Cancers are the first main cause of premature death in developed countries. Since brain tumors, especially gliomas, are the most lethal type of cancers, risk factors for their prevalence are still being discussed. Nearly 30–50% of all cancers could be prevented by proper nutritional habits and other lifestyle factors, but their influence on the tumors of the central nervous system has not been explained completely and still requires further studies. That is why we attempted to review the available research in this field, with a special focus on the factors with the proven protective activity observed in other cancers. Adequate vegetables and antioxidants (such as vitamins C and A) provided with a diet could have a protective effect, while other factors have shown no correlation with the incidence of glioma. However, further studies are necessary to determine whether fish, coffee, and tea consumption may prevent glioma. Maintaining proper body weight and undertaking a sufficient level of daily physical activity also seem to be important. Excessive body mass index (BMI) and higher attained height have increased the risk of glioma. In order to link more accurately the chosen factors to the prevalence of gliomas, it seems necessary to conduct large cohort, prospective, controlled studies in different world regions. Cancers are the first main cause of premature death in developed countries; this has been confirmed in 48 (28%) of 172 study countries among people under the age of 70, based on the epidemiological data of the World Health Organization (WHO). According to global cancer statistics assumptions in 2018, there were reported 18.1 million new cancer diagnoses and 9.6 million cancer deaths. Lung cancer and breast cancer were diagnosed most frequently—each one constituted 11.6% of new cases. The highest mortality of all cancer deaths was caused by lung cancer (18.4%). Regarding the available statistics for both sexes, brain tumors are not included in the 10 most common cancers and in the 10 cancers that cause the highest number of deaths. Despite this fact, the comparison of the age-standardized rate data for tumors incidence of the brain and the central nervous system (CNS)  and mortality  indicates extremely poor prognosis for this group of patients [However, in the group of brain tumors, in particular, malignant gliomas are the most lethal type of cancer. In 2019, a total of 296,851 new cases of cancers of the brain and the central nervous system were diagnosed, of which 52.5% were confirmed in Asia, 21.8% in Europe, 10% in Latin America and Caribbean, 9.1% in North America, 5.7% in Africa, and 0.82% in Oceania. The cumulative risk (CR) for the incidence of these types of cancers is the highest for Southern Europe and has been calculated as 0.65% and is the lowest for Western Africa, i.e., 0.11%. The CR in Australia and New Zealand has been established to be 0.56%, in North America as 0.52%, and 0.48% in South America, whereas, in Asia regions, the greatest CR has been determined in the Western Asia—0.49%. The CR of very high human development index countries (HDI) is higher than the CR of low HDI countries  [glioblastoma multiforme) and are considered secondary GBMs. Primary GBMs develop de novo and are molecularly distinct from secondary GBM. GBMs are the most common astrocytic glioma and are highly aggressive and lethal [Gliomas are often diagnosed in the advanced stage and are poorly responsive to currently available treatment. Glioma is the most prevalent type of CNS tumor in adults and constitutes 70% of all brain tumors . Based sd lethal . Fast-grd lethal . The medd lethal .The current treatment regimen varies depending on the tissue diagnosis and the selected molecular markers assessment. Younger age and better performance status are considered as therapy-independent prognostic factors. Whenever feasible, neurosurgical removal of the tumor with a safety margin for healthy tissue to improve neurological function is recommended in all newly diagnosed patients. The schedule of radiotherapy is also determined with regard to diagnosis and aims to improve the local control and preserve the patient’s function. Additionally, chemotherapy is included in the standard care for most glioma patients. The most commonly used cytotoxic agents are temozolomide, procarbazine, and lomustine. Due to difficulties associated with complete resection of glioma, the actions taken to prevent its progression play an important role in the treatment of this type of brain tumor . To date, 25 risk factor loci and several rare inherited mutations that may cause glioma in some families have been identified, but ionizing radiation is the only confirmed environmental risk factor . It is eThis paper was based on the articles published in English-language journals until December 2019, available in three electronic databases: Scopus, PubMed, and Google Scholar, which meet required quality standards in relation to the information on the dietary and lifestyle factors discussed in this paper and their association with the prevalence of gliomas. The articles were searched by using the following keywords: “glioma”; “glioblastoma”; “brain tumor” in collocations with “diet”; “nutrition”; “lifestyle factors”; “alcohol”; “tea”; “coffee”; “antioxidants”; “vitamin A”; “vitamin C”; “vitamin E”; “fruits”; “vegetables”; “meat”; “red meat”; “n-nitroso compounds”; “fish”; “fatty acids”; “physical activity”; “body weight”. We adopted the following inclusion criteria: the research in the study topic, specifying glioma cases, with the full text available. The individual studies incorporated in the meta-analysis on the selected topic were excluded from the overall review.Alcohol consumption is one of the strongest lifestyle-related carcinogens. It is considered to be a risk factor for brain tumors because of its ability to cross the blood-brain barrier and a neurotoxic effect. This may lead to structural changes in the hippocampus in adolescence and decreased brain volume (white and grey matter) in adulthood, probably contributing to the changes in the axonal integrity and myelination . The prop = 0.001) and overall CNS tumors  and alcohol intake [The negative impact of alcohol consumption has been demonstrated not only in the pathogenesis of selected tumors but also in cancer survivors. In the large meta-analysis study, focusing on the assessment of dietary factors in the patients cured of cancer, drinking alcohol was positively linked to the overall mortality (relative risk (RR): 1.08, 95% CI: 1.02–1.16) . Neverthl intake . The invl intake  on threel intake .Despite the fact that alcohol is an important risk factor, increasing the risk of developing certain types of cancer, based on the results above, alcohol consumption does not seem to enhance glioma risk significantly, but further investigations are required to confirm these conclusions.The mechanisms by which alcohol could induce cancerogenesis is still not clear; nonetheless, the cellular stress, the genotoxic effect of acetaldehyde, inflammation, and altered folate metabolism are considered . HoweverCoffee, a rich source of many health-promoting compounds, is the drink consumed most frequently in the world. However, it also contains dangerous substances, which are produced in the roasting process . Roasted coffee contains more than 1000 biologically active compounds, part of which could be beneficial for humans, characterized by the antioxidant, antifibrotic, antimutagenic, and anticancer activity . CaffeinCamellia sinensis leaves. Three basic types of tea are distinguished , depending on the process carried out. Green tea, unlike black and oolong, is not fermented. Tea is a rich source of polyphenols (including catechins). However, special attention should be given to green tea catechins amid which epigallocatechin-3-gallate (EGCG) is the most common, while epicatechin-3-gallate (ECG), epigallocatechin (EGC), and epicatechin (EC) are found in smaller quantities. Many mechanisms of green tea catechins have been described, which incorporate anti- and pro-oxidant activity, apoptosis induction, transcriptional factors inhibition, influence on the immune system, epigenetic alternation, inhibition of the receptor tyrosine kinase pathways, and anti-metabolic syndrome effects [Tea is another popular drink that is often consumed. Its production involves processing  effects . EGCG ma effects .p = 0.02) and in females (p = 0.05) but not in males (p = 0.22) [In 2013, Malerba et al.  conducte = 0.22) .2+ release channel— inositol 1,4,5-trisphosphate receptor (IP3R) subtype 3—and, consequently, slowed glioblastoma invasion and migration [The individual analysis indicated no unequivocal relationship between the consumption of coffee, tea, or combined drink of both beverages and risk of glioma. According to the data above, none of the studies has found that tea and coffee consumption increases the risk of glioma, but more research is required to prove whether they truly can prevent this type of cancer. As mentioned at the outset, coffee and tea ingredients generally have an anti-cancer effect. However, there are only a few studies regarding the anti-glioma activity. In the laboratory study on the mouse xenograft model, caffeine, which is the central nervous system stimulant, reduced the tumor mass and prolonged survival. In the in vitro model, it was demonstrated that caffeine inhibited the Caigration .Further studies are necessary to prove and explain the mechanisms of the inhibitory influence of bioactive compounds on glioblastoma development.High intake of fruits and vegetables  is one of the elements of a healthy lifestyle suggested to reduce the risk of cancer incidence. In the meta-analysis, taking into consideration the random relations, Aune et al.  observedp < 0.001). In the analysis of individual tumor histology subtypes, the researchers determined that these associations were strongest in astrocytomas and glioblastomas [Ying  conductelastomas .Protection and repair of DNA damage, modulation of DNA methylation, induction of phase II detoxifying enzymes, and promotion of apoptosis are mentioned among the mechanisms responsible for the protective effect of eating fruits and vegetables. The above mechanisms of fruits and vegetables action are attributed to the presence of numerous anti-tumor compounds, such as vitamins, flavonoids, isothiocyanates, glucosinolates, and dithiolthiones .Although the protective effect of eating vegetables has not been demonstrated in all individual studies, the results of the meta-analysis carried out by Ying deserve attention. A sufficiently high intake of vegetables could be an element of glioma prevention, especially among Asians. In turn, the beneficial influence of fruits intake has not been sufficiently documented for this type of cancer. However, it is vital to consume an appropriate amount of fruits because of their preventive role in reducing the incidence of other types of cancers.Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), included in polyunsaturated fatty acids n-3 (PUFAs), have numerous positive effects on the human body function. PUFAs should be provided with a diet because the human body can biosynthesize them only in small amounts. The dietary source of EPA and DHA is seafood .n = 1,262,104, glioma cases: 2313). Fish consumption had no effect on glioma risk, both in overall follow-up and after excluding the first 5 years of follow-up [Due to protective EPA and DHA effects on brain tissues, it can be suggested that fish intake could also reduce glioma incidence. In the meta-analysis, Lian et al.  looked follow-up , the datollow-up .The balance between PUFA n-3 and PUFA n-6 seems to play a crucial role in the formation of pro- and anti-inflammatory lipids. Recently, attention has been paid to pro-cancerogenic effects of omega-3 fatty acids, but this relationship could exist solely in individuals with specific genetic or metabolic predispositions. It is unclear whether the pro-cancerogenic effect is caused by pollution found in the marine food or by the imbalanced proportion of n-3:n-6 fatty acids in a daily diet .Having reviewed the currently available literature, it seems that fish consumption has no significant impact in the case of glioma development, but the protective effect of fish consumption in other brain cancer suggests a possible protective activity against cancerogenesis in the brain tissues. However, further research should be carried out to explain this mechanism.According to IARC, processed meat has been classified as cancerogenic to humans, and this effect refers to colon and rectum cancer. Red meat consumption has been determined as probably cancerogenic to humans, especially regarding colon, rectum, pancreas, and prostate cancer . The canA meta-analysis conducted by Wei et al. , taking Ward et al.  studied In turn, no relations were shown between the NOC precursors, such as nitrate and nitrite intake, and the glioma risk in the meta-analysis by Wang et al. . They inBased on the available literature, it seems that iron intake (both heme and non-heme) has no influence on the glioma incidence risk. However, taking into account the results of meta-analyses, some studies seem to show a relationship between the consumption of red processed meat and the risk of glioma development, while other studies have shown no relationship. The correlation between the red unprocessed meat consumption and the incidence of glioma has not been proved explicitly.Many compounds that could have a protective, delaying, or eliminating effect on oxidative damage in molecules are classified as antioxidants with different action mechanisms. Enzymes, such as catalase or glutathione peroxidase, and metal-binding proteins like ferritin and minerals  are involved in the prevention of free radicals formation. Another group of antioxidative compounds is focused on preventing oxidative chain reactions by capturing free radicals. Such a mechanism of action is observed, among others, in the case of glutathione, vitamin E, vitamin C, and carotenoids, while the damage caused by free radicals is repaired by lipases, proteases, DNA repair enzymes, or similar compounds .n = 2260 RR: 0.85, 95% CI: 0.63–1.16; cohort n = 920 RR: 1.00, 95% CI: 0.77–1.13), no significant effect on the level of vitamin E intake was determined, either [Vitamin E involves a group of compounds belonging to tocopherols  and tocotrienols. Antioxidative mechanisms of vitamin E  properties include the elimination of free radicals, induction of cell apoptosis, and an inhibitory effect on tumor growth and cancerogenesis . Besides, either . The dat, either , which w, either , median p < 0.001). However, no similar dependencies were observed in other countries [Vitamin A includes a group of compounds characterized by unsaturated hydrocarbons, in which retinol and its derivatives are included . Among antioxidative mechanisms of vitamin A activity, preventing lipid peroxidation, regulating cell differentiation, and inhibition of cancer cell proliferation are also described, besides its participation in the induction of detoxifying enzymes. Specific binding proteins for the retinol and retinoic acid could work as hormones controlling cell differentiation and influence the transport of these compounds across the nuclear membrane. Regarding provitamins A, the β-carotene is most effectively converted to retinol, and its main property is to reduce free radicals and prevent their negative effects . In the ountries .The next antioxidant vitamin, largely responsible for neutralizing free radicals, is vitamin C, which is most common in fruits and vegetables. One of the known anticancer mechanisms of vitamin C is the inhibition of nitrosamines formation by blocking the nitrosation reaction. Besides, vitamin C stimulates the immune system, participates in the protection against oxidative DNA damages, and inhibits the activation of carcinogenesis, especially, at the phase of tumor promotion . It was p = 0.05) [Zinc has also been proved to possess antioxidant properties. It takes part in protection against oxidative stress, DNA replication, and protein synthesis and is an important micronutrient for brain health. Zinc is a crucial element for human metabolism, being a part of more than 300 enzymes, which are responsible for several biological processes. Its deficiency could result in enhanced oxidative stress, leading to damage in DNA, proteins, and lipids. It is suggested that inadequate serum levels of zinc could be correlated with cancer incidence, especially in the development and progression of prostate, pancreatic, and breast cancer . In the  = 0.05) .With regard to the studies on the effect of antioxidants on the cancer patients’ survival, the previous studies have shown divergent results since it has been demonstrated that antioxidants can increase the tumor resistance to chemotherapy or radiotherapy, which could contribute to the shorter survival time or antioxidants might reduce the toxicity of treatment in relations to healthy tissues, which on the other hand, would improve the patients’ survival ,53. Brain = 497) compared to the controls in the case of the total antioxidant index (p < 0.05) and daidzein intake (p < 0.05) with a diet. While analyzing the data for all cases (n = 802), it was demonstrated that the total antioxidant index (p < 0.003), carotenoids (p < 0.05), and phytoestrogens—daidzein (p = 0.003), matairesinol (p < 0.05), secoisolariciresinol (p < 0.003), and cumasterol (p < 0.003)—were statistically significantly inversely associated with glioma risk [In turn, Tedeschi-Blok et al.  studied oma risk .p = 0.009). In the study group, tumor progression time was longer , though not statistically relevant. Moreover, after completing the research, Puri et al. [p = 0.05). It has been suggested that lycopene supplementation may be used as an adjunct to glioma treatment, but these findings require confirmation in the larger studies using the current treatment standard.Lycopene has been proven as an anti-invasion and pro-apoptotic compound, which could cross the blood-brain barrier . Nutritii et al.  found thThere is limited but positive evidence of the role of vitamin A and C in decreasing the glioma risk. This protective effect exists, especially among Americans, since most of the studies discussed in the meta-analyses above were carried out in the USA. Therefore, it seems rational to emphasize the role of proper nutrition in the prevention of gliomas, particularly the consumption of food products that provide adequate amounts of these antioxidants.Many dietary factors are related to the selected anthropometric indicators. Anthropometry is commonly used to measure the general nutritional status and predict health, performance, and survival of individuals. The indicators most frequently used are weight, height, and circumferences . Among determinants of adult’s height are genetic and environmental factors, of which the childhood nutritional status seems to play an important role. Higher levels of insulin-like growth factor (IGF) theoretically may be responsible for linking the growth achieved with the occurrence of individual cancers. Importantly, the height cannot be modifiable, in contrast to body weight. The results from the Million Women Study (MWS) showed that tallness could be seen as a potential risk factor for glioma development in adults; the risk of glioblastoma increased by about 20% for every additional 10 cm of growth . Moore e2 or over 30 kg/m2) is one of the causes of several cancers: colon and rectum, endometrium, ovary, esophagus, gallbladder, pancreas, liver, prostate (advanced), thyroid, multiple myeloma, and renal cancer [Excessive body weight is considered a risk factor not only for the development of several types of cancer but also for the survival and prognosis of cancer patients. Statements of two organizations—IARC and WCRF (World Cancer Research Found)—are consistent and indicate that overweight or obesity  was related to a slightly elevated glioma risk , whereas adult BMI and circumference of waist had no significant association with glioma incidence. Furthermore, the researchers determine no pre-diagnostic changes in body weight for 8 years before diagnosis [2) at the age of 21 in the study conducted by Little et al. [2) in women increased the risk of glioma , and a statistically relevant relationship was reported in the group of overweight females , while in the case of obese women, there was no such relationship. In the studies on men and the studies not taking into account the gender division, no significant relationship was found between overweight, obesity, or overweight and obesity combined and the risk of developing glioma [It must be highlighted that obesity was the strongest lifestyle factor in NIH-AARP Diet and Health study, enhancing the glioma risk to the greatest extend. Adults, who were obese at the age of 18, had nearly 4 times increased glioma risk in comparison to participants with normal weight  . Similariagnosis . Excessiiagnosis  meta-anaiagnosis . Howeveriagnosis . The metiagnosis . On the e et al.  was assoe et al. . Sergente et al.  meta-anag glioma .n = 68,132; observational study, n = 93,676), and compared the relationship between the anthropometric indicators and the risk of glioma in postmenopausal women aged 50–79. At the time of entering the study, all participants were subjected to anthropometric measurements carried out by the trained staff, while the data on anthropometric indicators from the years preceding the study were taken solely from the observational study . In Kabat et al.’s study [Kabat et al. , in turn’s study , there w’s study .Hence, based on the results above, the time of occurrence of excessive body weight seems to be important. Being overweight or obese before the age of 18  may increase the risk of developing glioblastoma, while the excessive body weight achieved in adulthood seems to have a little impact.The levels of physical activity for recommended adults (18–64) to maintain health include undertaking at least 150 min of moderate-intensity aerobic activity or 75 min of vigorous-intensity aerobic activity per week. Increasing the time of types of physical activity mentioned above could additionally be beneficial. It is also suggested to include strength training twice a week involving major muscle groups . The impAlthough we have no influence on height attained, it seems important to pay attention to maintaining proper body weight, especially in adolescence, to reduce the glioma risk. For this reason, taking an appropriate level of daily physical activity could also be beneficial in the prevention of glioma development.A summary of the aspects reviewed in this paper is presented in Considering the facts presented above, it is difficult to associate nutritional and lifestyle factors with the incidence of gliomas due to their complex etiology. Dietary factors that may significantly affect the risk of developing glioma are still not clearly identified and described. Based on the available literature, it can be assumed that coffee and tea consumption and the intake of adequate vegetables and antioxidants (such as vitamins C and A) could have a protective effect. While other factors like alcohol intake, red meat, and red processed meat consumption, as well as iron intake (both heme and non-heme), have shown no correlation with the incidence of glioma. In turn, the consumption of fish, coffee, and tea does not increase the risk, but further studies are necessary to determine whether they may prevent glioma. Maintaining a proper weight of the body, as well as undertaking a sufficient level of daily physical activity, also seems to be important, especially during adolescence. Higher BMI has increased the risk of glioma. The attained height, which cannot be modifiable in contrast to body weight, has shown a positive association with glioma development. However, there is still an insufficient number of studies in the field of nutritional factors and the risk of glioma. In order to link more accurately selected nutritional factors to the incidence of gliomas, it seems necessary to conduct large, prospective, controlled studies in the different world regions.


