toes axolotl mutant (met1 genomic region using offspring from axolotl x tiger salamander crosses. For example, it is well-established that all axolotl x tiger salamander F1 hybrids undergo metamorphosis. This suggests that dominant tiger salamander alleles at candidate genes are sufficient to induce metamorphosis. Conversely, this suggests that paedomorphosis is associated with the inheritance of recessive alleles that may be partially functional or null in regards to inducing metamorphosis. To test this hypothesis, it would be efficient to knockout candidate genes in F1 hybrids as only the tiger salamander allele would need to be edited. F1 individuals could be assayed for paedomorphosis or delayed metamorphic timing and thus in short order, each of the candidate genes could be tested functionally. We note that it would also be informative to knock out met1-3 candidate genes in other vertebrates, including frogs which do not present paedomorphosis.It is important to point out that none of the candidate genes in the tologies . Two genamanders , 35. Thyl mutant . In a simet1 genomic region that can be efficiently tested using CRISPR-Cas9 to knock-out gene functions. The recent development of essential genetic and genomic tools for the axolotl brings us closer to identifying mechanisms of paedomorphic development and understanding the role of thyroid hormone in development and evolution.Although the Mexican axolotl has been studied for over 150 years, the mechanism associated with its unique paedomorphic mode of development remains unknown. Endocrinology studies have established the importance of thyroid hormone in regulating amphibian metamorphosis. The axolotl does not show an increase in thyroid hormone during early development and thus fails to undergo metamorphosis. While many aspects of the HPT axis seem to be functional in the axolotl, and peripheral tissues are responsive to thyroid hormone treatment, endocrinology-guided studies have not resolved the basis of paedomorphosis. We argue the need to test candidate genes from genetic studies of axolotl paedomorphosis using new genomic resources available to the community. In particular, the new axolotl genome assembly has resolved a short-list of candidate genes for the AC, SK, DB, and SRV collectively wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.