Women with female genital mutilation or cutting (FGM/C) often suffer from physical and psychosexual problems related to FGM/C. As gatekeepers to the medical system, GPs are often the first to be consulted about these problems. It is as yet unknown if, and to what extent, Dutch GPs identify women with FGM/C or related health problems.To investigate how often Dutch GPs register FGM/C and related health problems.A case–control study of anonymised patient records was performed in the Netherlands.Medical records were checked for information on country of origin. Records of women, aged ≥15 years, from countries where FGM/C is practised were compared with those of a case-control.Although many migrants were registered with the participating GPs, information on country of origin was seldom recorded. Only 68 out of 16 700 patients were identified as women from countries where FGM/C is practised; 12 out of these 68 records contained information about the FGM/C status, but none on the type of FGM/C. There were no significant differences in health problems related to FGM/C between patients with FGM/C and the controls.FGM/C may be a blind spot for GPs and registration of information on migration background could be improved. A larger sample in a future study is needed to confirm this finding. Given the growing global migration, awareness and knowledge on FGM/C, and other migration-related health issues should be part of GP training. There is a need for more research on FGM/C in primary care such as general practice.‘all procedures that involve partial or total removal of the external female genitalia or other injury to the female genital organs for non-medical reasons’.2 The World Health Organization (WHO) has classified four main types of FGM/C: type I, or clitoridectomy, refers to excision of the prepuce, with or without excision of part or all of the clitoris; type II, or excision, which consists of the clitoris with partial or total removal of the labia minora; type III, the most severe form of FGM/C, which is known as infibulation, refers to excision of part or all of the external genitalia and stitching or narrowing of the vaginal opening. Type IV is unclassified. It includes all other harmful procedures to the female genitalia for non-medical purposes; for example, pricking, piercing, incising, scraping, and cauterising the genital area.3FGM/C consists of 4 and that an estimated 68 million girls are at risk of being cut between 2015 and 2030.5 As a result of increasing migration of women from countries where FGM/C is concentrated, FGM/C has become common in high-resource countries such as the Netherlands. It is estimated that approximately 41 000 girls and women with FGM/C are living in the Netherlands. About 37% of these girls and women are estimated to have been infibulated.6FGM/C is predominantly practised in countries in Africa, the Middle East, and Asia. Globally, it is estimated that more than 200 million girls and women have undergone FGM/C,2 and many countries have increasingly undertaken law reform to prohibit FGM/C.7 In the Netherlands, performing any form of FGM/C is forbidden and punishable by law, with an incarceration term of up to 12 years. The law also prohibits any Dutch nationals or permanent Dutch residents from performing FGM/C abroad.10 However, there is no indication of FGM/C being performed in the Netherlands and no suspected FGM/C case has, as yet, been convicted by a court.11FGM/C is recognised as a human rights violation12 Healthcare providers, social care professionals, and teachers who are concerned that a girl may be at risk of FGM/C have legal obligation to report this.The Dutch action plan against the practice of FGM/C was established in 2006. The rationale behind the Dutch approach against FGM/C is to prevent FGM/C by accomplishing a behavioural change towards the practice. The prevention policies in the Netherlands consist of, among others, awareness raising on FGM/C among communities concerned and the empowerment of women, as well as training of health professionals and healthcare workers to support women affected by FGM/C, and to identify and prevent girls from being cut.13 Healthcare providers in primary and secondary health care are also able to record FGM/C and they have been urged to practise good record-keeping. Unfortunately, to date, no reliable data are available in the Netherlands regarding FGM/C in primary and secondary care.Recently, the Dutch Society of Obstetrics and Gynaecology (NVOG) has developed guidelines on the management of FGM/C.16 as well as negative effects on mental health.19 The migration from countries where FGM/C is practised to high-resource countries will further increase.21 Consequently, in the near future, healthcare providers are expected to provide care for more women with FGM/C. There is increasing evidence that women with FGM/C receive lower quality of care, owing to ‘lack of knowledge, cultural sensitivities associated with the subject leading to silence, stigma and inaction'.1 Since most of these studies have been conducted in obstetric settings, 25 there is a need for more research on FGM/C in primary care, such as in general practice.FGM/C has been associated with adverse short- and long-term health consequences, including, severe pain, excessive bleeding, urinary tract infections, bacterial vaginosis, painful sexual intercourse, and adverse perinatal outcomes,In the Netherlands, the GP is often the first to encounter health problems of women with FGM/C. GPs function as a ‘gatekeeper’ to the healthcare system in the Netherlands and all residents are obliged by their (mandatory) health insurance to be registered with a general practice. However, it is as yet unknown if, and to what extent, GPs identify women with FGM/C, nor what care they provide to them. Insight into these aspects are vital in order to evaluate whether any inequities exist in primary health care for women with FGM/C. The aim of this study was to investigate how often in Dutch general practice FGM/C-related health problems were recorded in women from countries where FGM/C is practised, compared with women with other migration background of the same age.A case–control study of anonymised patient records of women with a migration background, including women from countries where FGM/C is practised, was carried out between November and December 2017.26 Therefore, the practices were recruited through the researcher's informal network. A purposive sampling method was used, aiming for practices with a high number of patients from very high FGM/C prevalence countries, striving for variation among GPs in terms of sex, size, and geographical location of the general practice.The study was executed in five general practices in the Netherlands, known to care for a large migrant community and trained to care for women with FGM/C. In the Netherlands, it is becoming more difficult to encourage GPs to participate in surveys, owing to their demanding work schedules and increasing frequency of being approached for surveys.27 Next, information on country of origin was searched through the first 10 contact records, including the first visit records. Then, notes were searched to retrieve information on country of origin, using the keywords 'origin', 'country of birth' or 'country of origin', 'Somalia', 'Eritrea', 'Egypt', 'Sudan', or 'Sierra Leone'. The data of these women were compared with those of a case-control: female migrant patients in the same age group from countries where FGM/C is not practised. Every next migrant woman from a non-FMG/C practising country was selected from the same age-group on the list after each woman from a country where FGM/C is practised was selected.Patient records of girls and women, aged ≥15 years, from very high FGM/C prevalence countries, including Somalia, Egypt, Eritrea, Sierra Leone, and Sudan, were included. The Netherlands has a large number of migrants from these countries in which more than 80% of girls and women of reproductive age have undergone FGM/C. Since country of origin is not routinely registered in general practice, all female patients with a foreign surname were first included. Identifying migrants by means of their surname has proved to be a reliable, second-best method.28 Hence, within the scope of this study, it was found to be more appropriate to compare the data within migrant subgroups, so data of women from countries where FGM/C is practised were compared with female migrant patients from countries where FGM/C is not practised.It has been well documented that migrants, in general, have at some point differing morbidity patterns and receive lower quality of care than native populations.The researcher (NK), a senior medical student, was allowed to access the complete patient records in each participating general practice, after signing the required confidentiality agreement, in line with national legislation on data protection. NK selected the records based on patients’ surnames and then anonymised the data by converting each patient’s personal details into an alphanumeric code. Records contained information on every contact with the GP (in person or by telephone), medication prescribed by the GP, and all letters to the GP from other  professionals and services.It was checked whether country of origin, FGM/C status of the patient, type of FGM/C, or health problems often associated with FGM/C were recorded in medical files. The files were searched for health problems, including recurring urinary tract infections (defined as two or more cystitis in 6 months), dysmenorrhoea, sub- or in-fertility, complications during childbirth, painful sexual intercourse, and fear of sexual intercourse. In addition, it was checked whether the FGM/C status of the patient was recorded according to the International Classification of Primary Care (ICPC). The ICPC code-X82 refers to injury to the female genital organs. Finally, women with unknown origin were excluded and the data were anonymised prior to analysis.2 test was used to compare the data from different groups and to determine whether the differences were statistically significant.The data were analysed using SPSS statistical software (version 22). The χA total of 16 700 patient records from five general practices were included in the analysis. Country of origin of patients from FGM/C-prevalent countries was mentioned in 68 cases. As shown in The FGM/C status of patients was recorded in 12 out of the 68 cases where country of origin was documented. Of these patients, 11 had undergone some form of FGM/C. In none of these cases was type of FGM/C classified and recorded. Moreover, the FGM/C status of one patient was recorded using the ICPC code-X82. Other cases were randomly recorded or found in the correspondence.n = 12), patients whose FGM/C status was not recorded (n = 56) and patients in the control-group (n = 61).There were no significant differences in recorded health problems related to FGM/C such as urinary tract infections, dysmenorrhoea, sub- or in-fertility, complications during childbirth, painful sexual intercourse, or fear of sexual intercourse between patients with FGM/C (In this first study on FGM/C in five general practices in The Netherlands known to care for a large migrant community, patient demographics such as country of origin were seldom documented in the medical charts. Only 68 out of 16 700 patients were identified as women from very high FGM/C prevalence countries; 12 out of these 68 medical files contained information about the FGM/C status of the patient, and in none of these cases was type of FGM/C classified.Although the participating practices were known to care for a large migrant community and trained to care for women with FGM/C, it is unclear why information on country of origin was seldom recorded and only 12 records contained information about FGM/C. It is believed that it is highly unlikely that only 12 out of 68 women from very high FGM/C prevalence countries had actually undergone FGM/C. FGM/C may be a blind spot in general practice in the Netherlands. Future research, preferably qualitative, is needed with larger samples to confirm this, and to explore the factors that encourage and hinder GPs in recording and discussing FGM/C with women who have undergone the procedure.The relatively small numbers of general practices included in the study is acknowledged, which may explain the non-significant difference in recorded health problems related to FGM/C between patients in the FGM/C group and patients in the control group. While healthcare providers are able to systematically record FGM/C, to date, no reliable data are available regarding FGM/C in primary and secondary care. Therefore, the authors believe that it may not be necessary to include more practices before concluding that there is an urgent need for improvement of recognition and recording of FGM in general practice; improvement could be achieved by discussing FGM/C with patients from FGM/C-prevalent countries, and by correctly classifying and recording FGM/C in the medical charts.1 Nonetheless, the present findings would be consistent with results from available studies among other healthcare providers caring for women with FGM/C in secondary care.29 Several reasons may explain the few recorded cases of FGM/C in general practice. In general, it is known that most women rarely proactively discuss FGM/C with their healthcare provider. At the same time, healthcare providers refrain from asking about it.33 The possible explanation could be owing to cultural taboos around FGM/C in women’s own community or fear of being judged, or being fearful as FGM/C is forbidden in their new country of residence.34There is limited evidence on FGM/C in primary care. Most studies have been conducted in secondary care and have focused on FGM/C in maternity settings.et al1 have explored factors influencing the provision of health care related to FGM/C from the perspective of health providers. Feeling of embarrassment, shock, disgust, and horror; uncertainty about how to frame the questions, or ‘anxiety about being perceived as culturally insensitive’, language barriers, and a lack of knowledge have been described as barriers to engagement with women with FGM/C.43In a comprehensive systematic review, Evans 1 As a result, FGM/C will probably never be discussed at all, hence not recorded. As mentioned, previous studies have reported expression of strong emotions by healthcare providers, including shock, disgust, and horror. Cutting of women was perceived as an alien and negative practice, with healthcare providers describing and perceiving these women as not ‘normal’, but mutilated. Also, lack of knowledge has been listed repeatedly as a barrier to identify and manage women with FGM/C.24 For instance, Chalmers et al22 reported dissatisfaction of women with care and a perceived lack of knowledge and ability by healthcare providers to care for women during pregnancy and childbirth. Relph et al23assessed the knowledge, attitude, and training on FGM/C among medical and midwifery professionals and found that although the majority of these professionals were aware of the practice, their ability to identify FGM/C and its associated health complications remains unsatisfactory. Similarly, Zaidi et al24 observed deficiencies in knowledge and adherence to guidelines on the management of health complications of FGM/C among healthcare providers. Zenner et al25 reported on the quality of obstetric and midwifery care, and noted major deficits in identification, management, and safeguarding.Because of the sensitive nature of FGM/C, talking about the subject can make health providers feel uncomfortable. Some healthcare providers reported they avoided discussing the topic with their patient, as they did not want to offend, stigmatise, or jeopardise their relationships, assuming that women would initiate a conversation about FGM/C if there was a problem.47 the subject of FGM/C is not included in the curriculum of most medical, midwifery, and public health training programmes.49 In addition, specific guidelines on the management of health complications related to FGM/C are not well known among healthcare providers.48The findings, along with those of previous reports, support the need for integration of FGM/C and culturally sensitive care in training programmes for healthcare providers. Currently, despite the growing number of books on migration health in primary care,et al,29 recognition of FGM/C is the first step in formulating a diagnosis, before providing information and treating a patient with FGM/C. However, FGM/C may be a blind spot in general practice in the Netherlands. Future research, preferably qualitative, is needed with larger samples to confirm this and to explore the factors that encourage and hinder GPs in recording and discussing FGM/C with women who have undergone it.As noted by Abdulcadir Further, registration of information on migration background could be improved and, as migration is on the rise and the population attending general practices gets more and more diverse, GPs should not only be aware and knowledgeable on FGM/C, but also on other migration-related health issues.


Underweight prevalence continues to be major public health challenge worldwide, particularly in developing countries like Pakistan. This study is focused on socio-economic and demographic aspects of underweight prevalence among children under-five in Punjab.In this study, several socioeconomic and demographic factors are considered using MICS-4 data-set. Only those variables which are usually described in the nutritional studies of children were picked. Covariates include: the age of children, sex of the children, age of mother, total number of children born to women, family wealth index quintile, source of drinking water, type of sanitation, place of residence, parents’ education and occupation. All Categorical variables are effect coded. The child’s age and the mother’s age are assumed to be nonlinear, geographical region is spatial effect, while other variables are parametric in nature.Since, the response is binary, covariate comprises linear terms, nonlinear effects of continues covariates and geographic effects, so we have use Geo-additive models (based on Fully Bayesian approach) with binomial family under logit link. Statistical analysis is performed on Statistical package R using Bayes X and R2 Bayes X Libraries.Underweight status of children was found to be positively associated with number of under-five children in household, total number of children ever born to women and age of mother when the child was born. Whereas, it negatively associated with place of residence, parent’s education and family wealth index quintile. On the regional effect, the Southern Punjab has higher prevalence of underweight compared to Central and Northern Punjab.Similarity of our results with several other studies demonstrate that the Geo-additive models are an ideal substitute of other statistical models to analyze the underweight prevalence among children. Moreover, our findings suggest the Punjab Government, to introduce target-oriented programs such as poverty reduction and enhancement of education and health facilities for poor population and disadvantaged regions, especially Southern Punjab.Supplementary information accompanies this paper at 10.1186/s12889-020-09675-5. Underweight prevalence is a major cause of child mortality worldwide. Underweight children have lesser immunity against infections and have higher chance to die from common diseases, and those who survive are exposed to recurring illnesses and slower growth. Such children are more likely to have lower IQ, which not only affect their educational performance but also reduce their working abilities , 2.According to MICS-2014 [To the best of our knowledge, not even a single research study has been conducted in Punjab so far, to explore and analyze the impact of factors influencing such a high proportion of underweight.Alasfoor et al. 2007), Mengistu et al. (2013) and Rayhan et al. (2006), use bivariate and multivariate analysis to identify the causes of under-five malnutrition –7. The k, MengistMajority of previous studies on child’s underweight have usually focused on different socio-economic, demographic or health related factors but mostly have ignored spatial and nonlinear effects of covariates. Considering these aspects, Kandala et al. (2008), Lasisi et al. 2014) and Lasisi et al. (2014) use Bayesian geo-additive models to observe the pattern of underweight among the children under five year of age  and Lasi, 18. TheWe aim to study the impact of several socio-economic and demographic factors on underweight status of under-five children in Punjab, considering the spatial and nonlinear effects of covariates. This study is based on data-set of the MICS-2014 conducted by Punjab Bureau of Statistics with the collaboration of UNICEF.Two-stage cluster sampling technique was used for selection of sample. At first stage 2050 clusters were randomly selected  from all 36 districts of Punjab through proportional allocation. On second stage systematic sample of 20 households  was randomly drawn from each of selected cluster . The infUWa), i.e. ‟weight-for-age” is taken as a dependent variable. In this regard, the weight and the height of each child in sample is calculated separately. These measures are then expressed as Z-scores from the median of the reference population.Underweight status of children  is binary with 0 & 1 refers to Normal weight and underweight children respectively.Where, WAPWe consider several socio-economic and demographic covariates as predictor, which are generally described in the nutritional studies of children. Covariates include; the age of children, sex of the children, age of mother, total number of children ever born to women, family wealth index quintile, source of drinking water, type of sanitation, place of residence , parents’ education and occupation. All categorical variables are effect coded; Children’s age and Mother’s age (when child born) are assumed nonlinear; ‟Region” is geographic effect, while other covariates are parametric in nature  is given in Table Model summary for our final model  is given in Table According to summary of results Table , going fUnderweight status of Children under-five in Punjab is negatively associated with education level of father and mother of children. The log odds of underweight decreased by 0.09, 0.17, 0.28 and 0.41 for children whose fathers attain primary, middle, secondary and higher level of education respectively w.r.t. those having no education at all. The log odds of underweight decreased by 0.08, 0.19, 0.25 and 0.59 for children whose mothers got primary, middle, secondary and higher education respectively, compared to those having no education  Table .Underweight status of Children under-five in Punjab is negatively correlated with household wealth index quintile. Going from the lowest wealth index quartile to second, middle, fourth and highest wealth index quantiles, decreases the log odds of underweight by 0.18, 0.35, 0.45 and 0.83 respectively Table .Total number of children ever born to a woman is positively associated with the risk of underweight. Addition of every single child into the total number of children ever born to a woman increases the log odds of underweight by 0.04 Table .The household’s access to improved sanitation facility decreases the log odds of underweight by 0.09 compared to unimproved sanitation facilities Table .Underweight status of children is inversely related to the age of their mothers. The risk of underweight found highest in the children whose mother’s age was less than twenty years at time of their birth, and it decline down gradually as the age of mother increase. The spread of underweight is almost same for mother’s age group of 20–42 years (when children born), relatively higher beyond this age group, and highest if mother age is grater then 45 years  as shown in Fig. Exposure of child’s mother to mass media reduces the log odds of underweight by 0.02 in comparison with no access to media at all , type of food he/she received, mother’s BMI, maternal and eternal care and use of extra food during pregnancy etc. In addition, there are three commonly used measures of child nutritional status: Stunting (height-for-age), Wasting (weight-for-height) and Underweight (weight-for-age). For the sake of conciseness only the last mentioned has been used, in this study.Since, quality, availability and accessibility of hygienic food, health care and other facilities  are considerably lower in rural Punjab, therefore, the children from rural areas of Punjab found more like to be underweight than their urban counterparts. This finding is consistent with findings obtained by other authors , 14, 17.Our finding discloses a strong positive association between underweight and education level of father and mother of children. It is because, the educated parents usually have more knowledge about diet and health of children. In addition, education may change their traditional beliefs about diseases and taking care of their children. It also found consistent with several other similar studies , 14–18. The risk of underweight found more severe in children belongs to families with lowest wealth index quintile. Because, it is impossible for parents with a very small income to maintain the increasing need of food as well as health expenditures of children, which results undernourishment. On the other hand, wealthier parents are more likely to afford hygienic food, healthier living conditions and better health-care that improves the child nutrition and overall health. Many other studies on child nutrition illustrate the similar results , 12–16.Our study has found positive relationship between the number of children ever born to a woman and underweight prevalence. Same findings are revealed in a study carried out by Islam et al. (2013) . GeneralThe underweight status of children found negatively associated with improvement in sanitation facilities. It is because, the improved sanitation reduces the risk of diseases; consequently, the risk of underweight is decreased. The source of drinking water revealed similar but comparatively lesser impact on underweight status of children. It might because, the 94% inhabitants of Punjab use improved source of drinking water while comparatively lesser proportion of population (66%) use improved sanitation facility . In addiThe underweight status of children is negatively associated with mother’s access to media Table . It coulRefer to Fig. The reasons for nonlinear association of underweight status of children with their age are that: usually children born with approximately normal anthropometric status; but afterwards, the health status of the most children became worse due to different socio-economic and biological factors and until it improved and become stable at a low level. The highest risk of underweight during the age group of 18–32 months is might because, during the aforesaid age group, kids are usually weaned from breast as well as their mothers do lose their ability to produce enough milk to fulfill the nutritional requirement of growing children. These results are consistent with findings obtained by other authors , 11, 14.The causes for such a high rate of underweight prevalence in Southern Punjab Southern Punjab also called Seraiki belt (consisting of 11 districts) could be:Firstly, Southern Punjab is more deprived region of province, 41% of people residing in this region have lowest wealth index quintile Table .Table 5Secondly, 72% of people living in Southern Punjab belongs to rural areas with poor health, living and educational facilities which is highest in entire province Table .Table 6Thirdly, the statistics of parent’s education of under-five children is worse in Southern Punjab. In this region, 64% of mothers and 41% of fathers of under five children are illiterate Table . This peFourthly, 45% of the inhabitant of Southern Punjab have no access to improved sanitation which is also highest in entire province Table .Fifthly, about one-half of resident of this region has no access to mass media at all. It is not only highest compared with rest of Punjab but horrible to see in this era Table .Table 8In addition, Southern Punjab is considered as most deprived and neglected part of Punjab. Since 1970, South Punjab has not been receiving its due share from provincial resources. Development schemes for said region were diverted to Central and Northern parts of province. According to recent data, poverty and unemployment are relatively higher in this region , 24–27. Therefore, to-a-days, for fair distribution of resources and administrative reasons, the demand for creation of new autonomous province in south Punjab, i.e. Seraiki Province (Seraiki is a native language of this region), is under national debate and focus.The results of this study reveal that the family’s wealth index quintile, mother education level, father education level, geographical region, locality and total number of children ever born to a woman (fertility) are significantly associated with underweight status of children under-five in Punjab. The results from this study are found to be consistent with other studies , 11, 13.In order to achieve the sustainable development goal Punjab needs to scale up target-oriented programs such as poverty reduction income-generating interventions and improvement of education and health facilities for poor population and deprived parts of province especially Southern Punjab.Recommendations for researchers:There are three commonly used measures of child nutritional status: Stunting, Wasting and Underweight. Only the last-mentioned measure has been used in this study. Hence, Stunting and wasting may also be used for said purpose. In addition, this study is specific to Punjab province; same may be don for other provinces/parts of Pakistan.Additional file 1. Supplementary information 1 [mation 1 –33Additional file 2. Supplementary information 2 [mation 2 –38Additional file 3 : Figure B1 ACF for step = 1.Auto correlation function plot at step = 1 created through R languageAdditional file 4 : Figure B2 ACF for step = 10. Auto correlation function plot at step = 10 created through R language


Biologists have reported on the chemical defences and the phenetic similarity of net-winged beetles (Coleoptera: Lycidae) and their co-mimics. Nevertheless, our knowledge has remained fragmental, and the evolution of mimetic patterns has not been studied in the phylogenetic context. We illustrate the general appearance of ~ 600 lycid species and ~ 200 co-mimics and their distribution. Further, we assemble the phylogeny using the transcriptomic backbone and ~ 570 species. Using phylogenetic information, we closely scrutinise the relationships among aposematically coloured species, the worldwide diversity, and the distribution of aposematic patterns. The emitted visual signals differ in conspicuousness. The uniform coloured dorsum is ancestral and was followed by the evolution of bicoloured forms. The mottled patterns, i.e. fasciate, striate, punctate, and reticulate, originated later in the course of evolution. The highest number of sympatrically occurring patterns was recovered in New Guinea and the Andean mountain ecosystems (the areas of the highest abundance), and in continental South East Asia (an area of moderate abundance but high in phylogenetic diversity). Consequently, a large number of co-existing aposematic patterns in a single region and/or locality is the rule, in contrast with the theoretical prediction, and predators do not face a simple model-like choice but cope with complex mimetic communities. Lycids display an ancestral aposematic signal even though they sympatrically occur with differently coloured unprofitable relatives. We show that the highly conspicuous patterns evolve within communities predominantly formed by less conspicuous Müllerian mimics and, and often only a single species displays a novel pattern. Our work is a forerunner to the detailed research into the aposematic signalling of net-winged beetles. The evolution of mimicry has been studied from various perspectives, but the considered numbers of interacting species and patterns under consideration have usually been low6. In order to investigate the evolution of mimicry from a long-term perspective, while simultaneously studying a high number of interacting species, we need to focus on ancient and highly diversified lineages8. Unlike the well-studied Heliconius system, some lineages of aposematically coloured insects contain up to several hundred species in a single geographic region and up to a hundred species in a single locality10. This means that unpalatable (or more widely unprofitable) aposematically coloured species dynamically interact within complex communities containing multiple patterns, imperfect mimics, as well as Müllerian and Batesian co-mimics. Here, we study the world fauna of net-winged beetles (Coleoptera: Lycidae), an elateroid family with pronounced chemical protection, diverse aposematic colouration, and ~ 4200 species11.Defence mechanisms are inextricably linked to biological fitness and among them Müllerian mimicry is one common anti-predatory strategy12. Most adults are inactive and sit on leaves under the forest canopy , and soldier beetles discharge protective droplets from cuticular abdominal and thoracic vesicles29. The haemolymph of net-winged beetles contains l-methyl-2-quinolone and 2-methoxy-3-s-butylpyrazine and is effective in repelling potential predators30. Net-winged beetles commonly form aggregations33 . Furthermore, the evolution of the signal might be affected by constraints which prevent high levels of similarity . We also need to consider how many aposematic patterns are present in a region. Multiple patterns in a single place, often within a single aggregation, have been reported66.The model of Müllerian mimicry is based on the benefit obtained by a protected prey which signals its unpalatability and shares a predation load with its co-mimicsAs described above, Lycids are a big group of putatively aposematic, conspicuously signalling species with a world-wide distribution, potentially taking part to several Müllerian mimicry rings. Investigating the diversity of aposematic signalling in ~ 4000 lycids on the scale of zoogeographic regions and over the period of the last hundred million years can provide invaluable information about the origin of aposematism and the evolution of aposematic signals. This work is a starting point for many future experiments focussing on specific local assemblages, but here we focus in discussing the differences between patterns, as well as their phylogenetic origins, i.e. how old are the principal patterns and whether highly conspicuous patterns rapidly evolved from their cryptic relatives or gradually evolved from the aposematically coloured relatives. These factors are also of interest to ecosystem-based studies because phylogenetic structure and signalling plasticity have consequences for the evolution of mimicry in close interactions within each locality.We assume that the complexity of local rings might partly depend on the regional signalling diversity and on the intensity of migration between neighbouring ecosystems. We thus quantify the diversity of lycid beetle coloration and patterning, focusing on their signal strength in the spectrum visible to humans, relationships, and distribution. Our main aims are to (1) describe colour pattern diversity of lycid beetles; (2) investigate their distribution and evolutionary origins ; (3) investigate how alpha-diversity, phylogenetic diversity, and abundance correlate with the number of aposematic patterns occurring in a geographic region.Field observations and voucher sampling were conducted by the majority of the authors from 1989 to 2019 in all zoogeographical regions and additional material was provided by colleagues. The studied specimens are deposited in the authors’ Voucher Collection. The total catching effort cannot be standardized as the number of collected individuals depends on the duration of sampling, the season, weather conditions, and habitat conditions. Therefore, the regional abundance is categorized as follows: (a) low ; (b) medium , high . Further data on aposematic patterns were obtained from museum collections (Supplements).Ficus lyrata Warb. , Hoya carnosa (Linnaeus filius) Brown , and Yucca guatemalensis Baker (light-green leaf).Two sets of photographs were used for colour evaluation and estimation of the contrast in the colour spectrum visible to humans. The photographs displaying net-winged beetles in their natural environment were taken by the authors  in linear intensity scale, calibrated with a IT8.7 calibration target  using a CoCa program with implemented Argyll Colour Management System (www.argyllcms.com), and finally converted to 24-bit sRGB IEC61966-2.1 colour space using perceptual rendering intent. The colour space characteristics of the bright and dark coloured elytral areas were quantified in the CIE La*b* absolute colour space . i.e., the colour spectrum visible to humans. The colour samples were taken five times from an area of 31 × 31 pixels at various points of measured body parts in Photoshop 13.0. The average colour values were computed and the colour distance DeltaE La*b* CIE 76 was counted as the Euclidian distance between colour positions in the 3D CIE La*b* colour space. The processing of photographs follows the methods used by the authors of an earlier study41. The DeltaE distances were counted between beetle dorsum and background  and distance between colours displayed on the dorsum, i.e. internal contrast, is designated as IC-DeltaE67.Images were converted from a Canon raw format (CR2) to a 48-bit tiff with dcraw program  and a white reflective panel. The white balance was custom set with a white target panel. The photographs were taken at various focus levels and the final photographs were assembled using Helicon Focus v.7.6.1.We mapped the geographic distribution of aposematic patterns through the examination of sequenced individuals, and we additionally, evaluated specimens in collections and searched the relevant literature. Nineteen categories were used for descriptions of patterns; aberrant patterns were described individually as non-categorized. We considered the uniform colouration displayed on the dorsal side of the body, the different colours displayed on the pronotum and the elytra, and a group of patterns with two colours displayed on the elytra. Additional patterns include striate, punctate, tricoloured, and reticulate types, and several unique patterns, e.g. green, green/black, black/amethyst; see Figs. cox1, nadh5, and rrnL mtDNA, SSU and d2 loop of LSU rRNA, and the transcriptomic and genomic data68. No data were specifically produced for this. We assembled a matrix of 751 lycid terminals and Iberobaenia minuta, a sister to the Lycidae69, as an outgroup  was obtained by applying UFboot75 with 5000 replicates. To obtain a tree with deep splits supported by the phylogenomic analysis, the relative position was fixed for 23 terminals whose relationships have been inferred from ~ 4200 orthologs . We dated the tree with the widely used rates for cox1 , nadh5 (0.0177s/l/my), and rrnL (0.054s/l/my78). Subsequently, we validated the results with the available records of Burmolycus minutus, Erotini indet., and Prototrichalus spp.80,unpubl data. The analysis was set to HKY+I+G model and the birth–death speciation prior; genes and codon positions were partitioned, and analyses were run for 108 generations with sampling frequency 10,000 generations. A maximum credibility tree was generated with TREEANNOTATOR v.1.8.176 discarding 25% of the trees as a burn-in after checking the ESS values and a plateau phase in TRACER v.1.681. Further, we used the database of biogeographic distribution data11 to relate the phylogenetic diversity (estimated as a number of tribes recorded in the region) and alpha diversity to the regional diversity of aposematic patterns.The complete dataset could not be reliably analysed using BEAST 1.8.1https://beastclassic.googlecode.com/files/ARv2.0.1.pdf for 108 generations with sampling every 10,000 generations. A burn-in was set to the first 25% of trees. The effective sample size values and plateau phase were checked in Tracer v.1.6 and the maximum credibility tree was generated using TREEANNOTATOR v.1.8.176. We set the age of the Lycidae root to 1 and dated the origins in relative time units (RTU), with 1.0 for the oldest and 0.0 for the extant splits.For the reconstruction of ancestral colouration, we used six categories: (1) the upper side uniformly black, yellow, orange, or red; (2) a different pronotal and elytral colouration resulting in a combination of dark and bright coloured body parts; (3) bicoloured elytra with a combination of dark and bright coloured parts of the elytra; (4) fasciate elytra; (5) striate elytra; and (6) non-categorized patterns . As only a few dimorphic species were present in the dataset, each specimen was separately coded and intraspecific polymorphism was expressed as a different code for specimens. The reconstruction of ancestral colour patterns was performed using the ‘discrete traits’ function in BEAST v.1.8.1. Each terminal was assigned to one of six states Figs. –S922. Th82. To calculate δ-statistics, we used the R package ape83 and the code provided in GitHub (https://github.com/mrborges23/delta_statistic82). We removed all taxa that had no information about adult colouration  and the tree was split into clades with more than 20 taxa as it is impossible to compute δ-statistics for subtrees smaller than this threshold. All parameters were set to default with the exception of sim = 1,000,000, thin = 100, and burn = 2500.The phylogenetic signal was calculated using the δ Bayesian approach, suitable for analysing categorical traitsThe net-winged beetles display a wide range of visually distinct patterns and we present photographs and distribution data for ~ 600 lycid species and ~ 200 species of non-lycid co-mimics from all zoogeographic regions Figs. –S922. ThThe monochromatic patterns include uniform black forms with low external contrast , even though the origin of net-winged beetles is earlier, about the mid-Jurassic. The deepest splits among constituent taxa in individual tribes are often delayed , whereas the most species-rich subfamilies Lycinae and Metriorrhynchinae express nearly twice stronger effect of the evolutionary history in their colouration pattern trait .Lycus F.; numerous Calopterini, a few Eurrhacini), and a few species of Metriorrhynchina. Similarly, the lycids with striate elytra are only known in some lineages . The patterns with punctate elytra are limited to a few lineages  low: Europe, the lowlands to lower mountain forests of most of China, the Russian Far East, most areas of Sub-Saharan Africa, North America, the Caribbean, some seasonally dry regions of South America; (b) medium: the lowlands of South East Asia and Indo-Burma, the Himalayas, mountain forests of China, Japan, the Philippines, Sulawesi, the Moluccas, the humid regions of Sub-Saharan Africa, the majority of the Nearctic region, eastern Australia; (c) high: the mountains of the Great Sundas, mountains of northern Laos, mid to high altitudes in the northern Andes, and New Guinea. The estimation of abundance excludes the local and temporal occurrence of some flower-visiting species in secondary habitats and savannahs. Unlike their relatives, some species from these genera aggregate on flowers outside the forest canopy (see Supplements for further information) and they are usually not present under the canopy, which is where most species occur. Based on field observation and museum collections, Lycidae are more common in areas without a pronounced dry season. Even in the humid tropics, a higher abundance was observed in elevations 1300–2000 m a. s. l. compared to adjacent lowlands. The highest numbers of species have been reported from Sundaland and the Philippines , continental Africa (656 spp.), South America (385 spp.), and New Guinea (341 spp.).The reflex-bleeding and pungent smell were confirmed for all adults when they were harshly disturbed. Haemolymph oozed from elytral ruptures, from intersegmental membranes of the thorax, antennae, and legs and was precipitated by alcohol when individuals were fixed Figs. , S764.The conspicuousness of the aposematic patterns depends on the beetles’ behaviour and is quite low under some circumstances. We found many individuals sitting on the under sides of leaves and they often sat parallel with the main veins or at the tip with their heads directed towards the base of the leaf Figs. A,B,E, 3QThe vivid aposematic colouration of Lycidae is often mimicked by other beetles Figs. –S922, bu90. We do not have data on putative predators, and therefore we will only discuss the conspicuousness of individual patterns in the colour spectrum visible to humans. It is highly probable that various patterns will have different levels of effectiveness in other colour spectra. Monochromatic perception can also provide a strong signal91, therefore we also consider the arrangement of differently coloured body parts as a component of conspicuousness.The signalling power of aposematic patterns depends on the conspicuousness of potential prey and its abundance, i.e. positive frequency-dependent selection93 and these colours are commonly found in the patterns displayed by net-winged beetles . Additionally, the internal contrast is higher than the contrast between brightly coloured body parts and the background alone . We might classify various fasciate forms as more complex signals Figs. K,L, 3Q. The effectiveness of the signal can also be increased by the greater area of the elytral surface. The body of a species can be slender, similar to its relatives, with only its elytra expanded or globulous in shape Figs. N–P, 3A. The conspicuousness of various forms depends on their positions on leaves Tables , S6. We,98. The uniform forms are the least apparent, and therefore potentially the least effective4 . To complicate the interactions leading to the evolution of multi-pattern communities, predators perceive lycids in different visual spectra, and they have abilities which allow them to learn and forget aversion. They have a differential perception of the salient components of a signal, and there is a potential for neophobia in the interactions with uncommon but highly apparent patterns104. These impact of these factors can only be investigated with dedicated experiments.Other characteristics of patterns could be considered if a single community was studied: different volumes of brightly coloured areas, body size, and gradual and/or sudden transitions between colours Figs. –S922. Ad108, Fig. 14 and the present study deals with all known patterns in the family. Based on the amount of data , we consider the present phylogenetic hypothesis to be sufficient for the investigation into the origins of aposematic patterns in lycids.We have produced a new phylogeny with ~ 570 species from 25 lycid tribes and each tribe is represented proportionally to its diversity . The indicated level of phylogenetic conservativism must be interpreted cautiously because the colouration is at least partly linked with the geographical origin and large clades from a single area are present in the tree Fig. . Therefo113 and the estimation would be prone to error. We hypothesize subsequent origins of further aposematic patterns such as the contrast between the differently coloured pronotum and the elytra, bicoloured elytra, and various characteristically mottled forms41 , we currently do not have data on the phylogenetic structures of net-winged beetle communities. Similarly, our estimation of alpha-diversity depends on the number of species already described11 and these are only a part of the real diversity84. Additionally, we dependent on an approximate estimation of abundance in various ecosystems .The highest phylogenetic diversity was identified in the Oriental region  and in Eastern Asia , although the interpretation is limited by the present state of the biodiversity inventory128. If we consider the real number of species identified in New Guinea , the R2 value doubles. Although we can keep the working hypothesis that the number of aposematic patterns and the number of species are positively correlated, the evidence is ambiguous and only future studies of a high number of mimetic communities where individuals are in real contact can produce reliable data.We can consider alpha-taxonomic diversity in separate regions Table . If we cAbundance is another factor, although it is potentially linked with alpha-diversity. Currently we do not have hard data to evaluate the abundance of net-winged beetles in various regions and, preferably, individual mimicry communities should be the subject of analyses rather than regions, as in this study. An estimation can only be based on typical collecting success in various regions, and then we could see that regions where net-winged beetles are most common also have the highest diversity of aposematic signals, i.e. New Guinea, the northern Andes, and South East Asian mountains such as Sumatran volcanoes and Mt. Kinabalu in Borneo.The main goals of our study were to assemble information on the aposematic patterns of > 4000 net-winged beetles, investigate the link between patterns and phylogenetic relationships, and identify the factors affecting aposematic signalling.9.We have documented numerous aposematic signals Figs. –S922. ThWe assume that regional signalling diversity will be taken in account when specific lycid mimetic complexes are studied in the future. We found the highest number of aposematic patterns in the areas with very high alpha-diversity and abundance but low phylogenetic diversity , as well as in areas with moderate abundance but high phylogenetic diversity (South East Asia as the whole). The relative importance of these factors needs further study limited to a strictly defined community of interacting mimics and predators. The lycid colour patterns are so diverse in some regions that continuous migration could affect convergence to a common signal. Although lycids species often occur in multi-species communities, it is possible that some of them face a different spectrum of predators and so their patterns are a compromise, which indicates the presence of more complex conditions than the traditionally considered simple prey/predator pairs.Supplementary information.


Temporomandibular joint disorders (TMD) is common in clinic at present, which seriously affects the mental health and quality of life of patients. With the development of society, the incidence of TMD is gradually increasing. At present, there are many treatment methods, Tuina as a characteristic traditional Chinese medicine therapy, clinical treatment of TMD has a significant effect. In recent years, there are many clinical studies on Tuina in the treatment of TMD, but the clinical efficacy of Tuina in the treatment of TMD has not been systematically evaluated. In this study, we systematically evaluated the relevant literature of Tuina in the treatment of TMD by using the method of evidence-based medicine, in order to provide reference for clinical research in this direction in the future.VIP Chinese database, China knowledge Network, Wanfang, China Biomedical Database, PubMed, Embase, Cochrane Library and Web of Science were searched for clinical randomized controlled trials of Tuina in the treatment of TMD from the establishment of the database to December 2020. The 2 researchers independently screened the literature and carried out quality assessment and data extraction for the included study, and used RevMan5.3 software for risk assessment and Meta analysis.In this study, the efficacy and safety of Tuina in the treatment of TMD were evaluated by effective rate, visual analog score (VAS) of temporomandibular joint pain, dysfunction index ((DI), palpation index (PI), craniomandibular index (CMI), maximum mouth opening (MMO), incidence of adverse reactions and so on.This protocol can provide evidence-based basis for the treatment of TMD, with Tuina to significantly improve the symptoms and function of patients with TMD.DOI 10.17605/OSF.IO/J75A8. It is reported that the prevalence rate of TMD is about 25%, seriously affecting the quality of life of patients. The pathogenesis of TMD includes mental, psychological, traumatic, occlusal, immune and other factors, but its cause is still not completely clear. Pain in patients with TMD is the primary symptom of patients seeking medical treatment.–4 At present, there are many clinical treatment methods for TMD, and Tuina is one of the very important treatment methods.–6Temporomandibular joint disorder syndrome (TMD) is a group of diseases with clinical symptoms such as pain, bounce and limited opening of temporomandibular joint, involving temporomandibular joint or masticatory muscle system. It is a common disease in oral and maxillofacial region.–8 At present, clinical studies show that Tuina has a significant effect in the treatment of TMD, which is worth popularizing widely. In recent years, there are many clinical studies on Tuina in the treatment of TMD, but there is no related systematic review. The purpose of this study is to objectively evaluate the efficacy and safety of Tuina in the treatment of TMD, and to provide reference for the clinical research and application of Tuina on TMD.Tuina is a characteristic therapy of traditional Chinese medicine that uses hands or tools to treat patients’ body surface. It is widely used in clinic and has the effects of relieving pain, relieving muscle fatigue, promoting blood circulation and so on. Modern studies have shown that pressing the special position of patients with pain can have analgesic effect, and the analgesic effect is positively correlated with the content of β-endorphin in blood. Tuina can relieve pain by reducing the content of substance P (substance P), in blood and increasing the content of β-endorphin.22.1This protocol of systematic review and meta-analysis will be drafted under the guidance of the preferred reporting items for systematic reviews and meta-analysis protocols (PRISMA-P). It will be registered in the open science framework (OSF) on December 6, 2020 (registration number: DOI 10.17605/OSF.IO/J75A8).2.2Since this is a protocol with no patient recruitment and personal information collection, the approval of the ethics committee is not required.2.32.3.1We will collect and search all the randomized controlled trials (RCT) of Tuina for TMD, regardless of the region of publication and blindness, but the search language is limited to Chinese and English.2.3.2Patients who can be clearly diagnosed as TMD, including gender, time of onset, age are not limited.2.3.3The treatment group was treated with simple Tuina therapy or Tuina combined with other therapy, while the control group was treated with other therapy except Tuina.2.3.41.Primary outcome: the wound healing rate;2.Secondary outcomes: ①visual analog score (VAS); ②dysfunction index (DI); ③palpation index (PI); ④craniomandibular index (CMI); ⑤maximum mouth opening (MMO); ⑥incidence of adverse reactions.2.3.51.only one article is selected for repeatedly published or repeatedly cited literature;2.studies in which abstracts or full texts are not available;3.studies in which the data are obviously incorrect or missing;4.documents that do not indicate the author.2.4The key words of “temporomandibular joint disorder syndrome”, “temporomandibular disorder”, “temporomandibular joint disorder”, “Tuina”, “Message” and “manipulation” were searched in Chinese database, while “temporomandibular joint disorders syndrome”, “TMD”, “Tuina”, “Message” and “manipulation” were searched in English database. Including China knowledge Network, Chongqing VIP Chinese Sci-tech Journal full-text Database, Wanfang, China Biomedical Database, PubMed, Embase, Cochrane Library, Web of Science, etc., clinical randomized controlled trials of Tuina in the treatment of TMD from the establishment of the database to December 2020. Take PubMed as an example, the retrieval strategy is shown in Table 2.5Referring to the Cochrane system evaluation manual, according to the flow chart of PRISMA, and according to the inclusion and exclusion criteria of the design scheme, the 2 researchers used EndNote X7 software to independently screen and extract data from the retrieval literature. If there is a dispute over the inclusion of the study, the 2 parties shall negotiate with each other or be decided by a third researcher. The 2 researchers used a data information extraction table designed in advance to extract the research content, including 1 basic data: title, publication date, journal, author, 2 research characteristics: number of cases, general demographic characteristics, intervention measures, follow-up, adverse events, etc. 3Indexes of outcome: effective rate, VAS, dysfunction index (DI), muscle tenderness index (PI), temporomandibular joint index (CMI), maximum mouth opening (MMO), incidence of adverse reactions. The screening process is shown in Figure 2.6The bias risk assessment tool in Cochrane5.1.0 is used to evaluate the methodological quality of the included trials. The corresponding risk degree is judged by the 2 researchers from the aspects of Randomsequencegeneration, Allocationconcealment, Blindingofparticipantsandpersonnel, Blindingofoutcomeassessment, Incompleteoutcome Data, Selectivereporting and Otherbias, and the assessment results are cross-checked. If disputes are discussed with each other, if no agreement can be reached, it will be agreed with the third researcher. Finally, RevMan5.3 software is used to evaluate the bias risk of the study.2.7I2. When there is statistical homogeneity among the studies , the fixed effect model is used for meta-analysis. When there is statistical heterogeneity among the studies , the source of heterogeneity needs to be analyzed. After excluding clinical and methodological heterogeneity, random effect model is used for Meta analysis. Subgroup analysis was used to deal with clinical heterogeneity, and if subgroup analysis could not be carried out, only descriptive analysis was carried out.RevMan5.3 software was used for statistical analysis. The measurement data are expressed by mean difference (MD). When the units or measurement methods of the same effect index are not unified, they are expressed by standardized mean difference (SMD). The counting data use relative risk ratio (RR) as the statistical quantity of curative effect, and the 95% confidence interval (95%CI) is calculated. Heterogeneity test: Chi-Squared test was used, and the test statistic was 2.7.1If there is a lack of data in the study, contact the author through the mailbox to obtain the relevant data. If there is no contact with the researcher or the research data has been lost, only descriptive analysis will be carried out.2.7.2According to the treatment group simple Tuina treatment of TMD and Tuina combined with other treatments for subgroup analysis; according to the course of treatment for subgroup analysis.2.7.3In order to ensure the stability of the outcome index results, the sensitivity analysis of each outcome index was carried out.2.7.4If more than 10 articles were included in the outcome index literature, the funnel chart was used to evaluate the publication bias. In addition, Egger and Begg test were used for the evaluation of potential publication bias.3–12 It is reported that Tuina can reduce inflammatory factors such as tumor necrosis factor α and interleukin-6, increase the content of dopamine and β-endorphin, and relieve fatigue, inflammation and pain by eliminating lactic acid and blood urea nitrogen in the blood.–15According to the traditional theory of traditional Chinese medicine and the guidance of modern anatomy and physiology, Tuina applies various techniques to patients meridians and diseased parts to achieve the purpose of treating diseases. Relevant clinical studies have shown that Tuina manipulation can promote blood circulation, reduce blood pressure by increasing the activity of sodium pump in the blood, and Tuina can also regulate the immune function of the human body as a whole through neuro-endocrine-immunity; Tuina can repair injured skeletal muscle cells. When the temporomandibular joint opening movement, it will produce pain or pain aggravation, resulting in limited opening of the patient. According to the disease spectrum study of Tuina, musculoskeletal disease is the dominant disease of Tuina, and Tuina has obvious curative effect on muscle pain, joint disorder and other symptoms. Therefore, for different types of TMD, different Tuina techniques can be used for targeted treatment to improve clinical symptoms. Relevant clinical studies have shown that Tuina can significantly relieve the pain of TMD patients, the number of joint bouncing, and restore the degree of opening of patients.–20The main symptoms of TMD include pain, joint bouncing and limited opening. Among them, pain mainly comes from joint trauma, inflammation, joint disc displacement and so on, involving muscle, fascia, bone joint and other tissue structure.Tuina has a good effect on TMD. And Tuina has the characteristics of simple operation and less pain, so it is very suitable for TMD, which is easy to have repeated attacks. At present, there are many clinical studies on Tuina in the treatment of TMD, but its clinical efficacy has not been systematically evaluated, so it is necessary to objectively evaluate the efficacy and safety of Tuina on TMD. Due to the influence of the quality and quantity of the literature included in the study, this study still has its limitations, and because of language ability, search is only included in Chinese and English studies. Therefore, it is necessary to have a large sample and multi-center.Data curation: Yongchao Li, Lirong Zeng.Funding acquisition: Wenjuan He.Funding support: Wenjuan He.Resources: Wei Sun, Ni Wei.Software: Lirong Zeng, Wei Sun.Supervision: Ni Wei, Hui Xie.Writing – original draft: Jing Chen, Yongchao Li.Writing – review & editing: Jing Chen, Wenjuan He.